?■ '"K JOURNAL OF SHELLFISH RESEARCH VOLUME 21, NUMBER 1 JUNE 2002 The Journal of Shellfish Research (formerly Proceedings of the National Shellfisheries Association) is the offlcial publication of the National Shellfisheries Association Editor Sandra E. Shumway Department of Marine Sciences University of Connecticut Groton, CT 06340 Standish K. Allen, Jr. (2002) Aquaculture Genetics and Breeding Technology Center Virginia Institute of Marine Science College of William and Mary P.O. Box 1346 Gloucester Point, Virginia 23062 Shirley Baker (2004) University of Florida Department of Fisheries and Aquatic Sciences 7922 NW 71- Street Gainesville, Florida 32653-3071 Brian Beal (2004) University of Maine 9 O'Brien Avenue . .»v..^«v-^---- " ■ Machias, Maine 04654 , . Peter Beninger (2003) Laboratoire de Biologic Marine Faculte des Sciences Universite de Nantes : BP 92208 ■ • . 44322 Nantes Cedex 3, France Andrew Boghen (2003) Department of Biology University of Moncton Moncton, New Brunswick Canada EIA 3E9 Neil Bourne (2003) Fisheries and Oceans Pacific Biological Station Nanaimo, British Columbia Canada V9T 6N7 Andrew R. Brand (2003) University of Liverpool Port Erin Marine Laboratory Port Erin, Isle of Man IM9 6JA United Kingdom EDITORIAL BOARD Eugene Burreson (2003) Virginia Institute of Marine Science P.O. Box 1346 Rt. 1208 Create Road College of William and Mary Gloucester Point, Virginia 23062 Peter Cook (2002) Department of Zoology University of Cape Town Rondebosch 7700 Cape Town, South Africa Simon Cragg (2002) Institute of Marine Sciences University of Portsmouth Ferry Road Portsmouth P04 9LY United Kingdom Leroy Creswell (2003) University of Florida/Sea Grant 8400 Picos Road, Suite 101 Fort Pierce, Florida 34945-3045 Lou D'Abramo (2002) Mississippi State University Department of Wildlife and Fisheries Box 9690 Mississippi State, Mississippi 39762 Christopher V. Davis (2004) Pemaquid Oyster Company, Inc. P.O. Box 302 1957 Friendship Road Waldoboro, Maine 04572 Ralph Elston (2003) Aqua Technics/Pacific Shellfish Institute 455 West Bell Street Sequim, Washington 98382 Susan E. Ford (2002) Rutgers University Haskin Shellfish Research Laboratory 6959 Miller Avenue Port Norris, New Jersey 08349 Journal of Shellfish Research Volume 21, Number 1 ISSN: 0730-8000 June 2002 www.shellfish.org/pubs/jsr.htm Raymond Grizzle (2003) Jackson Estuarine Laboratory Durham, New Hampshire 03824 Karolyn Mueller Hansen (2004) 1524 Barley Circle Knoxville, TN 37922 Mark Luckenbach (2003) Virginia Institute of Marine Science Eastern Shore Lab P.O. Box 350 Wachapreague, Virginia 23480 Bruce MacDonald (2002) Department of Biology University of New Brunswick Saint John, New Brunswick Canada E2L 4L5 Roger Mann (2002) Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Islay D. Marsden (2002) Department of Zoology Canterbury University Christchurch, New Zealand Tom Soniat (2002) Biology Department Nicholls State University Thibodaux, Louisiana 70310 J. Evan Ward (2002) Department of Marine Sciences University of Connecticut 1080 Shennecossett Road Groton, Connecticut 06340-6097 Gary Wikfors (2002) NOAA/NMFS Rogers Avenue Milford, Connecticut 06460 Joiinuil of Slwllfiih Rfst'iirch. Vol, 21, No. 1. 1-2. 2002. Honored Life Member John B. Glude . . -lOlocficgi rar)Ofa(o--v ' VVooas HoO Oceanograprnc Insiiiution Libr.3ry JUL 1 8 2002 John Glude was born August 2. 1918 to William and Florence Glud of Silverdale. Washington. William Glud worked at the Bremerton Naval Shipyard and Florence worked as an elementary school teacher. Growing up in the Silverdale area with his brother Clarence, John got his first taste of aquaculture when his father had two ponds on the property where trout were raised. The fact that he was able to be involved with raising trout got him interested in aquaculture, which he continued to pursue and later became known for. Along with his skills related to farming, building, and raising fish, he helped his father create an innovative system for generating electricity by taking advantage of the changing elevation of the stream entering their property. A wooden sluice carried water from the stream to the pond. There the water was released, falling into cups on the paddle wheel they had constructed. Through a series of axles and gears, enough force was created to generate all the electricity they needed. They were the first in the area to have electric lights. John attended grade school and high school in Silverdale from which he graduated in 1935 as class salutatorian at the age of 16. He then entered the University of Washington received his Bachelor of Science degree in Fisheries in 1939, with a major in Fisheries and a minor in Engineering. He then got his first job with the Washington Department of Fisheries (WDF). but left briefly during Worid War II. He put his education in engineering to work by taking a job as a naval architect draftsman at the Tacoma Naval Shipyard. His work was considered vital to the war effort. After the war was over, he returned to his position as a Fishery Biologist with the WDF. He was then involved with research to determine the effects of polluted waters from pulp mills on oysters. This work formed the basis for regulatory actions to reduce sulfite liquor waste from pulp mills, which set the stage for assisting in the preservation of the valuable oyster resources in the state of Washington. After the war. John was sent to Japan to inspect sea oysters for export to the United States to prevent contamination and/or introduction of undesirable organisms to the west coast oyster growing areas. Cedric Lindsay was a colleague of John's with the WDF who also went along with him on some of the early trips to Japan to inspect seed oysters for importation to the United States. These trips ultimately led to major seed importation to the Pacific coast of the United States and thus maintained the major oyster fisheries until recent years when seed shipment from Japan was no longer necessary. There John learned a great deal about the various Japanese methods for culturing oysters and other species which he brought back to the United States to share with growers. In 1948, John was offered a position at the Woods Hole Oceanographic Institute in Woods Hole. Massachusetts. The main emphasis during that time was research on the abundance and survival of soft shell clams throughout the entire U.S. east coast. The research project was moved the following year to a former fish hatchery at Boothbay Harbor, Maine. He continued his research on sofishell clam populations and other species found in the regiim. During this time, he became lab director of the Boothbay Harbor facility and instituted 2 Honored Life Member: John B. Glude further research on artificial propagation of chinis and other species. Some of ihe earher efforts were relati\e to wild catches of soft shell clams, but were also concerned with hatchery setting. He also studied the effects of green crab predation and their control. The research during those years on the soft shell clam was ground-breaking and is still refened to extensively. John continued his career in the federal government with the U.S. Fish and Wildlife Service and the subsequent National Marine Fisheries Services (NMFS) under the National Oceanographic and Atmospheric Administration (NOAA). He later became director of the NMFS Laboratory in Annapolis. Maryland where he moved in 1956 with his wife Jean, daughter Nancy and son Terry. This laboratory was primarily engaged in research to develop methods for farming shellfish (main emphasis on oysters). With the lab's closure. John was offered a position in the national headquarters of NMFS in Washington. D.C. While there, he was in charge of the shellfish research branch of the NMFS and responsible for seven regional laboratories. At that time, he developed the first National Aquaculture Plan through NOAA. During President Kennedy's Administration. John was appointed to lead a team of fisheries experts to assist Ireland in improving their fisheries resources. He spent one year on this project and recommended many changes to the Irish Department of Fisheries which were implemented to improve the economic situation. Yearning to return to the northwest and the state of Washington. John accepted a position of Assistant Regional Director of the Northwest Region of the National Marine Fisheries Service, which was headquartered in Seattle, Washington. His family mined back to the state where John not only oversaw federal fisheries research in the area but also pushed to promote and implement the NOAA National Aquaculture Plan. Upon retirement in Seattle, John started the Glude Aquaculture Consultants. Much of his work involved aquaculture. and he was a primary consultant for numerous aquaculture projects in various countries. He continued to encourage aquaculture enterprises and eventually organized a program in Puerto Rico to test the applicability of known culture methods for fresh water prawns. John also acted as a consultant for the United Nations Food and Agriculture Organization where he headed a team of scientists to determine how to increase fishery resources and revenues in a number of developing countries. One project was entitled "The South Pacific Fisheries Investigation" through which recommendations were provided for best approaches to increa.se fisheries activities for the regions. John has been a life member of several professional organizations. He served as Vice President and President of the World Aquaculture Society in 1977 and 1978. respectively. He also served for two years as president of the National Shellfisheries Association in 1963-65. Along with this service to the societies, he has published over 100 scientific papers related to his many areas of shellfish research. John is retired now. but he is most certainly a pioneer in the fields of aquaculture and fisheries, best known for his research on clam and oyster culture. An avid sportsman, his love for fly-fishing and duck hunting is well recognized. I have been on many duck hunting trips with John and his brother-in-law Dick Steele in Dabob Bay. Hood canal in Washington State and know of his obsession with bird hunting. John now resides at 6101 River Crescent Drive. Annapolis. MD 21401. Dr. Kenneth Chew College of Ocean and Fishery Sciences University of Washington, Seattle. Washington Jniiriuil of Shellfish Resi-aivli. Vol. 21, No. 1. 3-12. 2002. GROWTH OF THE NORTHERN QUAHOG, MERCENARIA MERCENARIA, IN AN EXPERIMENTAL-SCALE UPWELLER CRAIG L. APPLEYARD AND JOSEPH T. DEALTERIS Depariineut of Fisheries. Aiiinicil unci VeteriiuiiY Scieiue, Universitx of Rliode Island, Kiuiiston, Rhode Island 02HH1 ABSTRACT Upwellers have proven to be extremely effective as bivalve nursery units and their use is steadily increasing in North America. The re-analysis of previous work by others suggests an asymptotic relationship between growth (9c volume increase per day) and chlorophyll-(i effective flow rate (the amount food flowing past a unit biomass of northern quahogs. |jLg per minute per liter of northern quahog volume). An experiment field study was conducted to define the relationship between food flow and bivalve stocking density. Furthermore, this study was designed to investigate other significant environmental parameters influencing bivalve grovMh in an experimental-scale upweller system. Northern quahog, Merceimria mercenuria (Linnel, seed were grown from -2 (longest axis) to -13 mm in an experimental-scale floating upweller from June 21 to August 19, 1999 (four separate experimental periods) in Point Judith Pond, Wakefield. Rhode Island. Flow rates and stocking densities were varied in order to produce a chlorophyll-t; effective flow rate range of 360 to 1.500 jjLg ■ niin ' • r', and growth and environmental parameters were measured semiweekly. During the first two-week experiment (June 21 to July 7) an asymptotic relationship was observed between growth (% increase/day) and chlorophyll-^ effective flow rate. A significant difference in growth was found between the treatments. The difference in the functional relationship between experiments 1 and 3 was possibly related to lower DO values, which reduced differential growth in experiment 3. In experiment 1. the low-biomass treatments grew faster than the high-biomass treatments. A significant difference in growth between treatments was also observed in experiment 3. although the asymptotic relationship was less pronounced. In experiment 3. the high-biomass replicates grew faster than the low-biomass replicates. Experiments 1 and 3 both experienced similar environmental conditions; however, experiment 1 encoun- tered higher morning dissolved oxygen (DO) levels. In addition, the within experiment variability in experiment 3 was much less than the variability in experiment 1 ; therefore, accentuating growth differences in experiment 3. In both experiments 1 and 3 maximum growth occurred near treatment 2 in a range of chlorophyll-o effective tlow rates of 550 to 650 jig ■ min"' ■ T'. In experiments. 2 and 4 there were no significant differences in growth between treatments. Growth appeared to be limited by low oxygen. In order to eliminate the effect of food limitation on growth, the upper third of the replicates (the fastest growing animals) were u.sed to calculate the relative growth rate (RGR) during the two-month experiment. Growth was linearly correlated with morning-dissolved oxygen (R- = 0.42) and with chlorophyll-d (R- = 0.35). The critical DO threshold for growth in upwellers appears to be 5 ppm. below which growth is adversely affected. During this study, morning DO levels were less than 50 % saturated, indicating the potential for DO levels to be increased. Future research should investigate methods for elevating DO levels in upwellers. KEY WORDS: northern quahog. Mercenariu mercenarm. upweller. growth INTRODUCTION Over the last decade, the use of tipvvellers as bivalve nursery units has increased dramatically in North America (Man/.i & Castagna 1989). A number of studies have explored the relation- ships between tlow rate, stocking density, and growth in upwellers (Hadley et al. 1999; Baldwin et al. 1995; Malinowski & Siddall 1989; Malinowski 1988; Manzi & Hadley 1988; Manzi et al. 1986: Manzi 1985; Hadley & Man/i 1984; Manzi et al. 1984; Baye.s 1981; Claus 1981; Manzi & Whetstone 1981; Rodhouse & O' Kelly 1981 ). The majority of research on upwellers has focused on the northern quahog. Mercenaria mercenaria, because of its significant aquaculture potential. In particular, the northern quahog grows well at high densities, has adapted to a variety of geographic sites along the northeast coast, and has a lucrative market. Manzi et al. (1986) described a qualitative relationship between flow rate, stocking density, and growth in an experimental-scale upweller. In their experiment, stocking densities were varied while flow rates were held constant. The tlow rate was converted to an effective flow rate by multiplying the amount of food (|jLg/l of chlorophyll-fl) by the flow rate (l/min). The amount of food pass- ing by a unit biomass of clams was defined as the chlorophyll-(/ effective flow rate (p,g ■ min"' ■ kg"' ). During a period of optimal northern quahog growth in the fall 1982 the authors found that a maximum biomass increase of 267% (over 30 days) occurred at the highest chlorophyll-a effective flow rate of 1.929 |xg ■ min"' ■ kg"' and the most efficient growth (213%) occuired at an intermediate chlorophyll-a effective flow rate of 476 |a.g • min" ' • kg" ' . If growth (% increase/day) is plotted as a function of chlorophyll-u effective tlow rate, the data is represented by an asymptotic relationship; in particular, as the chlorophyll-a effective flow rate increases, growth increases steeply and then levels off with increasing chlo- rophyll-o effective flow rates (Fig. 1). Efficiency in this upweller system refers to economically optimizing both upweller space (density) and pumping capacity (tlow). Theoretically, growth will be optimized at some percentage of the maximum growth rate; as indicated in Figure 1, 80 to 90% of the maximum growth rate equates to a chlorophyll-n effective tlow rate range of 470 to 700 pg ■ min"' • kg"'. Manzi et al. ( 1986) concluded that tood supply was the primary limitation in their upweller system. Their data suggests that to obtain unlimited growth, northern quahog seed needed to remove approximately 150 p.g ■ min"' kg"'. The investigators deduce thai northern quahog growth was reduced if more than 20% of the ambient chlorophyll-^; concentration (p-g/l) was removed as water passed by the bivalves. Consequently, to supply the necessary ration of 150 p.g • min"' ■ kg"' without exceeding 20% removal, food must be supplied to the bivalves at a rate of 750 |jLg • min"' • kg"'. Malinowski and Siddall (1989) confirmed that ambient chloro- phyll-(( concentrations were reduced by -20% through an initial Appleyard and Dealteris 14.0 -, 13.0 ^ 12.0 1' 11.0 i 100 1 9.0 £. 8.0 u « 7.0 of f ^° 2 5.0 - (5 ^ 4.0 - 1 3.0 - °^ 2.0- 1.0 0.0 ' 90% 80% ^^ / D 500 1000 1500 Chlorophyll-a Effective Flow Rate (ng«min ' ' mg 0,/l (Stanley & Dewitt 1983; Hamwi 1969). Although there is a general disagreement as to the limiting parameter for growth in upwellers. in the literature growth is clearly related to both system operating parameters (flow rate and stocking density) and environmental conditions at the site (tem- perature and dissolved oxygen). Given the environmental conditions m the study area, an ex- perimental field study was conducted as to: (1) Define a relationship between food flow, bivalve stocking density, and growth so as to determine the chlorophyll-o effective flow rate that optimizes growth; and (2) Determine the most significant limiting parameter for bi- valve growth in the upweller system. The experiment monitored growth of northern quahog seed in an experimental-scale floating upweller at three ranges of nomi- nal chlorophyll-(/ effective flow rates of 350. 600 and 1.400 p.g ■ min"' r'. In addition, environmental conditions were moni- tored at the site. MATERIALS AND METHODS Experimental Detign Growth of northern quahog seed was studied over an 8-week period in an experimental-scale floating upweller system located in a nutrient-rich estuary. At the beginning of the experiment the ambient chlorophyll-^ concentration (|jLg/l) was measured at the site, and flow rates and stocking densities were adjusted to achieve three nominal ranges of chlorophyll-t; effective flow rates, includ- ing a low (-350 iJLg • min ), medium (-600 jjLg ■ min I ), and high range (-1,200 p.g ■ min"' • P' ). Each combination of effective flow rate (|j.g/min) and northern quahog biomass (1) or chlorophyll-(( effective flow rate (p-g • min"' P' ) represents a treatment, as shown in Table 1. The average chlorophyll-c; con- centration during the time period was 1 1 .70 ± 2.06 jjig/1 (S.E.) and the flow rates were set at 4 1/min, 6 1/min, and 8 1/min resulting in three effective flow rates of 38.36 (ig/min, 57.84 p.g/min, and 77.12 |jLg/min. The northern quahog seed were initially slocked at a biomass of 0.055 1 (density of 0.3 I/cm") and 0.109 1 (density of 0.6 1/cnr) resulting in the desired range of chlorophyll-o effective flow rates. The experiment was a two (density) by three (effective flow rate) factorial design with six treatments of chlorophyll-a effective flow rates. Each treatment was replicated in triplicate resultina in 18 observations (silos). Growth of M. mercenaria in an Experimental Upweller TABLE 1. Chlorophyll-fl effective flow rates and their corresponding treatment. Initial Stocking Biomass Effective Flow Rate ((ig/min) (liter) Low High Low Treatment 4 Treatment 1 Medium Treatmem S Treatment 2 High Treatment 6 Treatment 3 Silc Location The experiment was conducted iit Rum Point Marina. Inc.. at the head of Point Judith Pond. Wakefield. Rhode Island (4I°25.57'N; 7r'2Q..S7'W) (Fig. 2). Ram Point Marina. Inc is located on a spit between Silver Spring Cove and the Upper Pond. The site was selected to take advantage of the relatively high and consistent phytoplankton biomass (chlorophyll-^ concentrations >10 M-g/l. Rheault 1993) during the summer months. The experi- mental-scale floating upweller was situated at the northernmost comer of the marina to ensure a water depth >1.4 meters at mean low tide. Upweller System An experitriental-scale floating upweller was designed and con- structed to ensure the control of flow through each silo. The up- weller unit was 4.27-m long, 1.22-tii wide, and 1.35 m deep. Two 25 cm inside diameter (ID) polyvinyl chloride (PVC) pipes were positioned at the base and ran the length of the unit forming a tnanifold. Ten 15-cm (ID) silos were plutnbed into the top of each manifold. Flow was provided by a half horsepower axial flow 41''25' ] Ram Point Marine Silver Sprir g Cove // Upper Pond ^ ^ m A r- Figure 2. Location of the experimental-scale upweller at Ram Point Marina, Point Judith Pond. Wakeneld. Rhode Island. Appleyard and Dealteris pump (Ice Eater. Power House) mounted in each manifold. Water was pumped along the manifold, tlowed up each 15-cm silo, and exited through an S-cm (ID) ball valve plumbed into the top of each silo. The seed were placed on a Nytex screen 0.5 m above the silo"s base. When the unit was in operation, each ball valve lay approximately 8 cm above the water line. Flow through each silo was manipulated with the ball valve and was measured volumetn- cally with a graduated cylinder and a stopwatch. Northern quahog seed (300,000 at 0.6 mm) were purchased from Bluepoints Company. Inc., West Sayville, New York. The seed were held in the upweller until they reached >2 mm (longest axis). Data Collection At the beginning of each experiment the seed were pulled from the unit, sieved, and randomly distributed throughout the 18 rep- licates at a biomass of 0.055 1 (wet volume) and 0.109 1. In addi- tion, the valve length of a random sample (/; = 75) of seed was measured to the nearest O.Oi mm with vernier calipers. Five sub- samples of northern quahogs were also taken to develop a rela- tionship between wet volume (1) and wet weight (kg). Each ex- periment was terminated when the biomass in the slowest growing replicate doubled. This occurred approximately every two weeks during the summer. At the termination of each two-week experi- ment, the valve length of a random sample (/; = 25) of northern quahogs from each replicate was determined. Four two-week ex- periments were completed during the summer 1999. The change in volume of each silo was measured semiweekly resulting in 3- to 4-day growth intervals. Semiweekly flow rates to each silo were also measured in the morning or late at night to minimize wave activity. Care was taken to ensure that the upweller unit was not altered during measurements and flows were adjusted accordingly. Chlorophyll-c( (Chl-((). particulate organic matter (POM), tem- perature, salinity, and dissolved oxygen (DO) were measured semiweekly from an empty silo. With the start of the second two- week experiment (July 7) all environmental parameters were taken in the morning, midday, and evening to quantify daily fluctuations at the .site. Discrete chlorophyll-<7 samples (» = 3) were taken with a syringe. Samples were pre-filtered with a 150 |jim Nytex screen to remove particulates that bivalves are unable to filter (Defossez & Hawkins 1997). Samples (10 ml) were forced through a 25-mm diameter Whatman GF/F filter contained in a 25 mm Swinnex fdter holder. The procedure for chlorophyll-a analysis is slightly modified from the standard procedure outlined in Strickland and Parsons ( 1972). Filters were dissolved in acetone for 24 hours and read on a Turner Designs tluorometer (Model 10-005R, Turner Designs. Inc., Sunnyvale, CA). All samples were conected for phaeophytin-a. One-liter samples were also taken (;i = 2) for POM analysis. The samples were pre-filtered on a 150 p.m Nytex screen and later analyzed in the laboratory. In the laboratory, samples were vacuum pumped through a pre-ashed 47 mm What- man GF/F filter (normal pore size O.I |xm), rinsed with isotonic ammonium formate, and dried in an oven at 11()°C for 24 to 48 hours. Filters were then ashed for >6 hours at 450"C in a muffle furnace. Filters were weighed on an Ohaus electronic balance (Model AS 120) to the nearest 0.1 mg. Temperature, salinity were measured with an YSI (Model 30) probe and oxygen was mea- sured with an YSI (Model 55) probe. The oxygen probe was cali- brated prior to each measurement. When measuring the change in volume of northern quahog seed, each silo and screen was cleaned with freshwater. Once a week the remainder of the upweller manifold was cleaned by a di\er to ensure consistent flow through the svsteni. Data Analysis The chlorophyll-K effective flow rate (p-g • min~' ■ 1"') for each replicate was calculated as the product of the average chloro- phyll-K concentration ((j,g/l) during the period and the flow rate (1/min) to the replicate all divided by the average biomass (1) of the replicate during the same period. This study characterized growth as the relative growth rate (RGR) and was calculated as: RGR = {[(Volume,,-,,,,, - Volume, „„„,,|,)/Volume, „„,,_,,, ] x 1001/#ofdays where volume is measured in liters. RGR is expressed as a per- centage increase per day (9f increase/day). POM (mg/1) was cal- culated as the difference between total suspended particulate mat- ter (SPM) and particulate inorganic matter (PIM). The Effecl of Food Limilalion on GroHtli To elucidate differences in growth between treatments the total RGR C^ volume increase) was divided by the longest period avail- able, the length of each experiment. Since the RGR (% increase/ day) measures the change in volume over each two- week experi- ment, the average chlorophyll-^ concentration and average treat- ment biomass during the time was used to calculate treatment chlorophyll-(( effective flow rates. Prior to ANOVA analysis, the RGR (Vc increase/day) was arcsine transformed (Sokal & Rohlf 1995). Within each experiment, one-way ANOVAs were per- formed for each two-week experiment with the average RGR (% increase/day) as the dependent variable and treatment as the inde- pendent variable. Differences between treatment means were elu- cidated with the Tukey Honestly Significant Difference (HSD) test. When the one-way ANOVA proved significant, a within ex- periment two-way ANOVA was performed to funher investigate the effective flow rate and density as independent variables. Again, the Tukey HSD test was used to verify differences in means. The strength of the relationship was characterized by the standard omega-squared (w"), when appropriate. The to" was calculated as 0)= = [SSt;„eu - dfs,,x (MSs,,Ol/SS-r -I- MSs,a where SS^,,,.^,, is the sum of squares of the effect, dfs,^ is the degrees of freedom for the eiTor tenn, MS^/.^ is the mean squares for the error term, and SS,- is the sum of .squares total. A between experiment one-way ANOVA was performed to elucidate growth differences between the two-week experiments. The Tukey HSD test quantified differences between means. The Effecl of Environituiital Characteristics on Growth In order to illustrate the effect of environmental parameters on northern quahog growth, growth was characterized at the finest possible scale. In particular, RGR was calculated for each 3 to 4 day period (growth inter\al) between semiweekly volume deter- minations. The daily value of each environmental parameter was averaged over the concurrent growth interval. To eliminate the effect of food limitation on growth, RGR of the upper third of the replicates (upper 1/3 RGR) was calculated. A linear regression analysis (S.AS Institute, Inc.) of the upper one-third RGR was performed with temperature, salinity, chloro- Growth of M. mercenaria in an Experimental Upweller phyll-o. and dissolved oxygen to determine which independent variables were significant in determining growth. Dissolved oxy- gen concentrations were converted to percent saturation based on temperature and salinity measurements (Benson & Krause 1984). A step-wise linear regression analysis was also performed to elu- cidate the most significant parameter(s) for predicting growth in the experiment. RESULTS The Effect of Food Limitation on Growth There was no observed mortality during the course of the two- month experiment. Calculated mortality was extremely variable in experiments 1 and 2 because counts per ml were not replicated. In addition, counts were not made before and after sieving. In ex- periments 3 and 4, counts per ml were replicated (n = 3) and counts were made before and after sieving. Mortality was calcu- lated to be 1 11 ± 3<7f (S.E.) and 99 ± 2<7(- (S.E.) respectively. The first experiment began on June 21 and ended on July 7. 1999 ( 16 days) and the northern quahogs grew from 3.1 1 ± 0.06 mm (S.E.) to 3.95 ± 0.05 mm (S.E.). The average chlorophyll-(7 concentration was 16.42 ± 2.25 p,g/l (S.E.) and the average treatment biomass ranged from 165.8 to 85.5 ml. The chlorophyll-a effective flow rate ranged from 420 to 1,445 |ji.g • min ' • P' roughly correlating with a RGR from 4.76 to 9.32"* increase/day. As the chlorophyll-i;/ effecti\e flow rate increased, the RGR increased until -650 IJLS ■ inin"' ■ r' at which point growth leveled off (Fig. 3a). Growth, as measured by RGR. was subjected to a one-way ANOVA with six levels of treatment. This was found to be sta- tistically significant (f (5, 1 1) = 5.48, P < 0.05). The strength of the relationship was 0.57 as indexed by the standard omega- squared (aj~). The Tukey HSD test indicated that the mean RGR for treatment 1 (M = 5.54) was significantly lower than the means for treatment 4 (M = 8.14), 5 (M = 8,41), and 6 (A/ = 9.26). To investigate the effect of effective flow rate and biomass on growth, a two-way ANOVA was performed with three levels of seston flux and two levels of volume. Both effective flow rate (f (2, 11) = 5.13. P < 0.05) and biomass (F (\, II) = 13.36, P < 0.05) were statistically significant. The strength of the relationship (io~) was 0.21 and 0.31. respectively. The interaction between effective flow rate and biomass was found ordinal; therefore, the main effects were examined by the Tukey HSD test. The Tukey HSD test indicated that the low-biomass treatments (M = 8.60) grew faster than the high-biomass treatments (M = 6.99). The second experiment began on July 7 and ended on July 22, 1999 ( 15 days) and the northern quahogs grew from 3,46 ± 0.1 1 mm (S.E.) to 6.28 ± 0.07 mm (S.E.). The average chlorophyll-fl concentration was 1 1.83 ± 1.16 \i.°l\ (S.E.) and the average treat- ment biomass ranged from 205.6 ml to 91 .6 ml. The chlorophyll-a effective flow rate ranged from 231 to 977 jjig • min~' • 1"' roughly correlating with a RGR from 9.58 to 12.73 % increase/day (Fig. 3b). RGR was consistently high within the chlorophyll-a effective flow rate range specified. RGR was subjected to a one-way ANOVA and there was no statistical difference between treat- ments (f (5, 11) = 1.48. P > 0.05). The third experiment began on July 22 and ended on August 5. 1999 (14 days) and the northern quahogs grew from 7.04 ± 0. 1 1 mm (S.E.) to 9.96 ± 0.07 mm (S.E.). The average chlorophyll-^^ concentration was 18.55 ± 2.12 |j,g/l (S.E.) and the average treat- ment biomass ranged from 184.8 to 84.6 ml. The chlorophyll-c( effective flow rate ranged from 411 to 1,720 p.g ■ min"' ■ P' roughly corresponding to a RGR from 7.79 to 10.09 % increase/ day (Fig. 3c). The RGR increased slightly with an increase in the chlorophyll-(7 effective flow rate until -610 |a.g ■ min~' 1"', at which point growth decreased and leveled off RGR was subjected to a one-way ANOVA and was found to be statistically significant (f (5, 11) = 7.13, P< 0.05). The strength of the relationship was 0.64 as indexed by the u)". The Tukey HSD test indicated that the mean RGR for treatment 2 (M = 9.76) was significantly higher than the means for treatment 4 (M = 7.97), 5 (M = 8.62), and 6 (M = 8,38). In addition, the mean RGR for treatment 4 (A/ = 7.97) was significantly lower than the mean for treatment 3 {M = 14 - 13 - 12 - s 11 - «♦♦ ♦ b a^io- ♦ ♦ ♦ S S 6- li 5- ^ 3 - 2 - 0 - ) 500 1000 1500 2000 ChlorophyU- Effective Flow Ratcoigxmm'xr'l 14 n Relative Growth Rale (% Increase/day) % ♦ % % ♦ d 0 ) 500 1000 1500 2000 Chlorophyll-a Effective Flow Raie (pg x nun xr') Figure 3. Growth ( 'H increase/day) as a function of chlorophyll-o effective flow rate for (a) experiment 1 (June 21 to July 7, 1999 with an average chlorophyll-a concentration of 16.42 ± 2.25 (ig/l S.E.): (b) experiment 2 (Juh 7 to July 22, 1999 with an average chlorophyll-a concentration of 11.83 ± 1.16 (ig/l S.E.): (c) experiment 3 (July 22 to August 5, 1999 with an average chlorophyll-u concentration of 18.55 ± 2.12 fig/l S.E.); and, (d) experiment 4 (.\ugust 5 to ,\ugust 19, 1999 with an average chlorophjil-a concentration of 17.91 ± 3.17 pg/1 S.E.). Appleyard and Dealteris 9.20). A two-way ANOVA found both effective flow rate (F (2. 11) = 5.99, /'< 0.05) and bioniass(F(l, II) = 22.33. P < 0.05) were statistically significant. The strength of the relationship (to") was 0.21 and 0.45. respectively. The interaction between effective flow rate and biomass was found ordinal; therefore, the main ef- fects were examined by the Tukey HSD test. The Tukey HSD test indicated that the high-biomass treatments (M = 9.25) grew faster than the low-biomass treatments (W = 8.32). The Tukey HSD test also found that the replicates with an effecli\e flow rate of 1 1 1.3 jig/min [M = 9.19) grew faster than the replicates with an effec- tive flow rate of 74.2 |xg/min (M = 8.87). The fourth experiment began on August 5 and ended on August 19, 1999 (14 days) and the northern quahogs grew from 9.37 ± 0.12 mm (S.E.) to 11.47 ± 0.08 mm (S.E.). The average chloro- phyll-fi concentration was 17.91 ± 3.17 ^ig/l (S.E.) and the average treatment biomass ranged from 147.4 to 73.4 nil. The chloro- phyll-(( effective flow rate ranged from 491 to 1.905 |jLg ■ min^' ■ P' roughly conesponding to a RGR from 4.98 to 5.96% increase/day (Fig. 3d). RGR was consistently low within the chlorophyll-o ef- fective flow-rate range specified. RGR was subjected to a one-way ANOVA and there was no statistical difference between treat- ments (f (5. 11) = 0.76. P > 0.05). A one-way ANOVA was performed to compare the RGR be- tween the two-week experiments. This was statistically significant (f (3.64) = 135.34./'<0.05)with an a)-of0.80. The Tukey HSD test indicated that there was a significant difference between all the mean RGRs. with growth highest in experiment 2 (M = 11.89) and decreasing in experiments 3 (M = 8.81). 1 (M = 7.71). and 4 (M = 5.57). The Effect of Einironmeittal Charcuteristics on Growth The upper one-third RGR varied considerably during the course of the experiment from a high of 10.37 ± 0.43% increase/ day (S.E.) on June : low of 5.03 ± 0.41% increase/dav (S.E. on June 29 (Fig. 4a). During the course of the two-month experi- ment. RGR decreased sharply (June 22 to June 29). then increased (June 29 to July 17), and then gradually decreased (July 17 to August 19). Temperature during the experiment varied from 21.4 to 27.3°C (Fig. 4h). Other than a brief drop in temperature in mid July due to a rainstorm, temperature was consistent during the experiment. A linear regression analysis indicated that temperature was not sig- nificant in determining growth as indicated by the upper one-third RGR (F (I. 15) = 0.58. P > 0.05). Similariy, salinity during the experiment was relatively consistent ranging from 21.4 to 29.9%t (Fig. 4c). Linear regression analysis determined that salinity was not significant in predicting the upper one-third RGR (F(l, 15) = 0.70. P > 0.05). The seston concentration, as indexed by the chlo- rophyll-(( concentration, varied substantially during the course of the experiment from peaks of 21.2 ± 1.23 jxg/l (S.E.) on June 28 and 22.8 ± 0.86 |xg/l (S.E.) on July 3. to a nadir of 9.5 ± 0.09 p.g/1 (S.E.) on July 13 (Fig. 4d). Generally, as chlorophyll-(7 concentra- tions increased growth decreased. Similariy. as chlorophyll-o con- centrations decreased growth increased. Linear regression analysis indicated that this trend was significant (f (1. 15) = 7.46. P < 0.05) with a correlation coefficient (R-) of 0.35 (Fig. 5a). There was a minimal amount of variability between chlorophyll-70 i v^r^^x.] 25 70 1 S V ! 60 . \ /^ ^ "♦ ^2 50 v/ "3 - £40 1 30 — * — Upper 1/3 RGR 10 20 10 A Morning Salinity 6/20 im 6/28 7/2 7/6 7/10 7/14 7/18 7/22 7/26 7/30 8f! 8/7 8/11 8/15 8/19 DK A "°1 a 100 90 \ ■ -A .- . 250 -20-0 1 ^° S u 1 60 SS|,„ S £40 ■ ■ 15.0 i 100 _ 3 1 30 2.0 1-0 — • — Upper 1/3 RGR ■ Chlorophyll-a 50 6/ 20 fM Wi 7/2 7/6 7/10 7/14 7/18 7/22 7/26 7/30 8(3 sn 8/11 8/15 8/19 Dale 110 9.0 c 100-2 A 8.0 90 - 1 J70 1 '-:: ^ .^ '^■^.■T^} 60| V... •• ••• 40B 1 3.o| r 20^ 2.0 to • Morning DO ''° 6/20 6/24 W28 7/2 7/6 7/10 7/14 7/18 7/22 7/26 7/30 8/3 8/7 8/11 8/15 4^19 Dale Figure 4. Relative growth rate (% increase/day) for the upper third of rephcates (a) from June 21 through August 19, 1W» with (b) morning temperature ( Cl; (cl morning saHnity ['it); (dl morning chlorophyll-a l(ig/ll; and, (el morning dissolved oxygen (ppml. This trend is supported by the Tukey HSD test, which indicated that treatment 2 grew significantly faster than treatments 4. 5. and 6. In addition, treatment 3 grew significantly faster than treat- ment 4. Since treatment 3 and treatment 4 have nearly the same chlorophyll-a effective flow rate, a significant difference in growth indicates an effect of biomass on growth, with the higher bio- mass treatment growing faster that the lower biomass treatment. The two-way ANOVA also found a significant effect of biomass on growth with the high-biomass replicates growing faster that the low-biomass replicates. The two-way ANOVA also indicated a significant effect of seston flux on growth with the replicates with the intermediate effective flow rate of 1 1 1.3 pig/min growing faster that the replicates with the lower effective flow rate of 74.2 p.g/ min. Experiments 1 and 3 both experienced initially high morning DO values that decreased during the course of the experiment. Experiment 1 had higher initial morning DO values (8-7 ppni) than experiment 3 (5.5-6 ppm). The chlorophyll-o concentration also increased substantially during both experiments, with values peaking at 21.19 ± 1.23 |jLg/l (S.E.) in experiment 1 and 22.83 ± 0.86 jjig/1 (S.E.) in experiment 3. In addition, both experiments experienced the same range of chlorophyll-(v effective flow rates. The difference in the relationship between growth and chloro- phyll-(7 effective flow rate in experiment 1 and experiment 3 is probably the result of a number of factors. First, since experiment 3 did not experience the initially high morning DO levels observed in experiment 1, the treatments might not have had a chance to separate or grow differentially. Second, the spread in replicates in experiment 3 was considerably smaller than that in experiment 1 ; therefore, small growth differences between treatments in experi- ment 3 are essentially accentuated. In other words, the statistical difference between treatments in experiment 3 is a result of the relatively small within replicate variability. In both experiments 1 and 3. maximuin growth occurred near treatment 2 in a range of chlorophyll-a effective flow rates of 550 to 650 (j-g ■ min"' ■ I"'. In order to verify this result, growth should be investigated within the chlorophyll-a effective flow rate range of 0 to 500 p.g ■ min"' ■ 1"'. The benefit of defining a relationship between growth and the amount of food passing by a unit biomass of animals (chlorophyll- a effective flow rate) is apparent in the application to other grow- ers. The relationship can be easily applied to upwellers in a variety of locations, provided optimal environmental conditions persist. An aquaculture extension agent could characterize the water con- ditions at a site to determine that the minimum water quality stan- dards are met, such as temperature, salinity, and dissolved oxygen. The agent could then measure the amount of chlorophyll-a and estimate the average food concentration at the site. With this es- timate, the grower could determine the biomass and effective flow rate needed to optimize growth in the upweller. The Effect of EnviroiimeiUal Characleristics on Growth When environmental conditions were suitable for northern qua- hog growth, especially in the beginning of experiment 1, the effect of food limitation on growth was apparent. When environmental conditions were less than optimal, as in experiments 2 and 4, growth appears constant over a wide range of chlorophyll-a effec- tive tlow rates. In other words, growth was not controlled by food limitation, but some other factor. To quantify the effect of envi- ronmental conditions on growth, the upper one-third of replicates, the fastest growing northern quahogs, were used to determine 10 Applevard and Dealteris i (3 <2 ! 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 y = -0.1939x+ 10.436 03467 5.0 7.0 90 11.0 13.0 15.0 170 Chlorophyll-a (tig/1) 19.0 21.0 23.0 25.0 11.0 1 10.0 ♦ y = 0.7527x + 3.7366 ^ R^ = 04186 ♦ S 9.0 ♦ ^^^..^-""'''''''''^ r 1/3 Relative Growl (% increase/day) OS -J 90 ♦ S 5.0 ♦ 4.0 3 0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 70 7.5 8.0 85 Morning Dissolved Oxygen (mg O2/I) Figure 5. Linear regression of the relative growth rate (% increase/dayl for the upper third of replicates from June 21 through August 19, 1999 versus lal chlorophyll-o ((ig/l) and (b) morning dissolved oxygen Ippnil. growth. By eliminating the slowest two-thirds replicates, the effect of food limitation on growth was minimized; therefore, differences in growth were constrained by the en\ironmental conditions at the time. Manzi et al. (1986) concluded that food limited growth in their expenmental-scale upweller. Although there were signs of food limitation on growth in experiments 1 and 3. growth in experi- ments 2 and 4 were controlled by other factors. Malinowski and Siddall (1989) detemiined that the How rate limited growth in their upweller system. They surmise that flow through the upweller had to be above a critical threshold in order to create a uniform flow (distribute food evenly among the clam seed), maintain water qual- ity, remove wastes, and provide a sufficient chlorophyll-^ concen- tration to the northern quahogs. Although Malinowski and Siddall (1989) were unable to quantify the effect of water quality on growth, they eluded to the importance of environmental conditions on growth. 0\er the course of the iwo-month experiment, growth was positively correlated with morning DO and negatively correlated with chlorophyll-((. In late June and early July, the experimental site at Point Judith Pond experienced a pronounced algae bloom. The bloom was evident as an increase and peak in the chloro- phyll-o concentration (Fig. 4d). There was a clear relationship between chlorophyll-i; and morning DO. specifically as the chlo- Growth of M. mercenaria in an Experimental Upweller rophyll-<; concentration increased, morning DO levels decreased (Figs. 4d and e). The decrease in morning DO was a result of a combination of algae decomposition and algae respiration. At night, the algae were constantly respiring, converting captured energy into simple sugars, an o.xygen consuming and carbon di- oxide producing process. The algae were also continually dying off and decomposing, again an oxygen consuming process. A second algae bloom in the upper pond was apparent in mid August. Again, the same relationship between chlorophyll-i; and morning DO was apparent. In late July, the chlorophyll-i; concentration decreased substantially and morning DO levels increased. This decrease in chlorophyll-(( was most likely a result of zooplankton grazing de- scribed by Bengtson (1982). Alternatively, the decrease in chlo- rophyll-a could have been caused by a crash or die off of a par- ticular species of algae. The cychc pattern of algae in the upper pond could be further verified by quantifying the species of algae present as well as the amount of zooplankton at the study site. The relationship between primary productivity (chlorophyll-o concentrations) and low dissolved oxygen in shallow coastal and estuarine areas has received considerable attention, as this phe- nomenon appears to be increasing on a global scale. Diaz and Rosenberg (1995) reviewed the diversity of research characteriz- ing low DO events in the Chesapeake Bay. They found that the cause and ecological consequences of these events varied from tributary to tributary. Regardless of the specific controlling mechanisms, an increase in algae bioinass caused a distinct decrease in morning DO (<5 ppm) resulting in depressed clam growth. The relationship be- tween low DO and depressed clam growth has not been charac- terized in the literature: however, a number of researchers have successfully characterized changes in stress levels (Sparks & Strayer 1998) and predator behavior (Taylor & Eggleston 2000; Tallqvist 2001 ) of bivalves exposed to low oxygen concentrations. Hamwi (1969) determined that Mercenaria mercenaria were able to maintain a constant rate of respiration with decreasing oxygen levels until 5 ppm. The northern quahog is a classic oxygen regu- lator (Hamwi 1969), As the oxygen concentration decreases, bi- valves can increase their rate of oxygen consumption through two mechanisms: (1) increasing their pumping rate: or (2) increasing their percentage of oxygen utilization. Hamwi (1969) determined that the pumping rate of northern quahogs remained constant with decreasing oxygen concentrations; however, northern quahogs were able to regulate O, consumption by increasing the percentage of oxygen utilized. When oxygen levels reached 5 ppm or below, Hamwi (1969) found that oxygen uptake in northern quahogs de- creased continuously and an oxygen debt was incurred. Once con- ditions were favorable, the oxygen debt was rapidly repaid in a matter of hours and northern quahogs were able to function nor- mally. Although juvenile northern quahogs can survive in oxygen con- centrations below 1 ppm for up to three weeks (Stanley & Dewitt 1989). 5 ppin is the critical threshold for northern quahog growth. There have been a number of studies that have investigated the effect of low oxygen levels on survival and tolerance, yet none have investigated the effect of low oxygen levels on growth. Based on the work completed by Hamwi (1969). 5 ppm is the critical threshold for northern quahog growth. When oxygen concentra- tions fall below 5 ppm. the northern quahogs cannot maintain sufficient oxygen uptake and incur an oxygen debt. In essence, the northern quahogs shut down and stop growing until oxygen levels rise above this critical threshold. The results of this study stress the importance of sufficient oxygen concentrations for northern quahog growth in upweller systems. A number of methods could be used to ensure optimal oxygen levels in an upweller. The upweller could be moved to a site that experiences lower chlorophyll-(( values and higher morn- ing DO values, but food for the northern quahog would be com- promised. Alternatively, the oxygen concentration in the upweller could be increased. During periods of low morning DO (<4 ppm). the '7c saturation was below 60: therefore, during periods of low morning DO. oxygen concentrations have the potential of being increased. Future research should investigate the most cost effec- tive and efficient method of increasing dissolved oxygen levels in this upweller as well as in the more traditional passive flow up- wellers. With optimal DO levels, the effect of food limitation on growth can be further defined and replicated. SUMMARY AND CONCLUSIONS The hypothesized relationship between growth and chloro- phyll-fl effective flow rate was only apparent during the first two- week experiment (experiment 1). Although there were significant differences in growth between treatments in the third two-week experiment (experiment 3). these differences were most likely the result of small within sample variability. For the remainder of the experiment, northern quahog growth was limited by environmental conditions. Specifically, the relative growth rate of the upper one- third of the replicates was positively correlated with morning- dissolved oxygen (R" = 0.42) and negatively coirelated with chlo- rophyll-d (R" = O..^.^). The critical dissolved oxygen threshold for northern quahog growth in the experimental-scale upweller ap- peared to be 5 ppm. below which growth was adversely affected. Future research should investigate the most effective method for elevating DO levels in commercial floating upwellers. LITERATURE CITED Baldwin. R. B.. W. Mook. N, H. Hadley. R. J- Rhodes, & M. R. DeVoe. 1995. Construction and operations manual for a tidal-powered up- welling system. Charleston. SC: SC Sea Grant Consortium. Hayes. J. C. 1981. Forced upwelling nurseries for oysters and clams using impounded water systems. In: C. Claus. N. DePauw. & E. Jaspers, editors. Nursery culturing of bivalve molluscs. Eur. Maricull. Soc. Spec. Publ, Vol. 7. Belgium: Bredene. pp. 73-83. Bengtson. D. A. 1982. Resource partitioning by Menidia menidia (L.) and Menidia berylUna (Cope) in two Rhode Island estuaries. PhD disser- tation. Kingston: University of Rhode Island. 214 pp. Benson. B. B. & D, Krause. Jr, 1984. The concentration and isotopic fractionation of oxygen dissolved in freshwater and seawater in equi- librium with the atmosphere. Limnol. Oceanogr. 29:620-632. Castagna, M. & J. N. Kraeuter. 1981. Manual for growing the hard clam. Mercenaria. Special Report in Applied Marine Science and Ocean En- gineering No. 249. Gloucester Point. Virginia: Virginia Institute of Marine Science. Claus. C. 1 98 1. Trends in nursery rearing of bivalve molluscs. In: C. Claus. N. DePauw. & E. Jaspers, editors. Nursery culturing of bivalve mol- luscs. Eur. Maricult. Soc. Spec. Publ. Vol. 7. Belgium: Bredene. pp 1-33. Defossez. J. M. & A. J. S. Hawkins. 1997. Selective feeding in shellfish; size-dependent rejection of large particles within pseudofeces from 12 Appleyard and Dealteris Mytihis echilis. Riiditapes philippinanim and Tapes deciissalus. Mar. Biol. 129:139-147. Diaz. R. J. & R. Rosenberg. 1995. Marine benthic hypoxia: a review of its ecological effect and the behavioural responses of benthic macrofauna. Oceanogr. Mar. Biol. Anmi. Rev. 33:245-303. Hadley, N. H.. R. B. Baldwin. M. R. Devoe. & R. Rhodes. 1999. Perfor- mance of a tidal-powered upwelling nursery systein for northern qua- hogs (hard clams) (Mereeiiaria inercenuriu) in South Carolina. J. Shell- fish Res. 1 8:555-560. Hadley. N. H. & J. J. Manzi. 1984. Growth of seed clams. Mercenaria mercenaria. at various densities in a commercial scale nursery system. Aquaculture 36:369-378. Hamwi. A. 1969. Oxygen consumption and pumping rate of the hard clam Mereeiiaria mercenaria L. PhD dissertation. New Brunswick. New Jersey: Rutgers. The State University. 177 pp. Malinowski. S. M. 1988. Variable growth rales of seed clams. Mercenaria mercenaria (Linnel in an uptlow nursery system and the economics of culling slow growing animals. J. Shellfish Res. 7:359-365. Malinowski, S. M. & S. E. Siddall. 1989. Passive water reuse in a com- mercial-scale hard clam, Mercenaria inercenaria. Lipl1ov\ nursery sys- tem. / Shellfish Res. 8:241-248, Manzi. J. J. 1985. Clam aquaculture. In: J. V. Hunter & E. E. Brown, editors. Crustacean and nioUusk aquaculture in the United States. West- port, CT. AVI Publishing Company, pp. 275-310. Manzi. J. J. & M. Castagna. 1989. Nursery culture of clams in North America. Pages 127-147. In: J. J. Manzi & M. Castagna. editors. Clam Marieulture in North America. New York: Elsevier. 461 pp. Manzi. J. J. & J. M. Whetstone. 1981. Intensive hard clam marieulture: A primer for South Carolina watermen. South Carolina Sea Grant Consort 29401. Charleston. SC: Marine Advisory Publication. 81-01. Manzi. J. J.. N. H. Hadley. C. Battey. R. Haggerty. R. Hamilton & M. Carter. 1984. Culture of the northern hard clam Mercenaria inercenaria (Linne). in a commercial-scale, uptlow. nursery system. / Shellfish Res. 4:119-124. Manzi, J. J., N. H. Hadley & M. B. Maddox. 1986. Seed clam. Mercenaria mercenaria. culture in an experimental-scale uptlow nursery system. AquuciiUiire 54:301-31 1. Manzi, J. J. & N. H. Hadley. 1988. Recent advances in nursery culture of bivalve moUusks in North America. In: A. K. Sparks, editor. New and innovative advances in biology/engineering with potential for use in aquaculture. NOAA Technical Report National Marine Fisheries Ser- vice (NMFS) 70. Seattle. WA: National Marine Fisheries Service, 98115. pp. 4I-t6. Rheault. R. B. 1995. Studies on food-limited growth in luvenile shellfish using novel aquaculture approaches. PhD Dissertation. Kingston, RI: University of Rhode Island. 508 pp. Rodhouse, P G. & M. O'Kelly. 1981. Flow requirements of the oysters Ostrea ediilis and Crassoslrea gigas Thunbberg. In an upwelling col- umn system of culture. Aquaculture 22:1-10. Sokal, R. R. & F. J. Rohlf. 1995. Biometry. New York: W. H. Freeman and Company. Sparks. B. L. & D. L. Strayer. 1998. Effects of low dissolved oxygen on juvenile EUiptio compUmatu (Bivalvia:Unionidae). J. N. Am. Benthos. Soc. 17:129-134. Stanley. J. G. & R. DeWitt. 1983. Hard clam. Species profiles: life histories and environmental requireinents of coa.stal fishes and invertebrates (North America)— hard clam. U. S. Fi.sh and Wildlife Service. FWS/ OBS-82/1 1.18. US Army Corps of Engineers, TR EL-82-4. Stanley. J. G. 1985. Hard clam. Species profiles: life histories and envi- ronmental requirements of coastal fishes and invertebrates (Mid- Atlantic) — hard clam. Biological Report 82 (11.41). Strickland, J, D. H. & T. R. Parsons. 1972. A practical handbook of seawater analysis. Fish. Res. Board Can. Bull. 167. Second Edition. 310 pp. Tallqvist, M. 2001. Burrowing behaviour of the Baltic clam Macoma bal- thica: effects of sediment type, hypoxia and predator presence. Mar. Ecol. Prog. Ser. 212:183-191. Taylon D. L. & D. B. Eggleston. 2000. Effects of hypoxia on an estuarine predator-prey interaction: foraging behavior and mutual interference in the blue crab Callinectes sapidus and the infaunal clam prey Mya arenaria. Mar. Ecol. Prog. Ser.. 196: 221-237. Journal of Shellfish Research. Vol. 21. No. 1. 13-14. 2(102. FLOW CYTOMETRIC MEASUREMENT OF HEMOCYTE VIABILITY AND PHAGOCYTIC ACTIVITY IN THE CLAM, RUDITAPES PHILIPPINARUM BASSEM ALLAM, KATHRYN A. ASHTON-ALCOX, AND SUSAN E. FORD* Haskin Shellfish Research Laboratory, Rutgers University, 6959 Miller Avenue. Port Norris. New Jersey 08349 ABSTRACT The assessment of blood cell viability and defense abilities is a major concern in the study ol pathological processes. In this work, we devised and validated flow cytometric assays to measure viability and phagocytic activity of hemocytes from the clam Ruditapes pliilippinanim. a species susceptible to the bactena-caused Brown Ring Disease (BRD). Validated assays were subsequently used to measure hemocyte parameters following experimental contamination with BRD's etiologic agent Vibrio tapetis. Results show that clams that developed BRD symptoms had lower phagocytic rates and a higher percentage of dead hemocytes than those that did not. Ill vitro interactions between hemocytes and V. lapetis demonstrated that clam hemocytes are able to phagocytose formalin-fixed I', tapetis. but at lower rates than latex beads. Live V. nipetis were able to kill clam hemocytes in vitro. The in vitro assay also showed that phagocytosis increased with increasing temperature from 8' to 21°C. This work demonstrated the efficiency of flow cytometry for measuring inolUiscan blood cell activities during host/pathogen interactions and points the way lor further experiments using this analytical tool. KEY WORDS: bivalve, bacteria, phagocytosis, hemocyte viability, flow cytometry. Ruditapes plulippiiiaruiii INTRODUCTION Changes in overall hemocyte activity have been observed in parasitized bivalves or those experimentally exposed to pathogens (Nottage & Birkbeck 1990; La Peyre et al. 1995; Anderson 1996; Garreis et aL 1996; Allam el al. 2000u, 2000b). Techniques used to investigate these changes generally have serious drawbacks. Mi- croscopy is subjective and involves a considerable expenditure of time, especially when large numbers of samples need to be evalu- ated. Spectrophotometric inethods are based on the measurement of activity in the whole sample and not on a cell-by-cell basis. Flow cytometry, however, has proven to be extremely useful in overcoming these problems. In the field of molluscan research, flow cytometry has been used to quantify phagocytosis (Alvarez et al. 1989; Brousseau et al. 2000; Allam et al. 2001; Foumier et al. 2001) and hemocyte viability (Ashton-Alcox & Ford 1998; Ash- ton-Alcox el al. 2000; Fournier et al. 2001 ). In flow cytometry, particles are passed single-file through a laser beam. The light scattered by the particles indicates their size and internal complexity. Fluorescence, whether aulofluorescence or from a fluorescent tag, is measured by specific detectors. Flow cytometric phagocytosis ineasurements usually involve the use of fluorescent particles that are detectable by the flow cytometer even after phagocytosis has taken place (Alvarez et al. 1989; Brousseau et al. 2000). Specific fluorescent dyes that indicate membrane integrity and permeability, intracellular redox potential, or enzy- matic activity are available and can indicate cell viability using flow cytometry (Coinbrier et al. 1989). Although molluscan hemocytes have been studied by flow cy- tometry before, validation of the results is rarely reported. In this work, we refined and verified by microscopy, two flow cytometry- based assays to measure the phagocytic activity and the viability of hemocytes in the clam Ruditapes philippiminim. These allowed us to investigate hemocyte activity in clams experimentally infected with Vibrio tapetis. the bacterial agent of Brown Ring Disease (BRD) in Europe (Paillard et al. 1994). We further studied the ♦Corresponding Author. Phone: -h1-8.^6-78.')-0074; Fax: -I- 1-856-785- 1.^44; E-mail: susan@hsrl.rutgers.edu effect of in vitro contact between the pathogenic bacterium and hemocytes on hemocyte viability, and compared the uptake of V. tapetis with that of similarly sized fluorescent beads. We also investigated the effect of temperature on phagocytosis because temperature is strongly associated with the prevalence and inten- sity of BRD in nature and under experimental conditions (Paillard et al. 1994; Allam 1998). MATERIALS AND METHODS Experimental Animals Ruditapes philippinaruin were obtained from 2 locations: southern Puget Sound. Washington. USA (length = .39.4 ± 0.46 mm, mean ± SEM). and the Bay of Brest, Brittany. France (length = 37.7 ± 0.6.'i mm). Clains were shipped overnight to the labo- ratory where they were immediately placed in quarantine, aerated 35-L standing-water tanks (about 35 clams per tank al 1 3°C and 34 ppt), in which they were kept during the remainder of the study. Clams were fed daily throughout the experiments using a mixture of cultured algae. All the experiments reported here were con- ducted using clams from the USA, where BRD has never been reported, except for the infection experitiient. which was done using French clams. Clams affected with BRD were obtained by challenging them with V. tapetis as previously described (Allam et al. 2000a). Briefly, a 0.5 ml (5 x 10^ bacteria) aliquot of a suspension made with exponentially growing V. tapetis (ATCC 4600, strain PI 6) was inoculated into the pallial cavity of each experimental clam. Control clams were inoculated with the same volutne of sterile seawater. After 4 weeks of incubation under the conditions de- scribed above, hemolymph was collected and processed for phago- cytosis and viability assays. The clams were then shucked and BRD development on shells was recorded (Paillard & Maes 1994; Allam et al. 2()(IOa). Hemolymph Sampling Animals and hemolymph samples were kept on ice during pro- cessing to avoid hemocyte clumping. Hemolymph was withdrawn from the posterior adductor muscle as described by Auffret and 14 Allam et al. Oiihella (1995). Samples were immediately divided into aliquots and diluted in ditfercnt solutions as described below. Design and Validalion of the Phagocytic Assay In vitro Incubation of Heniocytes Hith Beads Fluorescent latex beads, 2.02 (im in diameter (Fluoresbrite Calibration grade, Polysciences, USA), were dispersed by expel- ling them through a 26-gauge needle and diluting them with fil- tered seawater (FSW) to give a final concentration of 6 x 10"^ beads ml"'. Two hundred microliters of this suspension were placed in each well of a 24- well microplate, which was centrifuged for 10 min at 200 x g at room temperature (-21°C) to form a uniform monolayer of beads on the bottom of each well. Hemolymph samples were immediately diluted with ice-cold sterile seawater (SSW) to give a final concentration of 5 to 7 x 10^ cells ml"' and 200-|xl aliquots of these suspensions were added to each well to give approximately a 1:10, cell:bead ratio. Following a 30-minute incubation in thermostatic chambers (Minifrige II®, Boekel Indus- tries, Inc., Philadelphia. PA) adjusted to 2I°C, conditions deter- mined to be optimal during preliminary assays, the cytoskeleton- inhibitor Cytochalasin B (10 (xg mP', final concentration) was added to each experimental well to stop hemocyte activity. At- tached cells were released by trypsinization (0A7c trypsin in 1% EDTA-saline solution for 10 min) followed by gentle sonication for one minute at room temperature (RT). Microscopic observation verified that this procedure detached cells from wells and also released non-ingested particles from the surfaces of hemocytes. Fomialin was then added for a final concentration of 2<'^i to fix the sample, which was transferred to a microfuge tube where it was held on ice until processed, within an hour, by flow cytometry. Because the flow cytometric profile for hemocytes from each clam was unique (see later), it was necessary to have a control (i.e.. no phagocytosis) profile for each individual. Thus, a control well was established for each clam in which Cytochalasin B was added at the beginning of the incubation period to prevent phagocytosis. After the incubation, the control wells received the trypsin and formalin treatments as described above. Flow Cytometry Flow cytometry was performed on a Coulter EPICS C equipped with an argon laser and operated at a wavelength of 488 nm. Gains and photomultiplier high voltage settings were adjusted to include all cell and bead particles. Forward light scatter (FLS) and green fluorescence (GFL) list mode data were collected. A total of 1 0,000 particles were counted for each sample. The percent phago- cytosis was calculated for each clam by bitmapping (electronic outlining) each of three particle types: (i) free beads; (2) non- bead-associated cells; and (3) bead-associated cells (Fig. 1). The bitmaps had been established previously by running beads alone, then non-bead-associated cells plus free beads, and then samples with phagocytosed beads. For each clam, the sum of counts in bitmaps 2 and 3 represented the total hemocytes in the flow cy- tometry sample. In each sample, the percent phagocytosis was computed as the ratio of bead-associated hemocytes to total hemocytes x 100. For each clam, the percent phagocytosis was calculated as the difference between the percent phagocytosis in the test wells and the percent phagocytosis in the control well. The percent phagocytosis was always below 0.5% in the control wells. For each sample, mean fluorescence intensity (channel number) was calculated within each bitmap. Assay Validation Epifluorescence microscopy was used to compare and correlate the percent phagocytosis results obtained from flow cytometry. Bead-associated and non-bead-associated hemocytes were counted using a Zeiss ICM 405 microscope equipped with a standard FITC filter set. A minimuin of 250 cells was counted in each sample. This comparison included control samples with added Cytochala- sin B. In addition, the reproducibility of the assay was tested by establishing duplicate experimental wells for each clam and com- paring flow cytometric results for the replicates using correlation analysis. Finally, to determine the effect of storage on the percent phagocytosis measurement, samples were collected as described above, processed by flow cytometry, stored in glass tubes at 4'C, and re-analyzed after 3 and 7 days. O (J G Forward light scatter (FLS) Figure I. Flow cytometry bivariate plots sboMiny the hitniups used to caliiiiate iHad-associated and non-head-associated cells in control (.\) and test (B) mixtures. Bitmap 1: beads alone, Bitmap 2: non-bead-associated cells. Bitmap i: bead-associated cells. Hemocyte Viability and Phagocytic Activity in Clam 15 Design and \'alidatioii of the Mabilily Assay III vitro Incubation of Hemoc) tes with the Fluorescent Vital Stain The percentage of dead cell^. was assessed using the fluorescent nucleic acid stain ethidium homodimer-1 (EHD) previously used by Ashton-Alcox and Ford ( 1998). EHD binds to nucleic acids by intercalation. It does not permeate cells with intact membranes; thus only dead cells, or those with damaged membranes, become fluorescent. Hemolymph samples were diluted in cold Alsever's solution (1:10. v:v) immediately after collection. Then, 2 |jlM EHD (Molecular Probes, Eugene. Oregon. USA), dissolved in DMSO according to manufacturer's directions, was added. The mixture was incubated at room temperature for 30 minutes. An unstained control sample was made for each clam. Flow Cytometry Flow cytometry was used to collect light scatter parameters and log red fluorescence (LRFL) signals for at least 5000 cells. The percentage of dead cells was determined by setting a cursor at the upper limit of the LRFL signal for the unstained control, which was used as the "zero" channel for the stained cells in the parallel treated sample (Fig. 2). The percentage of dead cells was calcu- lated as the ratio of cells above the "zero" channel to total hemocytes x 100. Assay Validation Fluorescence microscopy was used to compare and coirelate the hemocyte viability results obtained from flow cytometry. Fluo- rescent and non-fluorescent hemocytes were counted using an epi- fluorescence microscope as described above. A minimum of 200 cells was counted in each sample. The reliability of the assay was also tested by using hemocytes that had been killed by immersion in boiling water for 5 min. Hemocyte mortality was confirmed microscopically, after EHD uptake, to be lOO'Tf . whereas untreated cells were more than 96'7f viable. Five mixtures of untreated and heat-killed hemocytes were made using Q9c, 25%, 50%, 75%. and 100% heat-killed hemocytes. The percentage of viable and non- viable hemocytes was measured flow cytometrically as described above. Effect of Temperature on Phagocytosis Because BRD development is partially controlled by tempera- ture, the phagocytosis assay was used to quantify the effect of temperature on phagocytosis by R. philippinarum hemocytes /;; vitro. Hemolymph was withdrawn from clams and immediately diluted with ice-cold SSW to give a flnal concentration of about 5 to 7 X lO*" cells ml"'. For each clam, 6 test wells and one control well of hemocytes and beads were established. Two replicates were incubated at 8°C. two at 13°C, and two at 2rC in the Mini- frige 11'"' temperature chambers. These temperatures were selected because they are associated with the development of significantly different BRD prevalences and intensities in experimentally chal- lenged clams (Allam 1998). The single control well was incubated at 21°C since preliminary studies showed no evidence of phago- cytosis in controls at any tested temperature. All samples were processed for flow cytometry as described above. Ill vitro Interactions Between Hemocytes and I', tapetis The first experiment was devised to stud\ the uptake of fluo- rescently labeled V. tapetis by hemocytes. Exponentially growing V. tapetis, cultured on marine agar were suspended in phosphate- buffered saline (PBS) to obtain about 10'' cfu ml"'. The bacteria were then fixed in 2% formalin, washed 3 times with PBS. and resuspended in 1 ml PBS (pH 7.4) containing 1 mg FITC (Sigma). The mixture was incubated for 30 min at RT, washed twice in PBS. and finally resuspended in sterile seawater. Labeled bacteria were then placed in each well of a 24-well microplate. centrifuged to form a uniform layer on the bottom of each well ( 10 min. 500 X g, 2I°C) and used for measuring the phagocytic activity of hemocvtes as described above. Six wells were established for each a> 192 256 64 128 Log red fluorescence (channel) 256 Figure 2. Distribution of log red fluorescence (LRFL) in unstained control and sample stained with ethidium homodimer. A cursor was set at the upper limit of the I^RFL signal for unstained control, which was used as the "zero" channel for the stained cells within the sample. PDC: Percent Dead Cells. 16 Allam et al. clam using the same hemocyte:test particle ratio (1:10): 3 incu- bated with labeled bacteria (2 test, and 1 control well with Cyto- chalasin B added), and 3 incubated with standard lluorescent beads (2 test, and 1 control well). The second experiment investigated the elTect of live V. tapelis on hemocyte viability. Hemolymph samples were diluted with cold Alsever's solution (1:10, v:v) immediately after collection. Expo- nentially growing V. tapelis were suspended and diluted in sterile Alsever's solution to obtain about 10^ cfu ml"'. One milliliter of this suspension was transferred to a sterile plastic tube and mixed with I ml of diluted hemolymph to give approximately a 1:50, celhbacteria ratio. Ten (xg mP' of Cytochalasin B was immedi- ately added to the mixtures to prevent phagocytosis. They were then incubated with mild agitation on a rocker plate for 2 h at room teinperature. A duplicate tube containing only sterile Alsever's solution and Cytochalasin B was prepared for each clam and was used as a control preparation. After incubation, EHD was added and the percentage of dead hemocytes measured using the flow cytometer as described previously. Statistics Percent phagocytosis and viability values were arcsine trans- formed before the use of statistical tests, however tables show means and standard errors of non-transformed values. Correlation analysis was used in the validation of the phagocytosis and viabil- ity assays. Correlation analysis, as well as repeated measures ANOVA, was also used to test the effect of storage time on the percent phagocytosis. Mean values in clams with (symptomatic) and without (asymptomatic) BRD symptoms were compared using a Student's /-test. This test was also used to compare the /;; vitro effect of V. tapetis on hemocyte viability. Differences were con- sidered significant at a = 0.05. RESULTS Assay Validation Phagocytosis The percent phagocytosis of beads calculated by flow cytom- etry (19.9 ± 2.5) was significantly {P = 0.039) lower than that calculated by fluorescent microscopy (26.4 ± 2.6), but the two methods were highly correlated (N = 24, r = O.Sl, P < 0.0001 ). Microscopic observation confirmed that the beads associated with hemocytes were internalized. The percent phagocytosis in dupli- cate wells was also highly and significantly correlated (N = 72, r" = 0.67, P < 0.0001 ); consequently, two wells per individual was considered sufficient replication in all subsequent experiments. Although there appeared to be a slight decrease in the percent phagocytosis in samples stored for seven days at 4°C (Table 1 ), a repeated measures ANOVA showed no statistically significant ef- fect of time. Nevertheless, all further flow cytometric samples were processed within 24 hours because there was some micro- scopic evidence of an increase in broken cells and membrane fragments in stored samples. Fluorescence intensity (channel num- ber) was equal to 5.7 ± 0.3 in non-phagocytic cells (mean ± SEM), 12.6 ±4.1 in free beads, and 27.4 ± 7.3 in phagocytic cells. No significant changes in fluorescence intensity were observed in pre- served samples. TABLE I. Effects of storanc on flow cytometric counts of percent pliagocytosis In K. philippinanim. .Samples were processed al the day of collection and re-anal>zed after 3 and 7 da>s of storane at 4 C. Means, standard errors of the means, and correlation coefficients are presented (.V = 24 clams with 2 replicates eachl. No significant differences were observed among means (repeated measures ANOVA) which were highly correlated iP < 0.0001). Dav 0 Dav 3 Dav 7 Mean ± SEM 22.3 ± 1.3 lS.S + 0.9 14.9 ± 1.1 Dav 0 1 0.81 0.S5 Dav 3 1 n.77 Da} 7 1 Viability The percentage of dead hemocytes calculated by flow cytom- etry (3.7 ± 1.1) was not significantly different from values mea- sured microscopically (4.2 ± 1.0, P = 0.69). and the two methods were highly correlated (N = 10. r^ = 0.92. P < 0.001). About 99.97f of heat-killed hemocytes were located within the upper channels on the LRFL histograms, and thus detected by the flow cytometer as dead cells. Flow cytometric estimates indicated that the hemocyte mixture made without addition of heat-killed hemocytes contained 3.89;- dead cells, while those containing 25, 50 and 75'7f heat-killed cells contained 28. 1, 52.0 and 75.6% dead hemocytes, respectively. Disease Effects on Phagocytosis and Hemocyte Viability Flow cytometric measurements showed a somewhat lower per- centage of phagocytic hemocytes (7.2 ± 1.07f, N = 21 ) compared with those from asymptomatic animals (10.4 ± 1.3%. N = 23. P = 0.044) and no difference in fluorescence intensity among phagocytic hemocytes (33.2 ± 9.0 for diseased and 25.1 ± 5.1 for asymptomatic clams. P = 0.420). Symptomatic clams had a sig- nificantly higher (P = 0.0006) percentage of dead hemocytes ( 13.4 ± 1.0%. N = 54) when compared with asymptomatic clams (9.2 ± 0.5%. N = 20). Temperature Effects on Phagocytosis The percent phagocytosis was clearly related to the temperature at which the hemocyte-bead mixture was held (Table 2). This TABLE 2. Effect of temperature on in vitro phagocjtosis of heads bv hemocv tes from R. philippinarum (/V = 24 clams with 2 replicates each). For each parameter, letters (a. b and c; or x and yl represent significant differences among different temperatures (ANOVA, P < 0.05). Fluorescence intensity refers to the peak fluorescent channel of those hemocytes that had phagocv tosed heads. 8 C 13 C 2VC % Phagocytosis Mean ± SEM 7.9 ±0.7" 14,9 ± l.lf 21.1 ± 1,2' Range 2.7-19.3 4.4-25.5 12.5-41.6 Fluorescence Intensity Mean + SEM L'i.l ±3.0^ 24.0 ± 4.4^ 29.6 ± 8.3> Range 10-21 1 8-33 19-38 Hemocyte Viability and Phagocytic Activity in Clam percentage at 2rC was about 2lVc. at 13°C. it was 15%, and at 8°C, it was 8%. Similarly, fluorescence intensity within phagocyt- ic cells was also dependent upon the incubation temperature. This was about 30 and 24 in mixtures incubated at 21 and 13°C. re- spectively, and only 15 in those held at 8°C (Table 2). Uptake of V. tapetis versus Beads Clam hemocytes phagocytosed fluorescent latex beads at a sig- nificantly higher rate (22.3 ± 1.3%) than they did FlTC-labeled V. tapelis (12.8 ± 2.9%, P = 0.002, N = 24). Hemocyte Killing by V. tapetis The percentage of dead cells increased significantly after incu- bation of hemocytes with the pathogenic bacterium, V. tapetis. In control mixtures, the percentage of dead hemocytes was 6.8 ± 2.2 while it reached 20.4 ± 5.9% in mixtures with V. tapetis added (Student's f-test, P = 0.029, N = 11). DISCUSSION Flow cytometry has been used in several previous studies to quantify phagocytosis and hemocyte viability in marine bivalves (Alvarez et al. 1989; Ashton-Alcox & Ford 1998; Brousseau et al. 2000; Fournier et al. 2001 ). However, none of these studies re- ported validating the methodology by comparing the flow cyto- metric results against standard microscopic measurements. In this study, we quantified phagocytosis and hemocyte viability in indi- vidual clams by both flow cytometry and microscopy. The high correlation between the two methods shows that flow cytometry is not only a rapid and versatile method for analyzing these two important parameters, but that the results are very comparable to more traditional methods for assaying marine bivalve hemocytes. Notwithstanding the good correlation between flow cytometry and microscopy, differences in means between the two methods may be significant. Some of this disparity undoubtedly comes from uncertainty in drawing bitmaps to delineate presumed flow cytom- eter particle groupings. In our study, the fact that microscopy estimated a higher percent phagocytosis may be the result of the inclusion of some cell debris in samples analyzed by flow cytom- etry. Large, bead-free debris could fall above the noise discrimi- nator for forward light scatter and be counted as non- phagocytosing cells, which would decrease the calculated percent phagocytosis. Indeed, microscopic observation revealed the pres- ence of a limited quantity of such cell debris, which increased when samples were stored for several days. Such debris was not generated in the viability assay since the experimental protocol did not involve the addition of trypsin and the mild sonication used in the phagocytosis assay to detach adhered cells. Probably for this reason, the percentages of dead cells obtained using flow cytom- etry were not different from those measured microscopically. Critical to phagocytic assays is the detachment of cells from the incubation vials and the separation of non-ingested particles from the phagocytic cells. Trypsinization is generally used to detach cells (Alvarez et al. 1989) while a variety of methods have been developed to discriminate non-ingested particles (De Boer et al. 1996; Mortensen & Glette 1996; Lopez-Cortes et al. 1999). We found that a single procedure, trypsinization in the presence of EDTA followed by gentle sonication, was quite effective in both detachment and separation steps. The centrifugation step over a sucrose gradient (Alvarez et al. 1989) is not required here to sepa- rate non-bound beads from phagocytic cells since the forward light scatter allows easy differentiation between these particles based on size. In this study, fluorescent latex beads were used as standard experimental particles for phagocytosis, as in the work of Alvarez et al. (1989) and Brousseau et al. (2000) who studied phagocytosis by hemocytes in Crassostrea virginica and Mya arenaria. respec- tively. These commercially available, fluorescent beads are ex- tremely convenient for reproducible flow cytometric work due to their standardized sizes and fluorescence, qualities that are difficult to establish by labeling natural microorganisms. It must be recog- nized, of course, that latex beads may not e\ oke the same response from hemocytes as would foreign cells. Indeed, the present work demonstrated that the uptake of labeled V. tapetis was significantly lower than that of beads, despite precautions taken to ensure the same experimental conditions and hemocyte-to-pailicle ratios. Dif- ferences between the tw o test particles may be the result of specific interactions between hemocytes and beads or bacteria involving recognition factors that retard the uptake of formalin-fixed bacte- ria. Using microscopy, Lopez-Cortes et al. (1999) studied the phagocytic activity of /?. pliilippinanim against V. tapetis and noted that the uptake of bacteria depended on the V. tapetis strain used. They also noted that viable V. tapetis were more efficiently phagocytosed than were the formalin-fixed bacteria and concluded that this was related to the presence of "specific recognition mol- ecules" on the outer membrane of V. tapetis that combine with hemocyte receptors and that might be altered during fixation. Most work using labeled bacteria as test particles has been done after fixation of the microorganisms, which stabilizes the tag intensity because it prevents cell division or other processes that could alter intensity. Nevertheless, with appropriate controls, the use of live tagged bacteria should be included in cytometric assays to inves- tigate the role of bacterial epitopes in recognition processes. The development of BRD was associated with a significant decrease in phagocytic activity by hemocytes and an increase in the percentage of dead hemocytes. The lower phagocytic activity in diseased clams could be related to the percentage of dead hemocytes, since dead or moribund hemocytes are not capable of phagocytosis. Previous work has shown that clams with BRD have a high percentage of dead cells in the hemolymph compared to healthy animals and that this percentage increases with the devel- opment of the disease (AUam et al. 2000a, 2000b). The loss of phagocytic capacity and the death of hemocytes may result from deterioration of the physiological condition in severely infected clams (Plana et al. 1996), or from direct killing of hemocytes by V. tapetis. Indeed, results presented here demonstrate that V. tapetis is able to kill clam hemocytes /;; vitro. It is often of interest to know the number of particles a phago- cyte has ingested (phagocytic index) as well as the proportion of phagocytosing cells. The peak fluorescence intensity in the hemocytes that did ingest beads provided an index for the relative number of beads ingested by each cell. From this, it can be con- cluded that although clams with BRD symptoms had a smaller proportion of phagocytic hemocytes compared to asymptomatic clams, there was no difference in the number of beads each cell ingested. Our results show a positive correlation between temperature and both percent phagocytosis and the phagocytic index of latex beads and agree with previous studies of other marine bivalves (Feng & Feng 1974; Foley & Cheng 1975; Alvarez et al. 1989; Tripp 1992; Chu & La Peyre 1993). It is relevant that the devel- opment of BRD seems to be at least partially controlled by water 18 Allam et al. temperature (Paillard el al. 1^97; Allam I99S). Laboratory experi- ments have shown high prevalence and intensity of the disease when clams are incubated at 8°C and 13°C compared to clams incubated at 21°C (Allam 1998). Conversely, there are improved repair processes (recalcit'ication) at 2I'-'C as compared to the lower temperatures. It is possible that the low prevalence of BRD at 2 1 "C is related to better performance of the clam's defense system, including phagocytosis. The pathogen may also be less virulent at this temperature, although growth of V. lupctis is not inhibited al 21°C (Maes 1992; Paillard et al.. unpublished). In summary, we have described and validated flow cytometric methods to measure the phagocytic activity and \iability of hemocytes from the clam, Riulirapes pltilippiuaiiim. Additional types of cytometric measurements will undoubtedly be adapted from vertebrate systems for use with molluscs and other inverte- brates. For example, reactive oxygen species (ROS) generated by phagocytic cells represent a current concern of bivalve pathobi- ologisls and ecotoxicologisls (Winston cl al. 1996; Bramble & Anderson 1997; Lambert & Nicolas 1998). The successful use of How cytometry for ROS measurements in aquatic mammals (De Guise et al. 1995) and fish (Verburg van Kemenade et al. 1994) indicate that flow cytometric methods can also be adapted for this purpose in bivalves. As new cytometric assays come into use, the developmental protocol should include some type of verit'ication against a method that is considered the standard for that assay. ACKNOWLEDGMENTS The first author was supported by a fellowship from the French Government. The authors thank Dr. Joth Davis (Taylor United. Inc.) for providing us with clams from Puget Sound. We also thank Dr. Christine Paillard for valuable discussions. This paper is contri- bution No. 2002-9 from the Institute of Marine and Coastal Sciences at Rutgers University and New Jersey Agricultural Experiment Station Publication No. D-324(J5-2-02. supported by state funds. LITERATURE CITED Allam, B. 1998. The role of bivalve extrapallial lluids in immunological defense. The case of brown ring disease in the Manila clam, Ruditapes pbiUppinarum. PhD Dissertation. Brest. France: l_lniversite de Bretagne Occidentale. Allam, B.. C. Paillard & M. Auffret. 2000a. Alterations in hemolymph and extrapallial fluid parameters in the Manila clam. Rmlilapcs pbiUppi- narum challenged with the pathogen, Vilirio lupctis. J. Invertehr. Ptiihol. 76:63-69. Allam. B.. C. Paillard. A. Howard & M. Le Pennec. 2000b. Isolation of the pathogen Vibrio lapelis and defence parameters in brown ring diseased Manila clams, Ruditapes pbiUppinarum. cultivated in England. D/.v. Acptat. Org. 41:105-113. Allam, B., K. A. Ashton-Alcox & S. E. Ford. 2001 . Haemocyte parameters associated with resistance to brown ring disease in Riulitupes spp. clams. Dev. Comp. Immunol. 25:365-375. Alvarez. M. R.. F. E. Friedl. J. S. Johnson & G. W. Hinsch. 1989. Factors affecting in vitro phagocytosis by oyster hemocytes. / Invertehr. Pathol. 54:233-241. Anderson. R. S. 1996. Interactions of Perkin.su.s inariiuis with humoral factors and hemocytes of Crussostrea virginica. J. Shellfish Res. 15: 127-134. Ashton-Alcox K. A.. B. Allam & S. E. Ford. 2(.)00. Applications of flow cytometry to bivalve pathology. In: M. Fingerman & R. Nagabhusha- nam. editors. Recent Advances in Marine Biotechnology. Vol. 5: Im- munology and Pathology. Enfield. NH: Science Publishers Inc. 85- 124. Ashlon-Alcox, K. A. & S. E. Ford. 1998. Variahdily in molluscan hemocytes: a flow cytometric study. Tissue & Cell 30:195-204. Autfret. M. & R. Oubella. 1995. Cytological and cytometric analysis of bivalve mollusc hemocytes. In: J. S. Stolen et al.. editors. Techniques in Fish Immunology-4. Fair Haven: SOS Publications, pp. 55-63. Bramble. L. & R. S. Anderson. 1997. Modulation of Crassostrea virginica heniocyte reactive oxygen species production by Listonella anguil- larum. Dev. Comp. Immunol. 21:337-348. Brousseau. P.. J. Pellerin. Y. Morin. D. Cyr. B. Blakley. H. Boermans & M. Fournier. 2000. Flow cytometry as a tool to monitor the disturbance of phagocytosis in the clam Mya arenaria hemocytes following in vitro exposure to heavy metals. Toxicology 142:145-156. Chu, F.-L. E. & J. F. La Peyre. 1993. Perkmsus marinus susceptibility and defense-related activities in eastern oysters. Crussostrea virginica: tem- perature effects. Dis. Aquatic Org. 16:223-234. Combrier. E.. P. Metezeau. X. Ronot. H. Gachelin & M. Adolphe. 1989. Flow cytometric assessment of cell viability: a multilaceted analysis. Cvloicclinology 2:27-37. De Boer. E.G., R. F. M, Bevers, K. H. Kurth & D. H. J. Schamhart. 1996. Double fluorescent flow cytometric assessment of bacterial internal- ization and binding by epithelial cells. Cyiometiy 25:381-387. De Guise. S.. D. Flipo. J. R. Boehm. D. Martineau. P. Beland & M. Fournier. 1995. 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Fish Shellfish Immunol. 6: 111-121. Nottagc. A. S. & T. H. Birkbeck. 1990. Interactions between different strains of Vibrio alginolyticus and hemolymph fractions from adult Mvtilus edulis. J. Invertehr Pathol. 56:15-19. Paillard. C. P. Maes. J. Mazurie. S. Claude. A. Marhic & M. Le Pennec. 1997. Epidemiological survey of the brown ring disease in clams ot Atlantic coast: role ol Icmpeiaturc in variations of prevalence. Pro- Hemocyte Viability and Phagocytic Activity in Clam 19 ceecliiif; of I he Sth Sxinposnim of the Iinenuitiiels at Point Wells. .As a control, some large cockles were also returned to low on the shore at Lews Bay. Transplants of the New Zealand Little Neck Clam 23 Analysis The effect of shore level was essentially a separate investiga- tion at Point Wells. If those clams returned to Lews Bay had been planted out at mid-shore and low-shore levels as at Point Wells, this would not have served as a control, because clams would had to have been transplanted to different levels from which they were collected at Lews Bay. Because the "shore level"" treatment was not balanced between Point Wells and Lews Bay, the two shore levels at Point Wells were treated as separate sites, additional to Lews Bay, for statistical analysis. The names "Mid Point Wells" and "Low Point Wells" continued to be used in order to retain their site identity. Mortality Weekly visits were made to each site and visual searches un- dertaken for any dead shells (cluckers), which remained on the surface. This was used as an estimate of apparent mortality (Ar- nold 1984; Dobbinson et al. 1989), to minimize disturbance from frequent digging. Upon completion of all experiments (January 1999). plots were identified using the metal detector to locate the aluminum-tagged cockles (Stewart & Creese 2()()()). Plots had no external markings, which avoided interference with plots on public beaches. All clams were excavated and brought back to the laboratory. This enabled a direct measure of the number of clams remaining. Sediment in each plot was washed through a 2-mm sie\e and tagged individu- als were retained. A number of tagged cluckers were also retrieved during this process. The area (~ 1 m) immediately surrounding each plot was also sieved until no further tagged clams were retrieved in two successive sieves. In some instances, ""finger ploughing"" (James & Fairweather 1995) of the sediment was also used. When no additional clams were retrieved either through finger ploughing or sieving, the metal detector was used to locate any missed indi- viduals. The detector was passed over the surface for a distance of up to 5 m away and when a "hit"" was detected, the sediment was finger ploughed to retrieve the clam. This was repeated until no more clams were detected over the entire plot and surrounding area. For each replicate plot, the number of clams in each of four categories was recorded; alive, missing, dead as undamaged empty valves, and dead as empty valves with observable shell damage. Because the "summer transplant experiment"" ran for 1 1 mo and the "winter transplant experiment"" ran for 5 mo, a direct compari- son of mortality could not be made between seasons. However, data for the first 5 mo of the summer transplant experiment were analyzed to provide a legitimate comparison between seasons. For mortality, this was done using only the number of shells retrieved during visual searches, and therefore it is a comparison of apparent rather than actual mortality. At the end of the experiment, survival (estimated from the number of live clams retrieved) was analyzed separately for the two seasons. Data were tested for normality and homogeneity (using Cochran"s test) prior to performing analysis of variance (ANOVA). Growth Because clams were not numbered individually, growth was assessed by following changes in the modal length of size cohorts over time. Every 2 mo. a subsample of 20 clams was remeasured from each 0.25-m- plot. Subsampling avoided undue disturbance to plots. Plots and clams were relocated using the metal detector and clams were excavated, with care taken to minimize distur- bance to those clams not measured. To ensure that a random sample was taken every time, a strip was randomly drawn through the plot and clams were sampled in this area. Comparisons of growth over the full experimental period could not be made be- tween seasons. Interim calculations were made for growth after 5 mo for the summer transplant experiment. RESULTS Mortality Apparent mortality of clams was estimated from the nimiber of tagged cluckers retrieved after 5 mo for each season. A four-factor ANOVA was performed using the factors "season,"" "site,"" ""size," and "density."" There was no significant effect of season or density on apparent mortality (P > 0.05). nor any interaction involving these factors. The pattern of mortality between sites was not the same for large and small clams, giving a significant interaction {P < 0.01) between site and size. Mortality for large clams was high- est at Mid Point Wells, but for small clams it was highest at Low Point Wells (Fig. 3). Survival was significantly different between large and small clams (P < 0.01), with mortality higher for large clams than for small ones (Fig. 3). In addition, a significant dif- ference between sites (P < 0.01) was detected. Apparent mortality was lowest at Lews Bay, the site of origin for all transplanted clams (Fig. 3). Survival was assessed from the number of clams retrieved at the end of both summer (after 1 1 mo), and winter (after 5 mo) transplants. The average survival was 75%-90% for large clams transplanted in winter, and greater than 60'7f for large clams trans- Is sp Treatments Figure 3, Mean percent apparent niortalilv, estimated from the re- trieval of cluckers across treatments after 5 mo [from Marcli to July for the summer transplant experiment (a) and September to January for the winter transplant experiment lb(l. n = 3 replicated plots at each site. Ip = large cockles (25-32 mm shell length! at packed density (2()()/().25 m-|. Is = large cockles at spaced density (5(l/().25 nr). sp and ss = small cockles (1(1-18 mm shell length) at packed and spaced den- sities, respectively. 24 Stewart and Creese planted in summer, with the exception of Lews Bay (Fig. 4). Even for small clams, average survival was greater than 307f at all sites except Low Point Wells in summer (Fig. 4|. Survival at Lews Bay was confounded by storm events removing an entire shell bank containing the large clams in the winter transplant experiment, as well as many of the large clams in the summer transplant experi- ment. Therefore, data from Lews Bay were excluded from the analysis. Three-way ANOVAs were performed separately for each of the summer and winter transplants for the Mid Point Wells and Low Point Wells sites only, with the other factors being ""size" and "density."" With Lews Bay excluded, the two Point Wells sites effectively become "shore level"" treatments. For the summer trans- plant experiment, both the "size x density"" interaction (P = 0.04) and the "shore level x size"" interaction (P < 0.01 ) were significant. Survival for large clams was similar between Mid Point Wells and Low Point Wells, but survival of small clams was lower at Low Point Wells (Fig. 4). Significantly more large clams were retrieved than small ones (P < 0.01 ). and overall survival of large and small clams was highest at Mid Point Wells {P < 0.01 ). The significant "density x size"" interaction is due to fewer small clams in the spaced density treatment surviving than in the packed density treatment (Fig. 4). For large clams, survival was similar between density treatments (Fig. 4). Results for winter transplants are simi- lar, but the "size x density"" interaction was not significant (P = 0.08) (Fig. 4). 90 75 60 45 30 15 0 (a) Lews Bay Summer X I ' ■ I II I Ip Is sp ss control 9(1 - (d) Lews Bay Winter 75 - 60 - pS 45 - rn 1 30 - 15 - N/A 1 1 1 1 1 Ip Is sp ss control 90 75 60 45 30 15 0 (b) Mid Point Wells Summer i 90 - 75 - — -1 (e) Mid Point Wells Wmter 60 - -^ 45 - 30 - 15 - N/A 1 1 i Ip Is sp ss control Ip Is sp ss control 90 - 75 - 60 - T (c ) Low Point Wells Summer 45 - 30 - 1 15 - 0 - 1 1 ■ 1 Ip Because the storm events that affected experiments at Lews Bay affected only large clams planted low on the shore and not small clams planted higher on the shore, two-way ANOVAs on survival were run for small clams only. That is. the factor "size"" was removed and only "site"" (Lews Bay included) and "density"" were analyzed. For both summer and winter transplants, there was no significant "site x density"" interaction. However, both site and density were significant factors. In the summer transplant experi- ment, survival of small clams at Low Point Wells was significantly lower than at Mid Point Wells or Lews Bay (Student-Newman Kews test. P < O.O.'i). Survival was not significantly different be- tween Mid Point Wells and Lews Bay iP > 0.05). For the winter transplant experiment, survival of small clams was significantly different among all three sites (P < 0.05). with lowest values at Low Point Wells (Fig. 4). The majority of small clams not retrieved in the final sample (either as whole or damaged valves) could not be accounted for (Fig. 5). At Lews Bay, the majority of large clams not retrieved were also missing (Fig. 5a and b) presumably due to the storm events. However, at the Point Wells sites, the majority of large clams not retrieved in the final sample were accounted for by either whole valves or damaged valves, retrieved during visual searches carried out during the year. Most retrieved valves were still intact, with no evidence of damage that might indicate preda- tion (Fig. 5). Damaged shells were mainly crushed half valves or had chipped ventral margins. sp ss control Ip Is sp ss control Treatment Treatment Figure 4. Mean percent survival taken from the number oflive tagged cockles retrieved at the end of the experiment ( 1 1 mo for the summer transplant and 5 mo for the winter tran.splant). n = 3 replicate plots. Size and density treatments as in Figure 3. Disturbance controls (Low Point Wells site only! are shown in grey. 90 - 75 60 45 - 30 15 0 (a) Lews Bay Summer 90 - (h) Lews Bay 75 - Winter 60 J fi 45 - fl 30 - 15 - Ip Is control Ip Is sp ss control i 90 - (c) Mid Point Wells ij Summer ^ /s- ^ 60- c 45 - g_ 30 - p S 15- 2 0- ^t--^tL 90 - 75 - (d) Mid Point Wells Winter 60 - 45 - } i 30 - 15 - 0 - _^yj^ h~-^ k — 1 — Ip Is sp ss control Ip Is sp ss control 90 - 75 - (e) Low Point Wells -j Summer 60 - T 45 J 30 - 15 - 0 - k t t" X 90 - 75 - (f) Low Point Wells Winter 60 - 45- 30 -• li' ■ 1 missine 1 1 whole I^H damaged 15 - Ip Is sp Treatment Is sp ss control Treatment Figure 5. Mean percent of cockles missing, and mean percent of dam- aged and whole valves retrieved by visual searches at the end of the experiments ( 11 mo for summer experiment and 5 mo for w inter ex- periment). II = 3 replicate plots at each site. Transplants of the New Zealand Little Neck Clam 25 Growth Mean shell length was initially taken tVom random samples of 20 individuals taken from each plot every 2 mo. These individuals were considered representative of the experimental population. Inter-replicate variation was found to be virtually zero, so replicate treatments were pooled for graphical representation. Because there was little change in mean si^e until toward the end of the experi- ment (when water temperature increased), the difference in growth between treatments was analyzed in terms of the change between the initial mean shell length of transplants and the mean shell length at the end of the experiment. Growth was analyzed separately for each expermient using a three-way ANOVA with the factors "site," "size class," and "den- sity." For both seasons, there was a significant (P < 0.05) three- way interaction ("site x size x density"), but some patterns can be seen in the data. Large clams grew only 1-2 mm during the ex- periment (Fig. 6). Small clams, however, showed pronounced i 2 1 - (b) i I I Lews Bay ^M Mid Point Wells I ~1 Low Point Wells N/A sp ss control Treatment Figure. 6. Mean change in shell length (mm) after 11 months (March- January) for the summer transplant experiment (a) and alter 5 months (March-July and September-January) for the summer (b) and winter (c) transplant experiments, respectively. // = 3 replicate plots at each site. Control plots (Low Point Wells only) for disturbance are shown in the gray bars. growth in some treatments, particularly at the Low Point Wells site (Fig. 6). Interestingly, growth in the small clams was readily iden- tifiable by direct observation of the shell. A clear disturbance mark could be seen in the shell, indicating when they were first placed out in the field. No other clams in the area showed such as pro- nounced growth check. Clams taken from the high shore level at Lews Bay and transplanted to Point Wells grew more than those leturned to the site of origin at Lews Bay (Fig. 6). DISCUSSION Although the initial collection and tagging of clams did appear to cause a clear gi'owth check mark, the disturbance of clams for ongoing sampling during the experiment appeared to have no ef- fect on growth or survival. The absence of a digging effect is consistent with results from previous manipulations of these clams (Martin, unpubl. data). Apparent mortality of large clams was lowest when they were returned to their site of origin at Lews Bay. At the end of the experiment, however, no large clams transplanted in winter and few large clams transplanted in summer were retrieved from Lews Bay, due to storm events. Until this point, mortality, estiirtated both by the numbers of cluckers retrieved and by observations made during ongoing sampling, was lower than for the Point Wells sites. We are confident that this trend of lower moitality at the site of origin would have continued in the absence of storm inteifer- ence. Lower mortality for clams returned to the site of origin indicates that survival of transplanted clams was affected by en- vironmental characteristics of particular sites, because all clams underwent the same transplant process. The majority of small clams not retrieved at the end of the experiment could not be accounted for. These clams may have either died and their shells were lost, or they may have migrated out of the area. Small clams were observed to move away from the initial experimental plots more than large clams. If emigration is the main source of "loss," and these clams remain within the general vicinity of the transplant plot, then enhancement may still be considered successful, because these clams will still contribute to the population. If they have migrated away from the area en- tirely, then the effect on enhancement success is the same as mor- tality. Tethering of clams would allow better assessment of wheth- er these clams are dying (with subsequent loss of their shells) or if they are migrating. However, we suggest that a minimum survival rate for transplanted clams of 30% (as achieved in our experiment), although not great, may be a realistic management option and preferable to doing nothing and accepting the piesent situation of declining stocks and poor recruitment. The majority of enhancement projects are conducted using ju- venile shellfish, simply because large numbers can be produced in a hatchery and they do not require costly on-growing to a larger size before planting out. However, planting out of larger shellfish is often more successful (Peterson et al. 1995; Marelli & Arnold 1996), a result attributed to high mortality of juvenile shellfish, possibly from predation. This inverse relationship between size and mortality rate is often explained in terms of prey size refuge (Whetstone & Eversole 1977; MacKenzie 1979; Arnold 1984; Kraeuter & Castanga 1989; Peterson et al. 1995; Marelli & Arnold 1996). For this reason, Marelli and Arnold (1996) consider that broadcasting (i.e., the seeding out of very large numbers of un- protected juveniles) is not an effective stock enhancement tech- nique. 26 Stewart and Creese Our experiments enabled a comparison between the survival ot small and large transplanted clams. As predicted by the published studies cited above. surN ival of large clams was much higher than for small clams (generally >60'-f and up to 9()'/f ). The survival rates obtained in this study compare favorably with transplants of the hard clam Meaeiu'iUi iiwircuciia (mainly juveniles) in the United Slates. Peterson et al. (IW5) achieved 35% survivorship over 14 mo. Marelli and Arnold (1996) were less successful with 95% mortality after 80 d. hi earlier trials by Flagg and Malouf (1983), greater than 10% survival was only achieved for those clams larger than 20 mm. and even clams of this size suffered 100% mortality in areas with large whelk populations. A minimum survival for small clams of 30% could well be accept- able for enhancement, but a greater initial number of transplants would be required to compensate for subsequent losses. To achieve the desired level of enhancement, it may also be necessary to follow the initial transplant with a number of smaller secondary transplants. It would be laborious to collect such large numbers of A. snachhuni from the field, and enhancement may only be fea- sible if viable hatchery production, using local spawning stock, could be established. Damaged shells recovered in this study had either crushed valves, likely to be attributable to birds or crabs, or chipped ventral margins, likely due to whelks. No large crabs were observed in the area and the only birds observed were lone pairs of oystercatchers. Haeiustriipus ostralegiis. Other potential predators may have been stingrays or fish, because a number of feeding pits were observed in the area. However, predation by stingrays would have left a noticeable disturbance in the experimental grid, which v\as never observed. Peterson et al. (1995) found that survi\orship was greater for Mercenaria mercenaria clams planted in late fall/winter, but av- erage growth was greater for clams planted in late summer. How- ever, clams transplanted in summer were in the field for at least 2 mo longer than winter-transplanted clams. This temporal differ- ence is mentioned, but no adjustments were made for comparison of the two seasons. Thus, the period of exposure was compared, and not season. In our study, growth of transplanted A. Muichhiini was more dependent on the time of year than on the treatment. Large clams showed minimal growth, probably due to having already attained near maximum size of 35 mm (Larcombe 1971: Dobbinson et al. 1989). Small clams showed pronounced growth at some sites. This growth did not begin until late winter (September) for clams planted out in March, and was most noticeable in the spring (No- vember). For those clams planted out in September, an increase in size was already noticeable 2 mo later, in November. Small clams showed the greatest increase in shell length when transplanted, from high on the shore at Lews Bay (the original site) to low on the shore at Point Wells. This same shore level effect was found b\ Dobbinson et al (1989) in Otago Harbour, southern New Zealand. A. stutchburyi low on the shore are generally larger than those high on the shore (Wood cited in Morton & Miller 1973; Larcombe 1971: Dobbinson et al. 1989). Larcombe (1971) hypothesizes that this is due to poor grow th conditions, in particular food availability (due to increased exposure time), restricting growth at higher shore levels. Clams transplanted to low on the shore al Point Wells grew considerably more than those returned to the site of origin at Lews Bay, indicating that clams high on the shore at Lews Bay are unlikely to realize their full growth potential. If this is the case, then translocating these clams to areas lower on the shore at en- hancement sites may be a way of increasing the biomass yield (Dobbinson et al. 1989). Dobbinson et al. (1989) found virtually no effect of density on growth of A. sliilc-lihiiiyi. Density manipulations by Stephenson (1981) and Blackwell (1984), however, resulted in apparent in- traspecific competition, limiting growth and increasing mortality. Martin (1984) found higher growth rates in treatments where den- sity was experimentally reduced. The effect of density on growth and survival in our study is difficult to interpret because of an interaction with the effect of size. Higher retrieval of clams in densely packed treatments may have been because clam movement was restricted at the higher density, and therefore more clams were retained. If so. retention of transplanted clams may be improved if they are planted out in dense clumps rather than spaced over a beach. This would also make post-transplant monitoring easier. In conclusion, high survival for large clams and reasonable survival for small clams indicates that the transplant of New Zealand clams is a feasible technique for enhancement. It is rec- ommended that transplant of clams for enhancement should be undertaken with adult clams, because these show the highest sur- \ i\ al. are easier to collect, and are more likely to remain within an area. Recent studies have demonstrated that infaunal bivalve re- cruitment can be dependent on the adult density (Peterson & Sum- merson 1992; Peterson et al. 1996; Arnold et al. 1998). Large clams may therefore also have the added benefit of adding to the population sooner (through spawning and subsequent recruitment of new clams), thereby speeding the recovei^ of an area. ACKNOWLEDGMENTS This work was funded in part by the Auckland Regional Coun- cil. The transplant experiment was achieved with the help of many students at the Leigh Marine Laboratory. The manuscript was improved by the comments of an anon\ nious reviewer. LITERATURE CITED Arnold. W. S. ( 1984). The effects of prey size, predator size, and sediment composition on the rate of predation of the blue crab, CalUnectes sapidiis. on the hard clam Mercciiuiui mercenaria (Linne). / Exp. Mar. Biol. Eeol. 80:207-219. Arnold, W. S., D. C. Marelli, C. P. Bray & M. M. Hamson ( 1998). Recruit- ment of bay scallops Argopeclen irradians in Rorida Gulf of Mexico waters: Scales of coherence. Mar. Ecol. Prog. Ser. 170:143-157. Belton. R. J. 1986. The New Zealand clam Cliione (Aii.\lroveiius) .stutcli- hiini. Winston Churchill Memorial Trust Fellow. Wellington. New Zealand. Blackwell. R. G. 1984. Aspects of the population dynamics of Chiom- stutchburyi in Ohiwa Harbour, Bay of Plenty. New Zealand. Ph.D. thesis. University of Auckland, New Zealand. Browne. G. & M. Pawley. 1995. Trends in Auckland inlertidal shellfish surveys: 1993-1995. MAF Fisheries. Auckland. 79 pp. Creese. R. G. & R. G Cole. 1995. Marine conservation in New Zealand. Pac. Comerw Biol. 2:55-63. Dobbinson. S. J.. M. F. Barker & J. B. Jillett. 1989. Experimental shore level transplantation of the New Zealand clam Chione stutchlmryi. J. Shellti.sh Res. 8:197-212. Transplants of the New Zealand Little Neck Clam 27 Flagg. P. J. & R. E. Malouf. 1983. Experimental plantings of juveniles of the hard clam Menenaria mercenaria (Linne) in the waters of Long Island. New York. J. Shellfish Res. 3:19-27. Hancock, D. A. 1969. An experiment with overcrowded clams {Cardium ediile). In: Marine Biological Association of India. Proceedings of the Symposium on Mollusca — Part II. Symposiuin series 3. India, pp. 369- 402. James, R. J. & P. G. Fairweather. 1995. Comparison of rapid methods for sampling the pipi, Donax delloides (Bivalvia: Donacidae), on sandy ocean beaches. Mar. Freshwater Res. 46:1093-1099. Kassner, J. & R. E. Malouf. 1982. An evaluation of "spawner transplants" as a management tool in Long Island's hard clam fishery. J. Shellfish Res. 2:165-172. Kearny. M. B. 1999. Management oi Ausuovemis siuuhharyi resources: Contemporary and traditional issues. M.Sc. Thesis. University of Auckland. New Zealand. Kraeuter, J. N. & M. Castanga. 1989. Factors affecting the growth and survival of clam seed planted in the natural environment. In: J. J. Manzi & M. Castanga, editors. Clam mariculture in North America. Devel- opments in aquaculture and fisheries science. 19. Amsterdam: Elsevier, pp. 149-165. Larcombe. M. F. 1971. The ecology, population dynamics and energetics of some soft shore molluscs. Ph.D. Thesis, University of Auckland. New Zealand. MacKenzie C. L., Jr. 1979. Management for increasing clam abundance. Mar. Fish. Rev. October: 10-22. Malouf, R. E. 1989. Clam culture as a resource management tool. In: J. J. Manzi & M. Castanga, editors. Clam culture in North America. De- velopments in aquaculture and fisheries science. 19. Amsterdam: Elsevier, pp. 421-U7. Mason, J. 1969. Experimental transplanting of clams, Cardium edule Lin- naeus. In: Marine Biological Association of India. Proceedings of the symposium on Mollusca — Part 111. Symposium Series 3. India, pp. 824-83 1 . Marelli, D. C. & W. S. Arnold. 1996 . Growth and monality of transplanted juvenile hard clams, Mercenaria mercenaria. in the Northern Indian River Lagoon. Florida. /. Shellfish Res. 15:709-713. Martin, N. D. 1984. Chione (Austrovenus) stutchbuni (Gray): Population responses to exploitation. M.Sc. Thesis, University of Auckland, New Zealand. Morrison, M. A.. M. D. M. Pawley & G. N. Browne. 1999. Intenidal surveys of shellfish populations in the Auckland region, 1997-98, and associated yield estimates. New Zealand Fisheries Assessment Re- search Document 99/25. Ministry of Fisheries, Wellington. Morton, J., & M. C. Miller 1973. The New Zealand sea-shore. London. Auckland: Collins. 653 pp. Pawley. M. D. M., G. N. Browne & M. Cryer. 1996. Trends in Auckland intertidal shellfish between 1992 and 1996. Draft Fisheries technical report. Pawley, M. D. M., G. N. Browne & M. Morrison. 1997. Survey of inter- tidal shellfish populations in the Auckland metropolitan area (1996/ 1997) with a summary of previous sampling methodologies and results (1992-1996). Draft New Zealand Fisheries technical report. Peterson, C. H. & H. C. Summerson. 1992. Basin-scale coherence of population dynamics of an exploited marine invertebrate, the bay scal- lop: implications of recruitment limitation. Mar. Ecol. Prog. Ser. 90: 257-272. Peterson, C. H., H. C. Summerson & J. Huber. 1993. Replenishment of hard clam stocks using hatchery seed: Combined importance of bottom type, seed size, planting season, and density. J. Shellfish. Res. 14:293- 300. Peterson. C. H., H. C. Summerson & R. A. Luettich. Jr. 1996, Response of bay scallops to spawner transplants: A test of recruitment liinitation. Mar. Ecol. Prog. Ser. 132:93-107. Stace, G. 1991. The elusive toheroa. N. Z. Geogr. 9:18-35. Stephenson, R. L. 1981. Aspects of the energetics of the clam Chione (Austrovenus) stutchburyi in the Avon-Heathcote Estuary, Christchurch. New Zealand. Ph.D. Thesis. University of Canterbury, New Zealand. Stephenson, R. L. & P. E. Chanley. 1979. Larval development of the clam Chione stutchburyi (Bivalvia: Veneridae) reared in the laboratory. N. Z. J. Zool. 6:533-560. Stewart, M. J. & R. G. Creese. 2000. Evaluation of a new tagging technique for monitoring restoration success. J. Shellfish Res. 19:487—191. Turner, S. F. 1997. Community projects to enhance and restore degraded shellfish beds. Aijuacuhure Update. Autumn:7. Wada, K. T. 1993. Bivalve broodstock developments in Japan. World Aquacidture 24:54-57. Whetstone. J. M. & A. G. Eversole. 1977. Predation on hard clams, Mer- cenaria mercenaria, by mud crabs, Panopeus harhstii. Proc. Natl. Shellfish Assoc. 68:42-48. Jimnml of Shellfish Research. Vol. 21. No. 1. 29-32, 2002. MORTALITIES OF ENSIS ARCUATUS (JEFFREYS) (SOLENACEA) IN WESTERN IRELAND EDWARD FAHY,' MARIA LYONS ALCANTARA,' MARK NORMAN," RONAN BROWNE,^ VINCENT ROANTREE,- AND NICK PFEIFFER' 'Marine Institute. Ahhatstown. Dublin 15. Ireland: 'Taighde Mara Ted. Carna. Co Galway. Ireland: 'Atlantic Diving. 25 William St W., Galway. Ireland ABSTRACT Monalities of the razor clam Ensis arcuatus were widespread in western Ireland in the spring months of 2001 . Loss of biomass from one razor clam bed was estimated at 74%. Larger razor clams were more susceptible. Histological and bacteriological examinations and TEM were carried out on moribund and live individuals but no pathological cause was identified. Gonadal staging revealed that large Ensis arcuatus were partially or completely spent. Mortality is explained as a post-spawning phenomenon, which was unusually severe in 2001. KEY WORDS: Ensis arcuatus. razor clams, shellfish mortalities, western Ireland INTRODUCTION In 1997. a hydraulic dredge fishery for razor clams commenced in the Irish Sea where extensive beds of Ensis siliqiia had been discovered. A market developed, largely in Spain, and. when the Irish Sea fisheries became exhausted, additional beds were sought on the western Irish seaboard. The inshore waters of Cos Galway and Mayo contain thinly dispersed E. arcuatus. which form suf- ficiently dense concentrations to reward commercial exploitation only in special and limited circumstances. These patches are in- variably in the lee of reefs and islands that provide shelter from the Atlantic swell. Investigations were undertaken on these razor clam beds to provide biological data on the growth and reproduction of Ensis arcuatus (Fahy et al. 2001b); in the course of these studies we encountered the phenomenon described here, which is evaluated in the context of information emerging from the wider biological enquiry. Investigations on the local ecology of E. arcuatus had been carried out in Cill Chiarain Bay. Co Galway. in August 2000. In March of the following year, a mass mortality of the species was reported there by a local fisherman, prompting a second assess- ment. Cill Chiarain Bay |9°45'W: 53"20'N) is occupied by a major Irish shellfish cooperative. Comharchuman Sliogeisc Chonamara Teo, managing oyster and scallop, and concern for the possible involvement of a disease in the mortalities prompted further in- vestigations. Other shellfish species were not. however, similarly affected, and the fisherman who first reported the event also re- ported mortalities of Ensis arcuatus over a wide area south of Slyne Head (10°10'W:53°7'N), extending to Inishmann in the Aran Islands (9°36'W;53°24'N). Survey work by one of the au- thors (E. F.) in the vicinity since has confirmed that the phenom- enon was widely distributed and that it has affected large numbers of the species. MATERIALS AND METHODS The distribution of E. arcuatus in a part of Cill Chiarain Bay was first investigated in August 2000. Divers sampled by pouring 1 L of granular salt over a quadrat 0.33 m" and collecting all razor clams which emerged from the substratum within it. Initially, the area was investigated along north-south and east-west transect lines but. when heavier densities of the animals were located (the razor clam "bed"), sampling was concentrated on those areas. The length of every individual was measured on a fish measuring board and the biomass in each quadrat was estimated from a weight at length curve, the parameters of which were calculated in August 2000. Repeat sampling of the bed was conducted on April 23. May 29. and August 23, 2001. Samples of razor clams. Ensis arcuatus. taken froin Cill Chi- arain Bay in April (24 individuals) and May 2001 (29 individuals) were processed for histology. Animals were dissected and imme- diately fixed in Carson's fixative for 48 h. Cross sections were cut so as to include as many organs as possible. Tissue samples were processed in an automatic processor for 10 h. In the processor, the tissues were passed through different grades of alcohol and xylene and then impregnated and embedded in paraffin wax. Tissue sec- tions were cut at 3 p.m and stained in an automatic stainer with hematoxylin-eosin; they were then mounted on silinized slides and air dried. Some of the razor clams collected in April displayed postmor- tem tissue changes, so the following month a distinction was made between 13 moribund individuals that were lying on the surface of the substratum and 29 live razor clams that emerged from the substratum in response to granular salt having been spread within a quadrat. The stage of gonadal development was assigned to the samples collected in May following microscopic examination using the terminology of Caspar and Monteiro (1998): the six stages of gametogenic development progress from stage 0 (inactive) through stages 1 (early active gametogenesis). 11 (late active gatnetogen- esis). Ill (ripe), IV (partially spent), to V (spent). Tissues from two moribund individuals were fixed in Carson's fixative for 48 h for electron microscopy. They were wa.shed sev- eral times in 24 h in a bath of cold cacodylate buffer (at 4°C) and then fixed in 3% glutaraldehyde. Samples were washed in caco- dylate buffer then postfixed in 1% Os04 (for 1 h at 4°C) and washed again in cacodylate buffer, dehydrated in ascending grades of ethanol, and subsequently embedded in epoxy resin. Thin sec- tions were cut with a diamond knife using an ultramicrotome. stained with uranyl acetate and lead citrate, and examined under an electron microscope. Bacteriological culture plates were used for primary isolation of pathogens. The media employed included blood and seawater agars and thiosulfate citrate bile salt sucrose agar (TCBS) for detection of Vibrio. A BioNor (Norway) monoclonal antibody agglutination kit was used to delect the possible pre.sence of V. aiiguillarum. 29 30 Fahy et al. TABLE I. Single-factor ANOVA comparing razor clam biomasses in (ill Chiariiin on four occasions: August 200(1. and April, May, and August 2001. Groups 36982 37012 37104 Source of Variation Between Groups Within Groups Total Count 21 24 SS 11019.4 41477.1 428496.5 DF 50 52 Summary Sum 2502.274 606.9071 2657.337 ANOVA MS 5509.698 8349.542 Average 119.1 559 75.86338 110.7224 P-value 0.65988 0.521356 Variance 8031.401 2704.084 10344.37 F crit 3.182606 RESULTS When the razor clam bed was exainined in April 2001, dead and dying razor clams were much in evidence on the substratum or standing half-clear of it. together with freshly empty but undam- aged shells. Similar conditions obtained when the site was visited I mo later. Razor clams on the bed in Cill Chiarain Bay are not so densely concentrated when compared with parts of the Ensis sili- cjiia bed on the east coast, for example (Fahy et a!., 2001a) and there was wide variance in estimates of biomass on each occasion it was sampled. A single-factor analysis of variance (ANOVA) suggests that mean biomass altered highly significantly (Table I ) and more detailed comparisons revealed the differences in biomass to be highly significant between August 2000 and May 2001 (P < 0.001 ); biomass also differed highly significantly between August 2000 and August 2001. The reduction in biomass between August 2000 and May 2001 was 74% (Fig. 1 1. Length frequency distributions of live razor clams recovered by divers in August 2000, May 2001, and August 2001 are shown in Figure 2. along with the length frequency of moribund animals in April 2001. In May. there were signs that some of the larger animals recorded the previous August (corresponding with the moribunds recorded in April 2001), were absent from the popula- tion, although smaller size categories were not recorded either in May 2001. A notable difference between the years is the large incidence of juveniles, corresponding to recently spatted 0-group animals in August 2001. To compare the year-to-year changes in length distributions, therefore, only animals greater than 7 cm were considered. The two length frequencies differ significantly (P < 0.05) (Table 2); contrary to expectation, however (the mori- bunds had apparently been the largest size group), the greatest discrepancy is in the abundance of small- to medium-sized animals in the second year. Gonadal staging of live and moribund razor clams in May (Table 3) revealed that the majority were female, but all were either stage IV or V (partially or completely spent). Histological examination of razor clams sampled in April 2001 revealed that ciliates were occasional on the gills, and there was a low incidence of Nematopsis oocytes (Gregarines) in the connec- tive tissue of the mantle and gills of some animals. Low numbers of Chlamydia-Wke organisms were found in some epithelial cells of the digestive gland of some razor clams. The levels of infesta- tion of any pathogen were so low that they could not be associated with the mortalities. Some animals presented postmortem tissue changes. Abnormal nuclei were found in the connective tissue cells of some razor clams. Transmission electron microscopy (TEM) analysis of these samples confirmed that the abnormalities were related to necrosis and not to a pathogen. Some razor clams collected in May 2001 had Clilamydia-Uke organisms in the epithelial cells of the digestive gland and some had Neimilopsis oocytes in the connective tissue. One individual in May had a heavy infestation of metazoan sporocysts. But again, mortalities could not be attributed to any of these. The presence of an unexplained "deposit" in some digestive diverticular cell cyto- plasm was noted. Bacteriological analysis indicated the presence of Vibrio sp. (possibly V. fluvialis). the natural flora of the environment. Results of serological agglutination tests for V. anguilUiniiii were negative. DISCUSSION Occasional mass mortalities of razor clams. Ensis sp.. are at- tributed to several causes, among them storms (Tebble 1966) and 500 400 E CO 300 0} a 200 CTI 100 0 -100 -200 9 9 9 9 9 9 9 9 Q. ro 3 < 5 -> Figure 1. Mean values (±1 SD) of razor clam bioma.ss in Cill Chiarain Bav at times between August 2000 and August 2001. Razor Clam Mortalities 31 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 25 20 15 10 5 0 May-01 n ^x R n p 'f-^' i rr77 m i ■:». \ — 1 — 1 — 1 — "^ _ ^ J ,.,i: 8 10 12 14 16 18 >. 16 u S 14 i-12 t 10 Ol 8 c 6 01 !£ 4 0) Q. 2 0 15 / aiug-01 rn 1 i — - Tm ?/' ',<: _P3 - □EZ] - m ^, ■ s^ 0 12 3 4 5 6 7 9 10 11 12 13 14 15 16 40 30 20 10 Moribunds, April-01 w\ lai i£2 -.^j^ '<^ 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Length, cm Fijjure 2. Length frequency distributions of Ensis aniialiis in August 2UUU and in May and August 2001, and distributions of moribund animals In April 2001. "adverse environmental conLlitions" (Howard 1998). Mortalities of the Pacific razor clam. Siliiiiici puliiUi. explained by infection with Rick('ltsia-\ike organisms, referred to as nuclear inclusion X. have been reported in several locations in the United States (Elston 1986). Razor clam mortalities in western Ireland in the spring of 2001 were too widespread to be attributed to a local environmental explanation. They occuired within (and outside) a large shellfish cooperative and there was concern lest the oysters and scallops that are managed within the cooperative might be infected with a patho- gen, but simultaneous mortalities were not observed among these. TABLE 2. Length frequencies (above 7 cm) of razor clams in August 2000 and August 2001, compared by chi-square. Length .August August August August (cm) 00 01 00 01 Chi-square 7 4 1 8 7 6 12 8 2.0 9 9 3 9 5 3.4 10 11 5 11 5 7.3 II 12 13 12 I-"? 0.1 12 14 24 14 24 4..S 1,^ 21 2.^ 21 23 0.2 14 1(1 19 21 ")T 0.0 l.s 5 3 16 1 (1 100 IDU 100 100 17.6 N - \ = 6; at 6 degs of freedom, chi-square = 12.6. therefore. P < 0.05. The razor clams that made up the mortalities were apparently predominantly large E. arcuatus. the dominant bivalve species in this vicinity (Fig. 2). Although it is accepted that larger individuals would be more visible to a diver collecting specimens, the size groups which appeared to have been removed from the population are of small to medium size (Table 2). when August 2000 is compared with August 2001. although sampling in April and May 2001 suggested the largest clams were most affected. The question is why the small- to medium-sized razor clams were not better represented among the moribunds. Razor clams are highly mobile, probably the most mobile of the bivalves after scallops, and they are known, for example, to rapidly recolonize favorable ground after dredging operations, so some immigration to the depleted clam bed from the sunounding lower densities is likely to have taken place after the event. E. arcuatus is widely distributed in the vicinity and its length frequency distribution in the areas of great- est clam concentration (the "bed") was similar to that among the more thinly dispersed population in the waters surrounding it in August 2000 (Fahy et al.. 2001b). The razor clam bed in this part of Cill Chiarain Bay is understood to occupy more sheltered and hence more favorable conditions for the animals. Recorded mortalities were confined to animals of mature size (the smallest mature E. arcuatus recorded in the course of biologi- cal investigations was 8.5 cm, corresponding with an age of 2-3 y). TABLE 3. Gonadal staging for apparently healthy (livel and moribund razor clams from Cill Chiarain Bav in May 2001. Gonadal Developmental Nu liber of Status Sex Stages Ind ividuals Percentage Moribund Female Male V IV V IV 5 1 1 2 56 11 11 22 Live Female V IV 13 4 65 20 Male V IV 1 2 5 10 32 Fahy et al. Although it is possible that the smallest dead razor clams were undersampled. the mortalities appeared to be heavily skewed to- ward the largest, oldest individuals. There are signs that some spawn- ing by E. anuatus takes place in every month of the year, but it is more prevalent in the population from October to April; there were signs of only one spatfall. on the other hand, in June or July. The latter would coiTespond with spawning in March-April, which must therefore be the most significant spawning time in this species. If postspawning mortality is a normal occurrence in E. arciia- riis. the incident in the spring of 2001 must have been exceptional. A regular loss of 749?- biomass. although it was followed in this case by a considerably larger spatfall than in the previous year, would have .serious consequences for populations of a species with a longevity of up to 18 y. of which 21 Vc of the population on the bed and .^8.4% of the more w idely dispersed population in the Bay in 2000 were more than 10 y old. These are the age groups that contribute most to the biomass. A natural postspawning mortality is the proffered explanation for the razor clam deaths in Co Galway in 20(1 1 . ACKNOWLEDGMENTS The authors offer thanks to Frank Berthe and Bruno Chollet of the IFREMER EU reference laboratory for shellfish diseases for their continuous advice and for carrying out the electron micros- copy studies. Elston, R. A. 1986. An intranuclear pathogen (nuclear inclusion X (N1X|) associated with massive mortalities of the Pacific razor clam. Silicjuu patulci. J. Invert. Pathol. 47:93-104. Fahy. E. & J. Gaffney. 2001a. Growth statistics of an exploited razor clam (.Ensis siliijua) bed at Gomianstown. Co Meath. Ireland. In: G. Bumell (ed.) Coastal Shellfish - a sustainable resource. Hydrohiologia. 465:139-151. Fahy. E.. M. Nomian. R. Browne. V. Roantree. N. Pfeiffer, D. Stokes. J. Carroll & O. Hannaffy. 200 lb. Distribution, population structure, growth LITERATURE CITED and reproduction of the razor clam Ensis anuatus (Jeffreys) (Solenaceae) in coastal waters of western Ireland. Ir. Fish, hivest. 10:24 pp. Caspar. M. B. & C. C. Monteiro. 199S. Reproductive cycles of the razor clam Ensis siliquu and the clam Venus striatula from Barrinha. South- ern Portugal. J. Mar. Biol. Assoc. U.K. 74:939-954. Howard. A. 199S. Razor clam injuries at Paignton Beach. Shellfish News 6:6. Tehble. N. 1966. British bivalve seashells: A handbook for identification. London: Bntish Museum. Journal iif Shellfish Research. Vol. 21, No. 1. ?.^-«), 2002. ECOLOGICAL AND MORPHOLOGICAL FEATURES OF THE BIVALVE ASTARTE BOREALIS (SCHUMACHER, 1817) IN THE BALTIC SEA NEAR ITS GEOGRAPHICAL RANGE MICHAEL L. ZETTLER Baltic Sea Research Institute. Seestr. 15. D-181 19 Rostock. Germany ABSTRACT During 1444 and 2000 niacKvoobenthos surveys were made in the Mecklenburg Bight (western Baltic Sea). In total 1 16 stations were investigated between March and September. Astaite horealis showed a wide distribution at depths between 12 and 26.5 m. Mean abundance at these depths was 47 ind./m- with a biomass (AFDW) of 0.5 g/m'. Maximum densities observed at these depths were 541 ind./m" and 16 glrcr. respectively. In comparison to a data set of the 1960s, a decreased A. horealis population was found. A. horealis ranged from 1.2 to 28.7 mm in shell length. Most (78%) individuals in the population measured 20 mm) were observed only sporadically and in low numbers. Shell length to wet weight and ash free dry weight correlations are given. Mean wet meat yield was 14.5%. The individual ash free dry weight decreased with increasing shell length from 9.1% (<5 mm) to 5.6% (>25 mm) with a mean value of 7.4%. All results were compared with data from populations in Russian Arctic and adjacent waters. KEY WORDS: Astarte horealis. distribution, abundance, size, meat yield, Baltic Sea. Mecklenburg Bight INTRODUCTION The bivalve, Astarte horealis. is an arctic-boreal species that occurs in Arctic. North Atlantic and adjacent waters (Zettler 2001 ). Some data are known from the North Pacific waters such as Japan, Sea of Ochotsk. Behring Street, British Columbia and at the Aleu- tian Islands (e.g., Coan et al. 2000; Higo et al. 1999; Skarlato 1981). A. horealis find its most extensive distribution in Arctic waters of Russia (e,g,, Antipova 1978; Filatova 1957; Gagaev 1989; Matveeva 1977). It extends from the Barents Sea, via Kara and Laptev Sea to Chukchi Sea and to areas of northern Alaska. Greenland (Ockelmann 1938) and Spitzbergen (Hiigg 1904). At the Grand Banks off Newfoundland, it reaches high abundance in sandy bottoms around 130 m depth (Prena et al. 1999). In Europe this bivalve extends from Iceland (Thorarinsdottir 1997) and off Faeroes and Norway (Brattegart & Holthe 1997) to the northern North Sea (Johansen 1916) via Kattegat (Rasmussen 1973) into the western Baltic Sea and reaches there, its eastern limit of distribu- tion in the Bornholm basin (see Fig. 1) (Demel & Mulicki 1954; von Oertzen & Schulz 1973). Thus, the Baltic population repre- sents the most southern occurrence, an outpost of the mainly arctic area of distribution. The largest populations in the Balic are found in the Kiel and Mecklenburg Bights in depths below 15 m (KCi- hlmorgen-Hille 1963; Schulz 1969; Zettler et al. 2000). A. horealis is among the longest living species in the Baltic and is an impor- tant indicator of environmental conditions. Beside salinity and sediment structure, oxygen concentration has a strong influence on the composition of Baltic Sea fauna and flora. Although A. horea- lis is highly resistant to oxygen depletion (von Oertzen 1973; Oeschger 1990) frequent and long lasting periods of anoxic con- ditions finally diminish or kill the species. This has resulted in a severe decrease of the Baltic Sea population oi A. horealis during recent decades in the deeper parts of the Mecklenburg Bight (Gos- selck et al. 1987; Schulz 1968). Information on the population biology and morphological fea- tures (growth, age. size) of this species is limited. Some investi- gations on production, growth, population size, and morphological features were carried out in Russian Arctic Waters (Antipova Phone: -i-OO 49-381-5197-236; Fax: -l-OO 49-381-5197-440; E-mail: michael.zettler@io-wamemuende.de 1978; Gagaev 1989; Matveeva 1977). Within the framework of an autecological analysis of glacial relict species in the Baltic Sea. investigations on the reproduction of A. horealis were earned out in addition to experiments concerning its resistance and metabolic adaptations (von Oertzen 1972. 1973; von Oertzen & Schulz 1973). Schaefer et al. (1985) studied biometric features of A. ho- realis in Kiel Bight, the westernmost part of the Baltic Sea. They investigated several relationships between shell length and weight and their applicability for taxonomical distinguishing of species of the genus Astarte. The purpose of this study was to investigate the distribution, frequency and biomass of A. horealis in Mecklenburg Bight as the first extensive study on the population characteristics of this im- portant indicator species near its geographical range. A further aim was to compare these results with existing data of the 1960s com- piled by Schulz ( 1969). From one monitoring station (stn. 018). we have a long time data set to show the development of A. horealis within the last decades. .Area of Invesligalioii The Mecklenburg Bight is part of the Belt Sea and belongs to the transition area between North Sea and Baltic Sea (Fig. 1 and Fig. 2). It is connected with Kiel Bight via Fehmanibelt and with Kattegat via the Belts. To the East, the Kadet Trench crossing the Darsser Rise connects it with the Baltic proper. During 1999 and 2000 macrozoobenthos surveys were made in the Mecklenburg Bight. In total 116 stations were sampled be- tween March and September (Fig. 2). Station depth ranged from 5 to 29.6 m. The sediment varied from fine sand at the shallowest stations to sand mixed with silt and clay at the deepest stations. Sediment characteristics and current data for the area have been published by Lange et al. ( 1991 ). MATERIALS AND METHODS Profiles of salinity were recoi'ded throughout the water column using a CTD (conductivity/temperature/depth probe) system. Samples for bottom water oxygen were taken with a 5-1 water sampler (mounted on the CTD) at 0.5 m above the bottom and oxygen levels determined by Winkler titration. Benthic samples were taken with a 0. 1 m~ Van Veen grab. Due to sediment con- ditions, grabs of different weights were used. Three replicates of 33 34 Zettler 56,5 100 10.5 11,0 11 5 120 12.5 13.0 13.5 14 0 14.5 15.0 15.5 16.0 16.5 17.0 17.5 18.0 18.5 190 Longitude (E) Figure 1. Distribution nf Aslarle hiirealis within the Baltic Sea. The sources of the data are Deniel anti Mulicki (1454), Kuhlmorgen-Hille ( 1963), Liiwe (1963), von Oertzen and Schul/, (1973). Petersen (1918) and own observations. Due to a strou}; decrease and wide disappearance of this species mainly in the liornholni and Arkona ISasins not all dots represent recent locations. (KB-Kiel Bight, MB-Mecklenburg Bight, AB-Arkona Basin, BB-Bornholni Basin, SF-Slupsk Furrow, circled area refer to the present study and Fig. 2). grab samples were carried out at each station. The samples were sieved thi'ough a 1-mm screen and animals preserved with 4% formaldehyde in the field. For sorting in the laboratory a stereo- microscope with 10— lOx magnification was used. The shell length of all collected individuals was measured with a vernier calipers to the nearest 0.1 mm for the length-frequency distribution and the length-meat weight relationship. In total about 414 specimen were measured. The valves and the wet meat of the 54.5 - ■■ ■ ' ■ ' ' ' ' ■ ■ . :'.l 1 ] n 11 fl7 ^^ d n ] , ,ln=8 n=13 'JD n=13 n=6 n=2 ■ abundance □ biomass —i ' 1 1 ' hJ 0.5 1 1,5 Biomass (AFDW g/m^) 2,5 Figure 6. The vertical distribution of abundance (ind./nr) and bio- mass (.AFDW g/nrl iti. Aslarle borealis in the Mecklenburg Bight in 1999/2000 (n = number of included stations of each depth interval!). ASTARTE BOREALIS IN THE BALTIC SEA 37 28 26 24 22 I 20 ■~ 16 M 14 1 12 5 10 »} , . >ol ^20 mm) were observed only sporadically and in low numbers. The dominant 0-5 mm size class at most of the inves- tigated stations represents the survivors of the settlement during the last two years. Only at stn. 25 was the size structure dominated by older specimens. Relationships between shell length and individual weights (whole wet weight, wet meat weight and ash free dry weight) are shown in Figure 9. These graphs include all measured specimen (1.2 mm to 28.7 mm) from the 1999/2000 survey. The smallest meat wet weight was 0.09 mg at a shell length of 1.2 mm and the largest one was 1.17 g at 28.3-mm shell length. The estimated mean regression line indicates the average meat yield per unit shell length of y4. borealis. Differences between the station means were not statistically significant. The ash free dry weight \'aried between 0.08 mg (1.9 mm in length) and 0.29 mg (28.5 mm in length). The estimated regression lines of weights for different stations did not differ significantly. The results indicate that throughout the Mecic- lenburg Bight A. Iwrealis contained approximately the same meat per unit shell length for the range of length considered. The mean wet meat yield (percentage of total wet weight) of different size 00 n n% — 1 Cf^ . Stn. 25 27th Apr 1999 16.4 m n=31 ^ 40 ■ ■ 1 20 - ■ ■ , 1 0 1 1 — ^^Ma IJ Stn. 45 28th Apr 1999 16.5 m n=132 ^ N K ^ \ / ^/^^■^Tjir-v.,^^,:^ ^ -P , . / .f22 m) of the Mecklenburg Bight, oxygen depletion In late summer prevents successful re- cruitment and growth of the juveniles. However, A. horealis Is one of the most tolerant species to oxygen deficiency and to hydrogen sulphide (von Oertzen 1973; Oeschger 1990). Probably, the pos- sibility of the high tolerance to oxygen deficiency Is restricted to the adults, whereas the juveniles are more susceptible, causing settlement only In favourable years and depths. The lack of re- cruitment events Is described for populations of Kiel and Meck- lenburg Bight by Werner et al. (1974) and Kohn (1989). In shal- lower zones (between 15 and 20 m), no major hypoxic episodes have been observed in the recent past (Matthiius et al. 1999). Although recruitment can take place In the shallower areas, con- ditions for growth are suboptimal. probably due to the lower nu- trition supply, lower salinity and higher temperature (.Arntz et al., 1976; von Oertzen. 1973). In depths shallower than 1 1 m the mean salinity, 8-12 psu, is probably too low. The range of potency of A. horealis is 8-35 psu with an optimum between 14 and 30 psu (.laeckel 1952; von Oertzen 1973). ASTARTE BOREALIS IN THE BALTIC SeA 39 Population Siriicliire The largest living .4. borealis found in this survey was 28.3 mm long and 24.7 mm high. Valve material of the Zoological Collec- tion of the University of Rostock (sampled in the 1980s) had a maximum length of 31.2 mm (26.4-mm height). Lenz (1882) found specimen of 36 mm in length {31-mm height) in the Lijbeck Bay. The shell is quadrate to subtrigonal and compressed v\ith a total shell length of 38 mm at boreal outpo.sts (e.g., Baltic Sea. Jaeckel, 19?2) and 55 mm in the Arctic Sea (Coan el al. 2000. Filatova 1957) with a mean of 25-45 mm (Dance 1977). In gen- eral, the shell length exceeds the height and the height/length indices vary from 0.8 to 0.9 (e.g.. Ockelmann 1958). However. A. borealis is a variable species with several forms and varieties (see Zettler 2001). In the past, the great variability in morphological features resulted in a number of new species and subspecies de- scriptions (see Zettler 2001 ). Recent investigations of Hopner Pe- tersen (2001) show a large variety of shell morphology within the genus Astarte. The material discussed in the present study had a high constancy in respect to the relation between length and height (Fig. 7). Statements on population dynamics or size structure of A. bo- realis are very sparse in the literature. Some Russian studies (Gagaev 1989; Matveeva 1977) and few investigations on Baltic populations (Kohn 1989; Voigt 1991: Werner et al. 1974) have been carried out. During the studies of Kohn (1989) and Voigt (1991) in the Mecklenburg Bight the populations were dominated (89%) by individuals <6 mm in length. Larger sizes were observed only sporadically. In the present study, juvenile individuals were dominant at most of the stations investigated. Adult specimens (>20 mm) were only observed in high abundance in the inner part of the Mecklenburg Bight (Fig. 8). Kohn (1989) pointed out that only individuals >20 mm are reproductive. In the White Sea A. borealis reaches sexual maturity in sizes >17 inm (Matveeva 1977). The maintenance of the stock in the Mecklenburg Bight depends on few adults and/or probably on drifting of lecitothrophic (non-pelagic) larvae from regions nearby. In areas with strong currents, longer immigration distances are imaginable (Rasmussen 1973). In the Kiel Bight and in Russian Arctic waters however, the population structures are more balanced (Werner et al. 1974; Gagaev 1989). Meal Yield The mean wet meat yield of .4. borealis of 14.5% in the present study is similar to reported values of Kohn (1989). He found organic contents (wet) between 9% and 30% for a population in the Mecklenburg Bight. Ansell ( 1975) observed a soft tissue con- tent for/4, elliplica of 14.3-23.5% and dry tissue weights of 3-5% in British waters depending on the reproductive cycle. In the Mecklenburg Bight, in the present study, the mean individual ash free dry weight of A. borealis decreased from 9.1% to 5.6% with increasing size classes. The increasing mean wet yield and the decreasing ash free dry weight with increasing shell length indi- cates relatively higher water content of bigger bivalves. The length specific wet weight (shell length - wet weight relationship) in this study was similar to that reported for A. borealis of the Kiel Bight and Russian Arctic waters. In the Mecklenburg Bight, the calcu- lated wet weight for an individual of 25 mm shell length was 3.42 g. The wet weight of .4. borealis in the East Siberian Sea reported by Gagaev ( 1989) was 3.49 g and in Kiel Bight 4.22 g (Schaefer et al. 1985). According to Kohn (1989) the meat wet weight of a 25-mm specimen of A. borealis is 534 mg compared with 572 mg in the present study. In conclusion, the present study shows ecological and morpho- logical features of Astarle borealis in the Mecklenburg Bight near its eastern distributional boundary within the Baltic Sea. Further investigations must deal with growth rates and morphology of A. borealis within the Baltic and adjacent waters. The causes of the decline of this cold-adapted arctic-boreal species in much of the Baltic remain unclear. The comparative population dynamics at different water depths and/or within different sediment structures and the dispersion and settlement patterns of the larvae in this "border" area are of special interest. ACKNOWLEDGMENTS The author thanks Christine Peters, Ines Glockzin for helpful technical assistance, the Zoological collection of the University of Rostock for supplying valve material, and the referees for their helpful and interesting remarks. LITERATURE CITED Al-Hissni. Z. 1989. 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Box 5667 St. Johns. Nfld. Canada. AlC 5X1 ABSTRACT We monitored survival and allele frequencies at five enzyme loci in single year-class cohorts of cultured Mytilus spp. during a 14 month period from sleeving of seed to harvest at three commercial mussel farms in Notre Dame Bay, Newfoundland, Canada. Amona-site genetic heterogeneity at all five individual loci and over all loci combined was evident among the three seed sources. Significant temporal genetic heterogeneity occurred at all three sites. The observed intra-site temporal heterogeneity was not related to genolypic state smce neither the relative proportions of homozygotes and heterozygotes at each of the five loci individually, nor the mean heterozygosity over five loci, changed significantly within sites over time. Significant change in genotypic frequencies at the Gpi locus was the primary contributing factor to the overall temporal genetic heterogeneity within each of the three sites. Significant temporal changes in genotypic frequencies at each of the Mpi. Lap. Pgm. and Oilh enzyme loci inconsistently appeared among the three sites. A significant directional shift in genotypic frequencies, consistent across all three sites, gave evidence of genotype-dependent survival selection differentially favoring survival of Gpi genotypes with electrophoretically slower alleles in comparison to genotypes with electrophoretically faster Gpi alleles during sub-tidal rope culture of Mytihis spp. populations in Newfoundland. We also conclude that temporal genetic heterogeneity is a common occurrence in suspended rope culture of New- foundland blue mussel populations and is likely a significant contributory factor in the extensive geographic genetic population structuring previously reported among mixed M. ediilis - M. Irossiiliis populations. KEY WORDS: Mytihis. genotype-dependent, survival, genetic heterogeneity INTRODUCTION In northeastern North America, two separate mytilid species. Mytilus ediilis and M. trossiiliis are recognized (Koehn et al. 1984; Varvioetal. 1988; McDonald et al. 1991). In Atlantic Canada, the distribution of both species is widespread (McDonald et al. 1991; Mallet & Carver 1992) and the two form hybridized mixed-species assemblages (Saavedra et al. 1996). Newfoundland is. apparently, a zone of natural distributional overlap between these two mytilid species since most populations consist of mixtures of both species varying widely in relative frequency (Bates & Innes 1993; Penney & Hart 1999). Within the overlap zone in Newfoundland, sites in close prox- imity to each other display inter-site genetic heterogeneity on a magnitude scale comparable to sites much larger distances apart (Penney & Hart 1999). Such scales of genetic heterogeneity sug- gest the potential for significant stock- or site-related variability in growth, survival, or other characteristics among adjacent sites throughout the mixed-species zone. For commercial industry, fac- tors that significantly affect productivity within and among mussel farms is of great importance. Genotype-dependent temporal sur- vival selection processes have previously been noted in inter-tidal, mixed-species communities involving M. galloprovincialis and M. ediilis in England (Gardner & Skibinski 1991; Skibinski & Rod- erick 1991; Gardner 1994) and in a mixed M. ediilis and M. tros- siiliis intertidal assemblage in Nova Scotia (Pedersen et al. 2000). In cultured stocks, inter-site variation in mortality rates related to genetic (stock) differences are known to exist (Mallet et al. 1987, 1990) and may significantly affect production in suspended culture (Mallet & Carver 1989). There is also evidence that be- tween-site mortality differences are maintained when the stocks are transferred to new sites (Mallet et al. 1990; Myrand & Gaud- Corresponding author. R. W. Penney. Department of Fisheries and Oceans. Science. Oceans, and Environment Branch. P. O. Box 5667 St. John's, Nfld, Canada. AlC 5X1. E-mail: penneyrate'dfo-mpo.gc.ca reault 1995) suggesting that among-site survival variation is re- lated to stock genetics rather than environmental factors. Mortality and growth variation related to genotype {M. ediilis vs. M. tros- siiliis) was reported to significantly influence productivity at one fami site in Nova Scotia (Mallet & Carver 199.5). Since the com- bination of stock mortality and growth rale primarily define pro- duction in suspended culture, among-site stock and/or genotype- dependent variation in either parameter due to selective processes may be an important determinant of inter-site variability in fartn production indices. In this study, we investigate survival patterns in three hybrid- ized M. ediilis - M. trossidiis assemblages of cultured mussels in Newfoundland. Canada, over the latter phase of the production cycle, namely from sleeving of seed mussels to harvest. We use empirical observations of temporal change in allele frequencies at five enzyme loci to test the hypothesis that genotype-dependent selection processes occur within rope-cultured mixed M. ediilis- M. trossiiliis populations and. by inference, may be a significant contributory factor influencing genetic heterogeneity, survival, and production variability among mussel farms in Newfoundland. METHODS Seed mussels from collector ropes were sleeved in standard commercial plastic mesh on each of three farms in Notre Dame Bay. Newfoundland in August, 1998 (Fig. 1), In each case, the .seed mussels were derived from collector ropes set out on each farm in 1997. Seed mussels were mechanically de-clumped prior to sleeving to ensure random distribution across all sleeves. Each sleeve was 1 meter in length and was vertically suspended at 0.5-meter intervals from horizontal mainline ropes with flotation sufficient to ensure stable suspension at depths of approximately 10-12 meters. One week after gear placeinent, three replicate sleeves were retrieved from each farm site. From measured sec- tions of each sleeve, all the mussels were removed and counted and 100 individuals were randomly selected for allozyme analysis (to- tal of 300 from each site). The one-week delay in taking the first 41 42 Penny and Hart Burnt Arm \ Thwart Island ,r 55= 53", Notre Dame Bay Canada -Atlantic Coast Figure 1. Geographic location of the three farm sites in Notre Dame Bay. Newfoundland. sample was necessary to allow tor mussel accUmation and attach- ment to the sleeving material and thus eliminate drop-off of any mussels that failed to properly attach themselves via their byssal threads. Each farm site was similarly sampled in May. 1999 and again in October, 1999 at which time the majority of mussels had reached or exceeded the minimum commercial harvest size of 55 mm. Survival over time was calculated as the difference in mean number of mussels sleeve"' in May. 1999 and in October. 1999. compared to the initial sample. In this context, changes in survival do not distinguish between changes due to mortality versus drop- off from the culture gear. Hepatopancreas tissue was excised from each selected mussel, lyophilized. and stored at 5°C for later allozyme analysis. Five polymorphic loci were investigated: mannose phosphate isomerase {Mpi, EC 5.3.1.8). aminopeptidase-I {Lap. EC 3.4.1 1.-), phospho- glucomutase {Pgin. EC 2.7.5.1), glucose-6-phosphate isomerase {Gpi. EC 5.3.1.9), and octopine dehydrogenase (Odh, EC 1.5.1.1 1). A small amount of freeze-dried material was ground to a fine powder with 0.5M Tris HCL pH 8.0 buffer containing 20% glycerol and 0.2% NAD. Subsequent electrophoresis and staining on cellulose acetate plates followed the general procedures of He- bert and Beaton (1989). although a constant current of 2 mA per plate was used during the electrophoretic runs. The procedure for Lap was modified to run with Tris glycine pH 8.6 buffer. For Odh, we modified the stain for Cpi suggested by Hebert and Beaton (1989) to use octopine as the substrate instead of fructose-6- phosphate. Allele nomenclature is similar to that employed by previous authors (Koehn et al. 1984; McDonald & Koehn 1988) with the exception that alleles at the Odh locus were numbered consecutively from 0-9 in order of increasing electrophoretic mo- bility from the origin. The latter exception was necessary since we were unable to match Odh electromorphs on our plates with pre- viously published allele frequencies. Analysis of allele frequencies for population differentiation and probability estimation of log-likelihood G tests of genetic hetero- geneity within and among genotype classes and within and among sites utilized the software package F-Stat for WindowsT^. version 2.9.1 (Goudet et al. 1996). All other statistical analyses used the SAS software system (SAS Institute Inc. 1988). Significance lev- els of all test statistics involving multiple comparisons were Bon- ferroni-adjusted. Non-parametric analysis of variance of ranks and Rruskal-Wallis rank test statistics (SAS Institute Inc. 1988; Sokal & Rohlf 1995) were calculated to test for directional, intra-site, temporal trends in genotypic frequency distributions at the Gpi locus. For these analyses, the twelve observed Gpi alleles were assigned an ordinal rank score based on electrophoretic mobility, with faster alleles scoring lower than slower alleles (e.g., C/w' '" = rank 1 ; Gpi rank 2, etc.). Thus for diploid loci, the genotypic rank assigned each individual is the mean rank score of its two constituent alleles (e.g.. for an individual of genotype Gpi""'" . rank = 1 .5, etc.). For Kruskal-Wallis tests with n > 2 sample dates, SAS calculates a x" approximation of the Kruskal-Wallis test sta- tistic for significance testing (SAS Institute, Inc. 1988). RESULTS Seed mussels were collected from three farms located in Burnt Arm, Charles Arm. and at Thwart Island in Notre Dame Bay on the northeast coast of Newfoundland (Fig. 1 ) and transferred to com- mercial plastic mesh sleeves in August, 1998, for grow-out. Elec- trophoretic assays of the three seed mussel populations detected four alleles at the Mpi locus, six at the Lap locus, eight at the Pt;iii locus, eleven at the Odh locus, and twelve at the Gpi locus. Gene diversity per locus at the three sites was very low (<0.20l at the Odh locus, moderate (0.40-0.50) at the Mpi locus, relatively high (0.60-0.75) at Lap and Pgm. and highest O0.80) at Gpi. Log- likelihood G lest scores from inter-site comparison of the three Genotype-Dependent Survival in Mussels 43 seed mussel popiikitidiis in August 1998, revealed significant (p < 0.001) among-site population genetic heterogeneity at all loci in- dividually as well as o\er all loci combined (Table 1 1. At the Mpi locus, Mpi^"" predominated at Burnt Arm and Charles Arm while Mp/'"* was the allele at highest frequency at Thwart Island (Fig. 2). Pgm'°" was the most common allele at the Pgm locus at both Burnt Arm and Charles Arm while the frequency of Pgiii^ ' ' was highest at Thwart Island. At the Lap locus, Lcip''^ was the most common allele at Charles Arm but at Burnt Arm and Thwart Island the frequency of Lap'"^ was greatest. Gpr"^ predominated at Charles Arm while, at both Burnt Arm and Thwart Island, the frequency of Gpt"" was highest. At Odh. the three sites differed only in frequency of relatively rare alleles. The frequency of Odlr" was greatest for all three sites. In August, 1998, mean mussel density on the sleeves varied among sites from 450 mussels m~' at Charles Arm to 790 mussels m~' at Burnt Arm and 890 mussels m"' at Thwart Island. Survival to May, 1999 (calculated as within-site change in mean mussel density since August, 1998) was not significantly different among sites (ANOVA. P > 0.05). Mean survival during the first 9 months of grow-out (to May, 1999) was 81.2% at Charles Arm. 76.9% at Thwart Island, and 78.0% at Burnt Arm (Fig. ?<). After 14 months on the sleeves (October, 1999), mean survival at Charles Arm was significantly less than the other two sites (ANOVA, P < 0.05). Mean survival at Charles Arm was only 37.4% compared to 5 1 .6% at Thwart Island and 51.7% at Burnt Arm. Log-likelihood G test scores for among-site comparisons of population genetic structure after 9 and 14 months of grow-out (May and October, 1999 re- spectively) were also significant (Table I) indicating the initial among-site genetic heterogeneity was maintained during the course of the grow-out period. During the same 14 month grow-out period, significant intra- site temporal changes in allele frequencies at one or more loci were found at all three sites (Table 2). After the first 9 months of grow-out (to May, 1999), log-likelihood G test scores were sig- nificant over all loci combined at all three sites indicating signifi- cant temporal change in genetic structure had occurred over this period. Significant intra-site temporal population differentiation at the Gpi locus was evident at all three sites. Intra-site population genetic differentiation was also evident at the Mpi locus at Burnt Arm, the Pgm and Odh loci at Charles Arm, and at the Odh locus at Thwart Island. This pattern continued through to October, 1999 after 14 months of grow-out by which time further genetic differ- entiation at the Odh locus had become evident at Burnt Arm and at the Lap locus at Thwart Island. To determine whether the observed intra-site temporal genetic differentiation revealed by the log-likelihood G tests gave evi- TABLE 1. Log-likelihood G test (dloudet el al. 1996) probability estimates for inter-site population genetic differentiation al the time of socking (August, 19981, in May, 1999, and at harvest in October, 1999. Locus Date Mpi Lap Pgm Gpi Aug 98 May 99 Oct 99 ^^^ ***p<0.001 dence of selective survival patterns, we further examined the dataset for (a) non-random mortality related to genotypic state (e.g.. differential survival of homozygotes vs. heterozygotes) at either of the five loci individually or over all loci combined; and (b) non-random mortality favoring specific single locus genotypes. With respect to (a), for all five loci individually, the relative pro- portions of homo/ygotes vs. heterozygotes within all three sites did not significantly change over the 14 month grow-out period (Fisher's exact test; P > 0.05). Within-site variation in mean multi- locus heterozygosity was also not significantly related to time (sample date) and the interaction effect of time x site was not significant (ANOVA; P > 0.05) at all three sites (Table 3). Thus, we conclude the observed intra-site temporal variation in popula- tion genetic structure revealed by the log-likelihood G tests is not explained by selective survival related to genotypic state at either of the five loci individually or on the basis of multi-locus het- erozygosity (multi-locus homozygotes vs. heterozygotes). Multi- locus heterozygosity did significantly vary however among sites (ANOVA. P < 0.01; Table 3). Mean multi-locus heterozygosity was lowest at Charles Arm. highest at Burnt Arm and intermediate between these two at Thwart Island (Fig. 4). However, only the May and October means at Burnt Arm and Charles Arm were significantly different (Tukey. P < 0.05). With respect to (b). we then tested the hypothesis that the observed intra-site temporal genetic differentiation could be ex- plained by non-random mortality favoring specific single locus genotypes. To accomplish this, we tested for temporal patterns of change in genotypic frequencies (x^) with specific individual al- leles (homozygotes and heterozygotes combined) versus those without at each site x locus combination wherein the log- likelihood G test was significant. Homozygotes and heterozygotes were combined for this analysis due to the previously noted non- significant temporal change in frequencies of homozygotes and heterozygotes at all three sites. With respect to the Lap, Pgm. and Odh loci, the observed temporal changes in frequency of geno- types with or without all individual alleles were not significant after Bonferroni-adjustment of significance levels for multiple test comparisons. For the Mpi locus, a significant G test score was observed only at Burnt Arm (Table 2). Al this site, the relative frequencies of genotypes which included the Mpi""' allele were significantly reduced while the relative frequencies of genotypes which incorporated the Mpi"''' allele were significantly increased during the August. 1998, to October, 1999 period. These frequency changes remained significant after BonfeiToni correction of prob- abilities for multiple comparisons. Frequency changes at Burnt Arm for genotypes with all other Mpi alleles were not significant after Bonferroni correction of probabilities. At the Gpi locus, x' tests of temporal patterns of change in genotypic frequencies involving specific alleles varied across the three sites (Fig. 5). At Burnt Arm. significant (x", P < 0.05) fre- quency reductions occurred within genotypes which included the Gpi"", Gpi"'-, and Gp/'"^ alleles, while genotypic frequencies with — the Gp('\ Gp/^', Gpi^^, and Gpi" alleles all increased. At Charles Arm. a significant reduction in individuals with genotypes that qjIj ^11 included Gpi"" also occurred while genotypic frequencies with Gpi"'", Gpi"", and Gpi*^" all increased. At Thwart Island, a trend towards increasing frequency of individuals with Gpi''^ and G/x**' occurred with reductions in the frequency of individuals with the more electrophoretically mobile Gpi'"^ and Gpi'"\ Frequency changes over time for all other Gpi alleles at each of the three sites 44 Penny and Hart 09 08 05 04 < 03 0 1 . 04 < 02 ■ Burnt Arm n Charles Arm B Thwart Island 94 90 Mpi Allele ■ Burnt Arm D Charles Arm B Thwart Island 96 Lap 94 Allele 08 07 06 g 0.5 a> 3 S 04 « M 03 < 0,2 0 1 0 0.4 035 03 ^ 0.25 0} 3 IT S 0.2 u. a> M 015 < 01 0 05 [J_ 114' 111 ■ Burnt Arm DCharles Arm H Thwart Island Jk 106 100 Pgm Allele ■ Burnt Arm DCharles Arm H Thwart Island 107' 102 100 98 96 93 89 86 83' Gpi Allele *" ^ 1" — ■ Burnt Arm DCharles Arm 1 BThwart Island S »^ B-Ri -Tk, 09 08 07 u £ 06 O" S 05 u. I 04 < 0.3 02 01 0 0' 1 2 3 4 5 6' Odh Allele Figure 2. Inttr-site comparison of allelt frequencit's at the Mpi. Pfiin. Uip. Gpi, and Odh enzyme loci in seed mussel populations at Burnt Arm, Charles Arm, and Thwart Island at the start of the rearing trials in August, 1998. Allele frequency numhers with a ' (e.g., I'gm allelel 14' l include frequencies of adjacent rare alleles. were not significant (\^. P > 0.03) when significance levels were Bonferroni-adjusted for nuiltiple comparison tests. However, while the frequencies of the same individtial Gpi alleles were not consistently significantly differentiated among sites over time, the overall Gpi genotypic frequency distribution significantly shifted in favor of electrophoretically slower alleles at all three sites (Table 4). At the Gpi locus, mean genotypic rank scores within all three sites significantly increased over time. Also. Kruskal-Wallis H tests revealed the median genotypic rank score also significantly increased within all three sites over time. These results indicate a consistent directional change in the overall ge- notypic frequency distribution had occurred at all three sites. Gpi genotypes carrying alleles of higher electrophoretic mobility were significantly reduced in favor of those with alleles of relatively lower electrophoretic mobility at all three sites during the 14- month grow-out period. We infer this directional shift in Gpi ge- notypic frequencies which is consistent across all three sites is evidence of genotype-dependent selective survival processes dif- Genotype-Dependent Survival in Mussels 45 100 ^--^ - Charles Arm -Thwart Island Burnt Arm J 90 - - » - — -A- 2 80 I Survivorship 8 S 50 \ >■ 40 \ August 1998 May 1999 October 1999 Figure, i. Comparative survival from sleeving in August, 1998, to harvest in October, 1999 at the three farm sites. Plotted points are means of replicate socks with ±2 se. ferentially favoring alleles of relatively lower electrophoretic mo- bility at this locus during sub-tidal, rope culture of Mytihis spp. in Newfoundland. DISCUSSION Two recent studies {Gilg & Hilbish 2000; Pedersen et al. 2000) have provided evidence that both cohort niixuig as well as selec- tive survival processes work to produce significant temporal ge- netic heterogeneity in mixed-species, intertidal mytilid communi- ties. However, ours is the first to examine the potential for similar selection processes within suspended rope-cultured populations in commercial culture. In the present work, we have found significant within-site genetic heterogeneity among samples taken over a 14 month grow-out period for three mixed-species populations. The probability that our results were affected by cohort mixing seems remote. This would have required an undetected re-settlement on TABLE 2. l.og-likelihood G test ((loudet et al. 199A) probability estimates for intra-site temporal genetic differentiation during the 14 month grnwout period at all loci individually and over all loci combined. the culture sleeves with a new larval cohort some time after the original gear setup date in August. 1998. Such an occurrence would have been easily detected due to the disparity in length frequencies between the older mussels and the new cohort on the sleeves. Our length frequency data show no evidence of such cohort mixing. A more parsimonious explanation for the observed intra-site differences among sample dates is that temporal genetic heterogeneity occurred within the original populations from time of sleeving in August. 1998. through to harvest size in October. 1999. Both Gilg and Hilbish (2000) and Pedersen et al. (2000) re- ported genotype-dependent mortality patterns in Mytilus spp. populations that resulted in population structuring along species lines. In the case of the former, an intertidal mixed population of newly-settled M. edulis. M. galloprovincialis. and their hybrids in SW England became heterogeneous on the basis of tidal height. In the latter, a directional selective mortality pattern favoring M. tros- siihis at the expense of M. edulis was demonstrated in a mixed intertidal M. edulis and M. trossidiis assemblage in Nova Scotia. In our study, the mussel populations at all three sites could be clas- sified as hybridized, mixed-species (M. edulis, M. trossulus. and their hybrids) stocks based on their Mpi genotypes (Varvio et al. Interval (months! Loc us TABLE 3. Analysis of variance (ANOVA) test results of number of heterozygous loci (out of five) per ind variation ividual an Site Mpi Lap Pgm Gpi Odh All in the nong the Burnt Arm three sites and sample dates. F ratios without su perscripts are not 9 ** n.s. n.s. * n.s. ■* significant tP >0().S| 14 9 14 * n.s. n.s. n.s. n.s. n.s. n.s. * *** *** * *** *** *** Charles Arm Variable d.f. MS F * *** *** *** Date 2 0.62 0.56 Thwart Site 2 6.31 5.72** Island 9 n.s. n.s. n.s. *** * ** Date X Site 4 0.07 0.06 14 n.s. * n.s. *+* ** *** Residual 2652 1.05 * P < 0.05; * *P 9.28 ± 1 .00 1 .07 ± 0. 1 1 0.11 ±0.01 E 20.33 ± 1.28 14.34 ±0.06 5. 98 ± 0.46 0.30 ± 0.02 TPM: total paniculate matter: PIM: particulate inorganic matter: POM: particulate organic matter; f: organic content of suspended matter. Values are mean ± S.D. and faeces produced were collected continuously. The time when no more faeces and pseudofaeces produced was considered as the maximum evacuating time which was estimated al 50 min. After emptying the gut. all pseudofaeces and faeces produced in the first hour were removed. Each individual was then exposed to an ex- perimental diet for 150 to 180 min and faeces and pseudofaeces were collected with caution during and at the end of the experi- ment to prevent resuspension of faeces and pseudofaeces. After food supply was stopped, the mussels were maintained in the beakers for another 40 to 50 min until no more faeces and pseudo- faeces were produced. The minimum gut passage tiine rather than the mean gut passage time (Decho & Luoma 1991) was mea- sured and defined as the difference in time between initial filter- feeding by each individual and the first appearance of faeces dis- regarding extracellular and intracellular digestion. The total, inor- ganic and organic weight of pseudofaeces and faeces were determined by the same methods as those described for seawater samples. The following rates were then computed: total matter rejection (RR; mg h"'l, organic matter rejection (ORR: mg h"'), inorganic matter rejection (IRR: mg h~'), total egestion (defeca- tion) (ER: ing h~'). organic matter egestion (OER: mg h"') and inorganic matter egestion (lER: mg h"') (Hawkins et al. 1996). Calculation of the Feeding Parameters Food processing rates (feeding and absorption) were calculated following Iglesias et al. (1992). Assuming that absorption of in- organic matter through the digestive system was negligible (Cran- ford & Grant 1990). the sum of IRR and lER was considered to represent the rate of inorganic matter filtration (IFR; mg h~'). Clearance rate (CR; I h~'l was then estimated as CR = IFR/PIM. Filtration rate of total particulate (FR: mg h"') was computed as FR = CRxTPM and filtration rate of particulate organic matter (OER: mg h"') as OER = CRxPOM. Ingestion rates of total particulate matter (IR; mg h"') and of particulate organic matter (OIR: mg h"') were estimated as IR = FR-RR and OIR = OFR- ORR and hence, the organic content of absorbed food could be estimated as AR (mg h~') = OIR-OER. and absorption efficiencv (AE) = AR/OIR. Preingestive selection efficiencies for total filtered organic mat- ter (SEo) were estimated as: SEo = I - (p/f) (Navano et al. 1992). where p is the organic content of the pseudofaeces and f is the organic content of the suspended matter. In view of a very narrow size range of animals we used, no attempt has been made to weight-standardized the measured feeding rates. Statistical Procedures To obtain functional relationships between feeding responses and food parameters, a set of regression equations was fitted to experimental data, following standard least-squares procedures. Regression analyses were performed by simple linear and non- linear procedures, depending on the most appropriate function to be fitted in each case (Zar 1999). Multiple regression analysis was conducted when feeding behavior was correlated with more than one diet parameters, and the coUinearity between the independents was tested with coUinearity statistics of SPSS (Belsley et al. 1980; SPSS Inc. 1999a). When the independent(s) were highly correlated with others, they would be eliminated from the independent list and the model was reconstructed until all the intercorrelations between the independents were removed from the regressive model. Residuals were also analyzed to check the normality, con- Feeding Responses of Sept/fer virgatus 53 slant variance of predicted dependents and other necessary as- sumptions of the regression model. Data were transformed if nec- essary to meet the regressive requisites, i.e.. nomiajity of datum distribution and homogeneity of variances. Analysis of variance (ANOVA) was used for comparisons among different treatment groups. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of vari- ance with Kolmogorov-Smirnov test and Levene test, respectively. The specific function and procedures followed for each feeding response will be presented with the results. All the statistical pro- cedures were performed with software SPSS for windows, release 9.0 (SPSS Inc. 1999a.b). RESULTS Figure 1. The relalicinship between filtration rates (FR: mg li 'l and rejection rates (RR: nig h~'l in S. virgatus. Clearance Rales and Filtration Rates The relationship between CR and food availability was weak but significant and can be described by the multiple regression equation as: CR = 0.18-1- 0.09/TPM -i- 0.13 ftr" P < 0.001) 0.19. F,,,, = 6.56. There was no significant relationship between FR and f. FR. how- ever, was positively related to POM and the equation that describes the relationship is: FR = 0.79 -I- 0.57 POM (r- = 0.72, F, ^.^ = 185.57. P< 0.001) Pseudofaeces Rejection, Selection Efficiency and Ingestion of Filtered Food The organic content of particulate matter (f) was significantly higher than that of the pseudofaeces for Diet C. D and E of which pseudofaeces were produced (Table 2). As TPM increased, pseud- ofaeces production increased with the increase in FR (Fig. 1 ) and can be described by the equation: RR = -0.439 -f 1.014 hi(FR) (r" = 0.84. F.^^ = 365.04. P< 0.001). There was a significant positive relationship between TPM and RR (RR = 0.54 + 0.08 TPM (r- = 0.786. P < 0.001 )). No significant relationship, however, could be established between RR and f. showing that the rejection rate was related to the quantity (TPM) but not the quality (f) of suspended matter. As pseudofaeces were only produced for Diets C. D. and E. the lowest threshold of TPM for pseudofaeces production was about 10 mg P'. Selection efficiency (SEo) is a measure of the efficiency in selecting the organic fraction of food when pseudofaces are being produced. There was an inverse relationship between SEo and TABLE 2. Comparisons between the organic content of the diet (f) and that of the pseudofaeces (e) for diet C, D and E where pseudofaeces were produced. f e Diet (mean ± SD) (mean ± SD) value Degree of Freedom c (J.22 ± 0.02 0.12 ±0.058 5.10 D 0.11 ±0.01 0.071 ±0.016 6.91 E 0.30 ± 0.02 0.199 ±0.068 5.56 8 <0.001 7 <0.001 13 <0.001 TPM. No significant relationship between SEo and f. however, could be established. The corresponding equation is: SEo = 0.77 -t- 1.51/TPM (r" = 0.49. F, 66.34. P< 0.001) Ingestion rate (IR) is estimated as the difference between filtered food and rejected pseudofaeces. Both IR and OIR were not sig- nificantly correlated with f but IR was a positive power function of TPM and OIR a linear function of POM which can be described by the following equations: IR = 1.02 TPM""* (r- = 0.82. F, „« = 328.1 1, P < 0.001) OIR = 0.048 + 0.186 POM (r' = 0,91. F, ^g = 672.81. P< 0.001) The more the particles were filtered, the more they were in- gested, resulting in a positive linear relationship between IR and FR: IR = 0.50 + 0.64 FR (r- = 0.94, F.^g = 1013.45, P < 0.001) Absorption Rate and Absorption Efficiency AR was a positive linear function of f (Fig. 2). As OIR in- creased. AR increased rapidly until OIR reached about 0.4 mg h"' (Fig. 3). The rate of increase of AR. however, was slower when OIR increased further. The corresponding equations are: AR = 0.09 + 0.85 f(r- = 0.63, F, ^^ = 126.27. P < 0.001) AR = 0.45 - 0.05/OIR (r' = 0.74. F, 192.70. P< 0.001) 0.6 0.5 -~ 0.4 g 0.3 oi 0.2 0.1 0 0.1 0.2 0.3 0.4 0.S Figure 2. Absorption rates (AR: mg h ') as a function of organic content (f) of seston in S. virgatus. 54 Gao et al. 0.6 ♦ 0.5' ^ ♦♦ ♦ ^ 0-4' ^¥H 9f~ ♦ ♦♦ ♦ ■= 0.3 /* ♦ ♦ ♦ OX) E /<•♦ ♦ -S 0.2- k ck: ^ < 0.1- f 0 0 0.4 0.8 1.2 1 OIR(mg h^) Figure 3. The relationship between absorption rates ( AR: ni;; h ' ) and organic ingestion rales (OIR: nig h ') in S. rirgatiis. AE decreased exponentially with an increase in TPM in the water and was a negative quadratic function of both POM (Fig. 4) and OIR (Fig. 5). The corresponding equations are: AE = 0.962e-""-*''^™ (r- = 0.42. F, „, = 61.28. P < 0.01 1 AE = 0.726 + 0.037 POM - 0.017 POM' (r" = 0.73. F,. !.03. P < 0.001) AE = 0.77 + 0.07 OIR - 0.38 OIR- (r" = 0.74. F2.67 = '^6.36. P< 0.001) Gut-Passage Time Gut-passage time (GPT) for particles was a negative exponen- tial function of TPM (Fig. 6) and a negative quadratic function of OIR (Fig. 7). showing that the time the food particles stayed in the gut was prolonged when OIR was low. GPT. however, was much reduced when OIR increased. The respective equations are: 213.34. P< 0.001 = 0.42. GPT = jv._i^ e u - vj. /u, 1 , ,,x GPT = 21.28 + 49.07 OIR - 43 04 OIR' (r F,„7 = 24.16. P< 0.001) DISCUSSION Rates of Clearance, Filtration and Pseudofaeces Production Food availability is regarded as one of the most important factors affecting the feeding behaviour of bivalves. To compensate 1 1 0.8 0.6 < 0.4 0.2 0 3 4 POM(mgr') Figure 4. Absorption efficiency (AE) of .S. virgalus as a function of particulate organic matter (POM: mg I"') in the water. 0.4 0.8 OIR(mgh"') 1.2 1.6 Figure 5. The relationship between absorption efficiency (.\E| and organic ingestion rates (OIR: mg h') in .S. virgalus. for fluctuations in lV)od availability, various mechanisms have been de\ eloped which include regulation of feeding rate, pseudofaeces production, selection of nutritious particles and alterations to di- gestive efficiency (Hawkins et al. 1990: Bayne et al. 1993: Bacon et al. 1998). As particle concentration increases, ingestion is com- monly regulated by a reduction in clearance rate and the produc- tion of pseudofaeces in mussels (Widdows et al. 1979). scallops and clams (Navarro et al. 1992: Bacon et al. 1998). A similar reduction in clearance rate with food availability was also recorded for another locally dominant mussel, i.e.. Perna viridis. under both laboratory (Wong & Cheung 1999) and field conditions (Hawk- ins et al. 1998: Wong & Cheung 2001a.b). In contrast, the correlation between clearance rate and food availability (TPM and f) in Scptifer virgatits was weak, showing that ingestion of par- ticulate organic matter was not regulated through clearance rate. In M\tihis irussiiliis. CR was independent of increasing seston quality at relatively high seston loads (20 and 50 mg T') (Arifin & Bendell-Young 1997). Positive coiTelations between clearance rates and the TPM of natural seston have been reported upon for Mytilus edidis (Newell & Shumway 1993: Hawkins et al. 1996) and Cerastodenna edide (Iglesias et al. 1992). CR of Crassostrea gigas was maintained high and constant when TPM was 50 mg 1"' but decreased with further increases (Barille & Prou 1994). Nev- ertheless, all species should have an upper limit 10 the rate of panicle processing (Bayne & Newell 1983). It was expected, therefore, that the CR of 5. virgatiis would ultimately decrease when TPM increased to a level higher than that experienced in the present study. With clearance rate relatively independent of food quantity, filtration rate increases with POM, as shown here for Septifer 60 50 p 40 1 30 «5 20 ^ 10 10 15 20 25 TPM (mg r') Figure 6. Gut passage time (GPT: min) of S. virgatus as a function of total particulate matter (TPM: mg I"') in the water. Feeding Responses of Septifer v/rgatus 55 0 0.4 0.8 1.2 1.6 OIR(mgh"') Figure 7. The relationship between gut passage time K;PT: min) and organic ingestion rates (OIR: mg h"') in S. virgatiis. virgaliis. Reports on the relationship between fihration rate and food concentration, however, were variable. Winter (1973) showed that the filtration rates of M. ediilis decreased with increasing food concentration such that the bivalves could keep the number of filtered algae in unit time relatively constant within the range of 10 X 10'' to 40 X 10'' cells I"'. Contrary results, however, were re- ported by Winter (1978) and Riisgard and Randlov (1981), that is, that filtration rates increased quickly with increasing particle con- centration. Griffiths and Griffiths ( 1987) reviewed the relationship between food quality and quantity and filtration rate of various species of suspension-feeding bivalves and concluded that con- flicting experimental results were largely attributed to differences in particle concentration. At low concentrations, a positive rela- tionship between filtration rate and food quantity was established. When similar experiments were conducted at low to intermediate particle concentrations, and the concentration ranges were rela- tively narrow, no obvious relationship between filtration rate and particle concentration was observed. In the present study, four out of the five treatment groups had particle concentrations either equal to or lower than 10 mg I"'. A reduction in filtration rate, therefore, is expected at higher particle concentrations when CR decreases. Pseudofaeces production is regarded as one of the most Impor- tant regulative processes in bivalves to organically enrich ingested matter by the selective rejection of inorganic matter versus organic matter and thus, as a consequence, to optimize energy uptake. The production of pseudofaeces is strongly related to both the density and organic content of suspended seston In the water (e.g., Foster- Smith 1975; Bacon et al. 1998: Baker et al. 1998; Benlnger et al. 1999). Septifer virgatus started to produce pseudofaeces when TPM was -10 mg P'; its production rate was also highly corre- lated with TPM and FR. Ingestion rate, therefore, increased across the full range of food availability. This agrees with the observa- tions of Ki0rboe at al. (1980) and Hawkins et al. (1996) on Mytihis edulis but is not consistent with those of Widdows et al. (1979) who demonstrated that maximum IR coincided with the threshold TPM above which pseudofaeces were produced. Hawkins et al. (1996) anticipated that IR would reach maximal values in asso- ciation with reduced CR and/or an Increase In the ratio RR/FR, as observed for M. edulis feeding upon pure strains of cultured algae (Foster-Smith 1975). The SEo index represents the efficiency with which bivalves can organically enrich ingested matter by selectively rejecting In- organic matter via the palps. For both C. edide (Iglesias et al. 1992, 1996) and P. viridis (Wong & Cheung 1999), SEo was a curvi- linear function of f whereas in another study on C. ediile, SEo was positively related to f and negatively related to seston concentra- tion (Navarro & Widdows 1997). In S. virgatus. SEo varied from 0.74 to 1.0 and was negatively correlated with TPM but not sig- nificantly correlated with f. Such high values of SEo in S. virgatus can probably be attributed to low seston concentrations and it Is expected that SEo would decrease when seston concentration In- creased beyond that experienced by the individuals used in this study. A maximum selection efficiency of 0.60 was obtained for C. edule when fed at low seston concentrations, and this decreased to the lowest value of 0. 1 0 when seston concentration increased to 600 mg r' (Navarro & Widdows 1997). The SEo of Permi viridis was comparable to S. virgatus, with a maximum efficiency of 0.75 (Wong & Cheung 1999). The present study demonstrates inter-specific differences In pre-lngestive feeding behaviour between two mussels in Hong Kong. S. virgatus Is a dominant species on exposed rocky shores in the eastern waters of Hong Kong characterized by low TPM values (Morton & Morton 1983). Seasonal variations in TPM at Cape d'Aguilar, Hong Kong, where the animals used in the present study were collected from, have been studied for one year (unpub- lished data). TPM varied from 4.5 to 36 mg T' with an annual mean of 9.5 mg 1"'. With the relative independence of clearance rate from particle concentration, and positive relationships be- tween RR and FR and between IR and FR, the ingestion rate of organic particulate matter, therefore, could be enhanced under the low food concentrations which prevail In this environment. In contrast to S. virgatus. P. viridis occurs predominantly in sheltered environments in which the water Is often turbid and sometimes heavily polluted (Lee 1985; Cheung 1993). This species possesses large labial palps with strong ciliary rejection tracts (Morton 1987; Seed & Richardson 1999) to cope with the high sediment load- ings often associated with sheltered, low energy habitats. Ingestion of particulate organic matter by P. viridis was regulated through pseudofaeces production, as herein demonstrated for S. virgatus. although the rate was much higher at -9 mg h"' (Wong & Che- ung 1999) as compared with S. virgatus of a similar size (-1.6 mg h"'). Regulation was further enhanced by controlling clearance rate which has not been observed in S. virgatus. Absorption and Gut Passage Time The absorption efficiency of suspension-feeding bivalves has been shown to change over time scales according to variability In seston characteristics (Bayne & Newell 1983) and is mainly con- trolled by organic food quantity and/or quality when feeding on either natural seston or on suspensions resembling natural condi- tions (Bricelj & Malouf 1984). In the present study, the AE of S. virgatus was negative quadratic functions of both POM and OIR. Digestion in bivalves is biphasic involving the stomach and diges- tive diverticula as compartments for extra- and intra-cellular di- gestion, respectively (Purchon 1968). Food may undergo extra- cellular digestion in the stomach followed by intestinal absorption and is voided as "Intestinal faeces"". Some of the food may then also undergo intra-cellular digestion and absorption within the di- gesfive diverticula and eliminated as "glandular faeces'" (Bricelj & Malouf 1984). With higher rates of food processing, the pro- portion of particles by-passing the digestive diverticula would in- crease and result In reductions in both GPT and AE (Navarro & Iglesias 1993). In models combining gut passage time and feeding 56 Gao et al. behaviour. Willows (1992) also showed that GPT generally de- creased with an increase in the quality of food at high levels of food availability. These observations agree with the present study, i.e.. as food concentration increased (TPM). the GPT of S. virgatus remained at high levels and was relatively constant when OIR was low {<0.4 nig h"' ). Further increases in OIR resulted in a reduction in GET. The high and constant values of AE under relatively low OIR (up to 0.4 mg h"'), therefore, might be attributed to the prolonged GPT and resulted in a rapid increase in AR from 0. i to 0.3. Further increases in OIR. however, did not cause significant increases in AR. Similar observations were also reported upon for Placopecten magellanicus (Brillant & MacDonald 2000) and Aulacomya ater (Griffiths & King 1979) that AE of the latter species decreased rapidly with increasing algal concentration and AE approached zero when algal concentrations were >32 x 10'' cells r'. Using algal concentrations ranging from 50 to 100 x 10^ cells r' (equivalent to 1.25 - 2.5 mg 1"' POM). Gerdes (198.3). however, found that AE of the Pacific Oyster. Crassostrea gigcis. was independent of ration with AE of -75% being obtained for all three kinds of diet with different TPM and POM. As the range of POM used in our study on S. virgaius varied from 1 to 6 mg 1"' as compared to a narrower range ( 1.25 - 2.5 mg 1"' ) used by Gerdes ( 1983). AE of C. gigas is expected to decrease, as what has been observed in 5. virgatiis and other bivalves, when POM increases further. The ranges of seston quantity (TPM) and quality (f) used in this study were 20 mg 1"' and 0.4, respectively, and within the ranges recorded for the habitat where 5. virgciiiis was collected for this study. AR reached asymptote when OIR was only one third that of the highest value. This suggests that S. virgarns is particularly well-adapted to a low seston concentration environinent. Food quality was also an important factor controlling AR. For example, AR is a positive quadratic function of POM in P. viriiiis (Wong & Cheung 1999) and is positively linearly related to the different measures of diet quality in Placopecten inagellanicus. even when the organic content of seston (f) was as high as 80% (Cranford 1995). This agrees with the results obtained for 5. rirgatits in this study. The present study demonstrated regulative mechanisms in the subtropical mussel S. virgatiis which is dominant on locally clean and wave-exposed habitats characterized by low seston concentra- tions. As food concentration increases, particulate organic matter ingestion is enhanced through pseudofaeces production with pref- erential ingestion of particulate organic matter, although clearance rate is relatively independent of food quantity. GPT was a negative function of TPM and OIR such that AE is relatively constant and absorption rate reaches asymptote at low seston concentrations. 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E. 1978. ^ review on the knowledge of suspension-feeding in lamellibranchiate bivalves, with special reference to artificial aquacul- ture systems. Aquaculture 13:1-33. Wong, W. H. & S. G. Cheung. 1999. Feeding behaviour of the Green mussel. Pema viridis (L.): responses to variation in seston quantity and quality. / Exp. Mar. Biol. Ecol. 236:191-207. Wong. W. H. & S. G. Cheung. 2001a. Feeding rates and scope for growth of Green mussels. Pema viridis (L.) and their relationship with food availability in Kat O. Hong Kong. Aquaculture 193:123-137. Wong. W. H. & S. G. Cheung. 2001b. Feeding rhythms of the green-lipped mussel. Pema viridis (Linnaeus. 1758) (Bivalvia: Mytilidae) during spring and neap tidal cycles. J. Exp. Mar. Biol. Ecol. 257:13-36. Zar. H. J. 1999. Biostatistical Analysis (fourth edition). Prentice-Hall. New Jersev. Joiinml of Shellfish Rcsctnrh. Vol. 21. No. 1. 39-65. :002. COMPARATIVE SETTLEMENT DEPTHS OF MYTILUS EDULIS C. LINNAEUS, 1758 AND M. TROSSULUS GOULD. 1850: I. A MESOCOSM STUDY K. R. FREEMAN,' * E. KENCHINGTON,' AND S. P. MACQUARRIE" ^Department of Fisheries & Oceans, Bedford Institute of Oceanograpliy. P.O. Box 1006. Dartmouth, Nova Scotia B2Y 4A2. Canada: 'Department of Biology. Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia B3H 4J I, Canada ABSTRACT Production on many commercial mussel farms in Eastern Canada is hampered hy the presence of Mytilus trossulus. a comparatively thm-shelled but close relative of M. ediili.s. the principal species collected and grown. Exploitation of a species-based difference in depth preference could potentially increase the collection of M. edulis at mixed species sites. As a first step in the investigation, larval cultures of each species were reared to settlement in replicated mesocosms in a study conducted at Dalhousie University's Aquatron facility in Halifax. Nova Scotia. Depth preference for settlement was examined with and without a thermocline. Both M. edulis and M. trossulus had the largest percentage of larvae settling at the surface. Settlement occurred below the thermocline at 6 m in both species, although in significantly lower proportions. In mixed water, a significant species-depth interaction was detected. This was driven by a significantly large number of M. edulis spat settling at 8 m. a pattern not found in M. imssuliis- KEY WORDS: Myiilus edulis. M. trossulus, settlement, depth, mesocosms INTRODUCTION Farm recruitment 'of mussels in Eastern Canada depends on the timing and inter-relationship of a number of natural events coin- bined with judiciously applied husbandry practices. The deploy- ment of a suitable settlement medium, at an appropriate time, is key to maintaining an uninterrupted cycle of spat collection, grow- out, and harvest. Traditionally, spat collecting is performed on the farm site, usually by deploying plastic mesh (Vexar^^") on head- ropes near the surface several days before the anticipated onset of settlement. This technique has proven successful over the rela- tively short history of Eastern Canadian mussel farming. Never- theless, spat collections at some farms comprise two species {Myti- lus trossulus Gould, 1850 and M. edulis C. Linneaus 1758) and the presence of the former is cause for concern to many growers (Freeman 1996). Although it was realized early in the industry that certain farms often produced mussels of varying quality, it was not initially appreciated, at least in certain cases, that the problem was species- based. After this had been demonstrated (Koehn et al. 1984). in- vestigations into distributions of the two species showed that East- em Canadian Mytilus populations vary from purely one or the other species to varying proportions of each (Penney & Hart 1999; Mallet & Carver 1999). Although the two are often superficially similar, M. trossulus is characterized by comparatively fragile shells and generally lower ineat weight per shell length. For ex- ample, it has been found that the initial number of M. trossulus farmed would have to be multiplied 1 .7 times to achieve the same economic return as with M. edulis alone (Mallet & Carver 1995). Furthermore, anecdotal reports suggest that on some farms the proportion of M. trossulus to M. edulis is gradually increasing (Freeman 1996). Importation of pure M. edulis seed has been the sole means producers have to overcome production shortcomings associated with farm-collected M. trossulus. however, researchers have been looking for other options. It had been believed, for example, that comparison of early life events of both species might suggest alternate husbandry strategies at sites with both species. where growers inight wish to harvest only the more commercially desirable M. edulis (Freeman 1996) Examination of spawning times and duration of larval phases revealed close synchrony between M. edulis and M. trossulus (Freeman et al. 1994). indicating that any separation technique employed as part of routine husbandry would have to be based on some other criterion. Anecdotal references to different settlement depths for each suggested that looking for larval depth variations between the two would be an appropriate first step. It is presumed that initial settlement would "preferentially" occur at a depth cho- sen by the lar\ ae upon their attainment of competence, assuming the larvae had immediate accessibility to a suitable substrate at that depth. Following recent success of mesocosm studies of Pla- copecten magellanicus larvae at the Dalhousie University Aqua- tron in Halifax, Nova Scotia (e.g., Manuel et al. 1996; Gallagher et al. 1996). additional experiments were perfonned at the same fa- cility using laboratory-reared larvae of M. edulis and M. trossulus (MacQuarrie 1995; Freeman & MacQuarrie 1999). Here we present data on depth settlement preferences of both A/, edulis and M. trossulus as observed in the laboratory. Settle- ment behavior was examined in columns of mixed water, and when a thermocline was present at 6 m. METHODS Broodstock Selection *Corresponding author. Naturally conditioned broodstock animals were obtained from a commercial mussel farm near Lunenburg. Nova Scotia. The col- lection was made in early June 1995. a time corresponding to peak natural spawning at this site. Animals were selected on the basis of apparent shell weight (a completely subjective judgment) com- bined with shell length to shell height ratio derived from direct measurements. Those animals with a shell length to shell height ratio >2.2 and deemed 'light weight" for their length, were con- sidered M. trossulus. whereas those animals with a ratio of <2.0 and deemed 'heavy" for their length were regarded as M. edulis. Subsequent isoenzyme analysis in earlier work confirmed the ac- curacy of this visual identification method at this site (Freeman et al. 1994). Furthermore, a more recent shell analysis of mature. DNA-identified mussels has indicated that the length-to-height 59 60 Freeman et al. TABLE 1. Chronology of mesocosm experiment. 1995. M. editlis lar\ae produced on June 13, and M. trossiiliis larvae produced on June 20. Date & Age (days 1 of Mussels at Interval Collector Larval Age at Placement Larvae per Mesocosm (xlO") Species Replicate Placed Deployment Removal M. edulis El June 18 .i days 1.43 July 7 (24) July 31 (48) M. edulis E2 June 18 5 days 1.43 July 7 (24) July 31 (48) M. edulis E3 June 18 5 days 1.43 July 7 (24) July 31 (48) M. edulis NtE4 June 18 5 days 1.43 July 7 (24) July 31 (48) M. trossulus Tl June 22 2 days 1.60 Juh 11 (21) July 31 (41) M. trossulus T2 June 22 2 days 1.60 JuK 11 (21) Julv 31 (41) M. trossulus T3 June 22 2 days 1.70 Julv 11 (211 July 31 (41) M. trossulus NtT4 June 22 2 days 1.70 Jul) 11 (211 July 31 (41) Nt no thermocline mesocosm. proportions, as a guide to species identification at Corl F Power Sp. code 1 Depth 8 Sp. code X depth 8 Mesocosm |Sp, code] 4 Depth X mesocosm [Sp. code] 32 O.()0y075 I2.4378.'i() 0.364035 0.273,567 2.099770 0.2934 0.5903 0.083 50.2592 <0.0001 1.000 1.4710 0.1896 0.600 2.2109 0.0799 0.610 2.1212 0.0072 0.990 This analysis was based on dala from mcsocosms with no Ihermoclinc. Mytilus Settlement in a Mesocosm 61 TABLE 3. M. edulis: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test. Depth 0 2 3 1 4 5 6 8 7 II -0.35 -0.02 0.03 0.04 0.08 0.16 0.57 0.59 0.83 2 -0.02 -0..^5 -0.30 -0.29 -0.24 -0.16 0.24 0.26 0.50 3 0.30 -0.30 -0.3.S -0..34 -0.29 -0.21 0.19 0.21 0.45 1 0.04 -0.29 -0.34 -035 -0.31 -0.23 0.18 0.20 0.44 4 0.08 -0.24 -0.29 -0.31 -0.35 -0.27 0.13 0.16 0.39 5 0.16 -0.16 -0.21 -0.22 -0.27 -0.35 0.05 0.08 0.32 6 0.57 0.24 0.19 0.18 0.13 0.05 -0.35 -0.33 -O.OS 8 0.S9 0.26 0.21 0.20 0.16 O.OS -0.33 -0.35 -0.11 7 0.83 0.50 0.45 0.44 0.40 0.32 -0.09 -0. 1 1 -0.35 Positive values in bold show pairs of means that are significantly different at a = 0.05. A thermocline was present at 6 m. Depths are ordered to position significantly different pairs together. styrofoam floatation collar, and filled with 2.40 m' of 1.0-(jini filtered water. Hot and cold water circulating in vertically adjust- able piping circling the inside walls of the tank maintained a ther- mocline, in this instance at 6 m. The temperature above the ther- mocline was 19°C: below it 9°C. Of the M. edidis larvae. 1.43 x 1 0" were placed in each of four mesocosms suspended in the tower tank. For M. trossuhis, two mesocosms were charged with 1 .65 x 10'' larvae and two others with 1.70 x 10*" larvae. One of the latter M. twssulus and one M. edulis mesocosm was fitted with an outer polyethylene bag containing water and an airlift system, which kept the water stirred. This system eliminated the thermocline in these two mesocosms, and held their internal temperatures at 12°C, top to bottom. Logistic constraints eliminated the possibility of replicating the nonthermocline mesocosms. A 12-h day/night pho- toperiod established during the larval stages was continued to the end of the settlement experiment. The feeding regime established by the mid-larval period was also continued. Mixtures of Tahitian hochysis and Chaetoceros gracilis were distributed throughout the depths of each mesocosm by use of perforated hoses. Final con- centrations of 5.0 X lO' cells/mL of each algal species were main- tained to the end of the experiment. Algal counts were assessed and adjusted every 2 days. Collectors Duplicate, preconditioned. 13-inm diameter. 25-cm lengths of polypropylene rope were suspended by weighted monofilament lines in all eight mesocosms. The portions of rope were positioned more or less horizontally, at l-m depth intervals, from the surface to 8.0 m. The use of fine-diameter, smooth-surfaced monofilament line was intended to discourage newly settled spat from migrating vertically. The collectors were installed in the M. edidis meso- cosms when these larvae were 24 days old, and in the M. trossiilus mesocosms when these larvae were 21 days old. In each case, settlement began about 5 days after collector installation. On July 31, the 25-cm segments of polypropylene rope from these collectors with their attached spat were retrieved, placed in a container of 80% ethyl alcohol, and labeled by species, replicate, and depth. M. edidis collectors were removed at 48 days of age; those of A/, trossiilus were removed at 41 days. Spat were subse- quently separated from the rope segments with a light brushing, then counts of the total number of spat per seginent were done microscopically. The chronology of events is shown in Table I. Data Treatment Thermocline at 6 m Spat counts from the collectors (two replicates for each meso- cosm with a thermocline) were converted to percentages settled at each depth per collector, log,,, transformed, then tested for nor- mality using the Shapiro-Wilk W test. Normal Q-Q plots of the residuals were visually inspected for deviations. For each factor, homogeneity of variance was assessed with Levene's test. Data were then subjected to analysis of variance (ANOVA). All analy- ses were computed with JMP® version 3.1 software. The initial analysis was designed to test the hypothesis that there was no significant difference between species in settlement depth distri- bution in the presence of a thermocline at 6 m. The mesocosms were nested within each species. The model is expressed as (0 a (A Si E 3 z ■o « E o — therrr ocline 1.5- i - \ -*- T 1.0- I" S-- ■*~^ 0.5- - A ^ i - -Y — 0.0- I I 1 012345678 Depth (m) Figure 1. Log||,-transformed percent number of M. edidis spat. Mean, standard error, and standard deviation are indicated for each depth (0-8 m). The mean across all depths Is also indicated (solid line). A thermocline Has present at 6 m in this experiment. 62 Freeman et al. TABLE 4. M. trossitliis: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test. Depth 0 4 1 2 5 3 6 7 8 0 -0.46 0.00 (l.lll 0.02 0.07 0.21 0.53 0.59 0.67 4 0.00 -0.46 -0.45 -0.44 -0.39 -0.25 0.06 0.12 0.20 1 0.01 -0.45 -0.46 -0.45 -0.40 -0.26 0.06 0.12 0.20 2 am -0.44 -0.45 -0.46 -0.41 -0.27 0.05 0.11 0.19 S 0.07 -0.39 -0.40 -0.41 -0.46 -0.32 -0.01 0.05 0.13 3 0.21 -0.25 -0.26 -0.27 -0.32 -0.46 -0.15 -0.09 -0.01 6 0.53 0.06 0.06 0.(15 -0.01 -0.15 -0.46 -0.40 -0.32 7 0.59 0.12 0.12 tl.ll 0.05 -0.09 -0.40 -0.46 -0.38 8 0.67 0.20 0.20 0.19 (Ui -0.01 -0.32 -0.38 -0.46 Positive values show pairs ot means that are significantly different at a = 0.05. A thermocline was present at 6 m. Depths are ordered to position significantly different pairs together. X,ju = (X + A, + B, + AB, + C(B)„„ + AC(B),„„ + e,„j,) where A is depth with nine levels {0. 1. 2, 3. 4. 5. 6, 7. and iS in) treated as an ordinal value, B is species with two levels (M. ediilis and M. tros.siiliis). and C is mesocosm with three levels nested within species. Depth was orthogonal to both factors. Tukey-Kramer honestly significant difference (HSD) tests were perfomied in post-hoc analyses of all ANOVA results. Multiple comparisons of all pairs of means were performed. The signifi- cance level (alpha) in the one-way ANOVA for all comparisons was set at 0.05. The mean values of the log,o-transformed per- centages were plotted for each depth and species along with de- scriptive statistics. No Thermocline A second model was used to test the hypothesis of no differ- ence in the percent of spat of each species settling al depth, in the absence of a thermocline. Because there was only one mesocosm per species, a two-way ANOVA model was used; X„, = ix + A, + B, + AB„ + e,„ where A is depth with nine levels (0, 1, 2, 3. 4. 5, 6, 7, and 8 m), B is species code with two levels (M. cthilis and M. irnssiilus). and AB is the interaction term. The data were converted to percentage settled at each depth per collector rope and log,,, transformed. Normality, homogeneity of variance, and post-hoc tests were per- formed as above. RESULTS Spawning and Fertilization Of the putative M. edulis subjected to spawning inducement, 28 females and 27 males (55*"/^) spawned. Of the putative M. irossuhis subjected to spawning inducement, 18 females plus 22 males (43%) spawned. Two hours after batch fertilization, 84% of the M. edulis eggs had progressed to at least first divisions and for A/. trossiiliis. 86%. From past experience with broodstock frotii the same farm, the percentage spawning and the subsequent progress of fertilization and embryo development fell well within expecta- tions. Approximately 5.72 x 10'" M. edulis larvae and 6.60 x lO'' M. trossulus 2-day-old veliger larvae were taken to the Dalhousie University tower tank. Analyses (if Spat Settlement, Thermoeline Present at 6 m The transformed data produced a distribution that was not sig- nificantly different from normal at P = 0.01 (Shapiro-Wilk W test). Untransformed percentages did not meet the assumptions of the tests. The variances were homogeneous within depth and spe- cies according to Levene's test (P = 0.45 and 0.74, respectively). Normal Q-Q plots of the residuals indicated that the residuals were normally distributed. The ANOVA model explained approximately 80% of the vari- ance in the data set (adjusted R- = 0.81 ). Table 2 provides the test results. The.se indicate that both species had significantly different numbers of spat settling at different depths and that those distri- butions differed according to mesocosm. The spat were not uni- formly distributed by depth, however, no significant differences between the species were observed. (S Q. CO (0 3 "3 (0 It) o ^ .Q E ■a 4) E (0 c O) o thermocline Depth (m) Figure 2. Log|„-transformed percent number of M. trossulus spat. Mean, standard error, and standard deviation are indicated for each depth (0-8 m). The mean across all depths is also indicated (solid line). A thermocline was present at 6 m in this experiment. Mytilus Settlement in a Mesocosm 63 TABLE S. Two-way ANOVA of the transformed percentage of spat settled at depth by species. Source DF Sum of Squares F Ratio Probability > F Power Sp. Code Depth Sp. Code * Depth 1 8 8 0.007876 0.947826 0.609815 0.3184 4.7893 3.0814 0.5795 0.0028 0.0225 0.083 0.877 0.559 No thermocline was present. Post-hoc analyses of the depth distributions of spat of each species in the presence of a thermocline at 6 m depth were per- formed. For M. ediilis, the number of spat settling at the surface (depth 0 m) was significantly greater than those settling at any other depth except 2 m. All collections taken above 6 m were significantly greater than those settling below 6 m. where the thermocline was positioned (Table 3). The mean values of the transformed data are illustrated in Figure 1 for each depth. The general pattern is seen quite clearly. These results are mirrored in the depth settlement distribution of M. rmssuliis. however, the significance of the pattern is not as strong (Table 4. Fig. 2). In this species the surface samples (0 m) had significantly more spat than any other depth, whereas the shallower depths generally had sig- nificantly more spat than those below the 6 m thermocline. Analyses of Spat Settlement, No Thermocline The transformed data produced a distribution that was not sig- nificantly different from normal (Shapiro-Wilk W test, P = 0.873). The variances were homogeneous within depth and species according to Levene's test (P = 0.06 and 0.098, respectively). Normal Q-Q plots of the residuals indicated that the residuals were normally distributed. The results of this analysis are summarized in Table 5. Once again, there is a significant effect of depth, but no difference between the species. The interaction term is also sig- nificant and appears to be influenced by the differences in the means of each species below 6 m (see Figs. 3 and 4). Post-hoc analyses were performed on the data. M. ediiUs had large percentages of spat setting at the surface and also on the deepest collectors (8 m). This suggests that the thermocline was a barrier to settlement (Table 6, Fig. 3). because there was low settlement below 6 in when the thermocline was present, but high numbers when the water was mi.xed. However, the only signifi- cantly different pair of depths was between 0 and 6 m (Table 6). with a greater percentage of spat at the surface. In contrast. M. irossidits did not show an increase in settlement with depth when the thermocline was removed. The only significantly different pairs of depths were between 0 and 8 m (Table 7. Fig. 4l. This difference between the species at the 8-m depth accounts for the significant interaction term in the ANOVA (Table 5). CONCLUSIONS Larval depth distributions of M. edults and M. trossidus have been examined with a similar experimental design to that used in our study (MacQuarrie 1995; Freeman & MacQuarrie 1999). Half- way through the larval stage, vertical distributions over time in larvae of both species were similar to each other, with major concentrations found just above the 6 m thermocline. with slightly lesser amounts at the surface, and lower numbers elsewhere. This 3 ■o "5 I E 3 ■o « E c m ? Depth (m) Figure 3. Log,,|-transformed percent number of A/, ediilis spat. Mean, standard error, and standard deviation are plotted. The .sample mean across all samples is illustrated (solid linel. No thermocline was present in this experiment. (0 I a o n E 3 Z ■o 4) E (0 c o Depth (m) Figure 4. Log,„-transformed percent number of M. trossulus spat. Mean, standard error, and standard deviation are plotted. The mean across all samples is illustrated (solid line). No thermocline was present in this experiment. 64 Freeman et al. TABLE 6. M. cdulis: Comparisons for all pairs of depths (m) using Tukcy-Kramcr honestly significant difference (HSD) test. Depth 0 8 2 5 3 4 1 7 6 0 -0.56 -0,46 -0.36 -0.28 -0.26 -0.24 -0.20 -0.13 0.02 8 -0.46 -0.56 -0.46 -0.37 -0.35 -0.34 -0.30 -0.23 -0.08 2 -0.36 -0.46 -0.56 -0.47 -0.45 -0.43 -0.39 -0.32 -0.18 5 -0.28 -0.37 -0.47 -0.56 -0.53 -0.52 -0.48 -0.41 -0.26 3 -0.26 -0.35 -0.45 -0.53 -0.56 -0.54 -0.50 -0.43 -0.29 4 -0.24 -0.34 -0.43 -0.52 -0.54 -0.56 -0.52 -0.45 -0.30 1 -0.20 -0.30 -0.39 -0.48 -0.50 -0.52 -0.56 -0.49 -0.34 7 -0.13 -0.23 -0.32 -0.41 -0,43 -0.45 -0.49 -0.56 -0.41 6 0.02 -0.08 -0.18 -0.26 -0.29 -0.30 -0.34 -0.41 -0.56 Positive values in bold show pairs of means that are significantly different at a No thermocline was present. Depths are ordered to position significantly different pairs apart. 0.05. clustering of liirvae at the thennocliiie was not surprising, because the behavior has been noted in field studies of lamellibranch larvae (e.g.. Southward & Barrett 1983; Scrope-Howe & Jones 1986). In addition, M. rrossulus larvae lack a diel migration (MacQuarrie 1995), although Freeman and MacQuarrie (1999) report a modest ( 1.6 m) mean diel migration in 1 1 -day-old M. edulis larvae with a thermocline present. However, these diel migrations are unlikely to be statistically significant and are in contrast to observations made on scallop larvae (Manuel et al. 1996). Although thermocline accumulations of larvae decreased as the veligers matui-ed, the settlement patterns, as reported here (Figs. 1^), are incongruous, becau,se it was expected that a reflection of the earlier attraction to the thermocline would be clearly evident in the distribution of spat which were harvested immediately upon settling. M. eiliiUs and M. tiossuhis showed similar patterns of spat distribution with depth in the presence of a thermocline. Spat of both species settled below the thermocline. but at lower numbers than above and the highest numbers were recorded at the surface. In mixed water, M. edulis showed a strong preference for settle- ment at depth (8 m). a feature not seen in M. trossidus. although both had high numbers settling at the surface. Reasons for these different patterns are currently speculative, but a recognized weak- ness in the experiment was that we did not obtain genetic data to support the status of broodstock used. This leaves open to question whether these laboratory observations might have been affected by species contamination. Nevertheless, in subsequent field experi- mentation (see Kenchington et al. in this issue), DNA markers on field-collected mussels support the observation of behavioral dif- ferences by species seen in the mesocosm work. The industry custom of deploying spat collectors at or near the surface suggests that yields are better there, as was observed in this study. The high degree of similarity in the distribution of the spat of both species augurs little hope for growers wishing to prefer- entially select M. edidis over M. trossidus at mixed species sites. However, the significant species x depth interaction in the mixed water offers some basis for further hypothesis testing. Further- more, the coiTiplexity of natural conditions may provoke disparities between the species that are not seen under the controlled condi- tions of the tower tank. ACKNOWLEDGMENTS The authors are grateful for technical assistance provided by Mr. Ron Duggan and Ms. Natalie Randall, and for the broodstock animals supplied by Mr. Dale Cook, Corkums Island Mussel Farm, Lunenbuig. Nova Scotia. Prof. Ron O'Dor, Dalhousie University, provided funding and suppoil for this project through the Interim Funding Research Program, an Atlantic Canada Opportunities Agency-funded program in collaboration with Dr. E. Kenchington. The authors thank Mr. Dale Roddick for his critical review of the manuscript. TABLE 7. M. trnssulus: Comparisons for all pairs of depths (m) using Tukey-Kramer honestly significant difference (HSD) test. Depth 0 1 3 4 2 6 7 5 8 0 -0.68 -0.55 -0.47 -0.33 -0.25 -0.11 -0.09 -0.06 0.12 1 -0,55 -0.68 -0.60 -0.46 -0.38 -0.24 -0.22 -0.19 -0.01 3 -0.47 -0.60 -0.68 -0.54 -0.46 -0.32 -0.30 -0.27 -0.09 4 -0.33 -0.46 -0.54 -0.68 -0.60 -0.46 -0.44 -0.41 -0.23 2 -0.25 -0.38 -0.46 -0.60 -0.68 -0.55 -0.52 -0.50 -0.31 6 -0.11 -0.24 -0.32 -0.46 -0.55 -0.68 -0.66 -0.63 -0.45 7 -0.09 -0.22 -0.30 -0.44 -0.52 -0.66 -0.68 -0.66 -0.47 5 -0.06 -0.19 -0.27 -0.41 -0.50 -0.63 -0.66 -0.68 -0.50 8 0.12 -0.01 -0.09 -0.23 -0.31 -0.45 -0.47 -0.50 -0.68 Positive values in bold show pairs of means that are significantly different at a = 0.05. No thermocline was present in this experiment. Depths are ordered to position significantly different pairs apart. Mytilus Settlement in a Mesocosm 65 LITERATURE CITED Freeman. K. R., K. L. Perry. T. G. DiBacco & D. J. Scarratt. 1994. Ob- servations on two niytilid species from a Nova Scotian mussel farm. Can. Tech. Rep. Fish. Aqua!. Sci. 1969:47 p. Freeman. K. R. 1996. An examination of biological and other factors affecting mussel aquaculture development in the Scotia-Fundy Region of Nova Scotia. Can. Tech. Rep. Fish. Acjuat. Sci. 2125:32 p. Freeman, K. R. & S. P. MacQuarrie. 1999. Reproduction and prc- settlement behavior of Mylihi.t edulis and Mylihis trossiilus in con- trolled environments: Implications for mussel culture in mi.xed-species assemblages. Bull. Aquacult. Assoc. Can. 99: 1 7-2 1 . Gallagher. S. M.. J. L. Manuel, D. A. Manning & R. O'Dor. 1996. Onto- genic changes in the vertical distribution of giant scallop larvae. Plu- cvpecten magellanicus. in 9-m deep mesocosms as a function of light, food, and temperature stratification. Mar. Biol. 124:679-692. Koehn, R. K.. J. G. Hall, D. J. Innes & A. J. Zera. 1984. Genetic differ- entiation of Myiilus edulis in eastern North America. Mar. Biol. 79: 117-126. MacQuarrie. S. P. 199.5. The vertical distribution and depth of settlement of mussel larvae. Mytilus irossulus. in 8. .5 m deep mesocosms under controlled conditions. Honors Thesis, Department of Biology, Dalhou- sie University. Halifax. Nova Scotia. 55 p. Mallet. A. L. & C. E. Carver. 1995. Comparative growth and survival patterns of Mylihis Irossulus and Mytilus edulis in Atlantic Canada. Can. J. Fish. Aquat. Sci. 52:1873-1880. Mallet, A. L. & C. E. Carver. 1999. Maritime distribution and commercial production performance of Mytilus edulis and Mytilus Irossulus. Bull. Aiiuucul. Assoc. Can. 993:7-13. Manuel, J. L.. S. M. Gallagher, C. M. Peaice, D. A. Manning & R. K. O'Dor. 1996. Veligers from different populations of sea scallop Pla- copecten magellanicus have different vertical migration patterns. Mar. Ecol. Progr Ser. 142:147-163. Penney. R. W. & M. J. Hart. 1999. Distribution, genetic structure, and morphometry of Mylihis edulis and M. Irossulus within a mixed species zone. / Shellfish Res. 1 8:367-374. Scrope-Howe. S. & D. A. Jones. 1986. The vertical distribution of zoo- plankton in the Western Irish Sea. Estuar. Coast. Shelf Sci. 22:785- 802. Southward. A. J. & R. L. Barrett. 1983. Observations of the vertical dis- tribution of zooplankton. including post-larval teleosts. off Plymouth in the presence of a thermocline and a chlorophyll-dense layer. J. Plank- ton Res. 5:599-618. Journal of Slwllthh Racairh. Vol. 21, No. I, 67-73. 2002. COMPARATIVE SETTLEMENT DEPTHS OF MYTILUS EDULIS C. LINNAEUS, 1758 AND M. TROSSULUS GOULD, 1850: II. FIELD OBSERVATIONS E. KENCHINGTON, K. R. FREEMAN, B. VERCAEMER, AND B. MACDONALD Department of Fisheries & Oceans. Bedford Institute of Oceanoi;raphy. P.O. Bo.x 1006. Dartmouth. Nova Scotia B2Y4A2. Canada ABSTRACT Controlled niesocosm experiment', u^ing larvae of Mytilus ediilis and M. trossiilus have shown that the two species have different settlement patterns with respect to depth, and that the presence of a thermocline can modify the depth distribution. A field program was conducted over 3 y at one site, and at two sites during the final year to determine the settlement patterns of these species in the wild. In all analyses, there was a greater percentage of M. edulis postlarvae settling at ? m than of M. irossulus. In addition to opening up cenain ecological questions, these results show promise for direct application to husbandry practices at mussel farms having both species, where preferential collection of the M. edtili.<: is desirable. KEY WORDS: Mytilus ethilh. M. irossulus. settlement, depth, annual variability, spatial variation INTRODUCTION The relative depth at which Mytilus edulis C. Linnaeus, 1 758 and M. trossulus Gould, 1 850 settle is of direct concern to aqua- culturists in Atlantic Canada, and elsewhere, most of whom de- pend upon the collection of spat from the wild. Although similar in morphology, the lower yield and propensity for shell breakage observed in M. trossulus can cause significant production losses on mixed species farms (Mallet & Carver 1995). The possibility of using ecological infortnation (o preferentially collect M. edulis postlarvae over M. trossulus has been explored (e.g.. Freeman et al. 1994). Freeman et al. (2002) have shown that in experiments conducted an indoor tank at Dalhousie University (Halifax, Nova Scotia, Canada), the two species have different depth preferences for settlement. Both species had high percent- ages of postlarvae settling at the surface, however, in the absence of a thermocline, M. edulis showed a strong preference for settling at 8 m depth, which was not seen in M. trossulus. Should these results be confirmed in the field, they offer a practical solution to growers who have traditionally deployed surface collectors. In designing our experiment, we were particularly concerned with identifying patterns that were stable in time and space in order to generate advice to the mussel industry. Although our field ex- periments were limited in many ways, we were able to examine postlarval settlement patterns in M. edulis and M. trossulus with depth through the spawning season, across 3 y at one site, and between sites in 1 y. MATERIALS AND METHODS Stud\ Sites Two study sites were chosen to examine temporal and spatial variability in the depth preferences of the mussel spat. One site was located in St. Margarets Bay and the other in Ship Harbour, Nova Scotia (Fig. 1 ). Both sites were associated with mussel farms that collect, as pan of their operation, wild spat for culture, and were known to have both M. edulis and M. trossulus. Although both sites were located in coastal inlets, the oceanography of the two sites is quite different. Parrang Cove, St. Margarets Bay St. Margarets Bay is situated approximately 40 km west of Halifax, Nova Scotia. Our studv site was located near the head of Figure 1. Location of sampling sites at Parrang Cove in St. Margarets Bay and at Ship Harbour. Nova Scotia. the bay at Parrang Cove (44'-37'N, 65°56'W) on the eastern shore- line (Fig. 1 ). St. Margarets Bay is one of the larger bays along the Nova Scotian Atlantic coast and has an axis length of 23.7 km, a surface area of 138.0 knr, a total volume of 5191 x 10*' m\ and an opening of the sea that is -5 km wide. The bay is subjected to freshwater input from a watershed of 819.1 km"^ (Gregory et al. 1993). In addition to direct runoff, freshwater inputs come from five rivers and the tidal/freshwater volume ratio is 416.32 (Gregory et al. 1993). There is a large, deepwater basin of 80 m maximum depth, and there is a sill at a depth of 35 m (Piatt et al. 1972). As with all coastal inlets in Nova Scotia, the water in winter is well mixed. A thermocline establishes in late spring or early sum- mer between 4 m and 10 m, but in summer the warm surface layer may extend to 30-36 m, depending an direction and strength of winds (Piatt et al. 1972). The thermocline can be destroyed through sustained SE or NW winds and can also be pushed off- shore by the summer southwesterlies resulting in a cold upwelled nearshore area. 67 68 Kenchington et al. Ship Harbour Located 90 km east of Halifax, the much smaller Ship Harbour has an axis length of 8.3 km, a surface area of 6.6 knr. a total volume of 47 X 1 0'' m\ and an opening to the sea at the mouth of 0.6 km. It is subjected to freshwater input from a watershed of 444.7 km" (Gregory et al. 1993), including two rivers and two brooks. The tidal/freshwater volume ratio is 23.87 (Gregory et al. 1993). There is a small, deepwater basin of 27 m and an extensise sill at 7 m. As with other inlets along the coast, in Ship Harbour the water column turns over in the fall, with a theriin)cline reestablishing according to wind direction and velocity, the next spring. Ther- moclines are strongest in the summer months, establishing around 5 m (Strain 2002). Within Ship Harbour, the study site was located near the head of the harbour at 44 48.65'N, 62"30.53'W, along the northeastern shoreline (Fig. 1). Sampling Design Mussel growers at each site reported selllemenl beginning in mid- to late July and continuing through August and into Septem- ber. Settlement very late in the year occasionally occurs (Decem- ber), and may be attributed to either a second fall spawning or to a protracted spawning period. In the first year of sampling ( 1998), replicate collector ropes were deployed at Parrang Cove on July 24. These were removed on July 31 and replaced with a new pair of collectors. Spat collection continued through the spawning sea- son (Table 1). The schedule for the deployment and retrieval of collectors is given in Table 1 . Sampling continued at Parrang Cove at a reduced frequency in 1999 and 2000, according to the schedule in Table 2. In 2000, replicate collectors were also placed at Ship Harbour in order to examine geographic variation in the observed setting pattern. These were deployed and retrieved on the same dates as the Parrang Cove collectors (Table 2). During 1998, a small experiment was run to determine whether the active postlarval mussels would move along the collector rope after settlement. Two collectors were left in the water from July 24 until October 16 (85 days) at Panang Cove, in order to compare the depth distribution of older mussels with that of newly settled spat. Spat Collection Spat were collected using 5-m lengths of 13-mm-diameter yel- low polypropylene rope, weighted at the lower end and suspended at each site from buoyed, surface headropes. Prior to deployment, all collectors were soaked for 2 wk in running seawater to remove any chemicals that might inhibit settlement. As collectors were retrieved, they were cut into l-m lengths and TABLE L Deployment and retrieval dates of mussel spat collectors at Parrang Cove, Nova Scotia in 1998. TABLE 2. Deployment and retrieval dates of mussel spat collectors at the two experimental sites grouped according to three time periods used in the ANOVAs. Time Deployment Dale Retrieval Date .Inly 24 July 31 August 7 September 4 September 1 8 October 2 July 31 August 7 August 21 September 18 October 2 October 16 Deployment Retrieval Time Location Year Dale Date 1 Parranc Cove 199S July 24 July 31 1 1999 July 14 August 1 1 1 20()(J July 18 August 15 1 Ship Harbour 2000 July 19 August 15 T Parrang Cove 1998 August 7 August 21 "> 1999 August 1 1 September 8 T 2000 August 15 September 12 T Ship Harbour 2000 Au2ust 15 September 12 3 Parrang Cove 1998 September 4 September 18 3 1999 September 8 October 6 3 2000 September 12 October 10 3 Ship Harbour 2000 September 12 October 10 placed in polyethylene bags and sealed. Care was taken to avoid cross-contamination of the sample segments. In the laboratory, spat were preserved by adding SSCf ethanol to each bag. Subse- quently, each l-m rope portion was removed and the attached spat were separated from the rope by a gentle brushing, and then placed in vials with 80% ethanol. Although mussels from all five l-m lengths were saved, only those from 0-1, 2-3, and 4-5 m were analyzed (hereafter referred to as 1, 3, and 5 m), with one excep- tion. All five l-m rope segments were analyzed from the two collectors which were deployed at Parrang Cove on July 24, 1998, and left in the water until the fall. The number of spat at 1, 3, and 5 m were recorded for two time periods at each of two sites in 2000. Species Determination Approximately 30 individual spat from each sample depth rep- licate/time/site were subsequently decanted and examined under the microscope to ensure that the shell contained tissue. In some cases, there were too few animals to meet the sample size objec- tive, in which case all available animals were sampled. For the two collectors left in the water for 85 days, only animals greater than 9 mm were analyzed in order to ensure sampling of animals from the earliest settlement period. Individuals were then separately picked, crushed, and rinsed with high TE ( 1 M Tris 0.5 M ethylenediaminetetraacetic acid [EDTA], pH 8) and ddH,0 three subsequent times (Heath et al. 1995). Tissue was resuspended directly in a 20.4-|j.L extraction solution ( 19 |jiL H,0. 1 fjiL 10 x SFG buffer, 0.4 fjiL proteinase K) and digested overnight at 37"C. The reaction was stopped by boil- ing samples for 10 min before performing polymerase chain reac- tion (PCR) amplification. The internal transcribed spacer (ITS) region of the nuclear DNA was used as a species-specific marker (Heath et al. 1995). ITS primers were purchased from Operon Technologies, Inc. Twenty-two microliters of reaction mixture containing I x PCR buffer, 2 niM MgCK, 0.5 [xM of each primer, 0.2 niM dNTP, and 1 .0 U Taq polymerase were added to 2 p.L of DNA solution. The thermal cycling protocol was used as described in Heath et al. (1995). PCR-amplified ITS fragments (approxi- mately 1250 bp) were verified by running 8 |j.L of the amplifica- tion product on XJr agarose gels. Successfully amplified ITS frag- Mytilus Settlement in the Field 69 ments were digested with the endoiuieleiise Hhul (MBI Fernien- tas). Ten mieroliters of digestion mixture containing l(!)x en/ynie buffer and 5.0 U of Hhal was added to 13 (xl of amplification product. The Myuhis species-specific restriction fragment length polymorphisms (RFLPs) were then visualized on 2% agarose gels and samples were identified as M. edidis. M. trossiilus. or hybrid. Statistical Analyses The percentage of M. echilis spat was determined for each of the rope segments. Hybrids were rare and were not included in the percentage calculations, Arcsin-transformed data were tested for normality with the Kolmogorov-Smirnov (K-S) test. Levene's test was used to test the homogeneity of the error variance. Normal Q-Q plots of the residuals were visually inspected for deviations. Transformed data were subject to analysis of variance (ANOVA). A full factorial ANOVA was calculated froin the 1998 data series collected at Parrang Cove (Table 1 ) with two factors. Time (six consecutive sampling periods) and Depth (1. 3. and 5 m). using a type III sums of squares. For the two collectors which were left in the water froin July 24 through to October 16, a one-way ANOVA with Depth as a factor (1, 2. 3. 4. and 5 m) was per- formed. The results of these analyses inspired the subsequent field prograin to determine the temporal and spatial stability of our observations. To determine whether there were annual variations in the spe- cies-specific settlement patterns of Mytilns. a three-factor, full- factorial ANOVA was calculated using type III sums of squares. Specifically, the data collected from Parrang Cove (Table 2) were analyzed to examine whether the percentage of M, edulis spat varied in response to Year of collection (1998. 1999, 2000). Time of collection during the spawning season (three periods of collec- tion) and/or depth (1.3. and 3 m). Including the 1998 data created a lack of independence between this analysis and the previous ANOVA; however, excluding the 1998 results did not change the significance of the terms and so the full analysis is presented here for its greater interpretive value. A similar three-way ANOVA was calculated to examine whether the percentage of M. edulis spat varied in response to the Site of collection (i.e., Parrang Cove or Ship Harbour). Time dur- ing the season, and Depth (Table 2). Data for this analysis were collected in only 1 y. 2000. All interaction terms and main effects were tested, however, the three-way interaction term in the last two analyses was nonsignif- icant and so this term was removed from the equation and the analyses were rerun, testing only main and two-way effects. Tukey-Kramer honestly significant difference (HSD) pairwise multiple comparison tests between each pair of means were per- formed in post-hoc analyses of all ANOVA results with a signifi- cance level of 0.05. RESULTS The 1 998 data from Parrang Cove were analyzed prior to the 1999 field season in order to determine the design of subsequent fieldwork. The 1998 field season was designed to test the hypoth- esis, with higher power, that there was no significant difference in the percentage of M. edulis spat settling at different depths. The arcsin-transformed percentage of M. edulis spat data dis- tribution was not significantly different from a normal distribution (K-S Z = 0.628; P = 0.825). The variances were homogeneous and the residual plots supported a random distribution of the error term. The two-way ANOVA model explained 71% of the variance in the data set (adjusted R' = 0.71 1; F = 5.92; P = 0.00). There was no significant interaction between Depth and Time, however, the factors Depth and Time of collection were both highly signifi- cant (Table 3). Post-hoc analyses of the data using Tukey's HSD multiple comparisons test revealed highly significant differences between the transformed percentage of M. edulis spat settling at 1 m depth and the percentage settling at 3 and 5 m {P = 0.004 and 0.000. respectively). There was no significant difference between the per- centage of M. edulis spat settling at 3 and 5 m (P = 0.102). Examination of the mean values for each depth shows that there are significantly fewer M. edulis at 1 m, or conversely, that there are more M. irossulus. The nonsignificant interaction term indi- cates that this pattern did not change during the course of the setting season. The percentage of M. edulis spat settling also showed a sig- nificant difference with Time (Table 3). The post-hoc analyses (Table 4) did not have any clear pattern in these differences other than period 4 (deployed September 4th and retrieved on the 18th) which had a significantly lower percentage of M. edulis across all depths than most other sampling periods (except period 2). Al- though no pattern of species-specific spat settlement was known to exist during the setting season, a significant Time factor causing higher percentages of one species over the other through the sea- son can be explained by events such as differential spawning time, larval periods, and/or current changes. Did mussel spat change their position along the ropes when allowed to stay in the water for longer periods'? As in the above analysis, depth was a significant factor in the one-way ANOVA {F = 11.95; P = 0.009; power 0.93). Post-hoc analyses using Tukey's HSD test indicated that there was no significant difference (a = 0.05) between the percentage of W. edulis at 1.2. 3. or 4 m. but that significantly more M. edulis were found at 5 m than at these depths (P = 0.032. 0.008. 0.14. and 0.47. respectively). These animals were all over 9 mm in length, indicating that they had settled on the collectors early in the season. Therefore, the preference of M. edulis to settle at 5 m depth as observed in the newly settled spat, persists through 83 days to at least 9 mm shell length. Annual Variation in Setting Pattern The results from the 1998 program indicated that in the field. M. edulis prefers to settle at 3 and 5 m depth, while M. trossulus is dominant near the surface to 1 m. However, we felt that such results could not be generalized until the annual stabilitv of the TABLE 3. Two-way ANOVA of the arcsin-transformed percentage of M. edulis spat settling at Parrang Cove, Nova Scotia, by time (six periods during set) and depth (I, 3, 5 m) with two-way interactions. Observed Factor df SS F P Power" Time .s 1 .93 8.45 0.00 1. 00 Depth T 1.64 18.02 0.(» 1.00 Time X Deptli Id I.OI 2.:i 0.72 (1.72 Residual 17 0.77.'i ■' Computed at a = 0.05. 70 Kenchington et al. TABLE 4. Signincant (a = (t.05) Tukev's HSD nuiltipir comparisons test in a post-hoc analysis of the translbrnicd mean percentage of .1/. ediilis spat settinj; at six different periods at Tarran;; Cove, No>a Scotia, during the summer of 1998 (see Table 1 for dates). (ll Set Time (J) Set Time Probabiiil\ 0.034 0.003 0.008 0.045 0.034 0.045 0.002 0.004 0.003 0.002 0.008 0.004 pattern could be determined. Annual ditTerences in the percentage of M. edidis spat setting at depth during three time periods in each of 3 y were examined. The three-factor ANOVA model explained TO'^i- of the \ ariance in the data set (adjusted R- = 0.702: F = 7.66; P = 0.00) and identified a significant intei'action between Year of collection and Time of collection during the spawning season (Table 5). When the adjusted mean values of the transformed data are plotted (Fig. 2), it can be seen that this interaction effect is due to a much higher percentage of M. edulh sampled in 199S during the first collection period (July 14— August 15) than in a similar time period in 1999 and 2000. The second time period (sampled between August 7 and September 12) showed relatively high percentages of M. eduUs each year, whereas in the third time period (September 4-October 10) the percentage of A/, edidh was very low in 199S but lemained high in 1999 and 2000. These results indicate that the percentage of M. edulis vs. M. rmssidiis spat varies through the season and is not piedicted front year to year. Depth was the only significant main effect in this analysis (Table .'^). but was never significant in an interaction. Figuie 3 TABLE 5. Three-Hay ANOVA of the arcsin-transformed percentage of M. edulis spat settling al Parrang Co\e, Nova Scotia, by year (1998. 1999, 2U00), time (three periods during set), and depth (L 3, 5 m) with tHo-«av interactions. Observed Factor df SS F P Power" Year 2 0.044 0.69 0.51 0. 1 6 Time ~i 0.181 2.S5 0.07 0.52 Depth t 2.733 42.94 0.00 1.00 Year x Time 4 1.031 8. 10 0.00 0.99 Year x Depth 4 0.145 1,14 0.35 0.32 Time x Depth 4 0.254 1 .99 0.12 0.54 Residual 33 1.050 ■' Conipuled at a = 0.05. 3 0.9 0.8 0) O) CO c V S 0.7 V c (/) u < 0.6 c (C u 0.5 • » • / * \ / \ - 1998 - - - - 1999 — - -2000 1 0.4 1 2 3 Time of Set (Consecutive Periods) Figure 2. The interaction between year (1998, 1999, and 2(I(M)) with sampling period in the transformed adjusted mean percentage of M. edulis spat collected at Parrang Cove, St. Margarets Bay, Nova Scotia, shows the strong change in the median value of the transformed data with depth. Post-hoc analysis using Tukey's HSD identified significant differences between all paiis of means of each depth, with the greatest percentage of M. edulis spat settling at the 5 m depth and the least at 1 m. Geographic Differences The above-described anaKses confirmed that the ohser\ations first documented at Parrang Cove in 1998 were repeated in 1999 and 2000, with a greater percentage of M. edulis occurring at depth (5 ml and a greater percentage of M. trossuhis occurring at the surface ( 1 m). The final analysis w as designed to determine wheth- er this pattern was particular to Parrang Cove, or whether it also occuiTcd at other locations. The three-factor .ANOVA model explained 66% of the variance in the data set (adjusted R- = 0.660; F = 6.23; P = 0.00) and identified a significant interaction between the Site of collection and Time of collection during the season (Table 6). In all cases, there is a greater percentage of M. edulis spat at Parrang Cove than at Ship Harbour. However, in the first sampling period and in the last (third) sampling period, the relative difference between the sites is the same, whereas in the second period there are fewer M. edulis postlarvae (and therefore more M. trossulus) at Ship Har- bour relative to Parrang Cove (Fig. 4). LInlike the previous ANOVA, all three main effects were sig- nificant. The boxplots of the median values by depth and site are illustrated in Figure 5. As in the previous analysis, there is sig- Mytilus Settlement in the Field 71 a a. m (0 I 0) u 91 Q. (0 o Depth (m) Figure 3. Boxplots (median bar, upper and lower quartiles. and outer fences marking extent of data beyond the quartiles) of the arcsin- transformed percentage of A/, edulis spat setthng at Parrang Cove, St. Margarets Bay, Nova Scotia, at three depth intervals. 1 - 0.9 - s 0.8 - s \ s \ s edulis spat) o o CD ~J \ s * \ V \ (Percentage M. o o *> en • \ *\ \ * \ ft \ V \ ft \ ft \ V c \ 8 0.3- - - - - Time Penod 1 \ \ < — . —Time Period 3 \ 0.2 - \ 0.1 - \ 0 - ■ 1 1 Parrang Cove Ship Harbour Figure 4. The interaction between site with sampling period in the transformed adjusted mean percentage of M. edulis spat collected at the two locations in Nova Scotia in 2000. nificantly more M. edulis spat settling at 5 m depth than at the surface meter (Fig. 5). The third time period had the largest per- centage of M. edulis spat across all sites and depths. Therefore, as in the previous analysis, depth is an important factor in determin- ing the percentage of M. edulis spat, regardless of site or time period, or year of collection. The total numbers of postlarvae at the 1, 3 and 5 m depth intervals from a single collector rope from each site on two dates are provided in Table 7 in order to place the percentage data in perspective. As expected the total number of mussels collected was highly variable and ranged from 134 to over 10,000 per metre. The experimental design was not adequate to statistically analyze these data and so additional counts were not completed. However, it can be seen that large numbers of postlarvae did settle below the sur- face and that the total numbers of M edulis collected at depth was consistently higher than the number setting at the surface (Table 7). M. tnissulus was more variable being present in the highest numbers at the surface in two collections, at 5 m in one and TABLE 6. Three-way ANOVA of the arcsin-transformed percentage of M. edulis spat settling in 2000 by site (Parrang Cove, Ship Harbour), time (three periods during set), and depth (1, 3. 5 ni) with Iwo-wav interactions. Observed Factor df SS F P Power" Site 1 L55y 28.56 0.00 0.99 Time 2 0.747 6.84 0.01 0.88 Depth 2 1.199 10.98 0.00 0.98 Site X Time 2 0.434 3.98 0.03 0.65 Site X Depth -> 0.225 2.06 0.15 0.38 Time x Depth 4 0.256 1.17 0.35 0.31 Residual .^6 17.853 ° Computed at a = 0.05. ll i Depth (m) Parang Cove Ship Harbour Figure 5. Boxplots (median bar, upper and lower quartiles. and outer fences marking extent of data beyond the quartiles) of the arcsin- transformed percentage of M. edulis spat sampled in 2000 at three depth intervals (A), and at two sites in Nova Scotia, Canada (B). 72 Kenchington et al. TABLE 7. The absolute number of mussel postlanae per 1 m rope at I. 3 and 5 m depths collected at Parranye Cove and Sheet Harbour. Nova Scotia at two sampling dates in 2tM)(l. The percentage of mussels Identified as M. ediilis usinj; DNA markers is indicated, as well as the estimated numbers of M. edulis ( N , jnd I/. Irosiiiliis (N„ , postlarvae. Date Location Depth (m) % M. edulis ^IM. cdiilh Aug. 1?. 2(100 Parrang Cove 1 476 3 1 .3 3 IS4 61.9 5 367 60.7 Ship Harbour 1 569 6.7 3 831 10.7 5 6560 65.5 Ocl. 10. 2000 Parrang Cove 1 6030 71.9 3 10750 68.8 5 7470 80.0 Ship Harhiiur 1 741 15.2 3 134 64.7 5 140 100,0 149 114 223 38 89 4297 4336 7396 5976 113 87 140 327 70 144 531 742 2263 1694 3354 1494 628 47 0 homogenously distributed through the depth range in the fourth sample (Table 7). Hybrids Of the 24X5 animals scored using the DNA technique. 8.4% (208) were hybrids. The hybrids were not present in sufficiently high numbers to permit a robust analysis of their distribution. However, hybrids were relatively abundant in the October 2 rep- licates sampled at Parrang Cove in 1 998 (Table 1 ). with the per- centages ranging from 5% to 58%. In these two samples, the greatest percentage of hybrids was at the surface. DISCUSSION The mixing of seawater due to tides, winds, currents, and up- welling, and the seasonal establishment of thermoclines. charac- terize inshore marine waters around Eastern Canada and in similar temperate waters elsewhere in the world where Myiilus is found. The larval period is therefore one of great uncertainty, where the dynamic environment must be contended with while simulta- neously searching for food and avoiding predators. Under these conditions, it is remarkable that we were able to observe a con- sistently and significantly higher percentage of M. edulis postlar- vae at 5 m depth. This depth preference observed in the newly settled spat persists through 85 days to at least 9 mm shell length, despite the fact that postlarval mussels are very active and have an ability to repeatedly attach themselves to various substrata prior to choosing a final settlement location (e.g.. Bayne 1964; Dare & Davies 1975). Further, the total number of M. edulis collected at depth was greater than at the surface, to the extent that this was examined. Pineda (2(J0()) identified three processes affecting larval settle- ment in invertebrates; ( 1 ) processes influencing the larval pool; (2) physical transport; and (3) microhydrodynamics. substrate avail- ability, and behavior. Larger-.scale processes governing physical transport, and coupled to the onset and duration of spawning, likely explain the variation in the relative percentage of each spe- cies through the spawning season, across years, and between sites, observed here and elsewhere (e.g.. Seed & Suchanek 1992). How- ever, the significant difference in postlarval depth distribution be- tween the species is consistent with a behavioral differences. Freeman et al. (2002) observed the settlement of M edulis and M. trossulus separately in the laboratory, under mixed water con- ditions and with a thermocline. In all cases the greatest percentage of postlarvae settled at the surface, however, under mixed water conditions the postlarvae of M. edulis also had a large settlement at 8 m. a pattern not seen in M. trossulus. These data support an innate behavior as opposed to a response created through inter- specific competition (e.g.. Petersen 1984). Furthermore, the lim- ited data that we obtained on hybrids identified a settlement pattern similar to M. trossulus. We were unable to determine which spe- cies dammed these hybrids, however, the possibility of maternal inheritance of setting depth is intriguing. Mussel larvae are known to show behavioral responses to light, gravity, pressure, and substratum (e.g.. Bayne 1976; Seed & Suchanek 1992; Young 1995). To cite only a few examples: com- petent larvae of M. edulis migrate to deeper water in the North Sea (Bayne 1976) and prefer to settle subtidally below 3 m in the Wadden Sea (Pulfrich & Ruth 1993), although Dobretsov and Miron (2001 ) observed the opposite in the White Sea. with pedi- veligers migrating toward the surface to set primarily at 1 .5 m. M. galloprovincialis Lamark, 1819 is more homogeneously distrib- uted with depth than M. edulis and M. trossulus (as observed in this study). Hov\ever M. galloprovincialis shows greater settle- ment at 5 and 10 m depth in northwest Spain (Caceres-Martinez & Figueras 1998). reflecting the pattern seen in M. edulis. In addi- tion, variation in settlement depth preference of mytilid species can be influenced by the presence of phytoplankton and predators. With the literature confounded by difficulties with species identi- fication (cf. Johannesson et al. 1990). it is premature to determine whether the observations reported here are representative of the two species. Miron et al. (1999) have suggested that competent barnacle larvae position themselves at a depth in the water column corre- sponding to the position where they might settle on shore. If this were true for mussels, then photonegative and geopositive behav- ior would ensure a greater depth with less exposure when setting on a fixed substrate (e.g.. shore, pilings). Conversely, the photo- positive and geonegative behavior hypothesized for M. trossulus would ensure a distribution above mean low water spring tide (MLWS). with greater exposure to desiccation, freshwater, and Mytilus Settlement in the Field 73 temperature fluctuations associated with life in the upper intertidai environment. This tolerance is consistent with the more euryhahne nature of M. trossulus. For aquaculturists, positioning mussel collectors on the bottom or at 5 m depth would ensure the preferential collection of M. edulis over M. trossulus. at least in Nova Scotian waters. This is in contrast to the current practice of deploying plastic mesh on head- ropes near the surface for several days before the anticipated onset of settlement, a practice which may favor the collection of M. trossulus and increase the relative proportion of this species on the lease sites over time. The absolute numbers of spat were not re- corded for all collectors and density is expected to be much more variable because of large-scale processes and microhydrodynamic conditions. However, in the collectors examined, placing the col- lectors below the surface rarely resulted in reduced settlement, although the total number of post larvae was highly variable with respect to depth of collection. Nevertheless, we encourage mussel growers to experiment with subsurface and bottom deployment of collectors at sites where both M. edulis and M. trossulus occur. ACKNOWLEDGMENTS We thank Yonghong Shi, Melissa Cox, Angela Glass, Amy Chisholm. Shawn Roach, Amanda Ginnish. and Liqin Cao for their assistance in the laboratory; Amy Thompson for her help in the field. We thank Dr. Gareth Harding (Bedford Institute of Ocean- ography. Dartmouth, N.S.) for reviewing this manuscript. This work could not have been done without the co-operation and as- sistance of the mussel growers. Mr. Joe Levy {Parrang Cove), and Mr. John Stairs (Ship Harbour). This project was supported by the Department of Fisheries and Oceans, Canada, the Human Re- sources Canada Science and Technology Youth Internship pro- gram, and an NSERC research grant to EK. LITERATURE CITED Bayne. B. L. 1964. Primary and secondary settlement in Mytilus edulis L. (Mollusca). J. Aiiim. Ecol. 33:513-523. Bayne, B. L. 1976. The biology of mussel larvae. In: B. L. Bayne, editor. Marine mussels: Their ecology and physiology. Cambridge University Press: London, pp. 81-120. Caceres-Martinez, J. & A. Figueras. 1998. Mussel (Mytilus galloprovin- cialis Lamarck) colonization on artificial substrates in the Ria de Vigo of NW Spain. J. Shellfish Res. 17:153-157. Dare. P. J. & G. Davies. 1975. Experimental suspended culture of mussels (Mytilus edulis L.) in Wales using spat transplanted from a distant settlement ground. Aquaculture 6:257-274. Dobretsov. S. V. & G. Miron. 2001. Larval and post-larval vertical distri- bution of the mussel Mytilus edulis in the White Sea. Mar. Ecol. Prog. Sen 218:179-187. Freeman, K. R.. K. L. Perry. T. G. DiBacco & D. J. Scariatt. 1994. Ob.ser- vations on two mytilid species from a Nova Scotian mussel farm. Canadian Technical Repeport in Fisheries and Aquatic Sciences 1969:47 pp. Freeman. K. R., E. Kenchington & S. Macquarrie. 2002. Comparative settlement depths of Mytilus edulis C. Linnaeus, 1758 and M. trossulus Gould. 1850: I. Laboratory Observations. J. Shellfish Res. 21:000-000. Gregory. D.. B. Petrie. F. Jordan & P. Langille. 1993. Oceanographic. geographic, and hydrological parameters of Scotia-Fundy and southern Gulf of St. Lawerence inlets. Canadian Technical Report in Fisheries and Aquatic Sciences 143:248 pp. Heath, D. D., D. R. Hatcher & T J. Hilbish. 1996. Ecological interaction between sympatric Mytilus species on the west coast of Canada inves- tigated using PCR markers. Moi Ecol. 5:443-447. Johannesson, K., N. Kautsky & M. Tedengren. 1990. Genotypic and phe- notypic differences between Baltic and North Sea populations of Myti- lus edulis evaluated through reciprocal transplantations. II. Genetic variation. Mar. Ecol. Prog. Sen 59:211-219. Mallet, A. L. & C. E. Carver. 1995. Comparative growth and survival patterns of Mytilus trossulus and Mytilus edulis in Atlantic Canada. Can. J. Fish. Aquat. Sci. 52:1873-1880. Miron. G., L. J. Walters, R. Tremblay & E. Bourget. 1999. Intertidai bar- nacle distribution: A case study using multiple working hypotheses. Mar Ecol. Prog. Ser 197:205-219. Petersen, J. H. 1984. Larval settlement behaviour in competing species: Mytilus califimuanus Conrad and M. edulis L. J. Exp. Mar. Biol. Ecol. 82:147-159. Pineda. J. 2000. Linking larval settlement to larval transport: Assumptions, potentials, and pitfalls. Oceanogr. Eastern Pacific 1:84—105. Piatt. T.. A. Prakash & B. Irwin. 1972. Phytoplankton nutrients and flush- ing of inlets on the coast of Nova Scotia. Naturaliste Can. 99:253-261. Pulfrich. A. & M. Ruth. 1993. Methods for monitoring the spatfall of mussels [Mytilus edulis L.) in the Schleswig-Holstein Wadden Sea. ICES 199SCM 1993/K:42. Seed, R. & T. H. Suchanek. 1992. Population and community ecology of Mytilus. In: E. Gosling, editor. The mussel mytilus: Ecology, physiol- ogy, genetics and culture. Developments in aquaculture and fisheries science, vol. 25. New York: Elsevier, pp. 87-169. Strain. P. M. 2002. Nutrient dynamics in Ship Harbour. Nova Scotia. Al- njosphere-Ocean 40:in press. Young, C. M. 1995. Behavior and locomotion during the dispersal phase of larval life. In: L. McEdward, editor. Ecology of marine invertebrate larvae. New York: CRC Press, pp. 249-278. Joiirihil ,:t Shellfish R<'sc,inh. Vol. 21. No. 1. 75-79. 2(102. ROUTES OF INTRODUCTION OF THE MEDITERRANEAN MUSSEL (MYTILUS GALLOPROVINCIALIS) TO PUGET SOUND AND HOOD CANAL ANTHONY S. ANDERSON, A. LELANIA BILODEAU,* MATTHEW R. GILG, AND THOMAS J. HILBISH** Dcpartmcnl oj Biological Sciences, University of South Carolina. Cohunhia. South Carolina 29208 ABSTRACT To test alternative routes of introduction we describe the distribution o( Myliliis galloiymvincialis. M. Irossidus, and their hybrids in Puget Sound and Hood Canal. Native mussels. Myiihis imssniiis. dominate the blue mussel communities of Puget Sound and Hood Canal; at most sites M. i^idloproyincialis alleles were rare or absent. M. galloproYuicialis alleles were present but uncommon (-.')%) in mussel populations in the southern portion of Puget Sound, and were nearly absent in populations in the northern Puget Sound and the Hood Canal. The only locations where M. gallopiovincicilis alleles are locally common are sites where they are likely to have been repeatedly introduced. These include sites near mussel farming operations and near the Port of Seattle and Bremerton Naval Shipyards. The results indicate that both aquaculture and shipping activities have been responsible for the repeated introduction of M. aulloprovincialis to Puget Sound. Although hybridization between M. galloprovincialis and M. trossulus occurs, there was little evidence for advanced introgression between these two species. KEY WORDS: introductions, mussels. Myiiliis gtilloprovincialis INTRODUCTION The invasion of nonindigenous species has become of ecologi- cal and economic concern in the last few decades (Schiiiit/. & Simberloff 1997). The effects of species introduction range from localized and limited to those that severely degrade native ecosys- tems and have immense economic consequences (Geller et al. 1994; Ruiz et al. 1997; Ruiz, et al. 20001. The unpredictable eco- logical consequence of introducing nonnative species makes such introductions generally undesirable. Global commerce is responsible for the majority of both inten- tional and unintentional species introductions. In marine systems a major route of species introduction has been through the transport and discharge of ballast water from ships (Geller et al. 1994; Ruiz et al, 1997; Ruiz et al, 2000), For example, Carlton and Geller (1993) found that the ballast water from 159 cargo ships in Coos Bay, Oregon, contained a minimum of 367 marine species, A second, potentially major, source of species introductions is aqua- culture endeavors, particularly fartiis and fisheries. The decline of traditional fisheries has lead to a rapid increase in aquaculture as a source of aquatic products (Johnson 1998); presently 20% of the world's seafood conies from aquaculture. The cotnmercial culture of aquatic organisms has led to the intentional and accidental in- troduction of nonnative species in many parts of the world (Bartley & Subasinghe 1996). One marine species that has been both intentionally and acci- dentally introduced by these activities is the tiiussel Mytihis fial- loprovincialis. This mussel has been unintentionally introduced to South Africa (Grant & Cherry 1985), the Sea of Japan (Wilkins et al, 1983), and California (McDonald & Koehn 1988), Larvae of M, galloprovincialis are commonly found in ballast water of transport ships (Geller et al, 1994), making unintentional introductions likely, M. galloprovincialis is also the subject of large aquaculture farms in Europe and has been intentionally introduced to Puget ♦Present address: USDA-ARS. Catfish Genetics Research Unit, 127 Ex- periment Station Rd., Stoneville, MS .^8776. **CorTesponding author. Fax: (803) 777-4002. E-mail: hilbish@biol.sc,edu Sound and the Georgia Straits as pail of culture efforts in the northeastern Pacific (Heath et al. 1995). Mytihis galloprovincialis is a member of a complex containing two other species of blue mussel, M. ediilis and M. trossulus. M. galloprovincialis is native to the Mediterranean and the Atlantic coast of southern Europe, M. eduhs is native to temperate Atlantic waters of Europe and North America, and M. trossulus is native to cold-temperate waters of the northern Pacific and Atlantic (Gos- ling 1992). M. galloprovincialis is the most warm-adapted of the three species (Hilbish et al, 1994), which perhaps contributes to its propensity for introduction. All members of the blue mussel spe- cies complex will hybridize in nature. In particular, extensive hy- bridization between M. galloprovincialis and M. trossulus occurs in California (Rawson et al. 1999) and between M. galloprovin- cialis and M. edulis in Europe (Skibinski et al. 1983). The capacity to interbreed with congeneric species expands the possible conse- quences of introducing non-native species; they may genetically contaminate native species as well as ecologically displace them. The fate of hybrids, however, is uncertain. In Europe hybridization between M. galloprovincialis and M. edulis is extensive resulting in high levels of introgression between the species (Quesada et al. 1998; Rawson & Hilbish 1998). In contrast, hybridization between M. galloprovincialis and M. trossulus is prevalent, but results in very limited introgression (Rawson et al. 1999). This study describes the distribution of Mytihis galloprovincia- lis. M. trossulus. and their hybrids in Puget Sound. M. gallopro- vincialis has been intentionally introduced to Puget Sound through aquaculture efforts, but also may have been repeatedly introduced via shipping activities. If M. galloprovincialis is present in Puget Sound as a result of shipping activities, we expect high concen- trations of M. galloprovincialis alleles to be present around inajor shipping ports, particularly in the vicinity of Seattle, Alternatively, if aquaculture activities are responsible for the presence of M. galloprovincialis. we expect to find alleles indicative of this spe- cies in the vicinity of culture operations. In this study we also have used molecular markers that distinguish M. galloprovincialis and M. trossulus to determine whether genetic contamination of native mussels is occurring in Puget Sound and to infer the possible fate of hybrid mussels. 75 76 Anderson et al. METHODS AND MATERIALS Mussels were collected in the summer of 1997 and 1998 from 28 locations in Puget Sound and Juan de Fucu Strait and five locations in the Hood Canal (Fig. 1 ). Shell length of each mussel was measured from the anterior to posterior margin to the nearest 0.1 mm. We attempted to analyze both small and large mussels from each location, but this was not always possible because the size structure of the mussels varied widely among sites. We analyzed between 16 and 64 mussels from each location. Each mussel was dissected and a section from the margin of the mantle was removed and preserved in 95'7f ethanol. Total cellular DNA was extracted from each tissue sample as described by Rawson et al. ( 1999). The Glu-5' gene was amplified using the polymerase chain reaction (PCR) as described by Raw- son et al. (1996) using the primers of Inoue et al. (1995). PCR amplification of the Glu-5' marker produces species-specific prod- iM^?M^ Figure 1. Location ol'saniplin)> sites in Puget Sound and Strait of Juan de Fuca: Beliingham Bay = BH, Anacortes = AC, Deception Pass Marina= DP, Oak Harbor = OH. Keystone Ferry = KF, Freeland = FL, Mutiny Bay=MB, Possession Point = PO, Edmonds = ED, Poulsljo = PB, Silverdale = SD, Shilshole Bay = SB, Elliot Bay Marina = EB, Seattle Pier 91 = SE, West Seattle = VVS, Manchester Stale Parli = MC, Seahurst County Park = SH, Purdy = PV, Saltwater State Park = SV\ , Point Defiance = PD, Grape>ie« Marina = CV, Penrose Point Stale Park = PP. .Joemma Beach Slate F'ark = JB, Carlyon = CL, Tollen Inlet = TI, Taylor United = TU, Sleilacoom Marina = ST, Tolmic Slate Park = TL, and Priest Point = PR. Location of .sampling sites in Hood Canal: Seal Rock = SR, Mike's Marina = MM. Potlalch State Park = PL. Twanoh State Park = TVV. and Belfair State Park = BL. ucts which differ in size for Mylilus ediilis. M. trossuhis. and M. gcilloprovinicialis (Rawson et al. 1999). A second locus, internal transcribed spacer (ITS), was amplified using the procedures de- scribed by Rawson et al. (1999), cut with the restriction enzyme Sau96. and run on a 1.5% agarose gel. This produces a species- specific restriction pattern that distinguishes M. trossulus from M. gallopmv/iiiiali.s (Rawson et al. 1996). Mussels from three loca- tions (Silverdale. Totten Inlet, and Edmonds) were also assayed at a third nuclear locus. Mal-I. according to the protocol given by Rawson et al. (1999). RESULTS The M. galloprovincialis allele frequency for the Glu-5' and ITS genes at each site is presented in Table 1 . Alleles specific to M. galloprovincialis are not generally very abundant in Ptiget Sound. Outside of the Totten Inlet (where Taylor United. Inc., cultivates M. galloproviiulalis) the average frequency of the M. galloprovincialis allele was 0.032 for Glu-5' and 0.019 for ITS (counting only sites for which both genes were scored). The fre- quency of M. galloprovincialis alleles at Glu-5' and ITS was highly correlated (r = 0.775. P < 0.05. sites without M. gallopro- vincialis alleles at both loci and the Taylor United site were ex- cluded). In the central and southern Puget Sound. M. galloprovin- cialis alleles are relatively common in three locations. The Totten Inlet contained relatively high frequencies of M. galloprovincialis alleles. Mussels farmed by Taylor United in the Totten Inlet con- tained only M. galloprovincialis alleles at both loci. Wild mussels from a floating dock near the middle of the inlet (Totten Inlet) also had relatively high frequencies of M. galloprovincialis alleles. Mussels from the Carlyon Marina near the mouth of Totten Inlet. however, had low frequencies on M. galloprovincialis alleles, similar to that observed in other mussel populations in the southern Puget Sound (Table 1). Alleles specific to M. galloprovincialis were also common at Silverdale and at sites north and south ot Seattle (Edmonds. Shilshole Bay. and Seahurst) (Table 1. Fig. I). Silverdale is near the Bremerton Naval Shipyards, whereas the other three sites are near the Port of Seattle at Elliot Bay. Surpris- ingly. M. galloprovincialis alleles were absent from samples col- lected within Elliot Bay (Elliot Bay Marina. Seattle Pier 91. and West Seattle) (Table I ). Mussels with M. galloprovincialis alleles were absent from samples collected in the Hood Canal and from the northern portion of Puget Sound and Juan de Fuca Strait (Table 1 ). To evaluate the extent of introgression between the two species mussels from the three sites with the highest frequency of Myiihis galloprovincialis alleles (Silverdale. Totten Inlet, and Edmonds) were assayed at all three genetic markers and pooled into multilo- cus genotypic classes. Mussels homozygous for M. trossulus al- leles at all three loci were designated M. rrossulus. Those homozy- gous for M. galloprovincialis alleles at all three loci were desig- nated M. galloprovincialis. Mussels heterozygous for M. trossuhis and M. galloprovincialis alleles at all three loci were designated Fl hybrids. Those homozygous for M. trossulus alleles at one locus and M. galloprovincialis alleles at another locus were designated F2 hybrids. Of the remaining possible genotypes, those that con- tained four or five M. trossulus alleles were designated as M. trossulus back-crosses and those that contained one or two M. trossulus alleles were designated M. galloprovincialis back- crosses. Sorting individuals into the categories listed above does not necessarily indicate their genealogical origin; genuine F2 and back-cross matings should generate a wide variety of multilocus MyTILUS GALLOPROVINCIAUS IN PUGET SOUND 77 TABLE 1. Allele frequency of Clii-S' and ITS at each sample site in the Pujjel Sound and Hixid Canal: Sample size (;/) for each locus and the size ranye and habitat sampled are also indicated. Habitat Length (mm) Glu-5' ITS Site Frequency II Frequency II Puget Sound Bellinghani Buy Intertidal 25-40 0.00 22 0.00 18 Anacorles Suhtidal >40 0.00 32 0.00 28 Deception Pass Marina Suhlidal >40 O.fK) 30 0.00 28 Oak Harbor Intertidal 35-40 0.00 32 0.00 32 Keystone Ferry Intertidal >35 0.00 28 0.00 32 Freeland Intertidal >40 0.00 26 0.00 30 Mutiny Bay Intertidal 15-20 0.00 28 0.00 30 Possession Point Subtidal 20-25 0.00 28 Edmonds Subtidal >35 0.19 26 0.16 32 Poulsbo Subtidal >45 0.00 32 0.00 26 Shilshole Bay Subtidal <10and>35 0.12 68 Silverdale Suhtidal 5 to >70 0.35 126 Elliot Bay Marina Suhlidal <10 and >35 0.00 66 Seattle Pier 91 Intertidal 25-35 0.00 28 0.00 28 West Seattle Suhtidal <10 0.00 24 Manchester State Park Intertidal 3-35 0.00 48 0.00 20 Seahurst County Park Intertidal <5 and >40 0.1') 32 0.04 22 Purdy Intertidal <10and>40 0.04 52 Saltwater State Park Intertidal <15 and 25-35 0.04 28 0.04 26 Point Defiance Intertidal 20-40 0.07 28 0.04 24 Grapeview Marina Subtidal < 10 and >35 0.09 32 Penrose Point State Park Subtidal 5-20 and >40 0.02 60 0.00 22 Joemnia Beach State Park Suhlidal 10-40 0.02 58 0.00 32 Carlyon Suhlidal 35-40 0.08 26 0.07 28 Totten Inlet Suhlidal 35-50 0.21 24 0.33 26 Taylor United Subtidal >60 1 .00 58 1. 00 30 Steilacooni Marina Both 35 0.00 62 Tolmie State Park Intertidal 5-25 0.00 44 Hood Canal Seal Rock Intertidal 5-35 0.00 54 0.00 24 Mike's Marina Suhtidal 5-20 and 35-40 0.00 30 Potlatch State Park Intertidal 5-15 and 35-45 0.00 30 0.00 30 Twanoh State Park Intertidal 5-15 and 40-45 0.00 32 0.00 22 Belfair State Park Intertidal 5-10 and 35^0 0.00 26 0.00 20 genotypes, including individuals that are homozygous for alleles from one species at all three loci and mussels that are heterozygous at all three loci. It is important to note, however, that mussels homozygous for M. irossidus alleles at one gene and homozygous for M. galloprovwcUdis alleles at another can only be the product of F2 or other advanced introgressive crosses; they can not be the progeny of either Fl hybridization or first-generation back- crossing. Of the 55 mussels from Silverdale. Edmonds, and Totten Inlet assayed for all three genetic markers. 5 1 % had genotypes consis- tent with pure Mytihis trossulus and 22% appeared to be pure M. galloprovincialis (Table 2). Putative Fl hybrids were rare, com- prising only 9% of these samples. Putative back-crosses to M. trossulus appear to be about four times more common than back- crosses to M. galldpnivincUilis (15% versus 4%. Table 2). No individual was observed, in either the overall sample (Table I ) or this subsample (Table 2). that had a genotype exclusively compat- ible with F2 or other advanced hybridization. Silverdale was the only site with a high frequency of M. gal- loproriiwialis alleles where a wide range of size clas.ses was avail- able for analysis (Table 2). Mussels between 5 and 60 mm shell length were similar in genetic composition; the frequency of M. trossulus alleles was between 0.73 and I. depending on locus, and M. trossulus genotypes predominated. Mussels >60 mm shell length were dominated by M. galloproviiiciiilis genotypes and al- lele frequencies (0.75-0.77. depending on locus) (Table 2). M. galloproviiicialis may either grow faster or attain a larger size than M. trossulus. be selectively favored, or historical variation in re- cruitment success may have favored M. galloproviiuidlis some- time in the past, but not more recently. The presence of putative Fl and M. galloprovincialis back-cross genotypes among the largest mussels at Silverdale suggests the observed relationship between size and allele frequency is a long-term and recuning feature at this location and not the result of historical variation in recruit- ment. DISCUSSION Native mussels. Mytihis trossulus. dominate the blue mussel community in Puget Sound; at most sites M. galloprovincialis 78 Anderson et al. TABLE 2. Multilocus genotypes of mussels from Silverdale. Edmonds, and Totten Inlet: The number of mussels of each yenolype Is indicated. (Jenotype Trossulus Back-tross Fl Back-gallo Gallo Silverdale, length (mm) 0-20 3 -> 0 0 0 20-40 4 3 0 0 0 40-60 7 1 1 0 1 >60 1 0 3 T 6 Edmonds 8 1 0 0 -) Totten Inlet 5 0 1 0 3 Trossulus = mussels homozygous for M. irossuliis alleles at Glu-5'. ITS. and MalT, Gallo = mussels homozygous for M. f>aHoprn\incialis alleles at all three loci; and Fl = mussels heterozygous at all three loci. Back- tross and Back-gallo indicate mussels with genotypes consistent with M. trossulus back-crosses and M. galloprovincialis back-crosses, respectively, as specified in the text. No mussels were observed that were homozygous for M. Inissulus alleles at one locus and homozygous for M. galloprdvin- cialis alleles at another (i.e., F2 genotypes). alleles were rare or absent. The dislributioti of M. gaUiipiinincudis alleles varies regionally within Puget Sound. M. gaUoprovimudis alleles are present but unccMiimon (-59?-) in mussel populations in the southern portion of Puget Sound, and appear to be nearly absent in populations in the northern Puget Sound and the Hood Canal. The only locations where M. galloprovincialis alleles are locally common appear to be sites where they are likely to have been repeatedly introduced. These include the Totten Inlet where Taylor United. Inc.. maintains a mussel farm, and sites neai the Port of Seattle and Bremerton Naval Shipyards. Aquaculture operations appear to be the source of the high frequency of M. galloprovincialis alleles in mussel populations from the Totten Inlet. Taylor United, Inc., maintains a grow-out facility in the inlet that produces 1-1.5 millioti pounds (live weight) of M. galloprovincialis per year (Gordon King. pers. comm.). The frequency of alleles specific to M. galloprovincialis among "wild" mussels within the inlet ranged between 21% and .3.3%, depending on the locus assayed. However, the impact of the aquaculture operation is highly restricted; mussels sampled at the mouth of the inlet (Carlyon Beach) had a low frequency of M. galloprovincialis alleles that was not readily distinguished frotii the background frequency of these alleles found in other mussel populations in the southern Puget Sound, This result is surprising because the Totten Inlet is about 1 2 km long and mussel larvae could potentially disperse over much greater distances during their development in the plankton of several weeks. These results sug- gest that the circulation patterns in Totten Inlet retain larvae w ithin the inlet. Two additional lines of evidence suggest that the genetic im- pact of aquaculture operations on the genetics of indigenous mus- sel populations tnay be tniniinal. First, Taylor United has previ- ously cultured M. galloprovincialis at Freeland, on Whidby Island in the northern Puget Sound from 1992 to 1998, No M, gallopro- vincialis alleles were found among wild mussels at Freeland or at any other location on Whidby Island in 1997. Second. Taylor United tnainlains hatchery and nursery operations for the culture of M, galloprovincialis in Dabob Bay at the northern end of the Hood Canal. M. galloprovincialis alleles were viitually absent from all sites sampled within the Hood Canal, These lesults indicate that either cultured mussels are unlikely to escape aquaculture opera- tions or. if they do. they have relatively little impact on resident populations o( M. rrossnhis. Surveys conducted by Taylor United indicate that in 1996 and 1997, the frequency of M, galloprovin- cialis alleles among mussels on the shore near their mussel farm on Whidby Island was 2,8% and \3'7f. respectively (Gordon King, pers, comm,), Taylor United's mussel farming operations on Whidby Island were discontinued due to high mortality rates among M. galloprovincialis. These results all suggest that M. gal- loprovincialis is only marginally successful in northern Puget Sound. It is worth noting that 100% of the mussels obtained from Taylor United's grow-out operation in the Totten Inlet were ho- mozygotes for M, galloprovincialis alleles at both Glu-5' and ITS, This result indicates that Taylor United has been highly successful at maintaining pure cultures of M. galloprovincialis for several generations, despite the presence of resident populations of M. irossidas as potential sources of contamination at both the grow- out and the hatchery/nursery locations. Shipping also appears to be an important vector for the release of M. galloprovincialis into Puget Sound. The two greatest con- centrations of M, galloprovincialis alleles outside of the Totten Inlet are near major shipping areas. The highest frequencies of M. gall(}provincialis alleles outside of the Totten Inlet were observed at the Edmonds and Shilshole Bay Marinas north of Seattle. Seahurst County Park south of Seattle, and Silverdale. near the Bremerton Naval Base. Shipping appears to be the primary inecha- nism responsible for the introduction of nonnative marine species throughout the world (Carlton & Geller 1993; Ruiz et al. 1997: Ruiz et al. 2000). Geller et al. (1994) documented that larvae of M. galloprovincialis may be transported in ballast water of ships. The occurrence of M, galloprovincialis alleles near major shipping terminals in Puget Sound suggests that shipping may be respon- sible for the repeated introduction of this species to Puget Sound, It is also suggestive that M. galloprovincialis alleles are absent from the Hood Canal, which does not have any major shipping activity. Notably. M. galloprovincialis alleles were absent from within Elliot Bay near the Port of Seattle (Elliot Bay Marina. Pier 91 and West Seattle), which may indicate that ships release their ballast water before entering Elliot Bay. Although M. galloprovincialis appears to have been repeatedly introduced into Puget Sound by both aquaculture and shipping activities, the fate of these introductions is tenuous. Mussels from the southern Puget Sound with M. galloprovincialis alleles appear to mostly be the result of back-crosses with M. irossnlus. There was little evidence for pure M. galloprovincialis mussels outside of Totten Inlet and Dyes Inlet (Silverdale), Even in the three areas with the highest frequency of M. galloprovincialis alleles there was no evidence for advanced introgression. as indicated by the presence of genotypes that are exclusively compatible with being the progeny of F2 hybridization. These results indicate that when pure M. galloprovincialis are introduced, they occasionally hybrid- ize with M. trossulus and then Fl hybrids may subsequently back- cross to M. trossulus. Continued hybridization is not apparent within Puget Sound. Rawson et al. ( 1999) examined hybridization between M. galloprovincialis and M. trossulus in California, They also found that even though mussel assemblages often contained high frequencies of both species, hybrids were relatively uncom- mon and there was little evidence for advanced levels of introgres- sion. Although the mechanism is presently unclear, the results of Mytilus galloprovinc/alis in Puget Sound 79 Rawson et al. (1999) and of this study indicate that the "genetic pollution"" of native populations of M. irossiiliis by introduced M. guUopwvincialis is limited. Fuilher work is necessary to examine the mechanism(s) that determine the genetic fate of M. gallopro- vincialis alleles introduced by hybridization to resident popula- tions of M. trossidiis. It should also be noted that although intro- gression between these two species appears to be limited. M. gal- Inprovincialis has been successfully introduced in California and in the Sea of Japan, and the ecological impact of these introduc- tions on native mussel populations is presently unclear. ACKNOWLEDGMENTS We thank Gordon King and Taylor United. Inc.. for providing samples and information on local mussel farming operation. We acknowledge the support of NSF grants DEB-9807055 and OCE- 9731277 toT.J.H. LITERATURE CITED Bartley D. M. & R. B. Subasinghe. 1996. Historical aspects of international movement of living aquatic species. Rev. Sci. Tech. OlE I5(2);387- 400. Carlton J. T. & J. B. Geller. 1993. Ecological roulette — the global transport of nonindigenous marine organisms. Science 261(51 1 7):78-S2. Geller J. B., J. T. Carlton & D. A. Powers. 1994. PCR-based detection ot mtDNA haplotypes of native and invading mussels on the northeastern Pacific coast: Latitudinal patterns of invasion. Mur. Biol. 1 19:243-249. Grant W. S. & M. I. Cherry. 1985. Myiihis galloprovincialis Lnik. in southern Africa. J. Exp. Mar. Biol. Ecol. 90:179-191. Heath D. D., P. D. Rawson & T. J. Hilbish. 1995. PCR-based nuclear markers identify alien blue mussel (Mytilus spp) genotypes on the west coast of Canada. Can. J. Fish. Aquat. Sci. 52(I2):262I-2627. Hilbish T. J., B. L. Bayne & A. Day. 1994. Genetics of physiological differentiation within the marine mussel genus Mylihis. Evolution 48: 267-286. Inoue K.. J. H. Waite, M. Matsuoka. S. Odo & S. Harayama. 1995. Inter- specific variation in adhesive protein sequences of Mytilus edulis. M. galloprovincialis. and M. trossulus. Biol. Bull. 189:370-375. Johnson D. 1998. Seafood that never sees the sea: Improved aquaculture techniques are needed to feed the world. Futurist 32:14-15. McDonald J. H. & R. K. Koehn. 1988. The mussels Mytilus galloprovin- cialis and M. trossulu.s on the Pacific coast of North America. Mar. Biol. 99:111-118. Quesada H.. C. Gallagher. D. A. G. Skibmski & D. O. F. Skibinski. 1998. Patterns of polymorphism and gene tlow of gender-associated mito- chondrial DNA lineages in European mussel populations. Mol. Ecol. 7(8):1041-I051. Rawson P. D. & T. J. Hilbish. 1998. Asymmetric introgression of mito- chondrial DNA among European populations of blue mussels [Mytilus spp.) Evolution 52:100-109. Rawson P. D.. C. L. Secor & T. J. Hilbish. 1996. The effects of natural hybridization on the regulation of doubly uniparental mtDN.'K inheri- tance in blue mussels (Mytilus spp.). Genetics 144:241-248. Rawson P. D.. V. Agrawal & T. J. Hilbish. 1999. Hybridization between the blue mussels Mytilus galloprovincialis and M. trossulus along the Pacific coast of North America: Evidence for limited introgression. Mar. Biol. 134(1):201-2I1. Ruiz G. M., P. W Fofonoff, J. T. Carlton, M. J. Wonham & A. H. Hines. 2000. Invasion of coastal marine communities in North America: Ap- parent patterns, processes, and biases. A««. Rev. Ecol. Syst. 31:481-531 Ruiz G. M.. J. T. Carlton. E. D. Grosholz & A. H. Hines. 1997. Global invasions of marine and estuarine habitats by non-indigenous species: Mechanisms, extent, and consequences. Am. Zool. 37(6):62 1-632. Schmitz D. C. & D. Simberloff 1997. Biological invasions: A growing threat. Issues Sci, Technol. Summer:33— +0. Skibinski D. O. F.. J. A. Beardmore & T. F. Cross. 1983. Aspects of the population genetics of Mytilus (Mytilidae: Mollusca) in the British Isles. Biol. J. Linn. Soc. 19:137-183. Wilkins N. P.. K. Fujino & E. M. Gosling. 1983. The Mediterranean mussel Mytilus galloprovincialis Lmk. in Japan. Biol. J. Linn. Soc. 20:365-374. Joiinml of Shellfish Research. Vol. 21. No. 1. 81-86, 2002. TEMPORAL AND SPATIAL VARIABILITY IN DREDGING INDUCED STRESS IN THE GREAT SCALLOP PECTEN MAXIMUS (L.) JULIE A. MAGUIRE,' MAURICE O'DONOGHUE,^ STUART JENKINS,^ ANDREW BRAND,' AND GAVIN M. BURNELL' 'Aqiiaciilturc Development Centre. Dept.. of Zoolofiy and Aninuil Ecology. University College Cork. Lee Maltings. Prospect Row. Cork. Ireland: -Department of Microbiology, University College Cork. Cork, Ireland; ''Port Erin Marine Laboratory, University of Liverpool, Port Erin, Isle of Man, United Kingdom .ABSTRACT Temporal and spatial vanability in the stress caused to the great scallop Pecten maximus by dredge capture was determined using the adenylic energetic charge (AEC) of the striated muscle as an index. Samples were taken on a seasonal basis from dredge captured scallops of a single fishing ground in the north Irish Sea. All samples were taken from individuals under the minimum legal landing size. On one date samples were taken from scallops from three separate grounds with contrasting bottom characteristics. At all dates dredge captured scallops had a significantly reduced level of AEC compared to control scallops. This reduction was greatest in February when glycogen levels in the muscle tissue were shown to be at a minimum. There was little or no variability among fishing grounds in the effect of dredge capture on AEC levels. An additional aim of this study was to determine if scallops that encounter dredges but are not captured suffer a reduction in AEC levels. Scallops collected from dredge tracks immediately following the passage of dredges showed a significant reduction in AEC compared to control, scallops collected from outside the tracks. However, the reduction in AEC was not as great as in captured scallops. KEY WORDS: scallop. Pecten iihi.\iiims. dredging effects, adenylic energetic charge, seasonal effects, glycogen INTRODUCTION Most fishing techniques aim to capture a single or limited num- ber of target species over a minimum size. This size may be dictated by markets or by government legislation. In scallop dredg- ing, as in many other fisheries, a proportion of the captured target species is discarded owing to their small size. These undersized discards may be damaged during capture, for example suffering chipped valve margins or separation of the hinges, or may suffer stress from fatigue and desiccation. The level of mortality of these discarded scallops is unknown but may be considerable. Several studies have speculated on the levels of dredging induced mortality in relation to scallop size, sorting time and conditions on deck (Medcof & Bourne 1964), substrate type (Naidu 1988), catch weight (Chapman et al. 1977) and type and performance of gear (Gruffydd 1972). Damaged scallops that are left on the seabed, or are discarded, are likely to show high mortality owing to the aggregation of predators to dredge tracks (e.g„ Caddy 1973: Kaiser 1994: Medcof 1964) and to damaged bycatch (Veale et al. 2000). However, the majority of undersized discarded scallops show no signs of exter- nal physical damage. In the Irish Sea Jenkins et al. (2001 ) showed that less than I09c of scallops {Pecten moAiniiis} encountering dredges showed any signs of damage while Shepard and Auster (1991) estimated that damage to the scallop PUwopecten inagel- lanicus, that encounter dredges but are not captured, can vary between T7ir and 25"/^ depending on substrate type. Although dis- carded scallops may have low levels of datnage, they are likely to be highly stressed from the process of capture. Little is known regarding the effects of fishing induced stress on subsequent sur- vival of scallops. Dredging induced stress can be assessed in scallops using a Correspondmg author. Julie Maguire, Aquaculture Development Centre, Dept., of Zoology and Animal Ecology, University College Cork, Lee Maltings. Prospect Row. Cork, Ireland. Phone: +35.^-21-490-4053; Fax: ^-353-2 1-427-7922. E-mail; niaguirejuliels'hotmail.co variety of biochemical indicators. Maguire et al. (in press, a) dis- cussed the usefulness of various techniques for stress assessment in scallops and found Adenylic Energetic Charge (AEC) to be the most effective in the measurement of acute dredging stress. AEC was first proposed as a stress index by Atkinson (1968) and is defined by the ratio: AEC = (ATP + O..SADP) -H (ATP + ADP -i- AMP) where: ATP = adenosine tri-phosphate, ADP = adenosine di- phosphate and AMP = adenosine mono-phosphate. The AEC ratio varied from 0 to I i.e., 0 (all nucleotides were energy deficient AMP molecules) or 1 (all nucleotides were energy rich ATP molecules). In optimal conditions animals typically dis- played AEC levels >0.8 while under stress conditions values ranged from 0,5 to 0,75, Such animals had a reduced growth rate and did not reproduce, but recovered when returned to optimal conditions. Severely stressed animals yielded values of <0,5 and these animals had a negative scope for growth and did not recover (Duncan 1993: Ivanovici 1980), Subsequently many studies have been carried out using AEC as a stress index in scallops, for example P. imigellaiuciis (de Zwaan et al, 1980; Livingstone et al, 1981 ) and P. nui.\iiiu(\ (Fleury et al. 1997; Maguire et al. 1999a: Maguire et al. 1999b). Maguire et al. (in press a) used AEC as a stress index to investigate the effect of dredge capture on under- sized scallops. They found no difference in AEC levels between different lengths of tow (15, 30 or 45 minutes) but found that emersion following dredging had an added stress effect. In this study we aimed to investigate different aspects of dredging on scallops i.e., effects of different seasons, ground types and to as- sess the stress caused to scallops that come in contact with dredges but remain uncaught on the seabed. We aimed to exatiiine dredging induced stress in the great scallop Pecten maximus on a seasonal basis to determine if sea- sonal reproductive cycle in this species affected the extent to which it is negatively affected by dredge captui-e. Seasonal vari- ability in AEC levels has been examined in a number of inverte- brate species including the crayfish, Procamhanis acinus aculus 81 82 Maguire et al. (Dickson & Giesy 1982). the oysters Ostica ediilis and Crassos- trea gi,i;a.s (Moal et al. 1989b; Moal et al. 1991 ). the nuissel M\ri- his ediilis (Zaroogian et al. 1982) and the clam CanUiim sp. (Picado et al. 1988). Such variability has generally been associated with the reproductive cycle. Seasonal changes in biochemical com- position of the scallop P. maxhmis have been well documented (Ansell 1978; Maguire & Burnell 2001 ). Carbohydrate reserves are built up during periods of greater food availability dtiring the sum- mer and used up in the winter. In P. iiui.xiiiuis the main period tor gonadal proliferation takes place between November and February by the mobilisation of glycogen and protein reserves from the adductor muscle and lipid from the digestive gland. P. inaximiis may spawn over a long period from spring to autumn (Ansell 1978). Gametogenesis represents a period of high-energy demand in scallops and when external food supplies are limited, gamete production occurs at the expense of biochemical components in somatic tissues (Calow 1985). In this study on each sampling day we measured the glycogen content of both the striated adductor muscle and the gonad in order to assess the reproductive state of the scallops. Brokordt et al. (2000) showed that the reproductive state of the scallop Clilaiuys isUindka affected its ability to escape, presumably as a result of changes in the energetic state of the individual. Therefore, it would be reasonable to hypothesize that seasonal variability in the energetic state of scallops would affect the degree to which they are negatively affected by dredging. In addition to the major aim of this study, the determination of temporal variability in dredging induced stress, two further objec- tives were pursued. Initially, field trials were conducted at three different fishing grounds to assess the degree to which dredging induced stress varies spatially. Fishing grounds were specifically chosen with contrasting bottom characteristics. Diving was under- taken to collect scallops that encountered dredges but were not captured. AEC was measured to determine the extent to which these scallops were negatively affected by this experience. MATERIALS AND METHODS Seasonal Field Trial The field trial was carried out on the commercial scallop ground known as the Chickens, off the south west of the Isle of Man in the North Irish Sea (53°58.75'N. 04°52.7rW). The scal- lops were caught using a Newhaven type spring-loaded dredge (width - 75 cm) with a toothed cross bar (tooth spacing - 66 mm; tooth length - 100 mm), a collecting bag made from case hardened steel rings (diameter - 70 mm) and a net mesh cover (mesh size - 100mm). Eight dredges were towed at a speed of 5 kph for forty- five minutes on 4 dates, March 15th, June 5th, October 6th (2000), and February 14th 2001 . On each date two tows were made. At the end of each tow at least 15 scallops below the minimum legal landing size ( / m 4 E 30 - / CO / 13 J -D 20 - / > •o E 10 n 0.0 0.5 1.0 vertical and with phytoplankton depletion within the pearl nets {Claereboudt et al. 1994a; Claereboudt et al. 1994b). may have generated the height*group size interaction (Fig. 2A|. Because within pearl net phytoplankton depletion is required, this mecha- nism is different from the hypothesis of a straightforward negative effect of poor seston quality on assimilation efficiency which is usually invoked to explain near-bottom effects on growth of Ice- land scallops held in pearl nets (Wallace & Reinsnes 1984. 1985; Thorarinsdottir 1994). Clearly different group sizes should be used routinely in growth experiments to test for containment effects. The second mechanism tested, that is. a direct positive effect of higher current speed on clearance rate (Wildish et al. 1992). coupled with seston depletion within the pearl nets, also resulted in O) O O "D > C 20 10 0.0 0.5 1.0 Current speed ratio ^U ,/U 2) Figure 1. Simulated soft tissue growth «i(h(iul pliytoplanl^ton deple- tion in the hypothetical pearl nets. Diamonds: height : above bottom; squares: 2 m above bottom: solid symbols: 5 individuals per hypo- thetical pearl net: empty symbols: 1(1 individuals per hypothetical pearl net. Here empty symbols are hidden by solid symbols. \: effect of particle resuspension at the bottom. B: effect of positive relation between current speed and clearance rate. (Fig. 2A. B). An indirect effect of current speed, however, gener- ated a response with downward concavity (Fig. 2C). In all three cases, the difference between group sizes decreased with decreas- ing UJUy DISCUSSION Our simulations indicate that decreasing UJU2 ratio resulted in a height effect on simulated growth. However, phytoplankton depletion within the hypothetical pearl nets was required to gen- erate a height*group size interaction similar to that found in the actual growth experiment (Frechette & Daigle. in press). The chances of occuirence of a significant height*group size interac- tion increased with decreasing height c because the effect of group size on scallop growth decreased with decreasing current speed. The lower the UJU2 ratio, therefore, the better the simulations mimicked the experiment. The key factors implied here are seston depletion within the pearl nets and vertical differences in cunent speed owing to the BBL. Our simulations show that the three mechanisms tested might have generated the height*group size interaction. Vahl (1980) re- ported that seston quality had a strong negative effect on growth of Iceland scallops. According to our simulations, higher PIM near the bottom, coupled with homogeneous POM concentration in the 12 S 10 (/> CO E ^ 6 - to "D > c 0.0 0.5 1.0 0.0 0.5 1.0 O) o E o "O > C 0.0 0.5 Current speed ratio {U 2IU2) .0 Figure 2. Simulated soft tissue growth with phytoplankton depletion in the hypothetical pearl nets. Symbols as in Figure 1. A: effect of particle resuspension al the bottom. B: effect of positive relation be- tween current speed and clearance rate. C: effect of clearance rate regulation in response to seston concentration within the hypothetical pearl nets. 90 Frechettb and Daigle a heighI*group size interaction (Fig. 2B). It lias been argued, him- ever, that the positive relationship between clearance rate and cur- rent speed may be an artifact of flow removing seston depletion envelopes around individuals (Lenihan et al. 1996). There is in- deed evidence that phytoplankton concentration may be structured in space (cm scale) by exhalent jets of suspension feeders (Moni- smith et al. 1990; ORiordan et al. 1993; O'Riordan et al. 1993). In addition. Claereboudt et al. (1994b) found no evidence of positive effect of current speed on sea scallop growth. Although the ongo- ing considerations provide little support for a positive response ot clearance rate to cuirenl speed, it should be noted that Butman et al. (1994) found such a positive response in a mussel population contained in a flume. The third mechanism tested, that is. an indirect effect of flow mediated through changes in phytoplankton concentration within pearl nets coupled with clearance rate regulation (clearance rate is assumed to increase with decreasing phytoplankton concentration; e.g.. Bacon et al. 1998). also resulted in a height*group size in- teraction. The relation between growth and UJLK was curvilinear, with downward curvature. In the case of resuspension and ot a direct effect of flow, the relation was also curvilinear, but with upward curvature. Therefore, assuming that regulation of clear- ance rale in Iceland scallops resembles that of sea scallops and bay scallops, results of a growth experiment with different heights tested may provide information as to whether the height*group si/e interaction was attributable mainly to physiological regulation or to environmental forcing. We did not study the effect of inhibi- tory high flows on clearance rate (Eckman et al. 1989; Wildish el al. 1992) because it was inconsistent with lower growth near the bottom. In summary, our simulations suggest that the original seston quality hypothesis alone is insufficient to account for the decrease in growth of scallops near the bottom. An additional factor is required, that is. seston depletion within the pearl nets and its interaction with the BBL. Furthermore, two other mechanisms, independent of resuspension. may explain the growth pattern in the BBL. Both require food depletion within the pearl nets and inter- action with the BBL. The first mechanism is a direct positive effect of higher current speed on clearance rate and the second mecha- nism involves clearance regulation In response to food depletion. Because our simulations allow predictions about the profile of growth across height, growth experiments may provide evidence on the factor actually causing near bottom reduction in growth. pro\ ided that the processes simulated above act independently. LITERATLl Bacon. G. S.. B. A. MacD.mald & J. E. Ward. 1498, Physiological re- sponses of infaunal [Myu arciuina] and epifaunal {Plcuopecten imifiel- laniciis) bivalves to variations in the concentration and quality of sus- pended particles. 1. Feeding activity and selection. / Exp. Mar. Bid. Ecol. 219:105-12.';. Bayne. B. L. & J. Widdows. 1978. The physiological ecology of two popakitions of Mvf(7».v (-(/////v L. Oecologiii (Bcrl.). .'^T: 1.^7- 162. Butman. C, A.. M. Frechette. W. R. Geyer & V. R. Starczak. 1994. Flume experiments on food supply to the blue mussel Mytihis echilis L. as a function of boundary-layer flow. Linmol. Oceanogi: 39:1755-1768. Cahalan. J. A., S. E. SIddall & M. W. Luckenhach. 1989. Effects of flow velocity, food concentration and particle tlux on growth rates of juve- nile bay scallops Aii;iipecleii irnuliuns. J. E.\p. Mar. Biol. Ecol. 129: 45-60. Claereboudt, M. R.. D. Bureau. J. Cote & J. H. Himmelman. 1994a. Foul- ing development and its effect on the growth of juvenile giant scallops (Plucopecten magelhinnus) m suspended culture. Aqiuicultiiiv. 121: 327-.M2. Claereboudt. M. R.. J. H. Himmelman & J. Cote. |y94h. Field evaluation of the effect of current velocity and direction on the growth of the giant scallop. Placopecleii iiuif;i'lliinicu.s. in suspended culture. / E.\p. Mm: Bh>I. Ecol. 183:27-39. Dauvin. J.-C. & M. Joncourt. 19X9. Energy values of marine henthic in- vertebrates from the western English Channel. / Mar. Biol. Ass. U.K. 69:589-595. Demers. S.. J.-C. Therriault. E. Bourget & A. Bah. 19S7. Resuspension in the shallow sublittoral zone of a macrotidal estuarine environment: Wind intluence. Limiiol. Oceanogr. 32:327-339. Eckman. J. E.. C. H. Peterson & J. A. Cahalan. 1989. Effects of tlow speed, turbulence, and orientation on growth of juvenile bay scallops Ar- gopecten iinidians conccntricus (Say). J. E.xp. Mm: Biol. Ecol. 132: 123-140. Frechette, M. & G. Daigle. In press. Growth, survival and tluctuating asymmetry of Iceland scallops in a test of density-dependent growth m a natural bed. / £v/;. Mtii: Biol. Ecol. Grant, W. D., A. J. I. Williams & S. M. Glenn. 1984. Bottom stress estimates and their prediction on the Northern California continental shelf during CODE-1: the importance of wave-current interaction. ./. Phy.K. Oceangi: 14:506-527. RE CITED Lenihan. H. S.. C. H. Peterson & J. M. Allen. 1996. Does tlow speed also have a direct effect on growth of acuve suspension feeders; An experi- mental test on oysters. Linmol. Oceanogr. 41:1359-1366. Monismith. S. G.. J. R. Koseff, J. K. Thompson, C. A. O'Riordan & H. M. Nepf 1990. A study of model bivalve siphonal currents. Limiiol. Occanogi: 35:680-696. Muschenheim. D. K. 1987. The dynamics of near-hed seston dux and suspension-feeding benthos. J. Mar. Res. 45:473^96. Northby. J. A. 1976. A comment on rate measurements in open systems. Linmol. Oceanogr. 21:180-182. O'Riordan. C. A.. S. G. Monismith & J. R. Koseff. 1993. A study of concentration boundary layer formation over a bed of model bivalves. Linmol. Oceanogr 38:1712-1729. O'Riordan. C. A., S. G. Monismith & J. R. Koseff. 1995. The effect of bivalve excurrent jet dynamics on mass transfer in a beiithic boundary layer. Limnol. Oceanogr. 40:330-344. Parsons. J. G. & M. J. Dadswell. 1992. Effect of stocking density on growth, production, and survival of the giant scallop, Placopecten ma- gellanicns. held in intermediate culture in Passamaquoddy Bay. New Brunswick. Acpiaculrnre. 103:291-309. Pilditch. C. A. & J. Grant. 1999. Effect of variations in How \elocity and phytoplankton concentration on sea scallop (Placopecten fnngellani- cns) grazing rates. / E.xp. Mar Biol. Ecol. 240:1 1 1-136. Thompson. R. J. 1984. The reproductive cycle and physiological ecology of the mussel Myiilus eihilis in a subarctic, non-estuarine environment. Mar Biol. 79:277-288. Thomsen. L. & G. Gu.st. 2000. Sediment erosion thresholds and charac- teristics of resuspended aggregates on the western European continen- tal margin. Deep-Sea Res. I 47:1881-1897. Thorarinsd(ittir. G. G. 1994. The Iceland scallop. CliUimys isUunlica (O. F. Miiller) in Breidafjordur. west Iceland. 111. Growth in suspended cul- ture. Aqiiaculture. 120:295-303. Vahl. O. 1978. Seasonal changes in oxygen consumption of the Iceland scallop (Chlaniys islanilica (O.F. Muller)) from 70''N. Ophelia. 17: 143-1.54. Vahl. O. 1980. Seasonal variations in seston and in the growth rate of the Iceland scallop. Clilamys islandica (O.F. Miiller) from Balstjord. 70 N. ./, £a/;. Mar Biol. Ecol. 48:195-204. van Haren, R. J. F. & S. A. L. M. Kooijman. 1993. Application of a Near-bottom Effects on Iceland Scallop Growth 91 dynamic energy budget model to \'Iynhi\ Cihilis (L.). Neth. J. Sea Res. 31:119-133. Vogel, S. 1981. Life in moving fluids: the physical biology of flow. Bos- ton: Willard Grant Press. Wallace, J. C. & T. G. Reinsnes. 1984. Growth variation with age and water depth in the Iceland scallop iChlamys i.sUmdica. Pectinidae). Aquaculture. 41:141-146. Wallace, J. C. & T. G. Reinsnes. 1985. The significance of various envi- ronmental parameters for the growth of the Iceland scallop, Chlamys isUindIca (Pectinidae), in hanging culture. Aquciculnire. 44:229-242. Wildish, D. & D. Kristmanson. 1997. Benthic suspension feeders and flow. Cambridge University Press. Wildish, D. J„ D. D. Kristmanson. R. L. Hoar, A. M. DeCoste, S. D. McCormick & A. W. White. 1987. Giant scallop feeding and growth responses to flow. / Exp. Mar. Biol. Ecol. 1 13:207-220. Wildish. D. J.. D. D. Kristmanson & A. M. Saulnier. 1992. Interactive effect of velocity and seston concentration on giant scallop feeding inhibition.,/. E.\p. Mar. Biol. Ecol. 155:161-168. Wildish, D. J. & A. M. Saulnier. 1992. The effect of velocity and flow direction on the growth of juvenile and adult giant scallops. J. E.\p. Mar. Biol. Ecol. 133:133-143, Journal of Shellfish Rexfunh. Vol. 21, No. 1, 93-101. 2002. ULTRASTRUCTUR-\L CHARACTERISTICS OF SPERMATOGENESIS IN DIPLOID AND TRIPLOID CATARINA SCALLOP (ARGOPECTEN VENTRICOSUS SOWERBY II. 1842) ROSALIO MALDONADO-AMPARO AND ANA M. IBARRA Aqiuicidturul Genetics Laboratory. Centra de Investigaciones Biologicas del Noroeste. La Paz Mexico ABSTRACT Spermatogenic stages in diploid and triploid catarina scallop (Argopecten ventricosus) were studied by light and electron microscopy at two different ages. In nine month old triploid scallops, a reduced number of spermatogonia and pnniary spermatocytes were seen in the male acini, and although no spermatids were found, a few spermatozoa were observed. All triploid spermatogenic stages and their nuclei were larger in diameter than those in diploids. Ultrastructure analysis reveled that spermato- genesis in tnploids was almost completely arrested early, at the primary spermatocyte stage, indicating the halt occurs during the prophase stage of meiosis 1. before the first reductional division. At age 12 months a large number of hemocytes invade some of the remaining male acini of triploids. which possibly have a trophic role after development is halted. At this age. the male part of the gonad in most analyzed scallops is completely replaced by female acini, with some developing oocytes. Checkpoint mechanisms recently described as "meiotic checkpoints" are proposed as possibly being involved in the partial or total sterility resulting from the triploid condition. KEY WORDS: Argopecten renlricosiis. sterility. EM. triploid. pectinid. spermatogenesis, meiotic-checkpoint INTRODUCTION Triploid sterility has been reported for a number of mollusk species, but in most cases the sterility is not reflected in a complete lack of gametogenic stages, but rather on a delayed process and a reduced number of fully developed gametes (Allen et al. 1986; Komaru & Wada 1989, 1990; Allen 1987; Allen & Dov.'ning 1990: Guo & Allen 1994b; Co.x et al. 1996; Eversole et al. 1996; Kiyo- moto et al. 1996: Ruiz-Verdugo et al. 2000). Whereas the most common characteristic in triploid mollusks has been reported to be the reduced development of the gonad and gametes, the reversal of gonad sex in a known functional hermaphrodite has been only reported to occur in catarina scallop (Ruiz-Verdugo et al. 2000). In as much as several studies comparing diploids and triploids have been done on gametogenic stages by light microscopy, partial or total ultrastructure analysis of abnoimal gametogenesis in poly- ploid mollusks has been done only for triploid Pacific oyster Cras- sostrea gigas (Komaru el al. 1994), triploid Sydney rock oyster Saccostrea commercialis (Cox et al. 1996), and triploid and tetra- ploid Mediterranean blue mussel Mytihis giilloproviiicialis (Ko- maru et al. 1995: Kiyomoto et al. 1996). Spermatogenesis in dip- loid catarina scallop Argopecten ventricosus at the ultrastructure level has not been studied before, although it has been done for other pectinid species, Pecten mcLximits (Dorange & Le Pennec 1989a: Beninger & Le Pennec 1991). The catarina scallop, Argopecten ventricosus. is a functional hermaphrodite pectinid in which the gonad is divided into two parts, one female and one male. When triploidy is induced in this species, a gradual suppression of functional hermaphroditism has been noted to occur, with only female gametogenic stages devel- oping in the male part of the gonad in one-year-old scallops (Ruiz- Verdugo et al. 2000). As reported for oysters (Guo& Allen 1994a) and Manila clams (Utting et al. 1996), as well as for this panicular species (Ruiz-Verdugo et al. 2001 ), the number of oocytes formed was reduced when compared to diploids. In a continuance of those studies with triploid catarina scallop, we report here the effects of triploidy on spermatogenesis, studied as the comparative structure and ultrastructure of 9-nionth-old and 1 2-month-old diploid and triploid catarina scallops. Corresponding author. Ana M. Ibarra Aquaciiluiral Genetics Laboralon: Centra de Investigaciones. Biologicas del Noroeste. S.C. A.P. 128. Mar Bermejo # 195. Col. Playa Palo Santa Rita.La Paz B.C.S. 23000. /Mexico. FAX: -I- 52-612-125-3625: E-mail: aibarra@cibnor.mx MATERIALS AND METHODS Scallops Diploid and triploid catarina scallops about nine months age (6 cm in shell length), kept in a growout urea at Bahi'a Magdalena in Baja California Mexico, were transported to the Genetic Labora- tory of CIBNOR (Centro de Investigaciones Biologicas del No- roeste). and placed under inaturation conditioning for 20 days. A second group of scallops that were not conditioned for maturation in the laboratory was sampled from the field area at age 12 months. The triploid scallops were produced using cytochalasin-B (0.5 mg/ L) as described by Ruiz-Verdugo et al. (2001). and the diploids were from a control group the same age. Maturation Conditioning Each ploidy group was stocked (five scallops per 40-L tank) and kept under standardized controlled maturation conditions dur- ing 20 days (Ramirez et al. 1999). Feeding consisted of adding a mixture of Isochrysis gulhana. Monocluysis lutheri, and Chaeto- ccros muelleri at a total concentration of 4 x 10** cells / scallop / day. Water temperature was kept at 19-20°C. salinity at 36%f, and dissolved oxygen from 7-9 mg / L. The maturation room was provided with a 12-hour light and 12-hour dark photoperiod. Sampling and Histology for Light Microscopy After the 20-day maturation-conditioning period, the gonads from 20 scallops per ploidy group were fixed in buffered formal- dehyde ( 109r), dehydrated in a graded ethanol series, and embed- ded in paraffin. Duplicated sections, 5 (xin thick, were obtained from each gonad, and stained with hematoxilin-eosin (H&E) and Feulgen. Spermatogenic stages in diploid and triploid scallops were measured using image analyses. Images were taken with an Olym- pus BX-41 microscope, with an integrated camera (CoolSNAP- Pro). The images were analyzed with SigmaScan Pro 5, obtaining an area by digitalizing contours and estimating diameters from the 93 94 Maldonado-Amparo and Ibarra area for cytoplasm and nucleus of each spermatogenic stage. To correct for scale differences, for each microphotograph evaluated the image analysis program was calibrated accordingly to the mi- croscope objective used for those images. For each ploidy group and stage (spermatogonium, spermatocyte, spermatid, and sperma- tozoa), 30 cells were measured. Diameters of each spermatogenic stage for the two ploidy groups were analyzed with a single factor (ploidy) ANOVA, and mean differences tested with the F-test (Neter et al. 1985). Sig- nificance was set at P < 0.05. To establish the impact of triploidy on both nuclei and cell size, a ratio between nucleus diameter and total cell diameter was esti- mated by dividing the nucleus diameter by the total cell diameter. Additionally, the increase in area (Area = 3.1416 x radius") in triploids was estimated for each cell and nuclei from the following relationship; |(Area in 3n x 100 / Area in 2n ) - 100]. Electron Microscopy Five gonad samples from each ploidy group were taken 20 days after laboratory conditioning (9-month old) as well as five from each ploidy group from 12-month-old field scallops. The methodology described by Komaru et al. (1994) was fol- lowed for transmission electron microscopy (TEM). Gonad samples (2 mm") were fixed with a 4% gluteraldehyde solution in 0.1 M Sorensen's phosphate buffer, pH 7.5. for two hours at 4 'C. The pieces were then washed in Sorensen's buffer solution three times for 30 minutes, and post-fixed in \% osmium tetraoxide (OsOj) in the same buffer for one hour, at 4°C. Samples were then dehydrated in ethanol (25, 50, 70, 95, and 100%) for 20 minutes in each concentration, and twice in 1009f propylene oxide for 20 minutes. They were embedded according to the protocol of the commercial Kit Embed 812 Electron Microscopy Sciences (Luft 1961). Sections of 900A were obtained with an ultramicrolome (Reicher, Model 0MU3), mounted on a grid and stained with 29c uranyl acetate for 20 minutes and lead citrate for five minutes (Komaru et al. 1994). Photo microscopy was done using a JEOL JEM-1200EX1ITEM. Diameters of cell and nuclei of spermatogenic stages captured by photographic prints of TEM were measured after scanning the photomicrographs. To correct for amplifications, the size bar of each EM photomicrographs was used as a reference. All measure- ments were done using the image analyzer SigmaScan Pro 5. The number of stages measured varied, and depended on the number found in photomicrographs (6 spermatogonia, 15 spermatocytes and spermatids, 30 spermatozoa nucleus, mitochondria, and tla- gella, and 21 for acrosome length and invaginations in spermato- zoa). The low number of captured spermatogenic stages in the triploids did not allow for measurement. Scanning electron microscopy (SEM) gonad samples (4 mm") were fixed, post-fixed, and dehydrated in the same manner. Samples were dried in a SAMDR1-PVT-3P critical point dryer and coated with gold in an EDWARDS S150B sputter coating system (Komaru et al. 1994). Photo-microscopy was done using a JEOL JSM-5410LV SEM. All electron micro.scopy work was done at the Institute of Cellular Physiology in the National Autonomous Uni- versity of Mexico (UNAM). RESULTS Gonad stmcture of nine-month diploid and triploid scallops are presented in Figure 1. In diploid scallops the characteristic syn- chronized development of sperm and oocytes in the gonad was seen (Fig. I A). Compared to diploids (Fig. IC). gametogenesis in triploids was retarded (Fig. IB), and spermatogenesis was largely abnormal with only a reduced number of acini showing develop- ment of spermatogenic stages (Fig. ID). A summary of differences in mean size of spermatogenic stages between the two ploidy groups is in Table 1. The acini of triploid scallops were significantly larger (55%. P = 0.01) than those in diploids. There were also significant differences between ploidy groups in the diameter of all spermatogenic stages, as well as in their nucleus. Triploid scallops not only had larger nuclei area in spermatogonia and spermatocyte than diploid scallops (37% and 1 12% larger area in 3Ns. respectively), but also more cytoplasm area (78% and 1 19% more in 3Ns). Spermatids were not found in triploids, and comparative measurements between ploidy groups were not possible, but the few spermatozoa found indicated that triploids had a 50% larger area than diploids (Table 1 ). The nucleus/total cell ratio of spermatogonia was smaller for triploids (0.67) than diploids (0.76), whereas for spermatocytes it was ap- proximately the same ratio for both ploidy groups (0.88 for 2Ns, 0.86 for 3Ns). Diploid Male Gonad Structure and Vltrastructure The details of the male part of the gonad of a diploid scallop are shown in Figure 2 and Figure 3. Male germinal cells were grouped in acini ranging from 120 to 200 ixm in diameter. Each acinus contained a variety of developing stages distributed in a centripetal pattern from the inner acinus wall to the lumen (Fig. 2A). The spermatogonia (Spg) are oval shaped, and located nearest to the inner wall, whereas the spermatocytes (Spc) are smaller and loose from the acinus wall, positioned between the Spg and the lumen of the acinus. Mature spermatozoa (Spz) are almost exclusively con- fined to the central lumen of the acinus (Fig. 2A). The Spg had a diameter in ultrastructure measurements of 6.95 [jim. and a nucleus 4.39 p-iii. The Spc appeared to be more numerous than the Spg (Fig. 28). with a diameter approximately 3.92 |xm. and a nucleus 2.93 p.m. The Spc cytoplasm contained a complement of or- ganelles, Golgi bodies (Gb) and mitochondria (Mi), very similar to those in the spermatogonia. The nucleus of primary spermatocytes (Spcl ) exhibits scattered electron-dense chromatin (Fig. 2C), and its cytoplasm contains some Mi and Gb, Spermatids (Spm) in their early stages are spherical cells about 2.63 (jtm in diameter by ultrastructure measurements, with a dense nucleus, also spherical, about 1.95 |jLm in diameter (Fig. 2D). In the cytoplasm of the maturing Spm. Mi can be seen in what will be the basal pole of the future Spz. The Spz have a proximal acrosome (Ac), nucleus (Nu). and a maximum of five cross-sectioned Mi in its distal part (Fig. 3A). where the tlagellum (Fl) emerges. The anterior part of the sper- matozoa, where the acrosome is. appears slightly denser than the nucleus. The ultrastructure of the spermatozoa in diploid Ar- gopecten ventricosus can be divided into three main components: a sperm head consisting of the Ac and Nu, a middle piece con- sisting of two centrioles and the mitochondria, and a tail or fla- gella. The acrosome is invaginated toward the nuclear surface forming a conical structure about 0.51 [xni in height, and it is composed of an outer layer with electron dense material and an inner layer of electron lucent material (Fig. 3A). The spherical electron-dense Nu is 1 .82 |xm in diameter and presents an anterior Spermatogenesis in Diploid and Triploid Scallop 95 -^-J^f Figure I. Diploid and li iplcjid liLiht photomicrographs. A. Diploid female and male gonads in advanced stages of gametogenesis. B. Triploid female and male parts of the jjonad at 9 months old. C". Diploid male portion of the gonad in advanced stage of spermatogenesis. D. Triploid male part of the gonad with few spermatogenic stages developing. invagination (Ai) 0.23 [xm in depth (Fig. 3A). and a posterior invagination (Pi) 0.29 ^jim in depth (Fig. 3B). The distal mid-part of the spermatozoa head contains a ring of three to five cross- sections of mitochondria, each about 0.76 jjim in diameter around two centrioles (Fig. 3B. 3C). The proximal centriole (Pc) is joined to the nuclear envelope by a satellite body (not shown) found in the post-nuclear fosse. The distal centriole (Dc) is joined and forms the TABLE 1. Mean diameter (SD) of spermatogenic stages (n = 30) in diploid and triploid catarina scallop Argopecten veiitricosus measured bv image analyses — light microscop), and percent increase area in triploids with regard to diploids. Diameter (jini) Spermatogenic stages Diploid Triploid increase in 3N Acinus' 167(36)° 259(47)*" 141 Spermatogonia' 6.30(0.43)° 8.41 (1.64)'' 78 Spermatogonia nucleus" 4.81 (0.36)" 5.62(1.11)" 37 Spermatocyte" 3.81 (0.31)-' 5.64(0.44)'' 119 Spermatocyte nucleus" 3.34(0.32)- 4.86(0.36)" 112 Spermatid' 2.62(0.25) n.a. Spermatozoa' head 1.69 (0.08 )■■ 2.07(0.15)" 50 Number of sperm/ 1000 (im- 250.000 2-3 Superscript numbers in spermatogenic structures indicate magnification used for measurements: 1 = 4x; 2 = I Ox; 3 = lOOx. n.a. = not available for measurements. Different letters between pioidy groups indicate signifi- cant differences (P < 0.05). basal body of the Fl, which has a diameter of 0.31 |j.ni. Granules of glycogen (Gi) are delected between the mitochondria cross- section (Fig. 3B). Transversal cuts of the tlagellum show the clas- sical structure of nine external microtubule doublets (Md) and two internal single microtubules (Fig. 3D). Triploid Male Gonad Strucliire and Ultraslriiclure of Nine-Montli-Old Scallops Conditioned in the Laboratory Male acini of triploids were larger than in diploids, with a diameter varying from 197 to 387 p.m. On the acinus wall dark colored cells representing Spg can be seen, and detached from the acinus wall some Spc were present. In some of the triploid scal- lops. Spz were seen sparsely distributed in the lumen of the acinus when using the Feulgen stain (Fig. 4A), and also when using scanning electron microscopy (Fig. 4B). The overall structure of the spermatozoa produced by a triploid catarina scallop appears to be similar to those produced by a diploid. The spermatogonia were oval, and had the chromatin uniformly dispersed in small aggregations, with numerous mitochondria in the cytoplasm (Fig. 4C). As in diploids, the primaiy spermatocyte had a smaller size than the spermatogonia, and their cytoplasm contains a complement of oiganelles very similar to the spermato- gonia (Fig. 4D). In the nucleus, the chromatin, set out in clusters, indicates the spermatocytes were in zygotene-pachytene stages of prophase I. There was no evidence in transmission electron mi- croscopy of secondary spermatocytes and spermatids, although as already stated, some spei'inatozoa were observed by scanning elec- tron microscopy (Fig. 4B). 96 Maldonado-Amparo and Ibarra Figure 2. Diploid gonad structure and ultrastructure. A. Light microscopy (LM) of male acinus with spermatogonia (Spg) attached to the wall (W), spermatocytes (Spc), and spermatozoa (Spz). B. TEM of sperniatogenic stages: Spg. Spc. spermatid (Spni), and Spz. C. TEM of primary spermatocyte with multiple mitochondria (Mi) and Golgi bodies (Gb). D. TEM of Spm with mitochondria at posterior pole, transversal cuts of flagella (Fl). Triploid Male Gonad Strucltire and Vllrastructure of Field Scallops Sampled at 12 Months Old The male part of the gonad in some triploid catarina scallops at age 12 months had a mixture of male and female acini, with both male and female gametogenic stages occurring in different acinus. However, most of the male germinal cells were in the process of degeneration. Large numbers of hemocytes were invading the male acini (Fig. 5A, 5B). which were characterized by the presence of degenerating spermatogonia and primary spermatocytes (Fig. .SC). The hemocytes were of the basophilic granulocyte type, with a not centrally positioned oval-rounded nucleus 4.5 \x.m in diameter, condensed chromatin, and an entire cell approximately 8 p,m in diameter (Fig. 5D). In most triploid scallops a gonad sex change occurred, as the male part of the gonad (the most distal part) was completely sup- pressed and female germinal structures were replacing the male germinal structures (Fig. 6A). Oocytes developing in the male part of the gonad of triploids (Fig. 6B) were similar to those found in the female part of the gonad of a diploid (Fig. 6C). They contained numerous vitelline inclusions, cortical granules and a vitelline coat. DISCUSSION The ultrastructure of diploid spermatogenesis was similar to that described by Dorange and Le Pennec ( 19S9a) for Pecten ma.xi- mus. with some size and morphology differences which is not surprising, as closely related species are known to have similar but not identical ultrastructure of gametic stages (Le Pennec & Be- ninger 1997). Differences in cell size between triploid and diploid moUusks have been reported only for egg size (Guo & Allen 1994a, 1994c: Eversole et al. 1996; Utting et al. 1996; Ruiz-Verdugo et al. 2001 ) or sperm size (Komaru et al. 1994), but in triploid fish species an increase in size of different cell types has been reported (Valenti 1975: Ueno 1984: Small & Benfey 1987: Aliah et al. 1990). In the present study not only spermatogonia, but also all other sperniato- genic stages measured indicated a larger cell size in triploids than diploids. Fuilherniore. an increase in spermatogenic stage size was not necessarily a correlate of increase in nucleus size. For example, there is an expected increase in the nucleus/total cell ratio of sper- matocytes when compared to that same ratio in spermatogonia occurring as a consequence of nuclear distention (Dorange & Le Pennec 1989a). However, whereas the increased ratio in sperma- tocytes was observed in this study for both ploidy groups, the nucleus-total cell ratio in spermatogonia was smaller for triploids than diploids, and this occuned despite the larger nucleus in trip- loid spermatogonia than in diploids. This, as well as the increase in area estimated for triploid spermatogonia cell and nuclei indicates that a larger increase in cell size than nucleus size occurs in triploid spermatogonia of catarina scallop, and might be caused by a larger number of organelles (mitochondria, endoplasmic reticulum, ribo- somes) than tho.se found in diploids, as previously proposed by Guo and Allen ( 1994a) to explain the increased egg size of triploid oysters. These authors proposed that the increase egg size could be a consequence of a larger nucleus requiring a larger cytoplasm. Spermatogenesis in Diploid and Triploid Scallop 97 500 nm ^i^*^*^ 500 nm Figure 3. Diploid spermatozoa ultrastructure. A. TEM of section througli a spermatozoon head, sliowinj; airosome (Ac), anterior invagination (Ai), nucleus (Nu), and two cross-sections of mitochondria (Mi). B. TEM section of the spermatozoa mid-part; posterior invagination (Pi), proximal centriole (Pc), distal centriole (l)c), glycogen granules (Gi), mitochondria (Mi) and llagella (Fl). C. TEM of transverse section through the mid-part of spermatozoa showing five cross-sections of mitochondria at most. D. TEM of transverse section through flagella showing the classical structure of 9 external microtuhule doublets (Md). and 2 internal. such that allocation of nutrients and organelles is in adequate pro- portions during cell divisions. Unfortunately, because of the few spermatogenic stages found in triploids when electron micixiscopy was done, quantification of the number of organelles could not be accomplished. The spermatozoa of triploid catarina scallop was morphologi- cally similar to that in diploids, although as shown by Komaru et al. (1994) for the Pacific oyster, it showed a larger head than that in diploids. The diameter differences observed between diploid and triploid spermatozoa of catarina scallop correspond to an area for triploid sperm of 1.5 times that of a diploid sperm, and is most probably a consequence of an increased amount of DNA in the sperm produced by triploids. Komaru et al. (1994) found an in- crease area of 1.4 times in sperm from triploids when compared with diploids, and the increase in area in triploid spermatozoa of the Pacific oyster has been shown to agree with an increased amount of DNA (1.5 of that in sperm from diploids) by flow cytometry (Allen 1987; Guo & Allen 1994a). In spite of the size increase, the spermatozoa of triploid catarina scallop had the same number of cross-sectioned mitochondria than diploids. A larger number of organelles in gametic cells have been demonstrated only for tetraploid Mediterranean blue mussel, but not for sperm produced by triploid Pacific oyster. That is. Komaru et al. (1995) found that spermatozoa from tetraploid Mediterranean blue mus- sels had from 5 to 7 mitochondria with a mode of 6, whereas that from diploids had 5. Contrary to that, sperniatozoa from triploid Pacific oyster, although larger than that in diploids, had the same number of mitochondria as diploids (Komaru el al. 1994). The observation of spermatozoa in triploid catarina scallop, even if in low numbers was important because previously. Ruiz- Verdugo et al. (2000) were unable to observe sperm in triploids from this same species, but their histology techniques were limited to H&E staining and light microscopy. In the present study, the use of the Feulgen stain for light microscopy allowed for the finding of some spermatozoa in isolated acinus, an obser\ ation that was cor- roborated when spermatozoa were also found by scanning electron microscopy. However, even if present, the rare occasions in which spermatozoa were found indicates that the triploid condition in catarina scallop results for the most part in an anest of spermato- genesis early during the process of gamete formation, at the pri- mary spermatocyte stage. That is, the arrest occurs at meiosis I, when the secondary spermatocyte would be produced by the first meiotic reduction (Beninger & Le Pennec 1991). The nearly com- plete arrest of meiosis at the primary spermatocyte stage has been previously noted by ultrastructure analysis in other bivalves such as Saccostrea commercialis (Cox et al. 1996) and Mytilus gallo- provincialis (Kiyomoto et al. 1996). Contrary to that, in triploid Crassostrea gigas spermatogenesis has been reported to be re- duced, but not arrested al the primary spermatocyte as spermatid and spermatozoa production occurs (Allen & Downing 1990), the last one being capable of fertilizing eggs (Guo & Allen 1994a). The finding that in older triploid catarina scallop the male portion of the gonad was replaced with female acini is similar to that reported by Ruiz-Verdugo et al. (2000) after studying the annual gainetogenic cycle of diploids and triploids for this scallop. The few gametic stages still present in the male acini of scallops 98 Maldonado-Amparo and Ibarra Figure 4. Triploid structure and ultrastriiilure of male part of the gonad. A. \M of section througli a male acinus witli few spermatogonia (Spg) hv the Hall (\V(. spermatocytes (Spc). and spermatozoa (Spz). B. Scanning electron microscopy (SEM) of spermatozoa found in a male acinus, with electron dense nucleus (Nu), acrosome (Ac), mitochondria (Mi) at the base, and flagella (Kl). C-D. TEM of section through a male acinus with spermatocytes (Spcl), spermatogonia (Spg), and haemocyte (He). 12 momhs old were being lysed and possibly recycled through phagocytic activity, as demonstrated by the presence of laige num- bers of hemocytes. which are known to play a role in phagocytosis of degenerating pectinids gametogenic structures (Dorange & Le Pennec 1989a. 1989b; Beninger & Le Pennec 1991). The hemocytes were the typical ones described for other bivalves such as Mya areiiaria and Mytihis edulis (Cheng 1981 ). With this study we have demonstrated that the male gonad of triploid catarina scallop is almost but not completely sterile, as the lack of male gametes occurs until after spermatogenesis begins and is arrested, observing later oogenesis in male acini. The cause of the reversal of sex in male acini of catarina scallop is not known, but might be a consequence of the arrest of meiosis signaling, possibly through a neuroendocrine mechanism, an arrest of syn- thesis or release of some type of maleness factor required for spermatogenesis. Neuroendocrine factors are known to be neces- sary for development of male and female gonad in some mollusks (Feral et al. 1987), or only needed for development of the male gonad in functional hermaphrodites for which female gonad results from auto-differentiation (Gomot & Griffond 199.'?). The existence of those neuroendocrine factors has been demonstrated bv isola- tion in other mollusks (\an Minnen et al. 1989). but not in pec- tinids. With regard to the arrest of meiosis. it has generally been presumed that the primary cause for triploid sterility, measured as inability to fonn viable and large number of gametes as in diploids, is the inability of chromosomes to pair and complete synapses because of multivalent formation. However, it has been demon- strated at least for one species that this is not the cause of sterility. Guo and Allen (1994a) established that for the Pacific oyster. Crassostrea gigas. the normal synapse of chromosomes and nor- mal segregation was not a necessary event to obtain mature ga- metes in triploids, as they observed that the extra set of chromo- somes segregated randomly in spawned eggs from triploids. To explain the sterility induced in triploids it is interesting to point to new research being done in the molecular genetic field. During the last decade a series of "housekeeping" mechanisms or "check- points" operating in the cell cycle during meiosis have been de- scribed for a number of experimental organisms. Their function is to detain further de\elopment when problems during replication, recombination, or segregation are present in a cell, and it is pos- sible to explain some of the abnormal events occurring in triploids gametogenesis by considering them. These checkpoints act by means of protein complexes signaling abnormalities in chromo- some behavior, and effector proteins acting on those signals to delay or arrest the meiolic process (Roeder & Bailis 2000). For example, among the described checkpoint mechanisms for meiosis is a "S-phase or premeiotic replication checkpoint." which func- tions on detecting chromosomal lesions after replication, but be- fore recombination (Murakami & Nurse 1999). A second check- point mechanism, the "recombination or pachytene checkpoint." is known to act on gametogenic cells that have not completed cor- rectly the recombination between sister chromatids during meiosis I. or in which the synaptotemal complex is defective, and it will arrest meiosis at pachytene (Murakami & Nurse 1999. 20()0; Roeder & Bailis 2000; Tarsounas & Moens 2001). A third check- point mechanism, the "metaphase checkpoint," has been described for arresting meiosis in metaphase I. by blocking the metaphase to anaphase transition when chromosomes are misaligned on the spindle apparatus. An interesting aspect of this checkpoint mccha- Spermatogenesis in Diploid and Triploid Scallop 99 ►»f^i "^-^i^ %. T, .-^_ fi B He Dsc-^ «^ 45^ "• ftlf^ A ;« ^^ Mi Figure?. Triploid structure and ultrastructureshowingdegtniratioii ol male spermaliimiiii stages. \. I,M (it section through a male acinus with spermatogonia (Spgl and spermatocytes (.Spc) closest to the Hall, and acinus tilled with haeniocytes (He). B. LM of connective tissue (Ct) at acinus wall with He. C. TEM of Spc in degeneration process (Dsc), surrounded by multiple He. I). TEM at larger amplillcation of He and Dsc. *•'> Nu '••' Vv^. :?**•: ,-..^ ,vi ^ :i:.vv.-Ve V, 'i^h r ■*-. < • 5 pm , •'• s . * i.v : ■-*: •■.•.■; ••..;•. :#si." • • . "S' , - ^u'.v;.- \ .;: • !?*■.;; . ;i' :J^ \ J. ;-.^ 'Nu ; . %^?vM..-' ''\<^r\^ •^j'S. • ' S '' J-A . .*.- ' X ■ 4 *■ ' ..:,\-.^^5 $?*■ ■<^^-; ,' lOjum J Figure 6. Gonad structure and ultrastructure ol 12-ni(> old scallops. A. LM of male part of gonad in triploid occupied with female acini with oocyte development. B. TEM of oocytes in male part of the gonad of a triploid, showing nucleus (Nu), vitelline inclusions (Vi). cortical granules (Cg). and vitelline coat (Vc). C. TEM of oocytes from a diploid scallop with the same structures than that in triploids. 100 Maldonado-Amparo and Ibarra iiisni is that it is l. Zool. 241:339-342. Tarsounas, M. & P. B. Moens. 2001. Checkpoint and DNA-repair proteins are associated with the cores of mammalian meiotic chromosomes. Current Topics in Developmental Biol. 51:109-134. Ueno. K. 1984. Induction of triploid carp and their haematological char- acteristics. Jpn. J. Genet. 59:585-59 1 . Utting. S. D.. P. F. Millican & 1. Laing. 1996. The breeding potential and biochemical composition of triploid Manila clams. Tapes philippi- nantin Adams and Reeve. Atpiaciilriire Res. 27:573-580. Valenti. R. J. 1975. Induced polyploidy in Tilapia aurea (Steindachner) by means of temperature shock treatment. J. Fish Biol. 7: 519-528. van Minnen, J.. R. W. Dirks. E. Vreugdenhil & J. van Diepen. 1989. Expression of egg-laying hormone genes in peripheral neurons and exocrine cells in the reproductive tract of mollusc Lvninaea stagnalis. Neuroscience (England) 33:35-46. Joiirihil of Shellfish Reseiiivh. Vol. 21. No. 1. 103-108. 2002. REPRODUCTIVE CYCLE OF SPONDYLUS CALCIFER CARPENTER, 1857 (BIVALVIA: SPONDYLIDAE) IN THE "BAHIA DE LORETO" NATIONAL PARK, GULF OF CALIFORNIA, MEXICO MARCIAL VILLALFJO-FUERTE, MARCIAL ARELLANO-MARTINEZ. BERTHA P. CEBALLOS-VAZQUEZ, AND FEDERICO GARCIA-DOMINGUEZ Centni liuevdisiiplimirio de Ciencias Marinas, histihilo Folireciiico Nacional. Apartado Postal 592. La Paz. B.C.S. 23000. Mexico ABSTRACT The reproductive cycle of SpoinlYlux calcifcr lYom Bahia dc Loreto. Gulf ofCalitornia, was studied from January 1998 to March 1999. Microscopic analyses established that this species is gonochoric. The size at spawning occurred at 1 13-mm in shell height. S. calcifer e.xhibits a short spawning period from August to October followed by a long inactive period from November to February. Increase in water temperature initiates the gonadal maturation process, while decrease in water temperature inhibits gametogenesis. The threshold water temperature for spawning in S. calcifer. is 29°C. Nutrient reserves from the digestive gland are mobilized and used by the gonad during the developing stage. Meanwhile, reserves stored in the muscle are used only after gamete production has started, helping to support the energetic output during the ripe and spawning stages. In Bahfa de Loreto, the period of highest phytoplankton abundance in bottom waters coincides with the onset of gametogenesis in .S. calcifer. This suggests that S. calcifer also depends on food intake for oocyte growth. KEY WORDS: spawning season, histology, energy transfer. Spoiulyliis calcifer. Gulf of California FNTRODUCTION Spaiidylns calcifer Cuipenter. 1857 cotntnonly natned "Buitu clam." belongs to the Spondylidae Family. It is geographically distributed from the Gulf of California to Peiu. This clam can be found from intertidal to subtidal zones, attached by the right valve to exposed boulders or under rock ledges (Skoglund & Mulliner 1996). At present in Loreto, Baja California Sur. Mexico, this species is not commercially exploited; however divers capture it (only in small c|uantities) for human consutiiplion year around. This clam is the largest species of the genus Spondxlus. S. calcifer is considered in danger of extinction (Baqueiro et al. 1982). and is currently protected by Mexican laws (Norma Oficial Mexicana 1994). Despite the above, there are no published records on its biology. Few articles have been published about the genus Spondyhts. most focusing on the taxonomy, anatotny. tnorphology. habitat, and other topics ( Yonge 1973: Dakin 1928 a.b: Mata et al. 1990; Parth 1990; Okutani 1991; Skoglund & Mulliner 1996). There is only one study about the reproductive cycle and spawning season of Spondyhts leucacanthiis (Villalejo-Fuerte & Garci'a- Domt'nguez 1998). The present study describes the reproductive cycle throughout 15 months, and the spawning season of 5. calcifer. from histologi- cal analysis and measurements of the volumetric fraction of oo- cyte. Additionally, the relation between the reproductive cycle and gonadic. digestive gland and muscle yield indexes is analyzed. MATERIALS AND METHODS Sampling was conducted in Bahia de Loreto. Gulf of Califor- nia. Mexico (25°48'54". 1 1 1°15'45") (Fig. 1 ). Fifteen to .30 speci- mens of S. calcifer were collected monthly, from January 1998 to March 1999. by scuba diving at lO-m depth. Shell height and wet weight of the gonad, digestive gland, muscle and total soft body were recorded for each clam after fixation in a neutral 10% for- malin solution prepared with sea water. Water temperature was recorded at each sampling time. The photosynthetic pigment con- centration (mg chlorophyll/m') in Bahia de Loreto frotn January 1998 to March 1999 was obtained from Seawifs Project. NASA/ Goddard Space Flight Center, and this was considered as an esti- mation of the food availability for the clams. Becau.se 5. calcifer does not exhibit sexual ditiiorphism, indi- vidual sex was determined through histological analysis. The sex ratio for the total sample was obtained. The null hypothesis of a 1:1 sex ratio was established and its significance was tested using a chi-sqtiared analysis (Sokal & Rohlf 1979). For histological studies, gonads were dehydrated in an alcohol series and embedded in paraplast. Sections (7 |j.m) were placed on slides and stained with hematoxylin-eosin (Humason 1979). The classification of gonadal development was similar to that oi S. k'Hcacantlms (Villalejo-Fuerte & Garci'a-Domi'nguez 1998). which includes five development stages: undifferentiated, devel- oping, ripe, spawning, and spent. To facilitate the description of the reproductive cycle, the tnonthly relative frequencies for all gonadal development stages were calculated. The size at spawning in the population is defined as the small- est length at which 50% of females and males are spawning (Som- erton 1980). The size at spawning was estimated as the shell height at which the 50% of cumulative frequency of clams in the spawn- ing stage was attained. Three indexes were calculated: gonad index (GI). digestive gland index (DGI) and muscle yield index (MYI). Each index was calculated by dividing the gonad, digestive gland or tnuscle wet weight (respectively) by the total soft body wet weight, and ex- pressing the results as a percentage (Sastry 1970). Futtherniore. the monthly proponion occupied by developing and mature oocytes combined (volume fractions) were estimated by stereology (Lowe et al. 1982; MacDonald & Thompson 1988). The gamete volume fraction (GVF) was obtaitied frotn point counts in a Weibel eyepiece graticule mounted in an ocular microscope and applied to a gonadal section ( lOOX). Three repli- cates were done for each gonad. The algorithm of Lowe et al. (1982) was applied: GVF = No. of positive counts/total points 103 104 ViLLALEJO-FUERTE ET AL. 100 111"4'2" 26'6-23" Figure 1. Location of sampling station in Bahia dn Liireto, Gulf of California. Mexico. counted X 100. GVF was calculated as the sum nf the \alues for developing and mature oocytes in all females. The monthlv mean GVF values were plotted. A Spearman-rank correlation analysis was used to investigate the relationship between monthly mean values of GI. DGI. MYI. GVF. water temperature, and photosynthetic pigment concentra- tion. As GI. DGI. and MYI are percentage values, the arcsine transformation (Sokal & Rohlf 1979) was used to attain data nor- mality and homoscedasticity for statistical analysis. RESULTS A total of 22.S clams were captured, of v\hich 279c were fe- males and 32% were males. The remaining (41%) were undiffer- entiated. The sex ratio for the total sample was 0.8 F:! M and did not significantly differ from the expected ratio of 1:1 (P > O.O.'S). Shell height ranged from 70.5 mm to I6.'i.2 mm. with the mode at 1 15 mm. The reproductive cycle of S. cakifer was remarkably seasonal (Fig. 2). In January and February of both years, the majority of clams was inactive. Gametogenesis started in February in a small proportion (5.3%) of the clams. By March 52.9% of the clams were in the development stage. Development continued until Au- gust with the highest proportion occurring in April (93.7%). Ripe clams were found from April (6.2%) to July when the highest proportion was observed (80.0%). A small proportion of ripe clams was found in September (11.1%). Spawning was found in a higher proportion during August and September (40.0% and 33.3%, respectively) and dropped drastically in October (7.7%). From November to February, onlv undifferentiated and spent stages occurred. Figure 3 shows that the size at spawning in the population of S. calcifer is 1 13-mm in shell height: however, individual organisms may begin spawning at 86 mm in shell height. The GI shows its peak values during May. June, and July, J F 1998 Q] Undifferentiated ■ Spawning m Developing ^^ Spent jRipe Figure 2. Kepn)ducti>e cjcle u\ Spondylus cakifer in Bahia de Loreto, Gulf of California. Relative frequency of gonadal stages between Janu- ary IWS and March 1999. Observations of males and females are combined. coinciding with the occurrence of the highest frequencies of clams in the ripe stage. The GI declined from August to October, coin- ciding with the spawning season. A period of low values occurred from November to March, coinciding with the highest frequencies of spent and inactive stages (Fig. 4a). The DGI shows high values in February, declining from March to June lea\e as is coinciding with the developing and ripe stages. In August and September (months in which the highest proportions of clams in spawning stage occur), the DGI values decrease. Af- terwards, from October to March, DGI values start to rise again, coinciding with a reproductive inactivity of the specimens (Fig. 4b). The MYI values decreased according to the maturation process, beginning the decrease in April, two months after the onset of gonadal development (February). The lowest MYI value was found in January 1999 and the higher \alue was found in March 1999 (Fig. 4c). The volumetric fraction of gametes reflected the conad devel- 100 80 90 100 160 110 120 130 Height (mm) Figure 3. .Size at spawning of 50% of the population of Spondylus cakifer in Bahia de Loreto, (Julf of California, Mexico. Reproductive Cycle of S. calcifer 105 c o o CD (U E o JFMAMJJASONDJFM 1998 1999 Figure 4. Variation in tlie mean values of a) gonad index, b) digestive gland index, c) muscle yield index, and d) oocyte volumetric fraction. opment stages. Values increased when development and ripe stages were present, and they decreased concurrently with the spawning stage. Low values occurred when the inactivity and spent stages prevailed (Fig. 4d). Seawater temperature varied seasonally fruni 19'C to 29°C during the study period. Temperature increased from March to August, reaching its peak (29°C) from August to October, then decreasing from November to February. The lowest temperature was recorded in February, both years (19.5°C and 19°C) (Fig. 5). Photosynlhetic pigment concentration (mg chlorophyll/m'') in Bahia de Loreto was greater in the colder months than in the warmer ones. The ma.ximum \alue was in March 99 (2.1 mg chlorophyll/m') and the minimum was in September (0.2 mg chlo- rophyll/m'') (Fig. 6). GI and GDI values were negatively correlated, with statistical significance (P < 0.05). GVF had a significant positive correlation {P < 0.01) with GI and a significant negative correlation with photosynlhetic pigment concentration (P < 0.05). GDI showed a significant negative correlation with temperature (P < 0.05) and a significant positive correlation with photosynthetic pigment con- centration (P < 0.01). The photosynthetic pigment concentration showed a significant negative correlation with temperature {P < 0.01). No significant correlation was found between MYI and any other variable (P > 0.05). DISCUSSION In the Gulf of California low densities of S. calcifer have been reported, ranging from 1 clam per 100 m"^ (from 1 to 25 ni deep) to 1-5 clams per 25 m"(>25 ni depth! (Baqueiro et al. 1982). In this study, the number of organisms captured per month corre- sponds to these low population densities. The histological analysis revealed that S. calcifer is a gono- choric species as no hermaphrodite or sex-reversal specimens were collected. However hermaphroditism has been reported in 3.S'* of clams studied for S. leiicacanllms from the same locality (Villa- lejo-Fuerte & Garci'a-Domi'nguez 1998). The sex ratio of 5. calci- fer was not different from 1 : 1 , as also found for other bivalves like Mercenaria mercenaria (Heffernan et al. 1989), Venus siriatiila (Gaspar & Monteiro 1998). and Megapitaria squalida (Villalejo- Fuerte et al. 2000). No references regarding the sex ratio for this species were found in the literature. However. S. leucacanthus. a related and sympatric species, presents a sex ratio different from parity (Villalejo-Fuerte & Garcia-Dominguez 1998). The size at spawning in S. calcifer occurs at 1 13-mm in shell height, although specimens may start to spawn at 86-mrn in shell height. In contrast, in the sympatric species S. leucacanthus, the size at spawning is 75-mm in shell height with some spawning specimens being as small as 4()-mm in shell height. This difference in size at spawning may result from 5. calcifer being a the larger species within the genus Spondylus. The reproductive cycle of S. calcifer was remarkably seasonal. S. calcifer exhibits an annual reproductive cycle with a short spawning period from August to October followed by a long inactive period largely during the winter (November-February). The reproductive cycle of 5. calcifer was similar to the one de- scribed for 5. leucacanthus (Villalejo-Fuerte & Garcia-Dominguez 1998). In both species, recruitment occurs once per year with a similar short spawning period. These characteristics coiTespond to a conservative reproductive strategy, similar to the one reported for 5. leucacanthus (Villalejo-Fuerte & Garcfa-Dominguez 1998). Gametogenic cycles are generally ruled by external environ- inental factors that may trigger and synchronize the "timing" oi the different stages (Lubet 1983). The synchronization o'i the 106 VlLLALEJO-FUERTE ET AL. J FMAMJ JASONDJ FM 1998 1999 Fijjure 5. Variation of surface teniperatiiri' from January 1998 to March 1999 in Bahia de Loreto, Clulf of California, Mexico. gonadal cycles in ;i population is probably also the result of some kind of exogenous regulation (Gallardo 1989). Most studies consider that temperature is the important environmental factor in the regulation of bivalve reproduction (gametogenesis and spawn- ing) (Giese and Pearse 1974; Sastry 1979; Barber & Blake 19S1 ). However, in this study temperature was not correlated with Gl. Nevertheless, the increase in water temperature coincided with the beginning of gametogenesis. The above suggests that the rise of water temperature may be responsible for triggering the gonad maturation process, however, laboratory studies are need to confirm it. In this re.spect. it had been previously observed that sudden increases in water temperature appear to be the final cue for stimulating maturation and ovulation in some fish species (Bye 1990). In S. calcifer the inactive period (undifferentiated and spent stages) occurs from November to February, when water tempera- ture drops (from 29"C in October to 19°C in February). Thus, it is reasonable to state that the decrease in water temperature inhibits J F M A M 1998 J J A S O N D J F 1999 Figure 6. Variation of photosynthetic pigments concentration from January 1998 to March 1999 in Bahia de Loreto, Gulf of CaHfornia, Mexico. gametogenesis. but newly laboratory studies are necessary to con- firm this conclusion. Similarly, a cooler temperature inhibits ga- metogenesis in SpisuUi solidissiiiiii (Kanti et al. 1993). On the other hand, spawning in .S'. calcifer. only occurs when water tem- perature is at least 29'C (August to October). This fact suggests that 29 C is the threshold water teinperature at which spawning occurs in S. calcifer. A threshold temperature for spawning has also been reported for some oyster species (Burrell 1985). In con- trast, in other clam species, like Paphies donacina and Cerusto- dcvuia cdide spawning occurs within a relatively wide temperature range (Navarro el al. 1989; Marsden 1999). The bivalve gamete production is strongly infiuenced (set in a seasonal context) by environmental factors such as tempera- ture but also by food availability (MacDonald & Thompson 1985). However, both factors may be strongly related. In this work, the photosynthetic pigments concentration was negatively correlated with temperature. Then the higher food availability for clams (expressed as photosynthetic pigment concentration) were during the cold months coinciding with the resting period. On the other hand, the spawning season of S. calcifer coincide with the lowest food availability (August to October). A relation of spawn- ing time and food availability was found in Cldamys amandi (Jaramillo et al. 1993). whereas Hinnites gigaiueti.s showed no correlation between food availability and spawning (Malachowski 1988). The DGI had a significant positive correlation with photosyn- thetic pigment concentration, then the higher food availability and the higher DGI values were during the inactive period of reproduction. The trend observed for DGI suggests that a period of accumulation of nutrient reserve in the digestive gland takes place during the inactive period. These nutrient reserves are mobilized and utilized by the gonad during the developing stage. The significant negative correlation between DGI and Gl confirms the above for 5. calcifer. In mollusks. it has been observed that the onset of the oocyte growth phase is dependent upon the accumulation and transfer of nutrient reserves from the digestive gland to the gonad (Sastry 1968; Gabbott & Bayne 1973). Despite MYl and Gl not being coirelated, MYI values start to decrease in April, just two months after gametogenesis had started. This suggests that there is a delayed in energy transfer from muscle to gonad. The reserves accumulated in the muscle are used up after gamete production had started, helping to support the energetic output during the ripe and spawning stages. Similarly, in Ar- gopecten irradian.s it appeared that lipids from the digestive gland were used to fuel the beginning of gametogenesis whilst adductor muscle reserves becoine important later on to complete gonadal development (Barber & Blake 1981 ). A relationship between MYl and Gl has been observed in Pectea ma.ximus (Comely 1974; Fav- eris & Lubet 1991). Argopecleii irradian.s (Sastry 1966). Pari- iiiipecren ycs.soensis (Mori 1975). Plucopecten magellanicus (Rob- inson et al. 1981). and Argnpeclca circularis (Villalejo-Fuerte & Ceballos-Vazquez 1996). However, in pectinids and other bivalves, it has been proposed that oocyte growth is dependent on two factors; food intake and energetic storage in specialized organs (Sastry 1963. 1966. 1968; Barber & Blake 1983). In P. magellaidcit.'i and A. circidari.s. the energy for gamete maturation comes from both stored reserves and ingested food (Thompson 1977; Robinson et al. 1981; Luna- Gonzalez et al. 2000). Reproductive Cycle of S. calcifer 107 ACKNOWLEDGMENTS We are izratetui to The Iiistituto Pcilitecnicn Nacional (IPN) tor fimding this work, and tor the grants (EDI and COFFA) to the authors. We also wish to thank to Sofia Ortega for the data of photosynthetic pigments concentration and to Mari'a Elena Sanchez-Salazar for his editorial help on the English manu- script. LITERATURE CITED Baqueiro, C. E.. J. A. Masso & H. B. Guajardo. 1982. Distrihucicin y abundancia de moluscos de importancia comercial en Baja California Sur. Mexico. Inst. Nal. dc la Pesca. Mexico. Seric ilc Divitliiuvion. 11:1-32. Barber. J. B. & N. J. Blake. 1981. Energy storage and utilization in relation to gametogenesis in Ari^upcclen inadictus conccuiricus (Say). ./. Exp. Mar. Biol. Ecoi 5:121-134. Barber. J. B. & N. J. 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Temperature effects in reproduction of the bay scallop. Aecpiipccteu irradiaiis Lamarck. Biol. Bull. {Woods Hole. Mass.). 130: 118-134. Sastry, A. N. 1968. The relationship among food, temperature, and gonad development of the bay scallop. /Ac(/»//)c<7(v; irraduins Lamarck. Plns- /W. Zoo/. 41:44-53. Sastry, A. N. 1970. Reproductive physiological variation in latitudinally separated populations of the bay scallop Aeqiiipecten irradians Lama- rck. Biol. Bull. 138:56-65. Sastry. A. N. 1979. Pelecypoda (excluding Ostreidae). pp. 131-192. In A. C. Giese & J. S. Pearse (eds.). Reproduction of Marine Invertebrates. Academic Press, New York. Skoglund, C. & D. K. Mulliner. 1996. The Genus Spondylus (Bivalvia: Spondylidae) of the Panamic Province. The Festivus. 38:93-107. Sokal, R, R. & F. J. Rohlf 1979. Biometna. Principios y metodos esiadis- 108 ViLLALEJO-FUERTE ET AL. ticos en la investigacion biologica H. Blunie Ediciones- Madnd. Es- pana. 832 pp. Somerton D. A. 1980. A computer technique for estniiatnijj the si/e of sexual maturity in crabs. Can. J. Fish. Aqiuit. Set. 47:1488-1494. Thompson. R. J. 1977. Blood chemistry, biochemical composition, and the annual reproductive cycle ot the giant scallop. Placopecten magethiiu- cus, from southeast Newfoundland. J. Fisheries Res. Board of Canada 34:2104-2116. Villalejo-Fuerte. M. & B. P. Ceballos- Vazquez. 1996. Variacion de los I'ndices de condicion general, gonadico y de rendimiento muscular en Argopeeren eircularis (Bivalvia: Pectinidae). Rev. Biol. Trop. 44:591-594. Villale|o-Fuene. M. & F. Garcia-Domfnguez. 1998. Reproductive cycle of Spondyhis leiieaeanthiis Broderip. 1833 (Bivalvia: Spondylidae) at Isia Espiritu Santo. Gulf of California. / Shellfish Res. 17:1037-1042. Villalejo-Fuerte. M.. M. Arellano-Martinez. B. P. Ceballos-Vazquez & F. Garcia-Dominguez. 2000. Cicio reproductivo de la almeja chocolata Meaapiiana sqiialida (Sowerby. 18351 (Bivalvia: Venendae) en Bahia Juncalito. Golfo de California. Mexico. Hidrohiolvaia 10:165-168. Yonge. C. M. 1973. Functional morphology with particular reference to hinge and ligament in Spondyhis and Plicatula and discussion on re- lations within the superfamily pectinacea (Mollusca: Bivalvia). Proc. R. Soc. Land. B. 267:173-^208. Jnurmil of Shellfish Research. Vol. 21. No. 1, 109-112. 2002. BACULOVIRUS-LIKE PARTICLES IN EPITHELIAL CELL OF DIGESTIVE DIVERTICULA OF THE SCALLOP, PATINOPECTEN YESSOENSIS YOUNG JIN CHANG,'* MIN-DO HUH," MYUNG-JOO OH,' AND YOSHIO SUGAWARA^ ^Department of A National University. Busan 60H-737, Korea; -Department of Aquatic Life Medicine. Piikyong National University. Busan 608-737, Korea; ^Department of Aqualife Medicine. Yosu National University, Yosu 550-749. Korea; * Department of Biotechnology, Senslm University of Ishinomaki. Ishinomaki 986. Japan ABSTRACT Virus-like particles were encountered in one epithelial cell of digestive diverticulum in a chnically healthy scallop. Patinopecten yessoensis. Virions, showing typical rods, were found in the cytoplasm without marked cellular degenerative change and were recognized as ovoid or spherical bodies, depending on sectional orientations. The envelope was appro.ximately 1 2 nrn in thickness and the space between the envelope and nucleocapsid was about 10 nm. Average length and diameter of the complete virions were 520 and 130 nm. respectively. No occlusion bodies were observed. From this morphological evidence, it was considered that the virus particles observed in one epithelial cell of digestive diverticulum in the scallop are likely to be a species of nonoccluded, baculovirus type C, which are morphologically very similar to the baculovirus associated with white spot syndrome (WSSJ and baculoviral mid-gut gland necrosis (BMN) in shrimp. KEY WORDS: bivalve. Paimopeclen yessoensis, baculovirus. white spot syndrome (WSSl. baculoviral mid-gut "land necrosis (BMN) INTRODUCTION A number of infectious diseases have been observed in bivalves such as oysters, clams, mussels, and scallops. Because bivalve production represents a considerable proportion of fisheries indus- try in South Korea, much attention is now being paid to the cause of the massive deaths occurring in bivalve farms along the south- em coast. To date, diseases have been reported from 63 species of parasites, 7 strains of bacteria, 2 strains of fungi, and 3 strains of rickettsia or chlamydia. More recently, five kinds of viral diseases were reported from oysters (Fisher 1988; Sindermann 1990; Couch & Fournie 1993; Faisal & Hetrick 1994; Woo 1995). There are two other kinds of diseases with unknown cause. For most of the diseases cited, little is known regarding pathologic effects on the host and methods of prevention and control. Virus particles were, by chance, encountered in one epithelial cell of digestive diverticulum during a study on the relationship tif histological structures in digestive diverticula to nutrient accumu- lation. The morphological characteristics of the particles are de- scribed in comparison with other virus particles and discussed with respect to pathogenic potential. MATERIALS AND METHODS Thirty scallops were sampled every month from March 1982 to December 1983 from a bottom culture farm in Abashiri waters of Hokkaido, Japan. Each individual, ranging from 8.5 to 1 1.2 cm in shell height and from 62.9 to 197.4 g in total weight, was clinically healthy. For transmission electron microscopy, all scallops were necropsied and their digestive diverticula were carefully removed, then diced into 1-mm cubes. All tissue cubes were prefixed with a 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4, 4°C). After postfixation with 0.2 M cacodylate buffered 2% OSO4 for about an hour, tissue blocks were dehydrated in a graded series of ethanol and embedded with Epon 812. Ultrathin sections were obtained using an ultramicrotome (Porter Blum II, Agwani, Massachusetts, *Corresponding author: Fax: +82-51-628-7430; E-mail: yjchang@ pknu.ac.kr USA) and stained with uranyl acetate and lead citrate. The grids were examined with a transmission electron microscope (JEOL lOOB, Tokyo, Japan) under an accelerating voltage of 80 kV. RESULTS As shown in Figure 1, one epithelial cell containing vu'al par- ticles con-esponded to type I of the 3 types of cells that have been classified by Chang et al. (1989). Because of the shortness of apico-basal length, it appears to be embedded between the other types of cells. Cytoplasm of the virus-containing cell was com- pletely occupied basally by a densely packed mass of rough en- doplasmic reticulum (RER) and apically by a round area contain- ing a number of viral particles. A great number of virions had a fence-like array, which again formed several layers, roughly in parallel. Virions showed typical rods in shape but, depending on the sectional orientation, they were ovoid or spherical (Fig. 2). Virions were present among a great number of small vacuoles that appeared to be a network of proliferated, tubular smooth endoplasmic reticulum (TER). A layer of variably sized, unidentified vacuoles surrounded the virus- containing area (Fig. 3). The vacuoles, ranging from 520 to 1050 nm in diameter, were spherical in shape and clearly limited by double membranes. They also had thread-like or fuzzy materials with high electron density. No other morphological abnormalities were found in this virus-infected cell. Each virion had a nucleocapsid and an envelope of unit mem- brane that was quite similar morpholtigically to baculovirus (Fig. 4). The average thickness of the envelope was 12 nm and the average distance from the nucleocapsid was 10 nm. The average length and diameter of virions were 520 and 130 nm, respectively. No occlusion bodies were observed. DISCUSSION There have been few reports on viral diseases in bivalves, and most of these were reported for oysters. Since the first identifica- tion of birnavirus from clam, Telliiui tenuis (Hill 1976), retrovirus- like particles from clam Mya arenaria. iridovirus-like particles from oyster Crasso.strea aiif-iilula. and herpes-like particles from 109 110 Chang et al. Figure I. \ iius-inlfcttd tvpf I till in dijiestive diverticula epithelium. This cell was very similar to pancreatic exocrine cells of manunalian pancreas. Cytoplasm near the basement membrane (BM) of the cell contained many large flattened cisternae of the rough-endoplasniic reticula (RER) and relatively few mitochondria (M). The apical part of the cytoplasm was without marked degenerative change, but the cell was filled with numerous viral particles (VP). C. viifiinicd have been repurted (Perkins 1993). Snice oyster velar virus disease (OVVD) caused by iridovirus was reported from farmed C. i;igcis (Elston & Wilkinson 1985: Sindermann & Light- ner 1988: Bower et al. 1994). descriptions followed on small and nonenveloped virus particles from digestive diverticukmi of pearl oyster Piiicicuhi inaxiiiia (Pass et al. 1987), green lip mussel Perini canaliculus (Jones et al. 1966), herpes-like particles from hemocytes of flat oyster Oslrea angasi (Hine & Thome 1977) and enterovirus or calicivirus-like particles from digestive diverticula of scallop Pectc'ii ninxiezelandiac and loheroa Puphlcs vcntricn- sum (Hine & Wesney 1977). Virus particles in this study were moiphologically different from those particles previously reported, but were quite similar to baculovirus that has not been described from bivalves. In general, baculoviruses are rod-shaped DNA viruses, which are known to be 500 nm Figure 2. Fence-like array of viral particles. Numerous viral particles surrounded by tubular smooth endoplasmic reticula (TKRi were rod- shaped, spherical, or ovoid in morphology, because of the difference in viewing orientation. infective only in invertebrates. Baculoviruses are divided into the three subgroups of nuclear polyhedrosis \irus (NPV, type A), granulosis virus (GV. type B). and nonoccluded virus (NOV, type C) (Suidermann & Lightner 1988; Faisal & Hetrick 1994; Levy et al. 1988). From shrimps, six kinds of baculoviruses, including baculovi- rus penaei disease (BP virus disease), monodon baculovirus dis- ease (MBVD), baculoviral mid-gut gland necrosis (BMN), ple- bejus baculovirus disease, and yellow-head disease (YHD), have been described, although their pathogenesis is still poorly under- stood (Faisal & Hetrick 1994). The causative agent responsible for white spot syndrome (WSS). which was responsible for mass mor- tality of shrimps in Taiwan during the last 7 years, was identified to be a species of baculovirus (Wang et al. 1997). All baculoviruses reported in shrimps range from 1.^0 to .MO 11111 in length and 3.3 to 74 nm in diameter. Virions in the scallop were very similar in size to causative viruses responsible for BMN and MBVD in shrimps. The finding that they did not form occlu- sion bodies is consistent with the viruses responsible for BMN and WSS in shrimps. The causative virus of BMN belongs to NOV type C that is found only within the hypertrophied nucleus. BMN frequently brings about mass mortality in an early larval stage of shrimp. Histologically, BMN is characterized by necrosis of epi- thelial cells in the hepatopancreas and mucosal membranes with a variety of nuclear changes, including severe hypertrophy, margin- ation and loss of chromatin, and loss of nucleoli (Momoyama & Sano 1988: Momoyama & Sano 1989). In contrast, WSS is char- acterized by homogeneous nuclei with a variable degree of nuclear hypertrophy in nearly all kinds of tissue except for hepatopancre- atic epithelium (Wang et al. 1997). Even though grossly and micro.scopically no marked lesions were found in association with the presence of viral particles in the scallop, it was considered that the viral particles in this animal should be carefully monitored for the pathogenic potential. BaCULOVIRUS in DlGHSTIVE DIVERTICULA OF SCAI.LOP III lOOOnm Hfiuri' 3. Ihf cluster ot lirai piiiiicks ";is siirioundud by unidentified \aiii()lcs (\ al. 200nm JUKI. Figure 4. Higlier magnification of Figure 2. Single envelope (arrow! and a central nuclcocapsid (NC| were recognized in each viral particle. Tlie clear spaces between envelopes and nucleocapsids were recognizable from all orientations of viral particles. LITERATURE CITED Chang, Y. J.. Y. Sugawara & T. Nomura. Iy89. Structure and function of digestive diverticula in the scallop, Palinopecten ye.ssoensis (Jay). Tn- hoku J. Aj-hc. Res. 39:81-94. Couch, J. A. & J. W. Fournie. 1993. Pathobiology of marine and esluarine organisms. Boca Raton, FL: CRC Press. 552 pp. Elston, R. A. & M. T. Wilkinson. 1985. Pathology, management and di- agnosis of oyster velar virus disease (OVVD). Ac/iuiciilttire 48:189- 210. Faisal. M. & F. M. Hetrick. 1994. Annual review of fish diseases. Vol. 4. New York: Perganion Press, 433 pp. Fisher, W. S. 1988. Disease processes in marine bivalve molluscs. Am. Fish. Soc. Spec. Piihl. 18:315. Mine. P. M. & T. Thorne. 1997. Replication of hcipes-like viruses in haemocytes of adult flat oysters Osirea ani^iisi: An ultraslructural study. Dis. Aquat. Oiff. 29:189-196. Mine. P. M. & B. Wesney. 1997. Virus-like particles associated with cy- topathology in the digestive gland epithelium of scallops Pccleii im- niczelandiuc and toheroa Pupliies veiiliicusiim. Dis. 197-204. Ac/iiiil. Oifi. 29: Jones, J. B., P. D. Scotti. S. C. Deanng & B. Wesney. I96(i. Virus-like particles associated with marine mussel mortalities in New Zealand. Di.s. Aqiiut. Org. 25:143-149. Levy, J, A„ F. C. Heinz & R. A. Owens. 1988. Virology. 2nd ed., Engle- wood Cliffs, New York: Prentice Hall. 440 pp. Momoyama. K. & T. Sano. 1988. A method of experimental infection of kuruma shrimp larvae, Peiuieiis japonicus Bate, with baculoviral mid- gut gland necrosis (BMN) virus. J. Fisli Dis. I 1:105-1 I I. Momoyama, K. & T. Sano. 1989. Developmental stages of kuruma shrimp. Peiuieiis japonicus Bate, susceptible to baculoviral mid-gut gland ne- crosis (BMN) virus. J. Fisit Dis. 12:585-589. Pass, D. A., F. O. Perkins & R. Dybdahl. 1987. Virus-like particles in the digestive gland of the pearl oyster [Pinctada maxima). J. Iiwerl. Palhol. 51:166-167. Perkins, F. O. 1993. Infectious diseases of molluscs. In: J. A. Couch & J. j^2 Chang et al. W Fourn,e editors Pathohiology of maruie and estuarme organisms. Wang. C. S., K. F. J. Tang, G. H. Kou & S. N. Chen. 1997. L.ght and Boca Raton FL' CRC Press, pp. 255-287. electron microscopic evidence of white spot disease ,n the giant tiger Smdermann. C.' J. & D. V. Lightner. 1988. Disease diagnosis and control shnmp. Penaeus monodon (Fabricins,. and the k-ruma ^hnmp. Pe- rn North American marine aquaculture. New York: Elsevier. 431 pp. naeus japon,a.s (Bate), cultured in Taiwan. J. F,sh D,5 0.3-3-33L Smdermann, C.J. 1990. Pnncipal diseases of marine fish and shellf-ish, vol. Woo. P. T. K. 1995. Fish diseases and disorders. Vol. K Protozoan and ^ San Diego: Academic Press. 516 pp. nietazoan infections. New York: Cab International. 808 pp. Joiinuil ofShellfLsh Ri'.seanh. Vol. 21, No. 1. 113-118, 2002. HAPLOSPORIDWM COSTALE (SEASIDE ORGANISM), A PARASITE OF THE EASTERN OYSTER, IS PRESENT IN LONG ISLAND SOUND I. SUNILA,' N. A. STOKES,- R. SMOLOWITZ,' R. C. KARNEY,^ AND E. M. BURRESON" ^ State of Connecticut. Depurtincnl of Agriculture, Bureau of Aquaculture. P.O. Box 97. Milford. Connecticut 06460: 'Virginia Institute of Marine Science, College of William and Mary, P.O. Bo.\ 1346, Gloucester Point, Virginia 23062; Marine Biological Laboratory, 7 MBL St., Woods Hole, Massachusetts 02543: '^Manila's Vineyard Shellfish Group. P.O. Bo.x 1552, Oak Bluffs. Massachusetts 02557 ABSTRACT A haplosporidian parasite, Haplosporidium costale (seaside organism or SSO), is associated with high mortalities of eastern oysters (Crassostrea virginica) in seaside bays of Virginia and Maryland. Its presence in Long Island Sound has been tentatively suggested in several publications for the last 50 y. Positive identification of H. costale and differentiation from another haplosporidian parasite, Haplosporidium nelsoni (MSX), from histological sections is difficult and requires the presence of spores. We detected H. costale spores in 4 out of 5010 [Q.0V7c ) oysters collected from Long Island Sound in 1997-1999. In situ hybridization using an oligonucleotide DNA probe designed to detect small subunit ribosomal DNA from Virginia's H. costale reacted positively with tentative H. cosiale plasmodia in 5 oysters from Long Island Sound. In each case there was a coinfection of H. nelsoni. In Virgmia and Maryland. H. cosiale has historically sporulated in all infected animals in May-June. In Long Island Sound, the rare sporulating cases were detected in October-December, suggesting a different infection cycle. KEY WORDS: Haplosporidium costale. eastern oyster, Crassostrea virginica. in silu hybridization. Long Island Sound INTRODUCTION A haplosporidian parasite. Haplosporidium costale. was iden- tified as the causative agent of seaside organism (SSO) disease, resulting in high mortalities of eastern oysters (Crassostrea vir- ginica) on the Atlantic coast of Virginia (Wood & Andrews 1962). Prevalence, mortality and infection cycle of H. costale have been thoroughly studied in Virginia and Maryland, but information from other geographical locations is scarce. In seaside bays of the Del- marva Peninsula, the first plasmodia can be detected in the epi- thelia of digestive tubules in April-May. Plasmodia rapidly pro- liferate in May and .sporulate synchronously. Peak mortality of the oysters is in May-June, when dying oysters release spores in sea- water to initiate a new infection cycle. New infections remain subpotent until spring of the following year. Mortality of oysters has been 20% to 50% annually in the seaside bays of Virginia (Andrews 1988: Andrews & Castagna 1978). Reports of W. cosiale distribution north of Virginia's Atlantic coast are inconsistent. Several reports suggest the presence of plas- modia resembling H. costale in eastern oysters in Long Island Sound. According to Andrews (1984, 1988), SSO disease ranges from Cape Charles, Virginia, to Maine, but is important only in high-salinity bays (>25%t) from Cape Henlopen, Delaware, to the Virginia capes. He stated that the pathogen is regularly present from New York to Massachusetts, but mortality has not been a serious problem. However, a mortality event in Long Island Sound in 1953 may have been caused by H. costale (Andrews 1988). Sampling locations, dates, prevalences, or possible presence of spores were not mentioned in these reports. Newman (1971) studied 1,337 oysters from New Haven Har- bor. Connecticut, from 1966 to 1967. He found five specimens (0.4%) infected with plasmodia morphologically similar to H. cos- tale. with one of the oysters moribund with heavy infection. No sporulation was detected. Meyers (1981) described haplosporidia- like Plasmodia (4%) in juvenile oysters (n = 68) collected from Oyster Bay, north shore of Long Island. New York, from 1975 to 1976. He did not find similar organisms in adult oysters (/I = 145). No effort was made to distinguish between different haplosporid- ian species because sporulating stages were not present. In addi- tion. Plasmodia morphologically identical to H. costale were ob- served in oysters transplanted from the vicinity of New Haven, Connecticut, to Tomales Bay, California, in 1967-1968 (Katkan- sky & Warner 1970). Six specimens with tentative H. costale infection (four moribund and two living) were reported, one with spores. Total number of oysters studied was not mentioned. Reports of tentative H. costale infections in Long Island Sound reviewed above are based on histological examinations. Reliable diagnosis of H. costale on histological sections is nearly impos- sible when sporulating forms are not present. H. costale plasmodia can be easily misdiagnosed as Haplosporidium nelsoni (MSX). another haplosporidian oyster parasite enzootic to the area (for review, see Ford & Tripp 1996). During routine monitoring for oyster diseases in Connecticut, we found several oysters with spores and plasmodia consistent with descriptions of H. costale. We used a DNA probe, designed to detect H. costale from Virginia as the in situ hybridization (ISH) probe for these specimens to verify the presence of H. costale in Long Island Sound. MATERIAL AND METHODS The State of Connecticut. Department of Agriculture. Bureau of Aquaculture routinely receives oyster samples for histological diagnosis from Connecticut's commercial oyster companies. Most of the seed originates from natural seed beds, though some hatch- ery-raised seed is also used. Every oyster is transplanted an aver- age of four times before it is marketed, which exposes it to possible parasitic infections in several different sites. Seventeen oyster samples were studied in 1997. 63 in 1998. and 87 in 1999. Each sample consisted of 30 oysters, for a total of 5,010 oysters. Samples represented the entire Connecticut shoreline and the north shore of Long Island. New York. Of the 167 samples, 20 origi- nated from New York. Fifty-six of the samples originated from oyster nursery systems (5 from upwellers, 51 from suspended cul- tures). 30 from off bottom cultures, and 81 from natural oyster beds. Tissues were fixed in Davidson's fixative in 20%f artificial seawater. Six-micrometer-thick paraffin sections were stained with hematoxylin-eosin. Samples with H. costale or H. nelsoni pre- 113 114 SUNILA ET AL. spores and spores were stained also with Ziehl and Hanis' hema- toxylin according to Farley ( 1965). The ISH procedure was conducted with 27 oysters, two with presumed H. costale spores and 25 oysters with haplosporidian Plasmodia. Specimens were selected for ISH as follows: (1 ) speci- mens with haplosporidian plasmodia from samples in which H. costale spores were detected; (2) specimens with small plasmodia with central nucleoli, morphology that is considered to be charac- teristic for H. costale; and (3) specimens with plasmodia in the stomach, intestine, or digestive tubule epithelia, locations that are considered to be characteristic for H. costale. For the ISH proce- dure, 6-p.m-thick sections from these oysters were deparaffini/ed and ISH was performed on consecutive .sections as previously described (Stokes & Burreson 1995: Stokes & Burreson 2001). Two commercially synthesized digoxigenin-labeled oligonucle- otide DNA probes were used: a 22-base oligonucleotide (SS01318) specific for H. costale (Stokes & Burreson 2001) and a 2 1 -base oligonucleotide (MSXI347) specific for H. nelsoni (Stokes & Burreson 1995). A negative control was performed by substituting DNA probes with distilled water during hybridiza- tion. RESULTS HaplosporicUuiii costale was detected in three locations on Connecticut's shoreline: Norwalk. Branford. and Clinton (Fit!. I). H. costale was diagnosed in seven different specimens either by the presence of spores or by a positive ISH result. There were 17 oysters with haplosporidian spores among the 5,010 oysters stud- ied (0.3%). Four oysters had a mixture of H. costale and H. nelsoni spores (0.08%). The remaining 13 contained only H. nelsoni spores. Locations, dates, and seed origin of oysters with spores are listed in Table I. H. costale and H. nelsoni spores ditfered m size, form, and location in oyster tissues. H. costale spores (3 x 4 |j.m). the sporo- plasm of which stained bright red with acid-fast stain, were de- tected throughout the connective tissue. Prespores. which did not retain acid-fast stain, occurred inside sporocysts throughout the tissues. H. costale spores were found between vesicular connective tissue cells surrounding the digestive diverticula (Fig. 2A), in con- nective tissue of the gills, in the adductor muscle, in the heart, between neurosecretory cells in the ganglia, and between kidney tubules. On rare occasions, H. costale spores were detected inside digestive tubule or digestive duct cells, in the lumens of the di- gestive tubules and intestine, in epithelial cells of the intestine, or in the follicles. H. nelsoni spores (5x7 (j.m), which also stained bright red with acid-fast stain, were in most cases restricted to digestive epithelial cells (Fig. 2B). However, during intense sporulation in four speci- mens there was an overspill to digestive duct cells and the con- nective tissue surrouiidinsi the dicestive tubules. In cases with 73 eft' CONNECTICUT NEW YORK ATLANTIC OCEAN 20 Miles Figure. 1. A map of sampling stations with a positive diagnosis for H. costale in Long Island Sound. Haplosporidium costale in Oysters 115 TABLE 1. Haplosporidium costale (SSO) and Haplosporidium nelsoni (MSX) spores in eastern oysters in Long Island Sound. Sampling Sampling Sampling Shell Length Spore Seed Date Site Location (mm) Type Origin 09.28.97 Guilford, East River 41°16.05'N:72°39.62'W 104 MSX Natural 10.14.97 Clinton. Cedar Island mud Hat 4ri5.97'N:72°32.00'W 7S MSX Natural 10.14.97 Clinton. Cedar Island mud tlat 41°15.97'N:72°32.00'W 101 MSX Natural 12.17.97 Norwalk 1131 4r02.42'N:73''25.25'W 75 MSX Natural 12.18.97 Norwalk 162 41''03.36'N:73°25.I2'W 69 MSX Natural 01.08.98 West Haven Lot IB 41°15.47'N:72°55.26'W 42 MSX Hatchery raised 09.18.98 Stratford 709 41°07.4rN:73°09.13'W 70 MSX Natural 09.22.98 Milford 612 4I°1I.10'N:73''00.05'W 73 MSX Natural 10.17.98 Branford 3 1 6 4ri5.42'N:72°44.40'W 7.'i SSO, MSX Natural 10.17.98 Brantord 168 41°15.77'N:72°45.9.VW 42 MSX Natural 10.17.98 Branlord 179 41°15.73'N:72"45.63'W 64 SSO, MSX Natural 11.21.98 West Haven Lot IB 4ri5.47'N:72°55.26'W 48 MSX Hatchery raised 12.03.98 Clinton. Cedar Island Maruia 41''16.05'N:72°32.I0'W 24 SSO, MSX Hatchery raised 09.22.99 Clinton. Cedar Island Marina 4ri6.05'N:72°32.10'W 76 MSX Hatchery raised 09.22.99 Clinton. Cedar Island Marina 41°16.05'N:72°32.10'W 33 MSX Hatchery raised 09.22.99 Clinton. Cedar Island Marina 41°16.05'N:72°32.10'W 26 MSX Hatchery raised 11.16.99 Clinton. Cedar Island Marina 4r'16.05'N:72''32.10'W 29 SSO, MSX Hatchery raised extremely heavy sporulation. phagocytosed H. nelsoni spores were observed in the vascular system hetnolymph sitiuses and veins, in the mantle lobes and the gills, and being carried through stomach or mantle epithelia via diapedesis. Spores that occurred in the connective tissue were usually surrounded by aggregates of granu- lar hemocytes. H. nelsoni spores were detected inside the lumens of digestive tubules, digestive ducts, and the intestine. H. nelsoni prespores, which did not retain stain in acid-fast reaction, were detected exclusively inside digestive cells. It was as probable that spores would be found in oysters origi- nating from natural set as in oysters originating from hatchery- raised seed (x" = 0.68 [not significant] [NS]). Oysters with spores were detected in tnost areas of Connecticut's shoreline. No sporu- lating specimens were detected in the north shore of Long Island, New York. The size of an oyster with spores did not differ sig- nificantly from the average size of the sample from which it was taken {t = 0.08 [N.S]). The size of an oyster with spores also did not differ significantly from the average sizes of all oysters sampled for this study (/ = 0.97 [NS]). ISH results with H. costale and H. nelsoni DNA probes are summarized in Table 2. Five specimens had mixed infections, the rest were infected only with H. nelsoni. Two specimens with mixed infections had both types of spores present; three had only Plasmodia. (An additional two specimens were diagnosed positive for both H. costale and H. nelsoni on the basis of the presence of spores [Table 1]. with seven positive specimens altogether.) The two specimens with both spore types (Branford lots 179 and 316) that were subjected to ISH had prominent H. costale infections. Approximately 90% of the plasmodia hybridized with the H. cos- tale probe and 10% hybridized with the H. nelsoni probe in these samples (Fig. 3). Two other specimens with mixed infections (Clinton and Norwalk 1131) had very light H. costale infections (Fig. 4). More than 99% of the plasmodia hybridized with the H. nelsoni probe and the very rare H. costale plasmodia would not have been detected without the probe. H. costale plasmodia in the Figure 2. Sporulation of H. costale and H. nelsoni in Long Island Sound. (A) Digestive diverticulum of an oyster filled viith acid-fast H. costale spores (Ziehl and Harris' hematoxylin). .Scale bar 100 pm. (B) Cosporulation of H. costale and H. nelsoni. Small H. costale spores occur in the connective tissue surrounding digestive tubules; larger H. nelsoni spores occur inside digestive tubule. This is the same specimen as in item (A) (Ziehl and Harris" hematoxylin). Scale bar 20 (im. SUNILA ET AL. tablp: 2. ISH of Haplosporidmm coslale (SSO) and Haplosporidium nelsoni (MSX) in eastern oysters in Long Island Sound. Sampling Date Sampling Site ISH with SSO Sampling Slull Length and MSX Prohes Location (mm) (Positive Result I Location of Plasmodia 09.16.97 Norwalk Natural Bed 10.14.97 Clinton, Cedar island mud flat 12.17.97 Norwalk 11.11 12.17.97 Norwalk 162 4I'04.85'N:73°23.55'W 4I°15.97'N:72'32.00'W 41°02.42'N:73°25.25'W 41 03.36'N;73 = 25.I2'W 10.17.98 Branford316 10.17.98 Branford 316 10.17.98 Branford 168 10.17.98 Branford 168 10.17.98 Branford 168 10.17.98 Branford 179 10.17.98 Branford 179 10.17.98 Branford 179 11.10.98 Stony Brook Harbor, NY 11.10.98 Stony Brook Harbor, NY 11.10.98 Stony Brook Harbor. NY 11.12.98 Oyster Bay. H. NY 11.12.98 Oyster Bay. H, NY 11.12.98 Oyster Bay, W. NY 11.12.98 Oyster Bay, W, NY 12.01.98 Milford 30.«i 12.01.98 Milford 305 12.15.98 Northport Bay. NY 1 1.17.99 Clinton, Cedar Island Marina 11.17.99 Clinton, Cedar Island Marina 12.07.99 Oyster Bay, J. NY 12.07.99 Oyster Bay, J, NY 80 40°54.3O'N;73 10.70'W 40'54..30'N:73' 10.70"W 40°54.30'N:73° 10.70' W 40°54.33'N:73°30.22"W 40"54.33'N:73''30.22'W 40°52..59'N:73°32.irW 40°52..59'N:73"32.irW 4PI1.I5'N:7,3°04.80"W 4ril.l5'N:73°04.80'W 40°55.87'N;73°22.87'W 4r 16.05'N;72 32.10'W 4ri6.05'N:72°32.10'W 4O°53..5O'N:73°30.23'W 40"53.50'N:73"30.23'W 68 110 MSX 113 SSO. MSX SSO, MSX 66 SSO, MSX 12.17.97 Norwalk Manresa Island 4r04.42'N:73"24.55'W 70 MSX 41°I5.42'N:72'M4.4(1'W 68 MSX 41°15.42'N:72=44.40'W 75 SSO. MSX 4ri5.77'N:72'=45.95'W 41 MSX 41°15.77'N:72°45.95'W 41 MSX 41°15.77'N;72°45.95'W 6(1 MSX 41°15.73'N:72"45.63'W 62 MSX 41°15.73'N:72°45.63'W 70 MSX 41 = I5.73'N:72°45.63'W 64 SSO. MSX 123 MSX 127 MSX 107 MSX 76 MSX 87 MSX 92 MSX 84 MSX 78 MSX 24 MSX 92 MSX 67 MSX 65 MSX 9(1 MSX MSX Plasmodia throughout the tissues MSX plasinodia (>99'7f ) throughout the tissues, rare SSO plasmodia KlVr ) in the gills MSX Plasmodia (>99'/rl throughout the tissues, rare SSO plasmodia (' • •.•-' • » » • %.. • •• ».^' ^. • * •p • » • • ^ V|- • • • c • ': .♦ • ► t * .. ; •• r • . - V « Figure 4. Serial sections of an oyster with mixed haplosporidian plas- modial infection. (.A) Hematoxylin-eosin stain. (B) ISH with H. costale DNA probe detecting rare plasmodia in the gill. (Cl ISH with H. nelsoni DNA probe of the same area shown in item (B). Scale bars 100 (im. did not differ from those described in previous publications, H. costale in Long Island Sound appears to have a different patho- genesis than in the south. Gross signs as defined by Andrews (1988) (emaciation, failure of new shell growth in spring, high 118 SUNILA ET AL. epizootic mortality mid-May-iiiid-June. and discoloration of gap- ers by spores) did not apply to our samples. In Virginia, infected oysters sporulate synchronously in May-July, presenting an obvi- ous, easily diagnosed stage. All plasmodia develop into sporonts. and oysters die promptly after sporulation (Andrews 1984). H. costale is considered to be a well-adapted parasite that infects new oysters via spores that are released during mortality season (An- drews 1982). Spores are usually detected in moribund oysters and infection causes 20%-50% yearly mortalities (Andrews 1988). Cosporuiation with H. iielsoni was reported previously by Couch ( 1967) in oysters collected from Chincoteague Bay, Virginia. The six cases with both spore types were dead and dying oysters col- lected in May and June during the characteristic H. costale sporu- lation time for oysters in Virginia (Couch 1967). SSO disease in Long Island Sound differed from the above description in several ways. First, plasmodia and sporulation in the present material were found in October to December. During this time period, SSO disease in Virginia is subpatent and no plasmodia are detected before spring (Andrews 1988). However, in a recent paper describing H. costale probes also used in the present publi- cation, Stokes and Bun-eson (2001) reported the presence of a positive ISH to H. costale plasmodia in an oyster sampled in October 1994 in Virginia. In the pre.sent paper, spores were de- tected in live specimens with no evidence of a synchronous mor- tality event. Spores were very rare, and infection always occurred as a coinfection with H. nelsoiii. Because H. costale plasmodia were also detected in the samples, sporulation may occur infre- quently. Rare sporulation such as presented in this report cannot sustain a widespread infection. Actual prevalence of H. costale in the area is not known based on the results of the present study, but the rare sporulation and the difficulty of finding positive speci- mens with ISH suggest a low prevalence. Possible mortality associated with H. costale is impossible to estimate because of an H. iielsoiii epizootic that occurred in Long Island Sound in 1997-1998 (Sunila et al. 1999). Oyster production in Connecticut decreased from more than 300,000 bushels in 1996 to 170,000 in 1999, reflecting high MSX-associated mortalities. According to Andrews (1984), H. iielsoni kills oysters much quicker than H. costale, and depresses manifestation of SSO dis- ease during MSX epizootics. H. costale is usually restricted to high-.salinity bays with sa- linities >30%c. Its lower limit is 25%f and salinities <20%c appear to cause disease regression (Andrews 1979). It is possible that salinity in Long Island Sound's oyster beds is not high enough to sustain full epizootic H. costale activity. In Branford lots (Table 1 ). salinity varies between 26%r and 21%o: in the Clinton Cedar Island Marina (in the mouth of Hammonassett River), salinity varies from 139ff to 28'/ff. Oysters in the area are transplanted four times before they are marketed. This exposes them to even lower salinities, which may have provided a control for the disease. Classically, the diagnosis of H costale relies on the presence of sporulating stages and the site of initial infection, which for H. costale is the epithelium of the digestive system and for H. nelsoni is the gill epithelium. In addition, history of the sainpling area relating to past H. costale or H. nelsoni infections directs the diagnosis. In the present report, we were able to diagnose H. costale in a new geographic area with a deviating sporulation time by using DNA probes (Stokes & Burreson 1995; Stokes & Bur- reson 2001). Further research to study the infection cycle, preva- lences, and possible association with mortalities is under way. ACKNOWLEDGMENTS This study was funded in part by Sea Grant No. NA86RG0075; VIMS contribution number 2461. LITERATURE CITED Andrews, J. D. 1979. Oyster diseases in Chesapeake Bay. Mar. Fish. Rev. 41:45-53. Andrews, J. D. 1982. Epizootiology of late summer and fall infections of oysters by Haplosporidium nelsoni. and comparison to annual life cycle of Haplosporiditini costalis. a typical haplospiiridan. / Shellfisli Res. 2:15-23. Andrews, J. D. 1984. Epizootiology of diseases of oysters (Crussostrea virginica). and parasites of associated organisms in eastern North America. Helgoliinder Meeresunters. 37:149-166. Andrews, J. D. 1988. Haplosporidium costale disease of oysters. In: C. J. Sindemiann & D. V. Lightner, editors. Disease diagnosis and control in North American marine aquaculture. Amsterdam: Elsevier, pp. 296- 299. Andrews, J. D. & M. Castagna. 1978. Epizootiology of Minchinia ciisltdis in susceptible oysters in seaside bays of Virginia's eastern shore. 1959- 1976. J. Invert. Pathol. 32:124-138. Couch, J. 1967. Concurrent haplosporidian infections of the oyster. Cras- sostrea virginica (Gmelin). J. Parasitol. 53:248-253. Couch, J., C. A. Farley & A. Rosenfield. 1966. Sporulation of Minchinia nelsoni (Haplosporida, Haplosporidiidae) in Crassostrcci virginira (Gmelin). Science 23:1529-153L Farley, C. A. 1965. Acid-fast staining of haplosporidian spores in relation to oyster pathology. J. Invert. Pathol. 7:144—147. Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In: V. S. Kennedy, R. 1. E. Newell & A. F. Eble, editors. The eastern oyster. Crassostrea virginica. College Park, MD: Maryland Sea Grant Book. pp. 581-660. Katkunsky. S. C. & R. W. Warner. 197(1. The occurrence of a haplospo- ridian in Tomales Bay, California. J. Invert Pathol. 16:144. Meyers, T. R. 1981. Endemic diseases of cultured shellfish of Long Island. New York: Adult and juvenile American oysters (Crassostrea vir- ginica) and hard clams {Mercenaria mercenaria). Aqndcidtnre 22:305- 330. Newman. M. W. 1971. A parasite and disease survey of Connecticut oys- ters. Proc. Natl. Shellfish. Assoc. 61:59-63. Perkms, F. O. 1969. Electron microscope studies of sporulation in the oyster pathogen, Minchinia costalis (Sporozoa: Haplosporida). J. Para- sitol. 55:897-920. Rosenfield. A.. L. Buchanan & G. B. Chapman. 1969. Comparison of the fine structure of spores of three species of Minchinia (Haplosporida, Haplosporidiidae). / Parasitol. 55:921-941. Stokes. N. A. & E. M. Burreson. 1995. A sensitive and specific DNA probe for the oyster pathogen Haplosporidium nelsoni. J. Eukaryot. Micro- biol. 42:350-357. Stokes, N. A. & E. M. Burreson. 2001. Differential diagnosis of mixed Haplosporidium costale and Haplospinidium nelsoni infections in the eastern oyster, Crassostrea virginica. using DNA probes. / Shellfish. Res. 20:207-213. Sunila. I.. J. Karolus & J. Volk. 1999. A new epizootic of Haplosporidium nelsoni (MS.X). a haplosporidian oyster parasite, in Long Island Sound, Connecticut. / Shellfish Res. 18:169-174. Wood, J. L. & J. D. Andrews. 1962. Haplosporidium costale (Sporozoa) associated with a disease of Virginia oysters. Science 136:710-71 1. Journal of Slwlirtsh Research. Vol. 21, No. 1. 119-125. 2002. INFECTION INTENSITY, PREVALENCE, AND HISTOPATHOLOGY OF PERKINSUS SP. IN THE MANILA CLAM, RUDITAPES PHILIPPINARUM, IN ISAHAYA BAY, JAPAN KWANG-SIK CHOI,'* KYUNG-IL PARK.' KI-WAN LEE,' AND KAZUMI MATSUOKA^ ' File ill ty of Applied Marine Science. Cliejii National University, 1 Ara 1-Dong, Jejii City Jeju-Do 690-756. Korea: 'Laboratory of Coastal Environmental Sciences, Faculty of Fisheries, Nagasaki Universit}-, 1-14, Biinkyo-machi. Nagasaki 852-8521 . Japan ABSTRACT Infection intensity, prevalence of infection, and pathologic features of Perkinsus parasitism among Manila clams inhabiting in Isahaya Bay. Japan, were investigated. Ray's fluid thioglycollate medium technique followed by Choi's 2 M NaOH digestion assay was applied to determine the infection intensity and prevalence. The infected tissues were also microscopically examined from histological preparations. The prevalence of infection was STVr in clams sampled in February 2001. and the mean infection intensity was .^."i 1,603 Perkinsus cells per clam or 225.701 Perkinsus cells/g of tissue. Perkinsus sp. was abundantly distributed in the gill and vi.sceral mass, whereas it was rare in the adductor muscles and siphons. The total number of Perkinsus in the clams was linearly correlated with the number of Perkinsus cells in the gill tissues (r = 0.908), suggesting that gill could be a target tissue for efficient diagnosis of Perkinsus infection. Heavily infected clams exhibited white nodules on the surface of the mantle as a consequence of inflammatory response to Perkinsus infection. Numerous trophozoites were observed in the connective tissue around the gonads and gill filaments, suggesting that heavy infection with Perkinsus may exert potential deleterious effects on growth and reproduction by interfering with the reproductive maturation and filtration activities of the clams. KEY WORDS: Perkinsus. Ruiiitapes philippinarunt. infection intensity, histopathology, Isahaya Bay, Japan INTRODUCTION The protozoan parasite Perkin.siis sp. (Apicomplexa, Perkinsea) has been known to cause mass mortalities worldwide in commer- cially important shellfish, including oysters, scallops, clams, and abalones (Andrews & Hewatt 1957; Mackin 1962; Lester & Davis 1981; Navas et ai. 1992; Blackbourn et al. 1998; Canestri-Trotti et al. 2000; Park & Choi 2001; Liang et al. 20011. In particular. P. atlaiuicus has been associated with mass mortalities of the venerid clams of the genus Ruditapes (i.e.. Tapes or Venenipis) inhabiting the Mediterranean and Atlantic coasts (Da Ros & Canzonier 1985. Chagot et al. 1987, Sagrista et al. 1996). Perkinsus parasitism in the Manila clam ("short-necked clam"), R. philippinanim. was also recently confirmed in Japanese (Hamaguchi et al. 1998, Maeno et al. 1999) and Korean waters (Choi & Park 1997; Park & Choi 2001; Lee at al. 2001). Perkinsus sp. distributed in Korea is be- lieved to be responsible for the mass mortality of Manila clams. This has occurred yearly, in late summer, since the early 1990s. Park and Choi (20011 postulated that the decline in Manila clam landings for the previous decade in Korea could be caused by Perkinsus-associated mortalities occurring in every late summer. As many other studies have reported. Perkinsus infection is asso- ciated with high salinity, temperature, and density of clam popu- lations (Andrews & Hewatt 1957; Soniat 1996; Burreson & Ragone Calvo 1996; Cigarria et al, 1997). Perkinsus-hke microorganisms have been discovered in Manila clams distributed in Japan (Hamaguchi et al. 1998; Maeno et al. 1999) and China (Liang et al. 2001). Hamaguchi et al. (1998) reported on the occurrence of Perkinsus in the clams collected from Kumamoto and Hiroshima, in southern Japan. Comparison of the DNA sequence of Perkinsus with P. atlanticus and P. olseni indicated that the species of Perkinsus found in Kumamoto and Hiroshima. Japan, is taxonomically very close to P. atlanticus and P. olseni reported from Portugal and Australia (Hamaguchi el al. 1998). Manila clams are abundant in Isahaya Bay, southern Ky- *CorTesponding author. Fax: 82-64-756-3493; E-mail: skchoi@cheju.cheju.ac.kr ushu, Japan, where tidal flats are well developed and the clams are commercially cultured. Ishii et al. (2001) reported that the clam population in Ariake Sound has been declining significantly since 1987 due to overfishing, pollution, and predators. Because the presence of Perkinsus in clam populations inhabiting Ariake Sound was confirmed by Hamaguchi et al. (1998), detrimental effects of Perkinsus parasitism on clam growth, as well as on annual landings, cannot be Riled out in Isahaya Bay located on the west of Ariake Sound. However, infection intensity and prevalence of Perkinsus in the bay has not been reported previously. Infection intensity and prevalence of Perkinsus parasitism on Manila clams was investigated from a clam population distributed in Isahaya Bay in the present study. This paper reports diagnosis, histopathologic features of the infected clams, infection intensity, and prevalence of Perkinsus parasitism in the clams collected in February 2001. MATERIALS AND METHODS A total of 191 clams were collected from Isahaya Bay, Kyushu, Japan, in February 2001 (Fig. 1). In the laboratory, shell length (SL), width (SW), thickness (ST), and tissue wet weight (TWT) of individual clams were recorded. Condition index (CI) was then calculated for evaluating fatness of the clams as CI = [TWT/(ST X SW X SD] x 1,000 For evaluating Perkinsus infection, the clams were placed in two groups: one for histopathological examination and the other for measuring total body burden, which is the infection intensity of each clam as a total number of Perkinsus in a clam or number of Perkinsus/g tissue. For histopathology. a longitudinal section was made in the middle of the body, which included gills, digestive glands, gonads, mantle, and foot. A 5-|xm thin section was cut for each clam after dehydration. The sections were then stained with Harris' hematoxylin and eosin Y. For measuring total body bur- den, the whole fiesh of individual clams was immersed in fluid thioglycollate medium (FTMl. fortified with nystatin and chlor- amphenicol to prevent bacterial activity (Ray 1966). and placed in 119 120 Choi et al. Np^'^Fukuoka | • VD DsahayaBayX 130" 131° Figure 1. Location of the sampling site, Isahaya Bay, Kyushu, Japan. a dark area for a week. After immersion, the tissues were digested in 2 M NaOH, and the number of Perkinsiis cells was counted using a hemocytometer according to Choi et al. (1989). Total body burden was then standardized as the number of Perkinsiis/g tissue. To determine the distribution pattern of Perkinsiis per clam, the gills, mantle, adductor muscle, and body containing the visceral mass were excised separately from each clain and immersed in- dependently in FTM. After 1 wk of immersion, the number of Perkinsus cells in the tissue was measured as described above (Choi et al. 1989). The infection intensity was standardized and expressed as the number of Perkinsiis cells/g tissue. RESULTS Histopathological Observation of Perkinsus sp. Numerous trophozoites were observed in the clams collected from Isahaya Bay. Eccentric vacuoles, nuclei, and nucleoli were observed from histological sections of the infected tissues (Fig. 2A). The diameter of the trophozoites, estimated microscopically, ranged from 7.73 to 15.80 (j.m. with a mean of 10.98 p.m. Diam- eters of the nuclei varied from 4.20 to 6.59 )xin, with a mean of 5.44 jjim, whereas the diameters of the nucleoli varied from 1.90 to 2.67 ixm, with a mean of 2.27 |xm. Most trophozoites in the gills and mantle formed different sizes of clusters (Fig. 2A and B). Perkinsus was predominantly found in gill filaments, mantles, and digestive tubules, although a few of the trophozoites were observed in the foot (Fig. 2B, C, and D). Heavy infection with Perkinsus in the gill lamellae resulted in swollen connective tissue with severe hemocyte infiltration (Fig. 2B). An inflammatory re- sponse to the parasite was also observed in heavily infected clams in the form of nodules on the mantle surface (Fig. 2C). Those nodules appeared as white spots on the mantle surface and could be observed even with the naked eye. Numerous Perkinsus tro- phozoites were also observed around the digestive glands, indicat- ing that Perkinsus could inhibit the digestive activity of the clams in the visceral mass (Fig. 2D). Some clams exhibited mature eggs or sperm even in February because of the influence of the warm Kuroshio current in the bay. Perkinsus was also observed in the connective tissues of female as well as male gonads (Fig. 2E and F), indicating that Perkinsus infection in the Manila clam might disturb the reproductive processes. Sporocysts of a cercaria-like organism were also observed in the female gonad, although the prevalence was much lower than that of Perkinsus (Fig. 2H); only 2% of total clams investigated were infected. Prevalence and Infection Intensity of Perkinsus per Clam and in Different Types of Tissues The results of this Perkinsus infection survey conducted from a clam population in Isahaya Bay are summarized in Table I. A total of 191 clams with a mean SL of 31.0 mm and a mean TWT of 1.566 g were analyzed in the study. Prevalence, the percentage of infection in the clams investigated, was 57.4%. Total body burden, in terms of the total number of Perkinsus cells in individual clams, varied from 0 to 2,609,375, with a mean of 351,603. Infection intensity, as number of Perkinsus cells/g tissue, varied from 0 to 1,817,196, with a mean of 225,701 (Table I). No obvious corre- lation was observed between the infection intensity and size of the clams, as well as the CI. Table 2 shows prevalence and infection intensity in various types of clam tissues. Among the four types of tissues examined, the gills showed the highest prevalence and infection intensity, with 85.7I'7<- and 1.019.817 cells/g gill tissue, respectively. Preva- lence of infection in the visceral mass was as high as that observed in the gills, whereas the infection intensity as the number of Per- kinsiis/g tissue was much lower than the value observed in the gills. Infection intensity and prevalence was much lower in the adductor muscle and siphons compared to that in the gills and visceral mass. A positive correlation was observed between num- ber of Perkinsus cells/g gill tissue and the total body burden (Fig. 3, /- = 0.908). The number of Perkinsus cells in the visceral mass was also highly coirelated with the total body burden (;-^ = 0.893), suggesting that the gill and visceral mass are the main target tis- sues for Perkinsus infection in this species. Quantitative evaluation of Perkinsus infection among the various tissue types indicated that Perkinsus sp. is not evenly distributed in the clams; rather, it is concentrated in the gills and visceral mass. DISCUSSION Histopathological Features of Perkinsus Although histology is not widely used in the diagnosis of Per- kinsus infection, the technique provides valuable information on host-parasite cellular interactions (Hine & Thorne 2000; Diggles & Hine 2001; Lee et al. 2001). In the present study, pathogenicity of Perkinsus sp. was visually examined from histological prepara- tions of the infected clams. Figure 2A shows typical Perkinsus trophozoites displaying a Perkinsns-fipecifk "ring" structure (i.e., vacuole and nucleolus in a nucleus, Azevedo 1989; Azevedo et al. 1990; Auzoux-Bordenave et al. 1995: Perkins 1996; Park & Choi 2001). Trophozoite diameter measured in the present study was somewhat comparable to the size reported by Hamaguchi et al. (1998) and Maeno el al. (1999) in Japan. Trophozoite diameter measured in our study varied from 7.73 to 15.80 p.m, with a mean of 10.98 |xm. Hamaguchi et al. (1998) reported 5.3-32.5 jjim, with a mean of 14.8 |j.m, as diameter of trophozoites in Manila clams, and Maeno et al. (1999) reported 5.7-11.4 |a.m. In contrast, tro- phozoites of Perkinsus sp. found in R. plulippinanini on the north- em coast of China varied from 2 to 10 \i.m (Liang et al. 2001 1. The size of trophozoites estimated in our study is also similar to the size of P. olseni [which is taxonomically very close to Perkinsus sp. found in Japan (Hamaguchi et al, I998)|, discovered in the Australian black-ribbed abalone, Halioiis riilvci (Lester & Davis 1981). Some heavily infected clams exhibited numerous clusters of trophozoites on their gill plicae and digestive tubules with severe hemocytic infiltration (Fig. 2B and D). Such a heavy infection in Perkinsus Infection in Isahaya Bay, Japan 121 i^ai^:V/^:-'- '^^ « " !((■ Figure 2. Histopathological features of Perkinsus infection. (A) Mature trophozoites containing vacuole (V). Nucleolus (NL) occurs in nucleus (NS). l.OOOx, scale bar = 5 nm. (B) Severe inflammatory reaction (asterisk) occurs around trophozoites in gill tissues. lOOx, scale bar = 100 jim. (C) Early stage of nodule formation. Infiltration of hemocytes (asterisks) around trophozoites (arrows) results in the swollen connective tissue of the foot. 20(»x. scale bar = 20 pm. (I)) Trophozoites in connective tissues of digestive glands. 400x. scale bar = 40 pm. (E) Trophozoites in connective tissues of female gonad. \ oung oocytes (Ol are seen around the capsulated trophozoites. 400x, scale bar = 500 urn. (F) Grouped trophozoites in connective tissues of male gonad. Concentrated hemocytes of the host enclose the trophozoites forming a capsule around spermatogonia (SP). 400x. scale bar = 20 nm. (G) Inllanimation of host hemocytes (asterisk) in the mantle tissues. lOOx, bar = 100 fim. (H) Sporocysts containing germ balls (GB). No inflammation observed. 200x. bar = 40 pm. 122 Choi et al. TABLE 1. Survey results of Perkiiisus infection in the clams distributed on Isahaya Bay. .V Average SD Min Max SH (mml 191 21.4 2.1 15.6 25.9 SL (mml 191 31.0 3.1 22.3 39.9 TWT (g) 191 1.566 0.521 0.537 2.769 CI 89 0.167 0.028 0.059 0.2-32 Total PerlcinsLis (cells/clam) 89 351.603 549.046 0 2.609.375 Unit Perkinsus (cells/g twt) 89 225.701 365.002 0 1.817,196 Prevalence 150 57'7r SH = shell height; SL = shell length; TWT = total tissue wet weight; CI = condition index; SD = standard deviation; Min = minimum; Max = maximum. gill tissues may lower filtration efficiency and, in turn, cause re- tarded growth, although the effects of Perkinsiis infection on fil- tration activity of the clams has not been experimentally proven. Infestation of Perkinsus in digestive tubules would cause digestive tubule atrophy and exert deleterious effects on the food digestion of the clams, as reported by Lee et al. (2001 ). Clams more heavily infected with Perkinsus exhibited white nodules on their mantle surfaces as well as gills, as was reported in other studies (Azevedo 1989; Navas et al. 1992; Montes et al. 1996; Almeida et al. 1999; Lee et al. 2001; Park & Choi 2001). A cross-section of the nodules revealed that they are the result of hemocytic encapsulations of trophozoites and massive hemocytic infiltration around the mantle tissues, evoking tissue inflammation (Fig. 2C). Several studies also have reported that Perkinsus secretes extracellular enzymes that perturb the host's immune system, which, in turn, allows the host animal to become more susceptible to other pathogens (Gar- reis et al. 1996; La Peyre et al. 1996; Faisal et al. 1999; Ordas et al. 2000). Perkinsus was also observed among the connective tissues of female as well as male gonads (Fig. 2E and F), suggesting that Perkinsus infection also affects the reproduction of the clams in some way. Several studies have suggested that Perkinsus could ( I ) slow the gonad development process, or (2) reduce the reproduc- tive output of the host animals by consuming net energy produc- tion needed for gamete production (White et al. 1988; Wilson et al. 1988; Choi et al. 1993; Choi et al. 1994). In C. virginica. no obvious correlation was observed between estimated fecundity and P. mariniis infection (Choi et al. 1993). In contrast, the rate of egg protein synthesis was found to be slower in C. virginica heavily infected with P. marinus, whereas the rate was faster in oysters with relatively low infection (Choi et al. 1994). This suggests that the main effect of Perkinsus on host animal reproduction could be retardation of gonadal maturation rather than reduced gamete pro- duction. Reduced fecundity and impeded gonadal maturation were observed in Manila clams heavily infected with Perkinsus sp. in Korea (Park & Choi, in preparation). *" Infection Intensity and Prevalence per Clam and in Various Types of Tissues Numerous methods have been applied in the examination of Perkinsus parasitism since the first report on the occurrence of P. marinus in the American oyster, Crassoslrea virginica (Mackin et al. 1950). Perkinsus infection has been diagnosed using histology (Perkins & Menzel 1966; Azevedo el al. 1990; Navas et al. 1992; Sagrista et al. 1995; Sagristaet al. 1996; Montes et al. 1996; Bower et al. 1998; Mine & Thome 2000), FTM assay (Ray 1952; Ray 1966; Choi et al. 1989; Bushek et al. 1994; Rodriguez & Navas 1995; Fisher & Oliver 1996; Ford 1996; Almeida et al. 1999), immunological probes using Perkinsus-Hpecif\c antibodies (Choi et al. 1991; Dungan & Roberson 1993; Maeno et al. 1999), and polymerase chain reaction (PCR) techniques (Marsh et al. 1995; Hamaguchi et al. 1998; Robledo et al. 1998; Figueras et al. 2000; Coss et al. 2001 ). Among these methods. FTM assay has been the most widely and frequently used in all types of Perkinsus diagno- sis (Lester & Davis 1981; Azevedo 1989; Rodriguez & Navas 1995; Choi & Park 1997; Cigarria et al. 1997; Almeida et al. 1999; Liang et al. 2001 ), although FTM assay was initially designed for the detection of P. marinus (Ray 1953; Ray 1966). In the FTM assay, suspected tissues are immersed in 10 to 15 mL of FTM fortified with antibiotics for 1 or 2 wk. After immersion, hypno- spores of Perkinsus developed in FTM and stained dark blue or brown with LugoFs iodine and were readily identifiable under a light microscope. The number of Perkinsus spores in FTM-assayed tissues can be accessed after digesting the tissues with 2 M NaOH. according to Choi el al. (1989). FTM assay combined with 2 M NaOH digestion has been successfully used in the quantification of P. marinus (Choi et al. 1989; Bushek et al. 1994; Fisher & Oliver 1996), as well as other Perkinsus species (Rodriguez & Navas 1995; Choi & Park 1997; Park 1999; Liang et al. 2001; Park & Choi 2001). Infection intensity and prevalence of Perkinsus sp. in Manila clams in Isahaya Bay. Japan, is first reported in this study. Ray"s FTM technique combined with Choi's NaOH digestion technique, which has been used in many other studies, was successfully ap- plied in the quantification of Perkinsus in this study. Prevalence of infection and mean infection intensity of Perkinsus in the clams collected in February 2001 was 579c and 225,701 spores per gram tissue, respectively (Table I). Hamaguchi et al. (1998) also re- ported the prevalence of Perkinsus in R. philippinarum collected from Kumamoto. east coast of Ariake Sound, and Hiroshima. The prevalence measured from Kumamoto was 87.5% in commercial clam beds and 56.3% in natural clam beds. The prevalence values reported by Hamaguchi et al. (1998) are similar to those estimated from Isahaya Bay. The prevalence observed in Hiroshima. 93.8% in commercial beds and 84.4% in natural habitats, was somewhat higher than the prevalence measured in Kumamoto and Isahaya Bay. No data on the infection intensity of Perkinsus in the Manila TABLE 2. Prevalence and average infection intensity of Perkinsus sp. among various tissues of R. philippinarum in = 35). Average Infection Intensity Tissue Prevalence {Perkinsus Cells/g Tissue Types C/f) \\ et Weight p SD) Gills 85.71 1.019,817 ± 1,393,736 Siphons 45.71 66,579 ±152.537 Adductor muscle 42.86 56.331 ± 120.086 Visceral mass 82.86 120.918+ 164.124 SD = standard deviation. Perkinsus Infection in Isahaya Bay. Japan 123 7 x105- 6 xl05- 5x105- 4 xlQs- fl) -) 3x10!- rn CO ■*— 2 xl05- 0 z UJ D 0 LU I u_ JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTHS i I PREvrr VITEL POSTVIT Figure 3. Temporal variations In the mean frequency of occurrence of different t>pes of oocytes in Pinclada mazutUmica o>er an annual ga- met()j>enic cycle. Previt = previtellogenic oocytes; Vitel = vitellogenic oocjtes; Postvit = postvitellogenic oocytes. Bars denote standard de- viation. ANOVA detected highly significant differences in the mean area of oocytes over time (F = 102.97; P < 0.001). Biochemical Analysis Gonadal Tissue Preliminary tests made to measure the amount of carbohydrates in the gonadal tissue, digestive gland, and muscle indicated that over 80% was glycogen. Thus, glycogen is reported in this study as total carbohydrates (CHO). When water temperature rose from 20 to 2.*)' C. CHO were actively depleted during the first half of the year and decreased from 12 mg/g in January to 4 mg/g in July (Fig. 5A). Afterwards, CHO were not used for the production of gametes, yielding a slight increase in their concentration between 5 and 6 mg/g. The varia- tion in the concentration of this component showed significant differences over time (F = 35.83; P < 0.001 ), but not according to the sex of specimens (F = 0.414; P > 0.05). The decrease in the levels of CHO in the gonadal tissue was not significantly corre- lated with the increase in the area of postvitellogenic oocytes (/• = 0.102; P>0.05). Proteins (PRO) accumulated mainly in the female gonadal tis- sue during the first half of the reproductive cycle, resulting in a .sharp rise in their concentration from 52 mg/g in January to 100 mg/g in May (Fig. 5B). After a drop in June to July, the concen- tration varied between 58 and 65 mg/g for both sexes during the second half of the year. There were not significant differences in the PRO content over time (F = 0.954: P > 0.05), but differences were significant in relation to the sex of specimens (F = 8.16; f < 0.05). There were two peaks depicting the temporal variations in the concentration of total lipids (LIP; Fig. 5C) and triacylglycerides (TG; Fig. 5D) in the female gonadal tissue. The first peak was seen in April for LIP (20.4 mg/g) and in May forTG ( 13.5 mg/g). while the second peak was observed in August to September for both components (13.6 mg/g for LIP and 9.4 mg/g for TG). There were significant differences in the temporal variations of LIP (f = 13.23; P < 0.001 ) and TG (F = 2.84; P < 0.05) and also according to sex (f = 14.24; P < 0.001 for LIP; F = 43.58; P < 0.001 for TG). There was a positive and significant correlation between the variations of both elements over time (/■ = 0.973; P < 0.001). Similarly, variations in the concentration of LIP were significantly correlated to the increase in the area of postvitellogenic oocytes (,■ = 0.64; P < 0.005). Digestive Gland There was a condnuous accuiiiulalion of CHO m this tissue from 23.4 nig/g in March to over 32 mg/g until November (Fig. 6A). After the spawnmg. the concentration of CHO decreased to 22 mg/g in December. There were significant differences in the concentration of this component over time {F = 27.8; P < 0.001 ). However, differences were not significant in relation to the sex of oysters (F = 0.434; P > 0.05). PRO showed a gradual decrease from January (156 mg/g) to October (96 mg/g), especially in males (Fig. 6B). In females, this trend .showed two minimum peaks in May and October. After the spawning in late September, a shift in the concentration of PRO was recorded again. The variations in the concentration of PRO denoted significant differences over time (f = 24.86; P < 0.001 ), while differences were not significant according to the sex of specimens [F = 0.093; P > 0.05). LIP and TG in this tissue presented two peaks of concentration throughout time (Figs. 6C and D). The first peak occurred in April (25 mg/g for LIP and 15 mg/g for TG). The second peak was observed in August, with values of 17 mg/g (LIP) and 10 mg/g (TG). Unlike gonadal tissue, the concentration of both components during winter ( spent stages ) was the same or higher than that of the summer. There were significant differences in the variation of the LIP and TG content over time (f = 56.28; P < 0.001 for LIP; F = 26.82; P < 0.001 for TG), but differences were not significant 7C00 5000 "^ 4000 UJ 3000 a: < 2000 cti □p IE ±Sld Oev □ ±Sld Err, a Mean m _U C^Il m nan m m np JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTHS Figure 4. Box and Whisker for temporal variations In the mean area of oocytes in Pinclada mazallanica iner an annual gametogenic cycle. D5 Seasonal Changes of Gametogenesis in Pinctada mazatlanica A B 131 20 16 12 JAN FEB MAfl APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTHS MALE FEMALE Figure 5. Temporal and sexual variations in the mean levels of total carbohydrates (A), proteins (Bl. total lipids (C'l. and triacylglycerides lU) in the gonadal tissue of Pinctada mazallaiiica over an annual gametogenic cycle. Bars denote standard deviation. between sexes (F = 0.849; P > 0.05 for LIP; F = 0.267; P > 0.05 for TG). The temporal variations of both components were sig- nificantly correlated (r = 0.963; P < 0.001 ). Adductor Muscle CHO in the muscle showed high concentrations during the first half of the gametogenic cycle, varying between 95 mg/g (January) and 1 15 mg/g (June) (Fig. 7 A). Afterwards, a sharp drop to 50 mg/g in October was observed mainly during the mid and late- developing and ripe stages. After the spawning, a recovery in the content of CHO was noticed. The ANOVA found significant dif- ferences in the concentration of this component on time (F = 47.95; P < 0.001). Neither sex showed significant differences in their CHO content over time (F = 1.39; P > 0.05). During the first reproductive peak, the le\el of PRO in the muscle decreased from 235 mg/g in February to 128 mg/g in May. After a short period of accumulation in June, such levels gradually rose and reached 227 mg/g in December (Fig. 7B). The variations in the concentration of PRO denoted highly significant differences over time {F = 103.61; P < 0.001), but differences were not significant between sexes (F = QMS: P > 0.05). The decrease in the PRO content of the muscle was significantly correlated to the increase in the area of postvitellogenic oocytes ir = 0.632; P < 0.05). DISCUSSION Seasonal changes in energy storage and depletion in relati i- •■ gametogenesis have been well documented durins the las' liirct; 132 Saucedo et al. O 0 JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV OEC JAN FEB MAB APR MAY JUN JUL AUG SEP OCT NOV DEC FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC MONTHS MALE FEMALE Figure 6. Temporal and sexual variations in the mean levels of total carbohydrates (A), proteins (B). total lipids (C), and triacylglycerides (D) in the digestive gland of I'iiiclmla mazatkmica over an annual gametogenic cycle. Bars denote standard deviation. decades for some commercial-edible species of marine bivalves. such as scallops, mussels, and oysters (Giese 1969; Sastry & Blake 1971; Ansell 1974; Comely 1974; Gabbott 1975. 1976. 1983. Bayne 1976; Taylor & Venn 1979; Zandee et al. 1980; Barber & Blake 1981. 1983. 1991; Robinson et al. 1981; Bayne et al. 1982; Epp et al. 1988; Couturier & Newkirk 1991; Martinez 1991; Pazos et al. 1997; Racotta et al. 1998). Nevertheless, most of the aspects concerning the storage capacity and mobilization of nutrients to satisfy metabolic needs related to gametogenesis seem to be spe- cies-specific (Barber & Blake 1981. 1991) and .some important interspecific differences have been reported (Pazos et al. 1997). The present work couples for the first time both gametogenic and biochemical cycles for a species of pearl oyster. Both histological and biochemical analyses showed the exis- tence of two peaks of intense reproductive activity over the annual cycle; one in spring (March to May) when water temperature was rising from 21 to 23°C. and other in summer (August to October) when water temperature was about to reach a maximum value of 28-29°C. The first reproductive peak was clearly defined in terms of its high incidence of ripe specimens, high frequency of postvi- tellogenic oocytes, and high levels of protein, lipids, and triacyl- clycerides within the gonadal tissue. However, the histological analysis did not identify any spawning activity in the sample. This was confirmed by very low spatfall recorded in parallel field moni- toring (CIBNOR-Pearl Oyster Research Program database). Such result was probably ascribed to the low and abnormal water tem- Seasonal Changes of Gametogenesis in Pinctada mazatlanica 133 JAN FEB MAR APR MAV JUN JUL AUG SEP OCT NOV DEC < EC O o 250 200 150 - 100 - JAN FEB MAR APR MAY JUN JUL AUQ SEP OCT NOV DEC MONTHS MALE FEMALE Figure 7. Temporal and sexual variations in the mean \e\eh of total carbohydrates (A) and proteins (B) in the adductor muscle of Pinctada mazatlanica over an annual gametogenic cycle. Bars denote standard deviation. perature registered during March to May (21-23°C) and also to the drop-out in the same parameter detected in June (22°C) as a con- sequence of the presence of "La Nifia" phenomenon in waters of Bahi'a de La Paz during the first half of 1999, which caused gonads to undergo a partial lysis and resorption. Similar processes have been reported to occur in the scallops Argopecten irradians cou- centricus (Sastry 1966; Barber & Blake 1981. 1991: Epp et al. 1988) and Placopecten magellanicus (Robinson et al. 1981) under low temperatures conditions or during times of food shortage. In this study, the three major tissues related to gametogenesis (gonad, digestive gland, and adductor muscle) were utilized dif- ferently over time, but showed a clear seasonal cycle of gonadal development, spawning, energy storage, and mobilization of nu- trients. Gametogenesis started early in February, when food is commonly abundant from the phytoplankton (Signoret & Sanloyo 1980; Lechuga-Deveze 1997). and wild specimens had stored enough energy reserves in the adductor muscle (mainly) and di- gestive gland (secondarily). When gametogenesis had already reached the early-development stage in March, the first previtel- logenic and vitellogenic oocytes started to differentiate within acini. Because proteins in the muscle and digestive gland showed a downward trend from January to May, it seems evident that gonad growth took place at their expense during the first repro- ductive peak. Therefore, a substantial growth in the size of oo- cytes, an increase in the weight of gonadal tissue, and their levels of protein, lipids, and triacylglycerides was recorded. This was particularly evident in the female gonad, where the content of the last three elements doubled that of males, at least during the first reproductive peak, and partially during the second reproductive peak. On the contrary, as gametogenesis proceeded into the mid and late-developing and ripe stages (June to October), carbohy- drates (glycogen) from the muscle were the only substrate mobi- lized and used for the build-up of gametes. Following the spawn- ing in October-November, when gonadal tissue decreased in weight and their basic levels of lipids and triacylglycerides fell sharply, the condition of the digestive gland and muscle improved again. Carbohydrates obtained from ingested food were used as an energy-rich fuel for the build-up of gametes, via their conversion into lipids and triacylglycerides reserves (lipogenesis). This pro- cess has been reported to occur in some bivalves to satisfy the metabolic demands derived from vitellogenesis (Gabbott 1973. 1976, 1983), especially because lipids and triacylglycerides are the basic energetic reserves for sustaining embryonic and larval de- velopment of most species of marine bivalves (Holland 1978; Fraser 1989). However, there was a lack of correlation between carbohydrates of the gonadal tissue and the total area of postvitel- logenic oocytes. On the contrary, the correlation was significant between the area of oocytes and the content of lipids in this tissue. Several authors have discussed the relative contribution of food intake vs. energy reserves to satisfy the metabolic demands of growth and gonadal production in marine bivalves (Bayne 1976; Gabbott 1976; Barber & Blake 1981, 1983; Robinson et al. 1981; Epp et al. 1988; Racotta et al. 1998). However, because these processes are highly dependent on several e.xogenous and endog- enous factors, no pattern has yet been established. For example, in Pecten maximus (Comely, 1974), Chlamys opercidaris (Taylor & Venn, 1979), and Myiilus edulis (Gabbott, 1975), energy reserves are required for both the initiation of gametogenesis and subse- quent gonadal growth, while in others such as A. irradians con- ceiitricus. food intake is necessary to sustain gonadal growth since reserves from the muscle, digestive gland, and mantle are inad- equate. Parallel to their utilization for gametogenesis, ingested nutri- ents were also incorporated into the adductor muscle and digestive gland for storage. The muscle stored high levels of protein both at the beginning and end of the annual cycle to sustain the start of gametogenesis. No other study with bivalves has reported protein values as high as those presented here for the adductor muscle of P. mazatlanica. neither for scallops whose adductor muscle is associated with locomotion. Because the decrease in protein con- tent of the muscle was significantly related to the increase in the size of oocytes during vitellogenesis, the role of this organ as the most important site of energy storage is evident. In fact, a signifi- cant loss of weight in the muscle to less than half its maximun-, value was detected as both protein and carbohydrate reserves were 134 Saucedo et al. rapidly consumed in benefit ofganietogenesis. The cDnlnbiitiini ol the muscle has also been emphasized for other bivalves, such as ChUimys sp (Ansell 1974; Taylor & Venn 1979). M. ediiUs (Gab- bott 1975; Bayne 1976). A. inadians concentricus (Barber & Blake 1981, 1991; Epp et al. 1988), P. maximus (Faveris & Lubet 1991 ), A. purpumtus (Martinez 1991 ). and A. vciuriiiisus (Racotta et al. 1998). Unlike the muscle, the digestive gland appears to have a sec- ondary role in gonadal development of P. mazatlanica. This seems evident since protein was the only fuel transferred to the gonadal tissue during the first half of the year. On the contrary, carbohy- drates were progressively stored from March to November, indi- cating that despite the advancement of gametogenesis. these nu- trients are not used for the cycle in course and rather accumulated for further energy needs. This result is in agreement with the finding of Barber and Blake (1981. 1991 ) that the digestive gland acts as a short-term storage and transfer site of carbohydrates to meet the reproductive events in M. ediilis. Regarding the storage function, Saucedo et al. l2001b) found specialized vesicular con- nective tissue (VCT) cells — filled mainly with carbohydrates — surrounding excretory conduits among the digestive gland and gonadal tissue. We believe that VCT cells from both tissues are probably communicated via the interconnective tissue matri.\. which may be the vehicle for the transpoil of stored carbohydrates between the digestive gland and the gonadal tissue, and vice versa. Lipids and triacylglycerides reserves were stored and rapidly ino- bilized after each of the reproductive peaks detected. Therefore. P. muzalliuucu may be considered a species that uses a strategy of storing large quantities of energy for starting and sustaining ga- metogenesis. However, our knowledge about the metabolic control of reproduction in pearl oysters is still limited. More studies on seasonal cycles of energy storage and mobilization are required, especially involving the participation of the mantle tissue, which has been proposed to be an important site of storage of glycogen and lipids in some bivalves. It would also be interesting to obtain data similar to that from this study for tropical species of Pincladci. ACKNOWLEDGMENTS This study was done as part of two institutional projects of CIBNOR on pearl oyster culture and pearl induction in Bahia de La Paz (Projects ABM-7 and ABM-39). Additional grants were provided by the Consejo Nacional de Ciencia y Tecnologia (CONACYT-Me.xico, as a Ph.D. scholarship), the Consejo Nacio- nal para la Biodiversidad (CONABIO), and the Fondo Mexicano para la Conservacion de la Naturaleza (FMCN). The authors thank the following staff of CIBNOR: Diana Carreno and Carmen Rodriguez (for support during the biochemical and histological analyses, respectively) and to Horacio Bervera and Juan Jose Ramirez (for SCUBA diving assistance and collection of oysters). We also thank Dr. Paul Southgate (James Cook University, Aus- tralia) for his comments and suggestions during the revision of the paper and for editing the English-language text. LITERATURE CITED Ansell, A. D. 1974. Seasonal changes in biochemical composition of the bivalve Chlamys scplemrudiuui from the Clyde Sea area. Mar. Biol. 25:85-99. Arizmendi-Castillo. E. 1996. Ciclo reproductivo de las ostras perleras Pinctada mazadanica (Hanley, 1856) y Pleiia sterna (Gould. 1851) (Pteriidae). en el area de Guaymas. Sonora. Mexico. Tesis de Maestria. ITESM-Campus Guaymas. Mexico. Barber. B. J. & N. J. Blake. 1981. Energy storage and utilii:ation in relation to gametogenesis in Argopecten irradian.s idnceiuricKs (Say). J. E.x/'. Mar. 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J. Mar. Freshwater Res. 9:135-143. Tranter, D. J. 1959b. Reproduction in Australian pearl oysters (Lamelli- branchia). II. Pinctada albina (Lamark): Gametogenesis. Aiistr. J. Mar. Freshwater Res. 9:144-158. Wada. S. 1959. Biology of the silver-lip pearl oyster Pinctada maxima (Jameson). 2. Breeding season. Margarita. 1:15-28. Zandee. D. I.. J. H. Kluytrnans & W. Zurburg. 1980. Seasonal variations in biochemical composition of Mytilus edulis with reference to energy metabolism and gametogenesis. Neth. J. Sea Res. 14:1-29. Jminwl of Shellfish Reseurch. Vol. 27, No. K. 137-143. 2002. HYBRIDIZATION OF TETRAPLOID AND DIPLOID CRASSOSTREA GIGAS (THUNBERG) WITH DIPLOID C. ARIAKENSIS (FUJITA) HUAYONG QUE' AND STANDISH K. ALLEN. JR."* 'institute of Oceanology, Chinese Academy of Sciences, Qingdao. Shandong 266071, P.R. China: 'Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Science, Rutgers University, Port Norris, New Jersey 08349 ABSTRACT Three replicates of hybrid cros.ses ot tetraploid and diploid C. gigas (ThunbergI with diploid C ariakensis (Fujita) were produced with controls. Larval survival and growth were documented. Cytological events were also monitored in oocytes from hybrid crosses following insemination. Among the four types of hybrid crosses, diploid C. gigas (female) x diploid C. uiiakensis (male) (GA) was the most successful. Survival of GA was about the same as that of controls in two of three replications, although its growth rate was 25-30% lower. Crosses of tetraploid C. gigas (female) and diploid C. ariakensis (male) (GGA) had poor yield at day 2 post-fertilization (0.05%). but grew nearly as well as controls subsequently. The other two types of hybrids (i.e.. diploid C. ariakensis [female] and tetraploid C. gigas [male] [AGG]. diploid C. ariakensis [female] and diploid C gigas [male] [AG]) suffered very low yield at day 2 (0.01% and 0.003%) and grew very slowly. Spat were obtained from all replicates of GA crosses and one of three replicates of GGA, and proved to be hybrids by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) diagnosis. GGA hybrids were confirmed to be triploid by flow cytometry. No larvae survived to eyed stage in AGG or AG crosses. Cytological examination revealed that the vast majority (>99%r) of oocytes from hybrid crosses had a prolonged meiotic prophase I or metaphase 1 at least through 180 min post-insemination. KEY WORDS: Crassostrea gigas. Crassostiea ariakensis. diploid, hybrid, tetraploid. oyster, breeding, polyploidy INTRODUCTION There are numerous reports of attempted interspecific hybrid- ization in the genus Cra.'isnsirea (Gaffney & Allen 1993). How- ever, most should be viewed with caution because these reports were unaccompanied by genetic confirmation of putative hybrids. Even a modest amount of contamination may account for the ma- jority or all of surviving progeny in hybrid crosses in which fer- tilization rate and viability are normally low or nil (Allen & Gaffney 1993), One case seems clear: Pacific oyster C. gigas (Thunberg) and Suminoe oyster C. ariakensis (Fujita — formerly C. rividaris Gould) can be crossed to produce viable hybrids (Allen & Gaffney 1993). The production of hybrids is interesting because they may pos- sess qualities that improve commercial traits. Hybrids also could be back-crossed to introgress certain traits into either of the parental species; for example, disease resistance. Introgression of disease resistance into C. virginica (the Eastern oyster) from C. gigas was the rationale for the extensive hybrid trials under- taken by Allen et al. (1993). Later, many more hybridization trials were attempted, using bridging crosses between races of C. vir- ginica. using (only slightly) fertile C. gigas x C. ariakensis hybrids (GA), and using polyploidy (Lyu 1996). However, under no cir- cumstances tested in the lab did C. virginica hybridize with C. gigas or C. ariakensis. Although they failed as a bridging cross to C. virginica, GA hybrids are still of interest for several other reasons. First, no work has been done on the qualities of diploid GA hybrids as an aqua- culture product, although this work might more appropriately be carried out where there is on-going commercial culture of these two Asian species. For the East coast, they are nonnative. Second, because diploid hybrids are possible, production of polyploid hy- *Corresponding author. Fax: (804) 684-7717; E-mail: .ska@vims.edu Present address: Aquaculture Genetics and Breeding Technology Cen- ter, Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA 23062. brids should also be possible. Polyploid hybrids are potentially useful for improvement of commercial traits (Longwell 1986). Virtually no work on polyploid hybrids of shellfish has been done. Third, and most apropos to research on the East Coast, is the issue of testing nonnative species as an alternative to the native Eastern oyster because of the decline in the fisheries there. Trials of nonnatives were begun in Delaware Bay several years ago (Allen 1993) and have been conducted for C. gigas in the Chesapeake Bay (Calvo et al. 2000). Trials with C. ariakensis have been (Calvo et al. 2001) and continue to be conducted. C. gigas seems more suitable for higher salinity environments and C. ari- akensis seems suitable for more estuarine conditions. In all field trials up to this point, triploids have been used to effect population control because of their sterility (Allen & Downing 1990, Gaffney & Allen 1992, Guo & Allen 1994a). Triploid hybrids then are of interest because they are expected to be sterile, more so than diploids because of the added burden of gametogenesis in hybrids (Thorgaard & Allen 1986, Thorgaard & Allen 1992). Triploid hybrids may also have characteristics inter- mediate to the two parental species, for example, salinity prefer- ence. The genotypes that might be available for culture in an estuary as varied as the Chesapeake Bay, for example, could range from triploid C. gigas (GGG) through two types of triploid hy- brids— either tetraploid C gigas x diploid C ariakensis (GGA) or diploid C. gigas x tetraploid C. ariakensis (GAA) — to triploid C ariakensis (AAA), with phenotypes potentially encompassing the full range of estuarine and marine conditions. To date, all hybrid crosses between Crassostrea species have been made between diploids. Diploids are also used in the pro- duction of triploid hybrids using ploidy induction techniques (Allen et al. 1989). For example, triploid hybrids were attempted between C. virginica and C. gigas by inhibiting polar body 2 with cytochalasin B treatment (Allen et al. 1993). However, this hybrid seems to be inviable in any form. Triploid hybrids were also at- tempted (S. K. Allen, Jr., unpublished data) between C. gigas and C ariakensis. These, too. were unsuccessful for another reaso;;. fertilization in this cross is protracted, taking more than 3 h, .\s a 137 138 Que and Allen consequence, polar body 2 formation is asynchronous and treat- ments to inhibit polar body 2 are useless. The availability of tet- raploid C. f^i.i^as (Guo & Allen 1994b), tetraploid C. ariakeiuis. or both, provides a new opportunity to produce triploid hybrids by crossing (Guo et al. 1996), rather than induction and to further investigate the fitness of them. As a first step, we examined the feasibility of hybridizing tetraploid C. gii^as and diploid C. aria- kensis. At the same time, we repeated crosses of diploid C. gii;cis with diploid C. ariakensis as controls and also examined early development of eggs cytogenetically. MATERIALS AND METHODS Oyster and Gametes Sexually mature oysters used in this study were 2 years old and obtained from stocks held at the Cape Shore Laboratory, Haskin Shellfish Research Lab. Ploidy of tetraploid Pacific oysters was confirmed in all individuals by flow cytometry prior to spawning. Gametes were obtained by strip spawning. All surfaces and instru- ments contacting the oysters were cleaned with dilute bleach and rinsed with fresh water between handling and opening of different individuals. Sex was determined by gonad biopsy under a light microscope. Once the sex was determined, the animals from dif- ferent sexes were removed to separate containers. Gametes from each oyster were dissected into individual beakers. Eggs were passed through a 60-(ji.m Nytex screen to remove the large tissue debris and rinsed on a 25-jjim screen, then suspended in filtered (2 |j,m) seawater at 23-25°C for at least 30 min to confirm that the eggs were not self-fertilized. Sperm were separated from debris by passing the suspension through a IS-fjim screen. Experimental Design Abbreviations for gamete contributions of the two oyster spe- cies are as follows: G = diploid C. gigas: GG = tetraploid C. gigas: and A = diploid C. uriakensis. v\ ith female listed first. Eight types of crosses were conducted (Table I) o\'erall, although not all crosses were possible in all three replicates. For each rep- lication, an individual female and male were used. After spawning, TABLE L Experimental Design for Crosses Among 4n C. gigas. 2n C. gigas, and 2n C. ariakensis. G GG A 2 G GG GA GG GGA Rep 1 A AG AGG AA 9 G GG G/GG GA GG GG/G GGA Rep 2 A AG AGG AA 9 G GG G/GG GA GG GGA Rep .1 A AG AGG AA Individual females and males were used for each replication, and three replicates were made. Gamete contribution is represented by GG. G, or A, respectively, with female listed first. parents were frozen at -80°C for subsequent genetic confirmation of the progeny. Embryonic and luirval Development Insemination was conducted at 23-25°C and for hybrids, high densities of sperm were used (Lyu & Allen 1999). Fertilization rate was assessed by directly examining at least 100 oocytes under the light microscope at 60-90 min post-insemination for controls and up to 180 min post-insemination for hybrid crosses. After deter- mining fertilization rate, oocytes were transfeired to culture ves- sels whether fertilization was observed or not. Fertilization was considered successful if the oocyte was at or beyond polar body I formation. Yield at 48 h post-inseinination was estimated by directly counting straight-hinge larvae with normal appearance. Yield was calculated as (no. of straight-hinge x l(X))/no. of eggs incubated Temperature and salinity for lar\al cultures of crosses of GG, GA, GGA, G/GG, and GG/G, where C. gigas was the egg source, were 25°C. 22-23 ppt (Breese & Malouf 1975). For crosses of AA, AG. and AGG, where C. ariakensis was the egg source, tempera- ture and salinity were 26°C and 20 ppt (Breese & Malouf. 1977). Seawater in the larval cultures was renewed every 2 days. In all hybrid cultures, densities of larvae were sufficiently low to prevent density-related growth effects; densities in parental culture were within those used in standard larval culture, beginning at 10/mL and winnowing out to 1-2 /niL. During water changes, numbers of remaining larvae were estimated and shell length was measured for 20 individuals for each cross. When larvae reached eyed stage, eyed larvae were collected and treated with a solution of 10" M epinephrine for 16 h (Coon et al. 1986). Following treatment, melamoi-phosed larvae were held in a downweller system until they reached a shell length of approximately I mm, when they were transferred to an upweller silo. We took great care to elimi- nate all sources of contamination throughout the culture process. For cvtologlcal observations, eggs from each hybrid cross were sampled and fixed with Carnoy's solution ( 1:3 glacial acetic acid and absolute methanol) at 90. 120, 150, and 180 min post- insemination. Fixatives were changed twice following light cen- trifugation. Chromosomes were observed by acetic orcein stain (Guo et al. 1992). Genetic Confirmation We randomly sampled 28 spat from each replicate of GA crosses and all GGA spat. In progeny, the whole body was pre- pared for DNA extraction, whereas mantle tissue (2-8 mg) from corresponding parental species was prepared using a commercial kit (PureGene, Centra, Minneapolis, MN). An additional gill tissue sample from GGA spat was taken and stored in DAPI/DMSO (Sigma, St. Louis, MO) solution at -80°C for flow cytometric analysis. An approximately 550-bp region of the nuclear rDNA genome was amplified via polymerase chain reaction (PCR) using primer ITS-I. (The primer pair was designed by Dr. Patrick M. Gaffney. University of Delaware [Hedgecock et al. 1999]). Reaction volume of 25 jxL contained 50 MM MgCI,. 0.2 niM of each dNTP, 0.2 |j.M of each primer, 5 U/p.L polymerase (Taq DNA polymerase. Sigma) and 1 p-L DNA extraction. DNA amplifications were per- formed in a programmable thermal cycler (PTC- 100, M.J. Re- search, Inc.. Walthram, M.A) using a 2-min initial denaturation at Hybridization of Ckassostrea gigas 139 94°C and then 34 cycles of 45-sec denaturation at 94°C. 1-min annealing at 52"C, 1-min extension at 72°C. and finally a 5-niin extension at 72"C. Restriction enzyme digestion of PCR products was made with restriction endonuclease Hinf I (Sigma). Digestion volume of 20 fjiL contained 1 x buffer (supplied by New England Biolab Inc.. Beverly. MA). 5 units Hinf I. and 8 |jlL PCR product. Digestion mix was incubated at 37°C for 3—1 h. followed by 3 (xL lOx loading dye to stop the digestion. All PCR products and restriction digest fragments were elec- trophoresed in a 3% agarose (Sigma) gel in Ix TBE (0.089 M Tris-borate. pH 8.3. 0.002 M ethylenediamine tetraacetic acid (EDTAll buffer. A molecular weight marker (pUC 18. digested with Hae III. Sigma) was loaded along with the product of interest. The gel was run at 60-90 V. stained with ethidium bromide (0.2 mg/mL) for 10-13 min. and visualized by transillumination. Statistical Analyses All data were analyzed with the computer program SYSTAT (Wilkinson 1990). Fertilization rates and yield data were arcsine transformed prior to statistical analysis (Sokal & Rohlf 1981 ). To compare the performance of hybrids to their controls, a two-way ANOVA was used. Paired /-tests were conducted to compare cer- tain crosses to their reciprocals. RESULTS Larval Siinival and (irowth After 48 h post-insemination, survival of GA crosses was about equal to controls in two of three replicates (Fig. 1 ). Larvae of GGA crosses had high survival, although the number of eyed larvae was small (of 450 larvae on day two. 280 survived to eyed stagel. For AGG and AG crosses, mortality was severe and steady for 1 3 days, with no survival to eyed stage. In GG crosses, mortality was gen- erally density related, moderating at lower densities at about day 6-10. For AA crosses, survival was generally poor, making this cross a poor control. In general, survival ranked GG. GA. and AA. in descending order. Sur\ i\'al of G/GG was variable: better than GG in replicate 2 but worse than AA in replicate 3. Survival of Fertilization Rate and 48-h Yield Mean fertilization rates in the parental (nonhybrid) crosses were 94<7r (GG). 779f (AA). 88% (G/GG). and 83% (GG/G) (Table 2), with no statistically significant difference among them (F = 3.1 18, P = 0.132) by ANOVA. In hybrids, signs of fertil- ization did not appear until 180 min after insemination, precluding estimates of fertilization rate. Yield at 48 h post-insemination varied significantly among crosses (Table 2) (F = 3.964, P = 0.018). Yield in GG was significantly greater than that in AA (/ = 4.162, DF = 4, P = 0.014) but there was no difference between other parental crosses. Yields were similar in GGA and AGG (r = 1.010, DF = 4, F = 0.369), whereas GA had higher yields than AG (r = 3.364. DF = 4. F = 0.006). Yields of GA and AA crosses were about the same. GGA and AGG crosses produced many fewer larvae than controls. AG crosses suffered extremely low yield (0.003%). TABLE 2. Mean fertilization rates and yields + SD (h) at 48-h in parental and hybrid crosses combined from three replicates. Cross Fertilization Rate ( % ) Yield ( % ) GG GA GGA AA GG/G G/GG AGG AG 94 + 4.9(3) ND ND 77 ± 13.9(3) 85 1 1 ) 8X + . "5.0(21 ND ND 21 ±7.5(3) 4± 1.2(3) 0.05 ±0.01 (2) 3 ±2.5 (3) 4(1) 18 ±24.4 (2) 0.01 ±0.02(3) 0.003 ±0.006 (3) ND = no data. ' Fertilization rate was observed at 60-90 min post-insemination for pure crosses; 180 min post-insemination for hybrid crosses. DAY Figure 1. Mean survival of hybrid larvae and their respective controls from day 2 up to day 16 in crosses of diploid and tetraploid ('. gi^as with diploid C. ariakinsis. (A) Six matings were made in replicate 1. No larvae survived to day 2 in .AG, AGG, and GtiA. (B) Eight matings were made in replicate 2, No larvae survived to day 2 in AG, .AG(;, and A A. (Cl Seven matings were made in replicate 3. No larvae survived to day 2 in GGA. Counts were terminated when harvesting of eyed larvae was begun. GG ( ♦ ), GA (■), G/GG (A). GG/G (x), GGA (D), AG'; (A), AA (O), AG (•). 140 Que and Allen G/GG crosses was better than its reciprocal, GG/G. Larvae sur- vived to setting in all replicates of GG and GA. 2 of 3 replicates of G/GG and 1 of 3 replicates of GGA. Spat were obtained from all of these. Larvae of GG, G/GG. GG/G, and GGA crosses grew at similar rates. Crosses of GA grew slower than GG controls but faster than AA (Fig. 2). GA larvae were generally smaller than GG larvae. Both AG and AGG crosses grew very slowly (AG larvae died at day 10). AA crosses grew slowest of all controls. Mean size of eyed larvae was 350 |xm for GG, 336 |jim for GA, 360 \x.m for GGA, 361 iJLni for G/GG. and 361 |xm for G/GG. ANOVA showed no significant difference in eyed larvae size among crosses (F = 1.712, P = 0.199j. After 90 days post-setting, spat from GGA reached 12.0-17.5 mm in shell size compared with 2.87-8.0 mm in the corresponding GA cross (r = 8.49. DF = 6. P < 0.001 ). Cytological Observation of Eggs from Hybrid Crosses The vast majority of eggs from hybrid crosses were delayed at prophase I or metaphase I at least through 180 min post- insemination (Fig. 3A-E). In fact, of all eggs examined at 180 min post-inseminalion (a 150 observations from each of GA. AG. GGA. and AGG). only 2'7f of eggs from GGA had entered anaphase I (Fig. 3F). In GA. AG. and AGG. 10 bivalents were still observed at this time. Chromosome aggregation was much more complicated in eggs from GGA crosses. In general, eggs contained an average of 10 quadrivalents. although other types of synaptic chromosomes were also present, i.e.. univalents, bivalents. and trivalents (Fig. 3D. E). Genetic Confirmation Agarose gel electrophoresis of the PCR products consistently revealed two bands, one at around 587 base pairs (bp) and the other at around 527 bp (Fig. 4. top). Restriction digest fragments re- solved distinct bands in both parental species. Two bands were resolved, one at around 434 bp and the other around 138 bp in C. gigas females, both tetraploid and diploid. In contrast, two bands were resolved at around Iblllfil bp and 174 bp in C. ari- akensis males (Fig. 4, bottom). The hybrids (GA and GGA) ex- pressed all four bands corresponding to their parental species. An additional band was detected at around 458 bp in hybrids. Identical band resolution was observed in replicates 1 and 2 of GA crosses, whereas an extra band was resolved at 587 bp consistently in both parental species and hybrids in replicate 3 (Fig. 4. bottom, last four lanes). All progeny samples (28 individuals from each replicate of GA and a total of 4 individuals from GGA) were hybrids. Three of four GGA hybrids were Iriploid as confirmed by flow cytometry; the other hybrid was diploid. DISCUSSION In hybrid crosses, fertilization was not apparent even as late as 180 min post-insemination. These same observations were reported previously (Miyazaki 1939. Imai & Sakai 1961). both B Figure 2. Mean size (nm) of hybrid and control larvae from day 2-16 in crosses of diploid and tetraploid C. gigas with diploid ('. ariakensis. (A) Larvae from GG, GA, GGA, and \X in replicate 1; (B) larvae from GG, GA, GG/G, and G/GG in replicate 2; and (C) larvae from GG, GA. G/GG, AGG. AA, and AG in replicate }. Measurement of surviving larvae was made until eyed lar»ae appeared. QQ ( ♦ ), G.4 (■), G/GG (Al, G(;/(; (X), GGA (□). AGG (A). AA (O), .\G (•). Figure 3. Chromosome ohser\ations In crosses of diploid and tetra- ploid C gigas with diploid C. ariakensis at 180 min post-insemination. (A) Prophase I in oocytes from GA; (B) prophase I in oocytes from AG; (C) prophase I in oocytes from M'.C: (D) and (El prophase I in oocytes from (;(;.\: and (F) anaphase 1 in oocytes from (JGA. Scale bar on iDl: 10 fim. Hybridization of Crassostrea gigas 141 M123456789 llllllilL ^yyygyyy^ M 1 FiKure 4. Top: Electrophoretic separation of undigested DNA prod- ucts resulting from PCR ampliflcation of genomic DNA. using primer of ITS-1. rDNA originated from parental species and their hybrid progeny. Row 1: lane I = diploid ('. gigas I ; I; lane 2 = diploid C. ariakensis ( :' ); lanes i—X = G x A progeny: lane 5 = diploid C. gigas ( I ), lane 6 = diploid C. ariakensis ( 6 ), lanes 7-9 = (; x A progeny . Row 2: lane 1 = tetraploid C. gigas ( 9 ), lanes 2-5 = GG x A progeny: lane 6 = diploid C. gigas i J 1, lane 7 = diploid C. ariakensis ( :' ). lanes 8-9 = G X A progeny. Lane M = molecular weight marker. Bottom: Elec- trophoretic separation of PCR ampliried rDNA products from GA and GGA crosses digested with restriction endonuclease Hinf I. M = mo- lecular weight marker: lane 1 = tetraploid C. gigas I l ; lanes 2-5 = GG X A progeny, lane 6 = diploid C. gigas ( i ), lane 7 = diploid C. ariak- ensis ( d I, lanes 8-9 = G x A progeny. The molecular weights of bands resolved from the marker are. from largest (slowest migrating! to smallest: 587, 458, 434, 298, 267/257, 174, and U)2 base pairs, respec- tively. of whom found little or no fertilization between C. gigas and C. ariakensis in their studies of Japanese oysters. However, we observed 8%-9% fertilization rate in GA crosses in other experi- ments (data not shown). In a previous study, a mean fertilization rate of 12% was reported for GA ct'osses (Allen & Gaffney 199.^). Zhou et al. (1982) revealed that fertilization rates in C. gigas ( 2 ) X C. ariakensis { 6 ) were 0-52.6% and its reciprocal, 2.3^-1 8.8%. These data indicate that the fertilization rate in these hybrids varies widely. Success of hybridization should not be assessed solely on the fertilization rate. In contrast, fertilization between other species of Crassosirea occurred readily, and often showed relatively high rates, but larvae survived for only a short time before complete mortality (Menzel 1986; Allen et al. 1993). Despite the apparent lack of fertilization (observed up to 180 min post-insemination), replicates of GA yielded viable spat. which reconfirms the compatibility of the gametes from C. gigas (2) and C. ariakensis (6). Buroker et al. (1979) reports a rela- tively high genetic similarity between these two species. Other studies also demonstrate the feasibility of hybridization between these two species (Allen & Gaffney 1993; Downing 1988; Down- ing 1991; Zhou et al. 1982). although only Allen & Gaffney (1993) confirmed hybrids genetically. In contrast to the success of GA. the reciprocal AG failed to produce any spat, although no mor- phological deformities were observed in the larvae. AG larvae were previously shown to be much less viable compared with the reciprocal although a few spat were obtained (Allen & Gaffney. 1993). The diploid control AA (three replicates) consistently sur- vived poorly and grew slowly in this study. This may partly ac- count for the failure to obtain any spat from the AG cross. Ac- cording to published accounts (Breese & Malouf 1977; Langdon & Robinson 1996). the survival and growth of C. ariakensis are similar to C. gigas under appropriate culture conditions. It is not clear why larvae of C. ariakensis performed so poorly in this study, because we have routinely cultured C. ariakensis on other occasions. Only one of three replicates of GGA yielded spat. The failure of the other two replicates might be attributable to low fecundity of tetraploid C. gigas used here (data not shown), although generally tetraploids have shown high fecundity (Guo et al. 1996; B. Eude- line, Taylor United. Inc. and S.K. Allen. Jr.. unpublished data). In all three replicates, yield at day 2 was low. We suggest that the major barrier for GGA production on a pilot- or production-scale is low yield at day 2. Afterward, larvae of GGA crosses survived well (virtually no mortality). While it is clear that more GGA progeny could be obtained by using more parents, the real chal- lenge is to find factors that lead to high levels of fertilization for the gametes that are available. We also suggest that there is a difference in growth rate between triploid (GGA) and diploid (GA) hybrids. First, GGA eyed larvae appeared 5-7 days earlier than GA and right after those of controls GG. G/GG, and GG/G. Sec- ond, the size of spat from GGA was greater than the size of GA at 90 days post-insemination, although the number of GGA spat was small. All 28 progeny sampled from each of three replicates of GA were hybrids. With respect to GGA progeny, some eyed larvae attached to the culture containers, leading to loss of eyed larvae. Consequently, only four culchless spat were obtained. However, the fact that three of the spat were triploid hybrids demonstrates that hybridization between tetraploid C. gigas ( 2 ) and diploid C. ariakensis (S ) was successful. Triploid hybrids of C. gigas and C. ariakensis cannot be obtained in any other way. In particular, the use of cytochalasin B (or other polar body inhibitor! is pre- cluded in GA crosses because of the prolonged period leading to syngamy and polar body fomiation. It is not possible to create triploids without some level of predictability and synchrony among developing eggs (Allen et al. 1989). PCR restriction fragment length polymorphism (RFLP) diag- nosis was an effective means to verify putative hybrid progeny. ITS-1 ampIification/Hinf I digestion successfully distinguished among C. gigas. C. ariakensis. and hybrids, which show bands present from both parental species. Application of this method is based on availability of an appropriate primer: ITS-1 in this study. Other methods have been used to confirm hybrid status (Allen & Gaffney 1993: Allen et al. 1993; Nakamura et al. 1990; Jiang et al. 1988). Karyotype analysis has been useful for hybrids amonc ■.'■. 142 Que and Allen pearl oyster genus PinciaJa (Jiang et al. 1988). For species within the genus Crassusirea. the great similarity in their karyotype ren- ders this type of analysis useless in hybrid documentation until more discriminating markers are designed. Flow cytometry was equivocal in discriminating between hybrid and pure crosses using dissociated cells of pooled larvae (Allen et al. 1993). Cytological examination of newly fertilized eggs from hybrid crosses revealed that either fertilization was severely delayed or early development of oocytes was extremely slow. The duration of meiotic maturation in hybrid crosses was at least triple that of controls. The delay of meiosis was probably not due to the quality of gametes because eggs in controls were normal and reached first cleavage at around 6(J niin post-insemination consistently (data not shown). Another hybridization attempt between C. gigas and C. ariakcnsis indicated there was no apparent difference in the time of development from eggs to the straight-hinge larvae (Zhou et al. 1982). Apparently, fertilization was also highly successful in the study by Zhou et al. leading us to suspect contamination. An alternative explanation is that Zhou et al. used a different popula- tion of C. iiricikensis. The C. ariakensis used here are from a population derived from Ariake Bay in Japan. Those of Zhou et al. are likely from mainland China. In this study, despite the delay in fertilization, healthy larvae were obtained and cultured through metamorphosis in all replicates of GA and one replicate of GGA. Furthermore, hybrid spat were obtained. Success in obtaining lar- vae suggests that meiosis in GA and GGA crosses must have resumed at some time, despite being delayed for an uncommonly long time. A similar observation was made in C. angulata oocytes fertilized with sperm from C virginica. where 47% of them ex- hibited metaphase 1 nearly 4 h after insemination. However, no parental crosses were made for comparison (Stiles 1973). Oocytes in hybrid crosses of C. gigas and C. ariakcnsis with C. virginica proceed through meiosis and early mitosis in fairly normal fashion (Scarpa & Allen 1992). The timing of meiotic and mitotic land- marks was the same for all crosses, parental and hybrid. However, hybrids of C. gigas and C. ariakcnsis with C. virginica were in- viable (Allen et al. 1993) after a short larval period despite normal meiotic and early mitotic behavior. C. gigas x C. ariakcnsis crosses were not included in Scarpa and Allen's work. Overall, this study reveals a new potential application for oyster breeding: triploid C. gigas x C. ariakensis hybrids. We were lim- ited to relatively few tetraploid brood stock for this study, but since that time, tetraploids have become commercialized on the West coast of the United Staes. We were also limited to making the triploid hybrid in one direction; that is, using only C. gigas as the tetraploid, enabling experiments on GGA (4n$ ) or AGG (4nd ). We were unable to explore GAA (4nc5 C. ariakensis) or A AG (4n$ C. ariakcnsis). Presumably the former would behave like GA and GGA cultures, and the latter would behave like AG and AGG cultures. These experiments will have to wait for the tetra- ploid C. ariakensis that are currently under development. At present, GGA hybrids can be made only at a high cost of gametes from the parental species. Even with high fecundity, the losses are so severe that commercial production would be a problem. Studies on factors that promote higher fertilization rate, syngamy. devel- opment rates, or all of these would contribute to efforts to produce pilot-scale quantities of GGA for future testing. ACKNOWLEDGMENTS We thank Greg A. DeBrosse for care ol' brood stock, larval, and nursery cultures. We are especially grateful to Dr. Patrick M. Gaffney and Dr. Ziniu Yu for technical assistance in PCR/RFLP. This project was partly sponsored by the NOAA U.S. -China Ma- rine and Fisheries Science and Technology Agreement in Living Marine Resources. This is NJAES Publication Number D-32ll)()- 1-02 and VIMS Publication Number 2465. REFERENCES Alien. S. K.. Jr. 1993. Triploids for field study? The good, the bad and the ugly. J. Slu'llfisli Res. 12:125. Allen, S. K.. Jr. & S. L. Downing. 199(1. Performance of triploid Pacific oysters, Crassostrea gigcLs. Gamelogenesis. Can. J. Fish. Aqiiat. Sci. 47:1213-1222. Allen. S. K.. Jr. & P. M. Gaffney. 199.^. Genetic confirmation of hybrid- ization between Crassostrea gigas (Thunberg) and Crassostrea rivii- laris (Gould). Aquaculture 113:291-300. Allen. S. K., Jr., S. L. Downing & K. K. Chew. 1989. Hatchery manual for proiliicing triploid oysters. Seattle. WA: University of Washington Press, 27 pp. Allen, S. K., Jr., P. M. Gaffney, J. Scarpa & D. Bushek. 1993. Inviahle hybrids of Crassostrea virginica (Gmelin) with C. rivularis (Gould) and C. gigas (Thunberg). Aquaculture 113:269-289. Breese, W. P. & A. E. Malouf, 1975. Hatchery manual for the Pacific oyster: Corvallis, Oregon, Oregon State University Sea Grant College Program. 22 pp. Breese, W. P, & A. E. Malouf. 1977. 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Effect of sperm density on hybridization between Crassostrea virginica (Gmelin) and C. gigas (Thunberg). J. Shellfish Res. 2:459-464. Menzel, W. 1986. Hybridization in oysters and clams. In: K. Tiews. editor. EIFAC/FAO Symposium on Selection. Hybridization and Genetic En- gineering in Aquaculture of Fish and Shellfish for Consumption and Stocking. Berlin: Heenemann Verlogsgesellschaft. Vol. 2, 1987. pp. 47-59. Miyazaki. I. 1939. Some notes on cross-fertilization of Japanese oyster. Bull. Jap. Soc. Sci. Fish. 7:257-261. Nakamura. D., T. A. Tiersch. M. Douglas & A. W. Chandler. 1990. Rapid identification of sex in birds by flow cytometry. Cvtogenet. Cell Genet. 53:201-205. Scarpa, J. & S. K. Allen. Jr. 1992. Comparative kinetics of meiosis in hybrid crosses of Pacific oyster Crassostrea gigas and Suminoe oyster C. rivularis with the American oyster C. virginica. J. E.\p. Zool. 263: 316-322. Sokal, A. A. & F. J, Rohlf. 1981. Biometry. 2nd ed. San Francisco: W.H, Freeman. 859 pp. Stiles. S. 1973. Cytogenetic analysis of an attempted interspecies hybrid- ization of the oyster. Incompatihility Newslett. 3:41-45. Thorgaard. G. H. & S. K. Allen. Jr. 1986. Chromosome manipulation and markers in fishery management: In: N. Ryman & F. M. Utter, editors. Population genetics and its application to fisheries management. Seattle: University of Washington Sea Grant, pp. 319-331. Thorgaard. G. H. & S. K. Allen. Jr. 1992. Environmental impacts of inbred, hybrid and polyploid aquatic species. In: A. Rosenfield & A. Mann, editors. Dispersal of living organims and genetic materials into aquatic ecosystems. University of Maryland Sea Grant. College Park, MD. pp. 281-288. Wilkinson, L. 1990. Systat: The system for statistics. Evanston, IL: Systat, Inc., 676 pp. Zhou, M., Y. Kao & Y. Wu. 1982. Preliminary studies on hybridization of Crasso.strea gigas with Ostrea rivularis and Ostrea plicatula. J. Fish. China 6:235-241 (in Chinese, with English abstract). Journal of Shellfish Research. Vol. 21. No. 1, 14.'i-155. 2002. MICROSCOPIC ANATOMY OF GONADAL TISSUE AND SPECIALIZED STORAGE CELLS ASSOCIATED WITH OOGENESIS AND SPERMATOGENESIS IN THE CALAFIA MOTHER-OF-PEARL OYSTER, PINCTADA MAZATLANICA (BIVALVIA: PTERHDAE) PEDRO SAUCEDO, CARMEN RODRIGUEZ-JARAMILLO, AND MARIO MONTEFORTE Cenlw de Investigackmes Biulugicas del Noroesle. S.C. La Puz. Buja Cidifoniia Sin: Mexico ABSTRACT As part of a major experiment of (he reproductive biology of wild Phwtada muzatlanica. this study was aimed to characleri/.e the microscopic anatomy of the gonadal tissue and storage cellular elements involved in the seasonal advancement of oogenesis and spermatogenesis. Tissue samples were collected every fifteen days over an annual cycle and processed with histological and histochemical techniques. The heniato.xylin-eosin (cytology description). Blue Alcian-PAS (PAS. for carbohydrates), and Black Sudan (BBS) and Oil Red (OR. for lipids) techniques were employed. Temporal variations of the sex ratio and sexuality of specimens were also analyzed. Gonadal tissue developed synchronously over time at the expense of a matrix of interconnective tissue and reserves stored in the adductor muscle and digestive gland. The interconnective tissue communicates the gonadal tissue with the digestive gland and also serves as substrate for the differentiation of vesicular connective tissue cells and auxiliary cells. In fact, gametes (particularly oocytes) were exogenously nourished by both kinds of cells as gametogenesis advanced. The vesicular connective tissue cells, very abundant among acini, adenomeres, and peari sac, were observed surrounding excretory conduits in the first two tissues, and were identified as highly PAS++ and moderately BBS+ and 0R+. The auxiliary cells were found attached to previtellogenic and vitellogenic oocytes. The endogenous synthesis of lipids during vitellogenesis was associated to the Balbiani body, a storage compartment of the oocyte ooplasm not previously described for any species of peari oyster. The average female/male ratio is 0.35: 1 when specimens were obtained from cultured conditions, but females outnumbered males when collected from the wild. Several cases of protoginic specimens and a few functional hermaphrodite oysters were seen. KEY WORDS: pearl oysters. Pinctcie stage, with empty and collapsed ACI containing some phagocytes (Ph) and abundant ICT; Bl Activation stage, in which only stem cells (Sc» and oogonia (Ogl are attached to ACI walls, and. among them, large amounts of VCT: Cl Early development, dominated by previtellogenic oocytes (PrO) still attached to the acinus wall by the stalk region (Sr) and accompanied by AC and a few vitellogenic oocytes iVOl; Hi Mid-developing stage. with only a few Og and PrO present, while more VO and postvitdlogenic oocytes (PO) start dominating the ACI. The I5h and the mitochondrial clusters (Mc) are common during this stage: E) l.ate development, showing free PO tilling the ACI lumen, hut still leaving a wide inleroocyte space; F) Ripe stage, with large, free PO and the interoocyte space reduced to its mininuuii: Gl Partial spawning, presenting many scattered residual oocytes (RO), resorptive material (RMl. and a few Ph; H) Spent stage, showing empty ACI with atresic oocytes (AOl. RM, and more signs of Ph. Scale bar = 50 pm. Gonadal Tissue and Storage Cells in P. mazatlanica 149 nective tissue grows and appears distended and greatly diffused among acini (Fig. 2A). Sexual Activation Acini look small, round or oval, and easily distinguished from other acini. They are supported by interconnective tissue and nour- ished by VCT cells. Each acinus is lined up exclusively with small (3^ (xm diameter) spherical stem cells and larger oogonias (5-6 \x.m), either attached in clusters to the acinus wail or progressively expanding toward the lumen (Fig. 2B). Apart from these cells no other developmental stage has yet appeared. Early Development Acini begin to grow and enlarge, but their individuality is still clear. Oogonias rapidly proliferate by mitosis and give rise, by centripetal inovenients. to the first previtellogenic oocytes (Fig. 2C). These oocytes are easily distinguished from other types of oocytes by the dark blue-purple color of their ooplasma. They enlarge to a diameter of 14—23 (jim in diameter, lack yolk, and are still connected to the acinus wall by the stalk region, in which some AC are commonly observed. At the end of this stage, a few \ ilellogenic oocytes appear in the acinus, but the interoocyte space is still wide. Mid Development Acini continue to grow, adopting a more complex anastomosed shape. The proportion of interconnective tissue and VCT cells decreases, but AC are still observed. All developmental stages are present within acini, but vitellogenic oocytes — characterized by the growth of the yolk ooplasm and increase in diameter to 30 to 55 |j.m — are the dominant stage (Fig. 2D). This oocyte is still connected to the acini wall, presents a peduncle or pear shape, and shows a large nucleus and one or two dense, blue-stained nucleoli. Two different basophilic structures are observed in the ooplasm for the first time: the Balbiani body (seen as a black spot of variable size and shape) and many smaller, irregularly scattered black spots that probably correspond to mitochondrial clusters (Figs. IF and 2D). Late Development Acini continue to grow and stratify and interconnective tissue and VCT cells to decrease. AC tend to disappear or. if still occur- ring, they appear separated from oocytes. The interoocyte space reduces as previtellogenic oocytes diminish and vitellogenic and postvitellogenic oocytes progressively develop (Fig. 2E). Postvi- tellogenic oocytes are seen free in the lumen, have a polyhedral or polygonal shape, and increase ni diameter to 60-70 \xm. A dense ainorphous mass (probably chromatin) is observed in the nucleo- plasm. The nucleolus is dense, compact, and usually in a marginal position. The Balbiani body and mitochondrial clusters may still be present. Ripeness This stage marks the end of vitellogenesis. Acini are strongly anastomosed and packed almost entirely with mature, free postvi- tellogenic oocytes that attain a maximum diameter of 70-75 (xm (Fig. 2F). VCT cells in gonadal tissue almost disappear and inte- roocyte space is reduced to the minimum. The nucleus now occu- pies a large area in the middle of the oocyte. Many oocytes begin to enter the germinal vesicle stage, in which a gap between the nuclear envelope and the ooplasm appears, indicating a readiness for spawning. Partially Spawned After the germinal vesicle breakdown, oocytes are released outside the acini, which look distended and partially empty (Fig. 2G). Some types of phagocytes and granulocytes appear in the space between the free residual oocytes, which now look rounded or pear-shaped. Large amounts of resorptive material are notice- able. The nucleolus and chromatin disaggregate, but if still present, they may be associated with a new developmental process. This may be a strategy to recycle nutrients (proteins and lipids of the yolk) destined for the new oocyte. Spent Acini are collapsed and empty, with clear signs of phagocytic activity, oocyte degeneration, and much resorptive material. De- pending on the duration of this phase, residual oocytes, especially those of small diameter whose nucleus is not polylobed. undergo atresia (Fig. 2H). This process represents deterioration and disin- tegration of most of the major cellular constituents and thereby the breakdown of the oocyte. Spermatogenesis The step-by-step advancement of the annual spermatogenic cycle in P. mazatlanica is depicted in Figure 3. The stages are described as follows: Sexual Activation Acini initially are small, round or oval, and supported by vas- cular interconnective tissue and VCT cells. The germinal epithe- lium is formed exclusively by a single layer of small (3-4 {xm diameter) spherical stem cells that are strongly attached to the acinus wall and by some spermatogonias starting to differentiate. Development into spermatocytes, spermatides. or spermatozoa has not yet appeared (Fig. 3A). Early Development Acini grow and look enlarged and slightly branched, hut their individuality is still recognizable. Spermatogonias rapidly prolif- erate in a centripetal direction, giving rise to several layers of small (2-2.5 p.m diameter) primary and secondary spermatocytes ex- panding toward the lumen, which are the doininant stage present (Fig. 3B). A few isolated pockets of spermatids and spermatozoa can also be observed. The proportion of interconnective tissue and VCT cells decreases. Mid Development Acini begin to show stratification, causing a reduction of the interconnective tissue and storage cells. Starting from the outer layers to the center, all developmental stages are present: sper- matogonia, abundant spermatocytes, spermatids, and ripe sperma- tozoa (showing their acidophilic tails as pink lines radiating from the center of the lumen) (Fig. 3C). Cell diameter decreases to approximately 1-0.5 ixm. Late Development Acini continue their growth and stratification, appearing highly anastomosed. As a consequence, the matrix, previously occupied by interconnective tissue and VCT cells, has almost disappeared. Spermatogonia and spermatocytes have been reduced in thickness to a few layers of cells located at the periphery. Acini are p,hV,\i 150 Saucedo et al. Figure 3. Photomicrographs of male gonadal lissiu' i4(l\i, showing the sexual stages of spermatogenesis in Piinlada muzatlanica. A I Activation stage, showing AC! filled only with stem cells (St) and sperniatogonias (SPG) proliferating toward the lumen. Ahundant ICT with \ CT cells can he observed; B) Early development, with many SPG and primary and secondary spermatocytes (SPI and SP2| rapidly differentiating into the first pockets of sperniatides (Spl and spermatozoa (S); C) Mid development, characterized by the presence of all type of cellular stages, but more mature S showing their acidophilic tails (T) radiating from the center; Dl Late-developing stage, in which SPCi decrease in number and instead S sharply increase their frequency; E) Ripe stage, evidencing a dense volume of ripe S packing the acini: F) Partial spawning, showing distended but empty acini, residual spermatozoa (RS) and some Ph starting to appear: G) Spent stage, with collapsed ACI and evident signs of RS undergoing cytolysis. Scale bar = 50 nm. Gonadal Tissue and Storage Cells in P. mazatlan/ca 151 with a dense, dark-blue band of deep (Fig. 3D). Ripeness ripe spermatozoa several eel Acini look like a complex network of branched tubules that make distinguishing the boundaries between them difficult. The dominant stage is now the spermatozoa, which strongly pack the acini. Stem cells and spermatogonia are latent and restricted to a thin layer at the periphery of the acini (Fig. 3E). Only a small amount of interstitial connective tissue is evident at this stage. Partially Spawned After spermatozoa are expelled into the surrounding environ- ment, acini walls look broken but still distended (Fig. 3F). Many residual spermatozoa are observed scattered, with the first signals of phagocytic activity. Much residual material is also noticeable. A gap between acinus walls and the mass of residual spermatozoa appears. In some cases, redevelopment or sex reversal processes occur. Spent Because spawning is never complete, the spent stage may be skipped when new gonadal replenishment occurs off the main reproductive cycle. However, when gamete resorption starts, acini look collapsed and empty, with no evidence of active spermato- genesis taking place. This phase is characterized by the rapid pro- liferation of different kinds of phagocytes, granulocytes, and amoebocytes surrounding and destroying residual spermatozoa (Fig. 3G). The matrix of interconnective tissue starts to grow and develop again. Bisexuality (Hermaphroditism) This condition was observed in 8.3'7f of the sample |4() of 480 specimens). Bisexuality was classified as either consecutive sexu- ality or functional hermaphroditism. Consecutive Sexuality The individual functions as one sex when young and later changes to the opposite sex. As for bisexuality. two manifestations of this condition were observed: ( 1 ) Protandrous hermaphroditism: when sex reversal occurs, this is the normal condition reported for many bivalves and pearl oysters. In this situation both germinal lineages overlap in the same acinus, but the male gonadal tissue, which developed initially, undergoes regression and lysis. Only a few residual spermatozoa accompany oocyte development (Fig. 4A). This condition, which corresponds to the relation between stage 7 of spermatogenesis and stages 1. 2. or 3 of oogenesis, was observed in 5.89r of the sample (28 of 480 specimens). (2) Pro- toginic hermaphroditism: observed in only {.1% of the total sample (8 of 480 specimens). Again, both germinal lineages over- lap in the same acinus, but now the male gonadal tissue proliferates as female gametes gradually disappear. Therefore, only atresic oocytes are found in the acini together with developing male ga- metes (Fig. 4B). This condition corresponds to the relation of stage 7 of oogenesis and stages 1. 2. or 3 of spermatogenesis. Figure 4. Photomicrographs of hermaphrodite specimens of Pinctada mazatlaiiica stained with hematoxylin-eosiii. A) Protandrous hermaphro- dite, with active Ph surrounding and destroying RS. while PrO and VO present active development: B) Prologinic hermaphrodite, showing a few RO undergoing regression among mature spermatozoa (MSl filling the ACI; C) Functional hermaphrodite, presenting MS with tails and growing oocytes (PrO and VOl in equal proportion and apparent functionality; D) Functional hermaphrodite, in which both male and female lineages spawned at the same time, and only RS and RO can be seen within ACI. Scale bar = 50 pm. 132 Saucedo et al. Functional Hermaphroditism This condition was detected in 0.87f of tlie sample (4 oysters). Both sexes are present concomitantly within the same acinus in apparently equal proportion and display no sign of gonadal i-egres- sion in either gamete (Fig. 4C). Both lineages may be observed spawning and undergoing regression at the same time (Fig. 4D). Temporal Variation of Gamctogenesis, Oocyte Area, and Sex Ratio Temporal variations of the main developmental stages ot ga- metogenesis. oocyte area, and sex ratio of Pinctada imizatlanica are shown in Table !. Gonadal development started early in Feb- ruary and proceeded synchronously throughout the annual cycle, showing two reproductive peaks, one in March to May ( in which no trace of spawning activity was detected) and other in July to August (concluding with a massive spawning in September and October). Small oocytes were observed in February. June, and October to December. Large oocytes were observed in March. May. and August, corresponding to the two reproductive peaks detected histologically. There were significant differences in the mean area of oocytes over time (F = 102.97; P < 0.001 ). During most of the annual cycle (January to August), when cultured speci- mens were collected, males outnumbered females and led to an average female/male ratio of 0.6:1. From September until Decem- ber, when wild animals were collected, females doubled in number relative to males, yielding an average female/male ratio of 2:1. DISCUSSION In this study of P. mazatkmica, oogenesis and spermatogenesis were analyzed using a scheme of eight stages that adequately describe developmental variations in the microscopic anatomy of the gonadal tissues and storage cells. A 15-day sampling interval was selected as the best procedure for obtaining detailed docu- mentation of the step-by-step advancement of gametogenesis. The timing of sampling also avoided the major problem of missing partial spawns or sex reversal phenomena, and allowed us to ob- serve many unreported phenomena of the reproductive process ot P. mazatkmica. We introduced, for the first time, the activation stage to classify acini in which no other developmental stage rather than oogonias or spemiatogonias can be distinguished. This stage — not pi-eviously described for any marine bivalve — clearly marks the moment of sexual differentiation of germ cells and the very incipient commencement of gametogenesis. Both events occurred early in February to March, when phytoplankton is usually abun- dant in Bahia de La Paz (Lechuga-Deveze 1997) and wild oysters had stored sufficient energy reserves in the adductor muscle and digestive gland for starting gametogenesis (Saucedo et al. 200 Ih). TABLE L Temporal variations in the main developmental stages, mean oocyte area (± standard deviation), and sexual condition oi Pinctada mazatlanica during an annual gametogenic cycle. Oocyte Sexual Condition Shell M F I B F/M Height (mm ± sd) ORI Cult Cult Dev Stage TIM Area (*) (Jim ± sd) (%) (%) (% ) (%» SR 142 ± 7.4 151 + 11.8 Inactive Activ Jan Jan 4004 ± 928 60 50 25 25 15 5 0 20 0.4:1 0.5:1 0.2:1 0.7:1 140 ± 9.9 1 39 + 9 6 Cult Cult E-Dev M-Dev Feb Feb 3020 ± 649 80 50 15 35 0 10 5 5 146 ± 6.5 142 + 6.2 Cult Cult M-Dev L-Dev Mar Mar 4644 ± 868 50 45 25 40 5 0 20 15 0.5:1 0.9: 1 150 ± 10.7 143 -^ 9.5 Cult Cult L-Dev ML-Dev Apr Apr 4318 + 959 60 65 25 25 0 0 15 10 0.4: 1 0.4:1 130 + 7.6 139 + 8.5 Cult Cult ML-Dev L-Dev May May 4566 ± 1 085 50 65 40 25 0 0 10 10 0.8:1 0.4:1 141 +8.1 Cult L-Dev Jun 3009 ± 644 70 30 0 0 0.4:1 149 + 9.3 Cult M-Dev Jun 45 55 0 0 123 + 9.1 Cult L-Dev Jul 3806 ±931 75 25 0 0 0.3:1 148 ± 10.6 139 + 9.8 140+ 10.1 Cult Cult Cult L-Dev Ripe Ripe Jul Aug Aug 4037 ± 1132 45 50 50 45 40 40 0 0 0 in 10 10 1 :1 0.8:1 0.8:1 1.3:1 2.8:1 2.4:1 134 ± 12.2 148 + 10.3 Wild Wild Spawn Spawn Sep Sep 3259 ± 786 40 25 50 70 0 0 10 5 144 + 11.0 Wild Spawn Oct 2981 ±724 25 60 5 10 127 + 10.9 133 ± 10.2 137 + 9.6 Wild Wild Wild Spent Spent Spent Spent Oct Nov Nov 2602 ± 587 20 25 35 55 40 50 20 25 10 5 10 5 2.8:1 1.6:1 1.4:1 1.6:1 1.7:1 129 ± 12.3 125 ± 11.6 Wild Wild E-Dev E-Dev Dec Dec 1608 ±825 25 30 40 50 20 20 15 0 * Oocyte area was evaluated on a monthly basis only. c r, - i ORI = origin of specimens: DEV. STAGE = developmental stage: TIM = timing correspondence (Activ = activation stage: E-Dev -- early development: M-Dev = mid development: L-Dev = late development: M = male: F = female: 1 = inactive onesting: B = bisexual or hermaphrodite: F/M SR = female/male sex ratio). Gonadal Tissue and Storage Cells in P. mazatlanica 153 The developing stage was divided into three subcategories (early, mid. and late) as a strategy to identify, especially in previ- tellogenic, vitellogenic. and postvitellogenic oocytes, the presence of specific cellular structures whose expression is short lived. The Balbiani body and the mitochondrial clusters observed in the oo- cyte ooplasm are examples of these structures. The Balbiani body was initially described by Sastry (1968) in Aequipeaen irradians concentricus. and thereafter, it has been rarely cited (Guraya 1979; Dohem 1983; as cited in de Gaulejac et al. 1993 for the bivalve Pin. nobilis). This structure, not previously described for any spe- cies of pearl oyster, was mostly seen in vitellogenic oocytes and seldom in postvitellogenic oocytes of P. mazatlanica. According to Pipe (1987a. 1987b) and de Gaulejac et al. (1995). this body is probably filled with lipid droplets arising from the degeneration and transformation of mitochondria and other membranous or- ganelles, such as the Golgi complex. However, the mechanisms for the endogenous supply of lipids and other materials needed by the growing oocyte are poorly understood, although pinocytosis was proposed (Pipe 1987a; Pipe 1987b; de Gaulejac et al. 1995). The mitochondrial clusters (whose presence cannot be confirmed using light microscopy, but suggested because of their strong basophilic character), appeared initially near the stalk region and later scat- tered in the vicinity of the nucleus during the previtellogenic and vitellogenic stages of oogenesis. Certainly, their expression is as- cribed to the intense respiratory rate of early and mid-developing oocytes and the active synthesis of energy related to the formation of the yolk molecule. Several bisexual speciinens were detected during the annual cycle. Although sex reversal in pearl oysters is basically protan- drous (Gervis & Sims 1992), many protoginic and functional her- maphrodite specimens were found in this study, especially from September through December, when wild oysters were collected. This alteration in the sexual behavior of specimens was previously considered by Coe (1945) an accidental or abnormal mode of embryonic development, resulting from the failure of the heredi- tary sex-differentiating mechanism to function normally. How- ever, environmental influence is presently recognized as one of the most important factors controlling sexual differentiation of germ cells in the direction of maleness or femaleness. We believe that feinales appear more frequently in the wild population, while males predominate under culture conditions. Thus, oyster's age (as a biotic factor) and the density in which individuals are kept within the culture baskets (abiotic factor) may be important factors regulating sex re- versal from female to male in P. mazatlanica. Taylor ( 1999) reported a similar expression of sexuality in cultured P. iiuirgaritifera. In subtropical areas, such as in Bahia de La Paz. the range of temperature variation over an annual cycle is 1 1-1 2"C (Pearl Oys- ter Project database). Therefore, the energy to fuel gametogenesis. parallel to that arising from food intake, have to come from spe- cialized somatic cells involved in the storage of nutrients (Lubet 1959). According to Mathieu and Lubet (1993), there are three types of cellular elements commonly recognized as participating in these processes in bivalves: specific storage cells (adipogranular cells and VCT cells), intragonadal cells (follicular cells, Sertoli cells, and AC), and muscular cells. We identified the presence of two of them in P. mazatlanica: VCT cells (constituting an integral part of the interconnective tissue matrix, observed in the gonadal tissue, digestive gland, and pearl sac) and AC (within acini, always attached to developing oocytes). The muscular cells were not con- sidered in this study, since their active role upon gametogenesis has been clearly demonstrated for several bivalves (Ansell 1974; Gabbott 1975; Bayne 1976; Taylor & Venn 1979; Barber & Blake 1981. 1991; Epp et al, 1988; Faveris & Lubet 1991; Martinez 1991; Racotta et al. 1998), including the pearl oyster, P. mazat- lanica (Saucedo et al. 2001b). Follicular and Sertoli cells were not observed with light microscopy. Storage cells are expressed dif- ferently between families. For example, Mytilidae possess both types, VCT and ADG cells (Lubet 1959; Bayne et al. 1982; Pipe 1987a; Pipe 1987b; Peek & Gabbott 1989a; Peek & Gabbott 1989b). Glycymeridae only one type corresponding to ADG cells (Mathieu & Lubet 1993), and Ostreidae only VCT cells (Swift et al. 1988), but with an intermediate structure between ADG and VCT cells as described for Mytilidae. In contrast, Pectinidae. Ven- eridae. Cardidae. and Pinnidae possess none of them (Yongqiang & Xiang 1988; Dorange & Le Pennec 1989; Dorange et al. 1989: Rodn'guez-Jaramillo et al. 2001) since they rely mostly on energy stored in the adductor muscle for the synthesis of gametes. Storage cells also show important differences in their bio- chemical composition and ways of releasing nutrients. In this study. VCT cells were strongly PAS-h- and moderately BBS-H and OR-I-. demonstrating that although specialized in the storage of carbohydrates (glycogen), they also contain lipids. In M. edulis. nutrients are released by a progressive autophagic mechanism that involves the sequestration of small membrane-bound vacuoles from the large, stored glycogen vesicle, which causes a reduction in cellular volume (Bayne et al. 1982; Mathieu & Lubet 1993). As proposed by Pipe ( 1987a. 1987b). these vacuoles fuse with the cell membrane and release their glycogen content by eccrine secretion. VCT cells were commonly observed surrounding excretory con- duits in the gonadal tissue and digestive gland, indicating that both tissues must be communicating by the interconnective tissue ma- trix, which probably serves as the vehicle to transport the stored carbohydrates from the gonadal tissue to the digestive gland, or vice versa. Although it is difficult to determine the timing when carbohydrate reserves flow from one tissue to another, we believe that carbohydrates obtained from ingested food are initially used as an energy-rich fuel for the immediate build-up of gametes, and later incorporated into the digestive gland for storage. This pro- posal is in agreement with results of Saucedo et al. (2001b). who observed progressive accumulation of carbohydrates in this tissue, despite the advancement of gametogenesis in the same species (at least during the developing and ripe stages and later during the spawning occurring in October to November). This trend indicates that carbohydrates from the digestive gland are not transferred to the gonadal tissue for usage during the reproductive cycle in course, but instead stored and used to cover further energy demands. This argument also coincides with the finding of Barber and Blake (1981, 1991 ) that the digesti\e gland acts as a short-term storage and transfer site of nutrients to meet the reproductive events in M. edulis. In this study, another cellular storage component associated with the growing oocyte was the auxiliary cells. Although their presence has been associated mainly with the nutrition of previ- tellogenic and vitellogenic oocytes, an alternate function related to the resorption of residual oocytes was hypothesized by de Gaulejac et al. (1995). The first function was attributed to the intimate relation of these cells with oocytes by means of desmosome-like junctions and to their rich content of glycogen granules and lipid droplets. In con- trast, the presence of an active Golgi complex and large autophagic vacuoles suggests these cells may also have the ability to phago- cytize. and as such, auxiliary cells might be implicated in the growlli of oocytes by recycling nutrients originating by phagocytosis. With the results previously reported in Saucedo et al. (2001b. 134 Saucedo et al. 2001c) and those presented in this study, we believe that successful trials for the continuous production of P. imi:allaincii spat can be conducted if nutrients with the correct biochemical composition, energy profile, and quantity are supplied to broodstock during their gonadal conditioning. This is especially important to allow the vital energy storage process, either extracellular (in VCT cells and auxiliary cells) and intracellular (in the Balbiani body) to be ac- complished. More studies on seasonal cycles of expression, mo- bilization, and depletion of VCT cells and AC are needed to con- firm these findings, especially for the role of mantle tissue, which is an impt)rtant site of storage of glycogen and lipids associated with gametogenesis in bivalves. Ultrastructure studies of gameto- genesis are also required. ACKNOWLEDGMENTS This study was done as part of tv\(i institutional projects of CIBNOR on Pearl Oyster Culture and Pearl Induction in Bahi'a de La Paz (Projects PAC-7 and PAC-.^9). Additional grants were prinided by the Con.sejo Nacional de Ciencia y Tecnologia (CONACYT-Mexico. as a Ph.D. scholarship), the Consejo Nacio- nal para la Biodiversidad (CONABIO). and the Fondo Mexicano para la conservacion de la naturaleza (FMCN). The authors are grateful to the following .staff of CIBNOR: Horacio Bervera Leon and Juan Jose Ramirez Rosas for SCUBA diving assistance and collection of oysters. 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Journal of Shellfish Ri'si'tinh. Vol. 21. No. I, 157-162, 2002. GROWTH AND GAMETOGENIC CYCLE OF THE BLOOD ARK, ANADARA OVALIS (BRUGUIERE, 1789) IN COASTAL GEORGIA ALAN J. POWER AND RANDAL L. WALKER Marine Extension Service. Shellfish Research & Aqmwulttire Ocean Science Circle, Savannah, Georgia 3141 1-101 1 Lahoraiory, University of Georgia, 20 ABSTRACT We collected two cohorts ( 1994, 1995) of blood iirks, Anadara avails (Bruguiere 1789) from sets that had occurred on surf clam cages in Wassaw Sound. Georgia. Subsequent to transferal into pearl nets, the arks were suspended from the main dock at the Skidaway Institute of Oceanography on the Skidaway River, Georgia, between January 1995 and April 1997. To determine the reproductive cycle and growth rate, we collected arks monthly, measured each individual for shell length, and took a gonadal sample for histological analysis. Arks from the 1994 cohort grew from a mean shell length of 17.8 mm to 39.9 mm in 15 months, a rate of 1.47 mm per month. Arks from the 1995 cohort grew from a mean shell length of 9.9 mm to 42.6 mm m 16 months, a rate of 2.04 mm per month. Growth was most rapid during the spring and summer months. Of the 747 arks sectioned for histological examination, males dominated the population (66'7r) and 7.2% were sexually indeterminate. A minor spawning event occurred in the winter months of the first year for each cohort (0+ years) followed by a major spawning commencing in April and continuing through the summer months. In Georgia, it appears that blood arks recruit in summer-early fall, and that juveniles grow rapidly to reach sexual maturity at an early size {10-12 mm) and age (-8 months). KEY WORDS: Anadara oralis, arks, gametogenesis, growth, sex ratio, spawning INTRODUCTION A member of the ark shell family (Arcidae). the blood ark, Anadara oralis (Bruguiere 1789) is reported lo inhabit estuarine waters from Cape Cod, Massachusetts, to the West Indies and Brazil, at depths ranging from the low-tide line to >3 m (Abbott 1974; Anderson et al. 1984; Rehder 1981 ; Walker & Gates 2001 ). This bivalve is found over a variety of substrate types, but is most commonly found in sandy deposits (Alexander 1993), The blood ark inhabits Chesapeake Bay waters in area where salinity is above 15 ppt (Chanley & Andrews 1971 ). Blood arks are equivalved, and somewhat oval in shape, ranging in size (shell length) from 28 to 76 mm (Rehder 1981 ). Shell height is slightly less than the shell length, with a reported length/height ratio of 1.16 (Alexander 1993); shell depth reaches approximately 70% of height (McGraw et al. 1996). The blood ark is a short-lived species, which has up to 80% mortality in the third year of life, and a maximum life span of five years (McGraw et al. 1996; Walker 1998). Several ark shell species form the basis of economically sig- nificant molluscan fisheries and extensive culture operations throughout the world (Baqueiro et al. 1982; Broom 1985; Baqueiro 1989; Manzi & Castagna 1989; Nie 1990; Umezawa 1992). The 1997 worldwide harvest of arks iScaiiluirca. Area, and Anadara spp.) was 97,296 metric tons, landed in Cuba. Fiji, Indonesia. Korea. Japan. Mexico, Philippines, and Venezuela (FAO 1999). In the United States, ark resources have been largely ignored by the fishing industry until recently. Some interest was expressed in South Carolina in the 1980s, however, there were problems iden- tifying viable markets (Anderson et al. 1984; Anderson and Ever- sole 1985). Since then a small fishery, primarily for the blood ark. Anadara oralis (Bruguiere 1789), and the ponderous ark. Noeiia ponderosa (Say 1822), has developed in Virginia. Meats are sold primarily as an ethnic food in Chicago, New York. Los Angeles, and Washington D.C. or exported to Mexico (McGraw & Castagna 1994; McGraw et al. 1996; McGraw et al. 1998). The demand for arks has recently outpaced the numbers that can be supplied by the Virginia fishery. Since blood arks have been reported to grow up to twice as rapidly as ponderous arks in the first 2 years after settlement in Virginia waters, it has become the principal aquacultural species of interest (McGraw et al, 1996). Growth rates of Georgia stocks have been reported to almost double those observed in these more northern waters, reaching a marketable size in a 1-year period (Walker 1998). Consequently, the great potential of Anadara oralis as a new commercial re- source in Georgia has been recognized, and an aquacultural fishery is currently being investigated (Power & Walker 2001). Unfortu- nately, little life-history information other than from Virginia (McGraw & Castagna 1994; McGraw et al. 1996; McGraw et al. 1998) exists for this species in the United States. This study de- scribes the growth and gametogenic cycle of the blood ark from the coastal waters of Georgia, MATERIALS AND METHODS We collected blood ark cohorts in January 1995 ( 1994 cohort) and December 1 995 ( 1 995 cohort ) at the mouth of House Creek, Little Tybee Island. Wassaw Sound, Georgia. The clams had at- tached themselves to surfclam (Spisida solidissima similis. Say 1822) grow-out cages (6 mm vinyl coated, 1 x 1 x 0.6 m) that were partially buried (0.3 m) al the spring-low-water mark on a sand flat. Cages had been deployed in September of both years and when we stocked with surfclam seed in October, we noted that ark wild spat had settled and attached with byssal threads to the wire mesh sides of the cages. In the laboratory on Skidaway Island, we measured for shell length (longest possible measurement, i.e., an- terior-posterior) using Vernier calipers. Arks from the 1994 and 1995 cohorts had a mean shell length of 17.8 ± 1.52 (SE) mm (range: 1 1.9-28.3 mm) and 9.9 ± 0.19 mm (range: 4.3-17.7 mm), respectively. Each cohort was placed in a separate 3 mm mesh pearl net, and suspended from a floating dock on the Skidaway River. The 1994 recruitment was a small set (approximately 20-30 arks per cage), while the 1995 recruitment was a massive set with over 500 arks collected per cage. Between January 1995 and April 1996 we randomly collected arks (N = 10. 1994 cohort) from the pearl nets. We repeated this procedure for the 1995 cohort collecting arks (N = 30) moniliK between December 1995 and April 1997. We measured ihesc .nk^ 157 158 Power and Walker for shell length, and dissected a mid-lateral gonadal sample (ca. I cm") from each animal. Smaller speciinens with meats ni (he Kamiiv Arcidae. Shell Length at Initial Gametugenesis (mm) Shell Length at Sexual Maturity I mm) Species -Male Female Not Stated Male Female Not Stated Source Broom. 1985 21 Narasimhum. 1968 24-25 Broom. 1983 This study Natarajan & John. 1983 30 Baron. 1992 Tmg et al.. 1972 Walker & Power (in press) Broom. 1985 Yolove. 1974 Amiduni cornea Amuiiini i^ranosit Anadara grunosa Anadara avails Anadara rhombea Anadara scapha Anadara siibcrcnala Anadara transversa Anadara mberctdosa Senilia senilis 4 19 20 17.5 22 15 10 10-17 10 21-25 !l-25 36 present study (1.47 and 2.04 mm/mo) were comparable to earlier reported growth rates for natural populations in Virginian waters, and cultured arks in the coastal waters of Georgia. In Georgia, arks that were similarly grown in pearl nets for 36 ntonths exhibited rapid growth in the first year (<10 mm size class: 2.58 mm/mo; 10-20 mm: 2.01 mm/mo) with decreased annual growth for years two (0.83 and 0.89 mm/mo) and three (0.19 and 0.18 mm/mo) (Walker 1998). In Virginia, blood arks grew from an initial size of 14 mm to 30 mm in 1 1 months, a lower rate of 1.45 mm/mo (McGraw & Castagna 1994). Arks were noted in the present study to grow faster in the spring and summer months, therefore the slower growth rates in Virginia may result from colder water tem- peratures. Sexual maturity was attained at an approximate mean size of 10 mm in shell length for males and 12 mm for females (age 7 to 8 months) however: gametogenesis was noted to occur at minimum sizes of 4 mm and 7 mm, for males and females, respectively. Animals from the 1995 cohort were known to be at least three months old when gathered in December, since the cages were deployed in September. At this time. 20% of the 225 collected were ripe and 44% were in the late active stage. Because major spawning for blood arks starts in April-May. the maximum age of the new recruits on the cages is about 8 months. We believe that blood arks had already recruited to the sand flat prior to the place- ment of the field grow-out cages. Once the cages were in place, we postulate that the arks migrated up the sides and attached them- selves with byssal threads. Blood ark juveniles are commonly found attached to the upper ends of Dioptera tubes in the estuary (Walker, personal observations). An upward migration of juvenile arks has also been observed with transverse arks. Anadara trans- versa within the laboratory. Transverse arks contained within a northern quahog. Menenaria menenaria. seed shipment were ob- served to migrate to the top of the quahog seed mass and even up the sides of the holding tray to the surface/water interface (Walker & Power, in press). Members of the Arcidae are reported to initiate gametogenesis and reach sexual maturity at a small size (Table I ). The blood arks mature at a similar age to Anadara granosa (Lin- naeus 1758: Narasimham 1968) and Senilia senilis (Lamarck 1758: Yoloye 1974). but typically at a smaller si/e than most of the family (Table I ). Temperature is one of the main exogenous factors controlling reproduction in marine invertebrates (Giese 1959; Sastry 1975). In Virginia, blood arks are reported to spawn in the summer months when water temperatures reach abo\e I7°C (Chanley & Andrews TABLE 2. The sexual ratios and percentage hermaphroditism of various marine bivalves species from the Family Arcidae. Species Sample Sexual Ratio Size (Female:Malel 1040 1.00:1.00 300 1.00:1.00 185 1.00:1.98 693 1.00:2.44 1155 1.(10:1,27 235 1.00:1.47 100 1.00:0.34 1.00:1.00 1.00:1.00 199 1.00:0.53 1.00:1.00 218 1.00:1.25 1094 1.00:1.00 1.(10; 1.00 181 1.(10:1.26 Hermaphroditism Source Anadara anlicjuala Anadara granosa Anadara ovalis Anadara ovalis Anadara rhombea Anadara scapha Anadara senilis Anadara senilis Anadara subcrenata Anadara transversa Anadara trapezia Anadara tuberculosa Anadara tuberculosa Anadara tuberculosa Noetia ponderosa 0.003 0 0 0 0.004 Toral-Barza & Gomez. 1985 Broom. 1983 McGraw et al.. 1998 This study Natarajan & John. 1983 Baron, 1992 Yoloye, 1974 Broom. 1985 Broom. 1985 Walker & Power (in press) Broom. 1985 Cardenas & Aranda. 2000 Cruz. 1984 Dzyuba & Maslennikova. 1982 McGraw et al.. 1998 The Blood Ark in Coastal Georgia 161 1971; McGraw et al. 1998). In Georgia blood ark^ spawned earlier, from late spring ihrough summer, presumably due to the coastal waters warming earlier in the year than in Virginia. By April-May 1995 and 1996, water temperatures were already above 20°C in the Skidaway River. In general, spawning periods and gametogenesis in marine bivalves start earlier and last longer in southern geo- graphical areas than in northern ones (Eversole 1989; Thompson et al. 1996). In this study, males dominated the population of blood arks with an overall sex ratio of 2. 38; 1.00 (M:F). This is in agreement with the observed sex ratio (1.98:1.00 M;F) reported by McGraw et al. (1998) for a Virginia population of 1-year-old blood arks (Table 1 ). An equal sex ratio has been observed in Andara gnmosa (Pathansali 1966; Broom 1983) and Anadaia siibcreiiata (Lischke 1869; Ting et al. 1972): however, males dominated Senilia senilis populations (Yoloye 1974). For Anadara scapha (Linnaeus 1758), males were reported to dominate in the smaller size-classes while females were more frequent in the larger-size classes (Baron 1992). This may indicate a sex change but may also be a conse- quence of the differential energy requirements of males vs. fe- males. No hermaphroditic Anadara oralis were observed in the present study. Hermaphroditism is rare in the Family Arcidae (Table 2); Anadara scapha and Senilia senilis have been classified as a protandric hermaphrodite (Baron 1992; Yoloye 1974). The present study examined newly recruited blood arks, while in Vir- ginia 1-year-old indi\ iduals were examined. Males typically domi- nate protandric bivalve species in the first year; whereas older age classes are generally equal. Blood arks can reach a maximum of 5 years and thei'efore these older age classes need to be examined to determine if this species is protandric. ACKNOWLEDGMENTS This work was supported by the University of Georgia Marine Extension Service. The authors thank Ms. Dodie Thompson for processing the histological samples. LITERATURE CITED Abbott, R. T. 1974. American Seashells. Second Edition. Van Nostrand Reinhold Co., New York, 663 pp. Alexander, R. R. 1993. Correlation of shape and habit with sediment grain- size for selected species of the bivalve Anadara. Lethaia 26:153-162. Anderson. W. D., W. H. Lacey, III & A. G. Eversole. 1984. An investi- gation concernmg the feasibility of harvesting and marketing underuti- lized Anadara off the coasts of South Carolina and Georgia. Report prepared for the Gulf and South Atlantic Fisheries Development Foun- dation under Gram Contact No. CASAFDl 22-18-17320. Anderson. W. D. & A. G. Eversole. 1985. Arks - Is there a resource and a market?/ Shellfish Res. 5:31 (Abstract). Baqueiro, E. C. 1989. Clam culture in Mexico: Past, present and future. In: J. J. Manzi & M. A. 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Final Report submitted to Saltonstall-Kennedy Program Office of the National Oceanic and Atmospheric Administra- tion, National Marine Fisheries Service, NOAA Grant No. NA46FD0339. 62 pp. McGraw. K. A., M. A. Castagna & S. D. Dennis. 1998. The arkshell clams Noetia ponderosa and Anadara ovalis in the oceanside lagoon system of Virginia: A study of predation. reproductive biology and condition index. Final Report submitted to Sallonslall-Kennedy Program Office of the National Oceanic and Atmospheric Administration, National Marine Fisheries Service, NOAA Grant No. NA66FD0010. 59 pp. Narasimham, K. A. 1968. Studies on some aspects of biology and fishery of the cockle. Anadara granosa (Linnaeus), from Kakinada Bay. Pro- ceedings of the Symposium on Mollusca, Part II Symposium Series 3:407-117. Natarajan. R. & G. John. 1983. Reproduction in the edible ribbed clam Anadara rhomhea (Born) from the backwaters of Porto Novo. Induin ./. Marine Sci. 12:90-95. Nie. Z.-Q. 1990. 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Marine Fisheries Service 106:105-1 14. Walker. R. L. 1998. Growth and survival of the blood nrk. Anadara ovalis (Bruguiere 1789) in coastal Georgia. Georgia J. Sei. 56:192-205. Walker. R. L. & K. W, Gates. 2001. Survey of ark. Anadara ovalis. Anadara hrnsdiana. and Noetia ponderosa. populations in coastal Georgia. School of Marine Programs, Marine Technical Report No. 01-2. }4 pp. Athens, GA. Walker. R. L. & P. B. Heffernan. 1994, Temporal and spatial effects of intertidal exposure on the gametogenic cycle of the northern, Merce- naria niercenaria (Linnaeus. 1758), in coastal Georgia. 7. Shellfish Res. 13:479-486. Walker, R. L. & A. J. Power. In press. Growth and gametogenic cycle of the transverse ■dT\^. Aiiadioa transversa (Say. 1822) in coastal Georgia. American Malacological Bulletin. Yoloye, V. 1974. The sexual phases of the West African bloody cockle .Aiuidia-a seinlis (L.). Proceedings of the Malacological Society of Lon- don 41:2.5-27. Jounuil of Slu'lifish Research. Vol. 21. No. I. 16.V17I, 2002. TEMPORAL FLUCTUATIONS (1989-1999) IN THE POPULATIONS OF HALIOTIS FULGENS AND H. CORRIJGATA (GASTROPODA: HALIOTIDAE), AT ISLAS SAN BENITO (BAJA CALIFORNIA, MEXICO) J. A. RODRIGUEZ-VALENCIA* AND F. CABALLERO-ALEGRIA Institiito Naciunal dc la Pesca. CRIP-Euseiuuki. Carrctcni Tijiuiini-Eiisciuiclci Km 1(17. Paiyiic Iiulustriul Fondeport, El Sauzal. Baja Cullfornln. Mexico ABSTRACT In our study area, green abalone (Haliotis jidi^cns) was more abundanl than pink abalone (H. conui^atu) until the end of the 1980s, but nowadays, both .species have similar density levels and are close to values were recruitment failures may occur. In compiirison to pink abalone, green abalone showed higher abundance of legal-sized stock and condition factors. Abundance of the legal-sized stock of both species has significantly decrea.sed. but the density extracted by fishing remained the same. Overfishing of legal sizes and recruits, or poaching, may have not significantly contributed to the observed patterns. Only the proportion of recruits of pink abalone decreased significantly along the time and its fluctuation patterns support the idea that non-severe "El Nino" events may have a positive effect on recruitment. The lack of significant changes in recruitment of green abalone suggests that this effect may be species-specific. On the other hand, our results indicate that "El Niiio" events lead to poor physiological condition. Mean condition factors of both species decrea.sed significantly along the time, and are significantly related with the mean annual surface temperatures. The proportion of sexually mature organisms of both sexes, in both species, also decreased significantly along the time and could be a by-product of impoverished condition. Additionally, the sex ratio of green abalone varied significantly along the time, in favor to males. KEY WORDS: abalone surveys, long-term dynamics. Baja California. Hiilioii\ fi(li;eii.\. H. ctirnigala INTRODUCTION The quanlificatioii of abalotie and the assessment of its tempo- ral fluctuations are often difflcult tasks. Aggregated spatial distri- butions difficult the obtainment of precise abundance estimates at large spatial scales (McShane 1995), and even at small spatial scales estimates may have with high variance and low precision (McShane 1994). The first attempts to quantify abalone in Baja California were done between 1968 and 1970, covering wide spatial scales along the coast (Guzman-del-Proo 1992). Mean density estimates de- rived from these attempts had very low precision, due to habitat heterogeneity (Guzman-del-Proo 1992), After 1988, abalone sur- veys were restricted to each fishing area, in order to calculate local yearly catch quotes, and regulate the fishery. We present the re- sults of abundance surveys made between 1989 and 1999, for green abalone {Huliotis fulgens. Philippi 1854) and pink abalone {H. corntgata. Gray 1828), at Islas San Benito. These three islands are located at the current main abalone tlshing area of Mexico. This work brings information about one of the few long-term monitoring efforts applied to natural abalone banks in North America. Additionally to density values, we bring data that are rarely collected during surveys: namely, sizes, condition factors. sex ratios, and gonadal maturity of both species. We tried to assess the contribution of commercial extractions, overfishing of legal sizes, recruitment overfishing, and local climate forcing to the observed fluctuations. MATERIALS AND METHODS Study Area Islas San Benito are located at the Pacific coast of Baja Cali- fornia, 40 km northwest from Cedros Island (Fig. la). They are *Corresponding author. J. A. Rodriguez- Valencia. Alfred-Wegener- Institute, Wadden Sea Station Sylt., D-25992 List., Germany. E-mail: jrodriguez@awi-bremerhaven.de away from continental runoff and pollution, and strong seasonal upwelling favor the growth of Macrovyslis pyrifera, Eisenia arhorea, and Egregia menzie.ssi ( Gonzalez- Aviles & Shepherd 1996). The extension of the abalone fishing area is 6.862.155 m~ (Arano-Castanon & Uribe-Osorio 1998). The fishermen co- operative "'S.C.P.P. Pescadores Nacionales de Abulon" is the unique concessionaire of the abalone and lobster fishery in the area. Survey Methodology Surveys were conducted in the whole fishing area of the is- lands, when the fishery is closed (October to January), between 1988 and 1999. The coastline of the three islands has always been divided into 35 sections (=500 m width) placed in the same loca- tion during all the surveys, using floats as markers. The offshore extension of each section has been fixed at the 30 in isobath; therefore, the length of each section varied between 88 m and 1473 m. perpendicular to the shore (Arano-Castanon & Uribe-Osorio 1998) (Fig. la). Abalone divers from the fishermen cooperative collected all visible abalone inside each section, but the sampling procedure varied along the time (Fig. lb): 1989 to 1995. Six to 12 sampling stations were randomly distrib- uted inside each section, depending on the number of divers. A 4 nr-quadrat was used as a sampling unit from: 1996 to 1999, Each section was divided into three bathymetric strata: 0-10 m, 10-20 m, and 20-30 m. Three, five, and two sampling stations were randomly located inside each bathymet- ric strata, respectively. A 10 nr-quadrat was used as sampling unit. Density Estimations Only sampling stations located on rocky bottom were used for density estimations. The mean density was calculated consider!'': 163 164 Rodriguez-Valencia and Caballero-Alegria 1989-1995 1996-1998 6-12 sampling points randomly distributed Inside each section I- 3 sampling points randomly distributed inside this bathymetric strata Sampling unit: 4 m"! quadrats t 5 sampling points ' randomly distributed ,"~ - ^ « " 1^ -„ • ,. .^ \ J .J t. .1. _ . • ' \ « •'■*■*• 20-m isobath inside this bathymetric I x " \ ^^ " ^ '^30-m isobath strata ^^f__ 2 sampling points randomly distributed Sampling unit: 10 m*2 quadrats inside this bathymetric strata Figure. 1. (a) Location of the study area. The dashed line represents iVi-va isobath. (b( Scheme of the sampling method. all sizes. Estimations of the mean density of the legal-sized stock were done considering only sizes larger than the minimum legal size of capture (green abalone: 150 mm of shell length; pink aha- lone: 140 mm of shell length). Ninety-five percent confidence limits and standard errors of the total mean density, as well as 959r confidence limits of the mean density of the legal-sized stocks are shown. Significant temporal differences in the mean values were tested with ANOVA, in both species. We estimated the statistical power for the total mean density comparisons, at the given level of replication, and a = 0.05 (Zar 1999). Recruitment Estimations Our survey methodology is inadequate to estimate recruitment accurately, but serious recruitment failures due to recruitment overfishing or environmental changes should be reflected in our data (Shepherd et al. 1998). From the 1989 to 1998 for size-frequency data, we calculated the percentage of recruits of both species, using the size criteria of Shepherd et al. (1998). Overall, significant differences between percentages (inside each species) were tested with Chi-squared analyses (Zar 1999). Paired comparisons are shown, to estimate statistical power (Zar 1999). Assessing the Effect of the Fishery The density extracted by fishing, between 1989 to 1996. was estimated for each species. The following data were used: local captures provided by the fishermen cooperative, legal-sized stock densities, local area of suitable habitat for both species (Rodriguez- Valencia et al. 1998). and mean weight of muscle (as described later). Significant temporal changes were tested by Chi-squared analyses. Biomelric Data Biometric data of all captured abalone are available for the 1989 to 1998 surveys. Sizes and Weights Shell lengths of all captured abalone were measured to the nearest millimeter and muscles were weighed to the nearest gram. Each 5-mm size category contained at least three individuals. Since measuring and weighing is time-consuming, abalone were maintained in shadowed places and were covered with wet algae to prevent desiccation. Significant temporal differences in mean shell sizes were tested with ANOVA. Significant ascendant or descen- dant trends were tested by the significance of the slope (Beta) of simple linear regressions using the time as independent variable. Modal and maximum sizes of each species, at each survey, were compared using Chi-square tests. Condition Factor The condition factor of each sacrificed abalone was obtained dividing the muscle weight by the shell length, and the mean condition of each species at each survey was estimated. Significant temporal changes between mean condition factors were tested with ANOVA. Sex Ratio and Gonadal Maturity Sexes and gonadal maturity were determined to randomly se- lected abalone at each survey. Significant temporal changes in both variables were tested using Chi-square analyses. Fluctuations of H. fulgens and H. corrugata 165 Haliotis fulgens Haliotis corrugata a) Q TO < b) Isla Natividad (Shepherd at al. 1998) Islas San Benito (This work) 05 07 >, E w 0) c 5 n ?R 0) o Q CO n < 0 c) uu/ 'cO ^ 0 035 < d) 0) c Q. 1988 1990 1992 1994 1996 1998 1988 1990 1992 1994 1996 1998 1989 1991 1993 1995 1997 1999 1989 1991 1993 1995 1997 1999 Year Year Figure. 2. (a) Temporal patterns of total mean densities and their 95 "7^ confidente limits. In) represents the number of sampling stations located on rocky bottom: (b) Temporal patterns of the mean density of legal-sized stock and their 95% confidence limits; (c) Temporal patterns of the density extracted by fishing; (d) Percentages of recruits. RESULTS Patterns of the Total Mean Density Green abalone was 2.5 times more abundant than pink abalone at the end of the 1980s, but at the beginning of the 1990s its density decreased, while that of pink abalone increased. After 1995. both reached their lowest density levels and followed comparable abun- dance trends (Fig. 2a). Temporal fluctuations are significant (Table 1 ). Modifications in sampling methods were more precise after 1995. because the confidence limits and standard eiTors of the means decreased (Fig. 2a and Table 2). 166 Rodriguez-Valencia and Caballero-Alegri'a TABIE 1. Assessment of significant temporal changes in thf loUil iman {lensit\ of //. fiilgeiis and //. coiniifula and istimates of the statistical poHcr. H. fiilgeiis H. iiirnigata Comparison ^(df.Kflett. dr.Error) Effect MS El rror MS * Power r,di.l-.rfi- Ll. dl.I-;rr.)n Effect MS Error MS <> Power 1989 10 1991 15.3 0.7 2.3 0.9 F,,,4.M = 5.5 = 1.3 0,2 1,0 0.6 19X9 to 1993 F,,,„„„ = 16.9** 9.9 0.6 2.8 1.0 F„,,„„, = 14.9** 8.4 0.6 2.6 1.0 1989 to 1995 F,,,,„ = 24.7** 12.0 0.5 4.2 1.0 •"11,785) = 23.1** 11.1 0.5 4.2 1.0 1989 to 1996 F,4.M60, = 27.7** 11.3 (1,4 5.2 1.0 ^(4.1 Ihll) = 34.0** 11,3 0,4 5.1 1.0 1989 to 1997 F„.„c„, = 25.0** 9.4 0.4 5.5 I.O F|?.l4y7( = 30.3** 10.2 0.3 5,5 1.0 1989 to 1998 F„,„„ = 28.6** 9.9 0.3 6.4 1.0 P(h. 17491 = 25.7** 8.6 0.3 6.0 1.0 1989 to 1999 •|7,207til -''-- 9.6 0.3 7.0 1.0 F,7,:,m„ = 11.9** 1,9 0,2 3.9 1.0 * = P < 0.05. *" P <0.0\. Patterns of the Mean Density of Legal-Sized Slocks and Density Exlrueted by Fisliing The pfopoi'tmn of har\ esl;ible sizes of green iibalone has been twice as high as that of pink abalone (Table 3), and its density of legal-sized stock is significantly higher (f, 1.3499, = 189.3**). The mean density of the legal-sized stock of both species has signifi- cantly varied and decreased along the time (green abalone: ^(6, 1744, = 34.0**; pink abalone: F,f,.i744, = 8.0**) {Fig 2b). On average, the fishery extracted 24% and 14% of the legal-sized stocks of green and pink abalone. respectively, coiresponding to 15% and 7% of their total mean densities. The extraction by fish- ing has remained constant, since the densities extracted by fishing of both species remained constant along the time {green abalone: ^-^^^ = 0.02: pink abalone: x',^, = 0.004). although that of green abalone seems to have increased {Fisi 2c). Patterns of Sizes and Condition Mean sizes of both species showed significant temporal varia- tions {Table 3) without trend (green abalone: F,, j,, = 1.2: pink abalone: f , , ,,, = 3.5). Modal and maximum sizes showed non- significant tempoial variations (Table 3). The mean condition of green abalone was significantly higher than that of pink abalone during the studied period (1.6 vs. 1.1; f(i 11771 = 196.7**). No significant differences were detected between sexes, inside each species. Significant temporal changes in the mean condition of both species were detected (green aba- lone: f,,,.io.i2, = 10- 1**: pink abalone: F,f,^^^, = 3.0**). and both species showed significant decreasing trends (Fig. 3a). The mean condition of both species was significantly related with the mean annual surface temperature (Fig. 3b). which increased 2.rC be- tween 1989 to 1998. in the studv area. Recniilment Estimations Recruits of pink abalone have been more abundant than green (Fig. 2d). Overall comparisons between the percentages of recruits of green abalone indicated non-significant changes (x"(6i = 1-8). but those of pink abalone varied significantly (x'tb) = 19.5**). with increasing percentages until 1995 and decreasing percentages after. Paired comparisons indicated non-significant changes for green abalone, and significant changes for pink abalone (Table 4). The average power of the paired comparisons was 0.81 and 0.83. for green and pink abalone. respectively. TABLE 2. Standard errors of the total mean density estimations (Abalone*m"-). Haliolis eornigala 0-030 0.039 0.042 0.027 0,017 0,017 O.OIX 0,021 urvey Haliolis fnlgens 1989 0.06 1 1991 0.045 1993 0.046 1995 0.028 1996 0.026 1997 0.028 1998 0.025 1999 0.027 Patterns of Sex Ratio and Gonadal Maturity The sex ratio of green abalone showed significant temporal changes in favor of males, while that of pink abalone remained close to 1:1. without significant changes (Table 5). The proportion of mature organisms of both sexes, inside each species, decreased significantly along the time (Table 4). In 1995. a conspicuous peak of mature organisms of both sexes was detected, in both species. Relationships between the proportion of mature organisms of green abalone and its mean condition factors and mean annual surface temperature were suggestive, but non-significant (Fig. 3 c-d). On the other hand, these relationships were significant for pink abalone (Fig. 3 c-d). DISCUSSION The Surveys We have tried to follow the survey approach proposed by Hil- born and Walters (1992), Van der Meer ( 1997), and Gorfine et al. ( 1998). distributing sampling points in the whole capture area and applying low sampling effort at each point. The method used to estimate abalone abundance seems to be appropriate. However, main disadvantages are: (a) The small portion of potential habitat sampled (().01%-0.02% between 1989 to 1995; 0.05%^^-0.07% be- Fluctuations of H. fulgens and H. corrugata 167 TABLE 3. Sizes (minimum, maximum, mean and its 95% confidence limits, and m MSC Min. Max. Mean Mode > MSC 1989 1991 1993 1995 1996 1997 199S 1999 Temporal 721 303 269 137 1030 1089 364 721 comparisons 47 31 .56 99 23 S-) 40 87 2113 196 193 190 197 200 197 212 M = 0,6 152, 7± 1 148 8 ± I 152,3 ±2, 151.2H 151.9± 1. 153,2 + 150,2 ±; 146,7 ±; ,4,47, = 5,4** 15(1 140 150 160 150 160 156 138 ,,, = 1,2 66,4 269 73 169 51 8 311 65 173 64 9 350 (i6 175 65,0 159 75 180 63,9 827 29 187 66.7 847 46 190 55,2 598 54 180 42,4 269 48 177 = 19 F,.: 131, 8± 1,8 130 125,9± 1,8 120 1 3 1 ,2 ± 1 6 130 133,0 ±2,6 130 131, 6± 1,2 135 134,2 ± III 140 131,1 + 11 135 135.5 ±2.0 128 ,,,74, = 11.3** X^M = 6.0 28.3 19.6 29.7 38.4 30.5 39.6 23.4 42.4 tween 1996 to 1998); and lb) The lack of use of specific methods to properly quantify Juveniles (e.g.. anesthetics (Prince & Ford 1983); sub-aquatic amplifiers (Shepherd & Turner 198.3); suction method (McShane & Smith 1988). Differences in efficiency among divers were surely low. since only on-duty abalone dixers participated, and they are highly efficient locating abalone (Prince & Gu/.man-del-Proo 1993). Additionally, the use of quadrants and di\ ing transects prevents over estimations and competition among divers (Findlay & Willerton 1996. McShane 1996). Shepherd et al. (1998) stated that Mexico is the only country making serious ef- forts to quantify the abundance of its abalone banks; nevertheless, surveys are frequently criticized by local cooperatives and aca- demics. TABLE 4. Paired comparisons of the percentage of recruits of H. fulgens and H. corrugata belween surveys and their statistical power. Pair iA isons H. fulgens H. corrugata Compar Z-value Povier Z-value Power 1989 vs. 1991 0.21 0.96 0.73 0.86 1989 vs. 1993 1.03 0.76 1.70 0.60 1989 vs. 1995 0.06 0.62 4.23* 0.99 1989 vs. 1996 1.40 0.68 0.42 0.92 1989 vs. 1997 0.24 0.94 0.72 0.85 1989 vs. 1998 1.70 0.60 1.26 0.70 1991 vs. 1993 0.62 0.81 0.86 0.84 1991 vs. 1995 0.21 0.95 3.62* 0.95 1991 vs. 1996 0.93 0.81 0.40 0.91 1991 vs. 1997 0.11 0.95 1.96* 0.55 1991 vs. 1998 1.05 0.71 2.42* 0.71 1993 vs. 1995 0.60 0.87 2.86** 0.83 1993 vs. 1996 0.22 0.95 1.70 0.60 1993 vs. 1997 0.89 0.74 3.34** 0.91 1993 vs. 1998 0.01 0.94 3.70** 0.96 1995 vs. 1996 0.74 0.90 4.91** 1.00 1995 vs. 1997 0.01 0.96 6.50** 1.00 1995 vs. 1998 0.89 0.82 6.60** 1.00 1996 vs. 1997 1.55 0.60 1.90 0.55 1996 vs. 1998 0.26 0.91 2.43* 0.73 1997 vs. 1998 1.61 0.63 0.66 0.88 * = P < 0.05. ** = P < 0.01. Interpretation of Abalone Dynamics Both species reflect the decline of the Mexican abalone fishery. observed since 1984 (Guzman-del-Proo 1992; Shepherd et al. 1998). and their abundance patterns are similar to those reported for Isla Natividad (Shepherd et al. 1998). Their most recent density levels are close to values where recruitment failures may occur (Shepherd & Partington 1995) and a clear tendency to reco\er is still absent. Vega et al. ( 1997) and Shepherd et al. ( 1998) proposed causing factors for the decline. Here we discuss the local role of some of these as follows: (a) overfishing of legal sizes and/or recruits; (b) negative effect of "El Nino" events over recruitment (indepen- dently of their intensity); and possible increases of recruitment during non-severe "El Nino" events; (c) physiological weakening durino "El Niiio" events. Overfishing of Legal Sizes and/nr Recruits Overfishing can happen by fishermen cooperatives not respect- ing authorized fishing quotes, poaching, cooperatives fishing at erroneously calculated quotes, or the mixture of all of them. The first two choices have not occurred at Islas San Benito, since the local cooperative has been exemplarily adhered to the regulatiims and authorized quotes. Additionally, the location of the islands, and strict surveillance programs applied to owners and strangers resulting in rare poaching. The absence of significant temporal changes in the recruits of green abalone support our suppositions, and although that of pink abalone changed significantly, there is no reason to believe that only this species has been affected by poach- ing or recruitment overfishing. Changes in the percentage of re- cruits of pink abalone may be related to climatic changes, (see later). It seems that fishing quotes were eiToneously estimated, since densities extracted by fishing remained constant, while the abundance of the legal-sized stock decreased significantly in both species. Catches of green abalone have been higher than that of pink abalone. at Islas San Benito (Fig. 4). Pink abalone was the dom- inant species in the local catches between 1957 to 1984 and green abalone became dominant until 1985 (S. A. Guzman-del-Proo. pers. comm.). Nowadays, green abalone is preferably fished for having a higher proportion of harvestable sizes and being more 168 Rodriguez-Valencia and Caballero-Alegri'a a) o C o o c CC b) c) d) Haliotis fulgens Mali Otis corrugata 1.4 0.8 n=471 -3: n=65 n=137 |n=93 I •I' n=97r n=62 n=115 ,x. '■ f^= 0.53 b= -0.034 (F(, .5)=7.9; p<0.05) 0.8 b=-0.02(F,i. 0,70 5,=15.1;p< 0.05) n=268 n=110 n=109 n=59 "X- - i- -^ n=159 .:: n=103 n=44 -- 1989 1991 1993 1995 1996 1997 1998 ' 1989 1991 1993 1995 1996 1997 1998 Year c O 1.5 § 1 O i 05 /^= 0.94 b=-0.2(F,, 5)=75.9;p<0.01) 1.5 0.5 Year i^= 0.93 t)=-0.15(F|i.s)=45.5;p<0.01) 18 19 20 Temperature (°C) 21 18 19 20 Temperature (°C) 21 50 0) s § 25 O q5 CL i^= 0.60 b=0.78(F(i 4,=6.1;p>0.05) 50 25 r^=0.74 t)=0.86(F,i.4)=11.5;p<0.05) 2 0 Mean condition 50 Q) O) CO ^ 25 O 0) Q. i^= 0.56 b=-0.74(F,i,4,=5.1;p>0.05) 50 25 Mean condition i^= 0.80 b=-0.89(F,i,4,=16.6:p<0.01) 18 19 20 Temperature (°C) 21 19 20 Temperature (°C) 21 I eriipeidiuic V ^; ' > ^ ' Fiaure ^ (a) Temporal patterns of the mean condition factors. (») represents the number of analv/.ed organisms; (b» Relationship bet« condition factors and mean annual surface temperatures; (c» Relationship between the percentage of sexual mature organisms . condition factors; (d) Relationship between the percentage of sexual mature organisms and mean annual surface temperatures. een mean and mean revenue yielding by having larger sizes and shells that are more valuable. Fishing has been surely more important tor the decline ot green abalone than for that of pink abalone. It would be desirable to have estimations of mortality by fish- ing, but this is out of our reach. We can only speculate that they would be lower than those at Isla Natividad (Shepherd et al. 1998), since captures of both species have been much higher at Isla Na- tividad (Fig. 4). but Isla Natividad and Islas San Benito have similar density levels. Additionally, quotes at Isla Natividad have been fixed considering an extraction of 30% of the legal-sized stocks (Shepherd et al. 1998) and this percentage at Islas San Benito has been lower. Fluctuations of H. fulgens and H. corhi'gata 169 TABLE 5. Sex ratio ( 9 : c? ) of //. fulgens and H. cornigata. at Islas San Benito. Surve> H. fulgens H. cornigata 1989 1991 1993 1995 1996 1997 1998 Temporal variations 1:0.8 1:0.7 1:0.7 1:1.3 1:1 1:1 1:1.5 = 20.7* 1:1 1:1 1:0.9 1:1 1:1.5 1:1.5 1:0.9 S.5 "" P <0.Q\. Negative Effect of "El Nino" Events over Recruitment (Independently of their Intensity); and Possible Increases of Recruitment During Non-severe "El Nii'io" Events According to Vega et al. (1997), increased water temperatures during "El Nifio" events reduce the extensions of Icelp beds and negatively affect the recruitment, because larvae are taken away from the banks by the currents. The frequent occurrence of "El Nifio" events during the last decade (Fig. 5) should have continu- ously affected the recruitment, but our results of green abalone do not support this. The recruitment patterns of pink abalone support the hypothesis about a possible positive effect of non-severe "El Nino" events (Shepherd et al. 1998). Increasing recruitment was detected until 1995. because between 1981 and 1988 larvae settled under the influence of non-strong "El Nino" events (Fig. 5). but the 1981 to 1986 "El Nifio" was one of the strongest ever observed (Amtz & Fahrbach 1996). Decreasing percentages were detected after 1996. because after 1989 larvae settled under the influence of frequent strong "El Nirn)" events (Fig. 5). It seems that the "El Niiio" events determine a positive or negative effect over recruit- ment, but differences between species suggest that this could be species-specific. Data series longer than a decade are still neces- sary in order to have evidence that is more reliable. Physiological Weakening During "El Nii'io" Events Vega et al. (1997) and Shepherd et al. (1998) suggested that the reduction of food during warm periods could have lead to poor physiological condition and greater susceptibility to predation. The association between decreased physiological condition in both spe- cies and increased water temperatures support this. Local fisher- men affirm that the local extensions of kelp beds declined due to the incidence of warm events, but we have no data to prove this. Kelp beds are also exploited by the local chemical industry, but records about the crop are rare. The relationship between impoverished physiological condition and reduced proportions of sexually mature organisms was sig- nificant for only one species and suggestive for the other. Again, longer time series are needed. The remarkable peak in maturity detected in 1995 could have been produced by delayed spawning and/or shift in the reproductive season. Changes in the sex ratio of green abalone could be a by-product of changes in physiological condition, but it is hard to find a clear explanation. Guzman-del- Proo (1992) also reported inter annual variations and deviations from the normal 1:1, in favor of females, in both species at other locations. This indicates that the sex ratio in abalone is also a dynamic parameter (Table 6). Finally, as Shepherd et al. ( 1998) proposed, climatic variability, represented in our case by the surface water temperature, contrib- uted to the decline of the populations by their weakened physi- ological condition. These effects have been underestimated, and although the environmental effect of sea temperature anomalies is considered in using the modified version of the Schaefer surplus production model (Shepherd et al. 1998). it seems that this caution has not been enough. Fluctuations in physiological condition should be taken into account when deciding the fishing quotes. 300 250 «■ 200 c o t 150 - O 100 50 H. fulgens Islas San Benito (This work) ^ -H. corrugata Islas San Benito (This work) ■••»■■ H. fulgens Isia Natividad (Shepherd et al. 1998) •x- H. corrugata Isia Natividad (Shepherd et al. 1998) ■jfc^.-^iP".-'^'^--.ijji_ _ — 1 r- "b^ "b^ -^ <^ 4> <§"' <^' %*' <§>' 4"' 4-' cF 4^ q^

I5 cells I++++) = >?0 cells RESULTS Figure 1 shows a diagraniatic drawing of the gross anatomy of adult H. asiniini. The nervous system consists of a pair of cerebral ganglia, a pleuropedal ganglion and a visceral ganglion. Figure 2 shows the frontal sections of the cerebral (Fig. 2A), pleuropedal (Fig. 2B) and visceral ganglia (Fig. 2C) in adult H. asinina. There are 10 types of nerve cells in all ganglia of H. asinina (Figs. 2D. 3A), i.e., three types of neurosecretory cells (NS,_3), four types of neurons (NR,„,) and three types of neuro- glia (NG,_,) (Upatham et al. 1998; Kruatrachue et al. 1999; Thongkukiatkul et al. 2000). The NS cells were identified using special stains, i.e. chrome-hematoxylin- phloxine (Gomori I '■Ml) and paraldehyde-fuchsin (Gomori. 1950). The shape, size, and type of cells and their number in ganglia during various ages of developing abalone are summarized in Tables 1 . 2 and 3. Cerebral Ganglia In 1 -month-old abalone, the cerebral ganglion appeared as an elongated bean shape whose size was approximately 121 x 47 1 x 100 \xm (Fig. 3B). Most of the ventral, dorsal and lateral parts of the ganglia had a thick cortex that contained 3-4 cell layers, while the medial part contained only 0-1 cell layers (Table 1 ). NS, cells first appeared in 1 -month-old abalone; there were 1-2 cells per section. These cells were concentrated in the dorsal horn of the ganglion (Table 1 ). Most types of neurons (NR, 4) were present, but NR, were the most numerous. NR, and NRj were moderate in number, while the NR, or the giant neurons were rarely found but when present were usually located in the dorsal horn similar to NS cells (Table 1). All types of NG were present but in a small number. At 2—1 months, the ganglia appeared bean shape similar to those in I -month-old abalone. The number of cell layers increased with age. The number of NS cells increased to about 2-5 cells per section (Table 1 ). Most of these were NS,. while NS, were ob- served in 3-month-old and NS, in 4-month-old abalone. Most NS cells were concentrated in the dorso-lateral and dorso-medial, ven- tral and ventro-medial parts of the ganglia (Table I ). NR cells were similar in type and number to those in 1 -month-old abalone. NG cells increased in number from one month onwards. At 5 months, the size of the ganglion increased to 203 x 632 x 200 |j.m (Table 1 ). From 5 months onwards, the ganglion assumed a sickle shape (Fig. 3C). The number of cell layers in the cortex increased, especially in the ventral and dorsal parts. The number of NS cells increased to about 10 cells per section, and although all types of NS were scattered in all parts of the ganglia, most were still concentrated in the dorsal and ventral horns (Table 1 ). The number of NR increased with age. and the NR, count was ap- proximately 11-20 cells per section (Table 1). They were present in the dorsal and ventral areas. At this age, the number of NG slightly increased. From 6 to 10 months, the ganglia appeared sickle shaped but were larger and more elongated than those of 5-iTionth-old abalone. The cortex in all areas thickened and the quantities and distribution of NS and NR cells were similar to those of 5-month-old abalone (Table 1). At 1 1 months, the cerebral ganglia increased in size to about 377 X 810 X 300 ptm (Fig. 3D). Other appearances were similar to those of 5- to 10-month-old abalone. However, the numbers of NS and NR, cells increased (Table 1 ). NG also increased with increas- ing age. When abalone were 12 months old, their ganglia (377 x 901 X 325 p.m in size) were fully developed and appeared similar in all aspects to those of the adult abalone (Fig. 2A). Pleuropedal Gaiialia 111 1 -month-old abalone, the pleuropedal ganglion appeared butterfly-shaped and about 189 x 418 x 150 (xm in size (Fig. 4A). In the ventral and lateral parts of the ganglia, the cortex was thick and contained 2-5 cell layers (Table 2). The remaining parts of cortex were relatively thin. There were only about 1-2 NS cells per section. These cells were confined to the dorsal-sulcus of the gan- glion; most of them being NS, (Table 2). There were all types of NR, but a few NR, and NR4 were present in the dorso-medial part (Table 2). All types of NG cells were found in the ganglion at this age. At 2-3 months, the size of the ganglia increased from 273 x 497 X 155 to 289 x 522 x 170 |jim, but the shape was not altered (Fig. 4B). The number of cell layers in the cortex. NS and NR cells appeared to increase, and most cells were found in all parts of the cortex. However. NR cells were concentrated in the dorsal and dorso-lateral parts, while NS cells were concentrated in the dorso- medial and lateral sulci (Table 2). At 4-6 months, the pleuropedal ganglia was still butterfly- shaped but increased in size from 337 x 556 x 200 |xm to approxi- mately 488 X 707 X 250 p.m (Fig. 4B). and the cortex became much thicker. The number of NS cells (mostly NS,) increased to about 20 cells per section and a larger number were found in the dorso-lateral and ventro-lateral parts (Table 2). NR cells increased in number with increasing age and were found in the \entro- medial. ventro-lateral and ventral sulci (Table 2). At 7 months, the ganglia were H-shaped and increased in size to about 544 x 1 6 1 5 x 3 1 5 p.m (Fig. 4C). The number of cell layers increased and NS cell (mostly NS, ) number was about 30-40 cells per section; these cells were distributed in all areas (Table 2). NR cells (mostly NR,) increased in number in comparison to earlier stages. From 8 to 10 months, pleuropedal ganglia were similar in shape to those of 7-month-old abalone (Table 2). Development of the Nerve Ganglia of Abalone Cerebral commissure 175 Appendage tentacle Appendage tentacular nerve- _ Cerebro-pedal connective ^ _ Cerebro-pleural connectives^ Osphradium nerve- Osphradium- Esophagus--" Gill Intestine- Visceral ganglion Digestive gland Cephalic tentacle Cephalic tentacular nerve -Eye - Optic nerve % Cerebral ganglion ^i- — Buccal ganglion .>>^. -Buccal nerve ■Vj-Pleuropedal ganglion Epipodium tentacle Pedal ner\e ^S^- -Epipodium ■=^ tentacular nerve Pedal ganglion Pedal nerve cord Gonad 0-4cm Figure 1. Diagramatic drawing of the gross anatomy of adult abalone showing the ganglia and their peripheral nerves (modified from Crofts, 1929). At 1 1 months, the ganglia increased in size to 589 x 2508 x 470 |j.m; the ventral and dorsal horns were elongated (Fig. 4D). The number of cell layers in the cortex increased. The number of NS cells was about 60 cells per section (Table 2). NR cells and NG cells were distributed in all areas; their numbers increased with increasing age. At 12 months, the pleuropedal ganglia (589 x 2543 X 500 p.m in size) were fully developed and appeared similar to those of the adult abalone (Fig. 2B). Visceral Ganglia In 1 -month-old abalone. the visceral ganglion was as small as 37 X 72 X 30 p.m and bean-shaped (Fig. 5A). The cortex had only one layer of cells (Table 3). NS cells and NR, cells had not yet appeared. In contrast, the remaining types of NR (NR^j) were present but still few in number (Table 3). All types of NG were observed. From 3 months onwards, the ganglion was dumbbell-shaped (Figs. 5B-5D) and its size increased with increasing age (Table 3). The cortex was thicker, especially the lateral part. NS, cells first appeared in 2-month-old abalone, and their number was about 1-2 cells per section (Table 3). They were present in the left lateral, left lalero-dorsal and left latero-ventral parts. There were all types of NR, but NR, and NRj were rarely observed. At 3-10 months, the visceral ganglion increased in size from 1 18 X 488 X 50 (xm to about 160 x 770 x 110 ixm (Fig. 53). The number of cell layers in the cortex increased. The number of NS cells increased to about 20 cells per section, and they were dis- tributed in the right lateral part (Table 3). NR cells were similar in number and distribution to those at 3 months (Table 3). At 1 1 months, the ganglion ( 160 x 889 x 1 10 \xm) increased in length, but still had a similar width to that of 10-month old abalone (Fig. 5C). NS cells (mostly NS,) increased in number and were 176 Kruatrachue et al. Figure 2. Photomicrographs of the frontal sections of ganglia of H. asinina. I A) A low-power micrograph of a cerebral ganglion, showing thick cell layers on the ventral (\ ) and dorsal (D» sides. Ca-capillary. t'o-cortex, 1,-lateral. M-medial. Me-medulla. Mu-niuscle. (Bl A low-power micrograph of a pleuropedal ganglion showing thick cell layers on the dorsal (Hi and lateral (I,) sides. Co-corte\. M-medial, Me-medulla, V-ventral. (C) A low-power micrograph of a visceral gangli(m showing thick cell layers on the ventral side. 1,1. -left lateral, Rl. -right lateral. (J- gill, Co-cortex, Me-medulla. (D) .\ high-power micrograph of a pleuropedal ganglion showing various types of nerve cells in the cortex region. NGi-type 2 neuroglia, NG,-type 3 neuroglia. NS,-type 1 neurosecretory cell, NS,-type 2 neurosecretory cell, NS,-type 3 neurosecretory cell. distributed in all areas of thie ganglia (Table }<). but were concen- trated mostly in the right lateral part (Fig. 5D). At 12 months, the ganglion ( 163 x 939 x 150 jxm in size) appeared similar to those of adult abalone (Fig. 2C). DISCUSSION During postembryonic development of the nervous system of gastropods, the nerve cells and neurosecretory cells increase in size Development of the Nerve Ganglia of Abalone 177 NR2-|PSV^ "V m^^^ *' •^^ , ^^ jjR ^ ^p v^^tk' ^prV • "^M f-NRj M^ r • '•"4 0 a -"S, 10U "r^ ^A Figure 3. (Al High-poHer micrograph of cerebral ganglia showing type 1 neuroglia iNCJ,), types 1-4 neurons |NR,_,1 and type 3 neurosecretory cell (NS,). (B) Frontal sections of cerebral ganglia in I- to 4-month-old abalone that appeared bean-shaped. (C) In 5- to l(l-nionlh-old abalone, the ganglion appeared as a sickle shape. iDl In ll-month-old abalone, the ganglion greatly increased in size. D-dorsal, L-lateral, M-medial, V-ventral and number (Bullock & Horridge 1995). for example, the neuro- endocrine cells of L. stagiuilis increase in number and size with increasing shell length (Roubos et al. 1988). In addition. Lever et al. (1965) showed the same result in the cerebral and parietal ganglia of Bioinphalaria i;lahralu (Say). Similarly. Kruatrachue et al. (1994) reported that the number and size of neurosecretory cells in the cerebral ganglia of A. fiilica increased with increasing age. A similar trend was observed in H. asinina in the present stud\. Furthermore, our histological study indicates that the cerebral, pleuntpedal and visceral ganglia appeared as definite organs with specific shapes in 1 -month-old abalone. Later there were changes in the size but not so much shape of these ganglia; and the numbers of neurons and neurosecretory cells in all ganglia markedly in- creased with increasina aae. 178 Kruatrachub et al. f- E Q. O u f^ , o C/3 u Z ^ £ c 3 Z y; CA z z i* > a "Z X X Z o c ra ij rj .s k. = Z -3 > a =S " N E X 3 Q Q Q d d d > > Q -J ^- > d + + + + t + + + + + + + I 1 § 1 5 I J J < J < — Q Q > Q > ::) a d d d ^ n U-, ir-, _, III 2 — ri rj (V-, r^, r*-, -+ rf III I I r^l — — ri — — r*-, r^. r*~. r^, I -1 J. I I o o o o o rj oc oc ijj u u D D (L) U 1) TJ S £ E E c E S 73 ra rt ra :^ n c:: c/^ c/: c/: c/1 c/^ cn 00 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + c. o o o o -f 7 (-1 rj ri ri '^. r^ J. r<~, (-*-, I/-, U"i It r*-, n r<-, r^, r^, rr. r<-, I { I I i I I o — o o o — — >: r- r- r- I I I I nC ir-i ir, ir, I/", U-) ir, (L» 1/; E ^ "^ c3 -J o 1) (U £ ^' s: 'J '-J 'c ■-J o C C3 ■u II II It O ? 1! O, 1 T 11 X £. ^ y Development of the Nerve Ganglia of Abalone 179 •= OS a Z. B 3 ^ a! 0^ Z CQ c H = e -J) tt. s a« o . 1> U 1j -— E £ 5 c r3 "rt C^ S n c cy^ ^ 00 c/^ ■^ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ^ + + + + + + + + + + + + + + + "" + + + + + + + + + + + + + + + + + + + + + + -4 ~ r) c: — n + + + + + + + + + + + + + + + + + + + + + + + + + + + ;: + + + + + + + ':: _i -J _j a q q c/l 00 v^ Q V3 ^ cjn fA _1 -J J a C3 S S > S > J3 ^ rt rt q _i Q _i Q _i — ■^ — — ^. > M > !/5 > "^ ~ ~ ~ c:; o Q Q c c S c ^ o o o o o c; o o r^. r*-i r*~j r*~, — I r*". "^ — rj n-. r*-, r*-, — — ri TT W", I/"; \C n rt -4 sC 'y~. w. '/". ^C _ ir-- O O O' O O ™ -^ 0^ r^-i r- U~i r*-, r^. r*-, -rf -rt ■"' X X X X X •'^ v, ri — O cc r- — v~, ^o o o o >£i ^ *o r-- I/", -t ^C' <-t o c> -f -t r^ :>c oc Cu C Cl D. D. Z.li 1) 1/ '■''- y, I I I X X ^ A A II II ^ t t t t + • 1 1 II II + + + + Z - o — " C oil c5 II II X T T II " i 180 Kruatrachue et al. -J-l-'-^-iH p i _)_]_] < < -z. - -^ 4* ^ c > _ X B -I + + + + + + I I I I _J C/3U^C/0C/^C/5 — — + + + + + + + + + + + + I t 4- 4- + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + _: _i — — — -4 >' > > I J -1 -J C/5 on t/5 — — — — t .- r^, rf, r*-, <■*", r^, r*", n"i O i^i */"' '^' I/", o o — — '<^. oo X X X oo oc r^. O O O O 3^ O- ri T T = l~ :c rf, ^ ^O *c r^ r~- 30 o^ X X X X X X X w-, oc — O O O u^ 1^, vC ^ ^ ^ ^ *^ a. Q- D- o. a. D- a. c D- — _ — CO aa C D o A 1 n — II + + + + O _l_ z a: z II II H >. 5 QQQQQQQ ^ r^ cc :;^ O' — + i S -H 2 § g : s i 111^ i I. II 2 ■* " II Dr I I " X Development of the Nerve Ganglia of Abalone 181 Figure 4. Low-ixiuir mkni<;r;iplis of frontal siiiiiins sical Stimulation The ani)iials were exposed to air for 15-20 minutes. Other Stimuli A combination of chemical and physical stimulation was used, tiigether with the addition of macerated gonad tissue to the sea- water. Stimuli for the induction of spawning were applied to groups of 4 to 10 limpets, males and females separately, each group being maintained in 4 liters of filtered seawater gently cir- culated (0.5 1/min). Any liiTipet failing to respond to a spawning stimulus within two or three days was replaced. The nonparametric Wilcoxon test ^P < level 0.05) was used to compare responses of male and female limpets to the various spawning stimuli, using the program Statistica (Windows version 4.2), Ferlilizalion and Devclopiiient After the initiation of spawning. i)idi\ idual limpets were placed in small containers with 1 liter filtered seawater. Triplicate samples of 1 ml were taken from each suspension of gametes. Oocytes were TABLE 1. Effect of ditTerent inductors on spawnings in Fissurella picta. Inductor No Successful No Successful No of No of Trials with Trials Individuals Trials Males with Females 78 Temperature Stimuli Temperature ( 10-24"C) Chemical Stimuli KCl (injection) 0.3 mM 10 mM 20 mM 100 mM 8 mM (seawater) 50 niM (injection) Dopamine 2 mM (injection) Dopamine -i- H^O-, (seawater) Dopamine + seawater treated with UV PG (injection) PG (injection) + seawater treated witli UV PG (seawater) -^ seawater treated with UV Other stimuli KCl (seawater) + macerated gonad KCl (seawater) -i- exposed to air Seawater treated with UV TOTAL Date Oct 1^)96. Dec 19%. Feb 1997 22 3 0 0 Feb 1997. Apr 1997 40 5 3 1 Oct to Dec 1996, Oct 1997 16 -) 0 0 Oct 1997 16 2 0 0 Oct 1997 Dec 1996. Oct Nov Dec 1997 96 12 1 1 Jan to Apr 1997 16 -> 1 1 Nov 1997 9 1 0 0 Nov 1997 5 1 0 0 Nov 1997 26 4 1 0 Nov 1997 24 3 1 0 Apr to Nov 1997 ^2 4 0 0 Feb to Nov 1997 24 3 1 1 Nov 1997 24 3 2 1 Nov 1997 8 1 1 0 Nov 1997 15 -> 0 0 Dec 1996, Oct to Nov 1997 451 57 13 5 Spawning and Early Development of F. picta 187 Figure 2. A. Oocyte in \i\(i ol I'issunlki picui Hith its membranes, at the light microscope, am: albumen membrane; gc: gelatinous coat; o: oocyte; vm: vitelline membrane. B. ■"Micropyle" inside the gelatinous coat of the oocyte of Fissurella picla. am: albumen membrane; m: micropyle; o: oocyte; vm: vitelline membrane; gc: gelatinous coat. counted under a stereomicroscope. a mean value obtained, and the total number of gametes calculated. Sperms were counted with a Malassez camera. Sub-samples of oocytes taken during spawning were fixed in 3% glutaraldehyde. washed in phosphate buffer and distilled wa- ter, and mounted under cover slips for examination and photomi- crography (Zeiss Axiomat microscope). The remaining oocytes were screened (150 |jLm mesh), washed several times in sterile filtered seawater and maintained in the 1-liter containers to await fertilization. The sperm suspensions were also screened (45 )xm) before use to get fertilization, gamete suspensions from individual limpets were used in ratio 10-15 sperms per oocyte. After fertilization had been confirmed by the appearance of the first polar body, each batch of eggs was washed several times in sterile seawater to remove the excess of sperms, then transferred to a 2 liter container of sterilized filtered (0.45 |j.m) seawater and kept in darkness with constant aeration. Every 24 hours afterwards, two thirds of the water was replaced. Temperature was maintained at 17,5°C ± V'C and salinity at 29-30%6. Replicate samples were taken every 15 minutes for the first 3 hours after fertilization, every 30 minutes from 3 to 24 hours post-fertilization, and every hour thereafter until the liberation of the larvae. The embryos and larvae were screened with different mesh sizes appropriate for their size. The samples were examined under a light microscope (Olympus) and obtained from a camera (Sam- sung NF-E80SN) which replaced one of the eyepieces of the mi- croscope. Selected images were captured (ATI program), digitized (BMP format) and processed by image analysis (Scion Image PC). The criterion for determining the stage of development for any given sample was that at least 60% of the individuals should have attained the stage in question. RESULTS liidiiclion of Spawning Data from the 57 spawning induction trials, undertaken princi- pally in October and November 1997 are presented in Table 1. More males responded than females (Wilcoxon test; N = 16; Z = 2.201; P < 0.05). Potassium chloride was the most effective in- ducing agent (10 mM), whether administered by injection, added to the medium with macerated gonad tissue, or combined with air exposure. A combination of UV-in-adiated seawater with either dopamine or prostaglandin (PG) also induced spawning in males. On one occasion with hydrogen peroxide injection, and on another with PG added to the medium together with UV- irradiation of the seawater. as many males responded as females did. Only once did a male spawn in response to PG. Dopamine was not effective for inducing spawning in males, except when com- bined with UV-irradiated water. Characteristics of Gametes Mature oocytes had two external membranes, the vitelline membrane and the albumen membrane, outside which was a ge- latinous coat (Fig. 2A). The gelatinous coat was often lost, but when it was present the micropyle originating from the albumen membrane was clearly visible {Fig. 2B). The albumen membrane, which in Figure 28 had yet to expand, was derived from the oocyte and had the appearance of a clear gelatinous fluid. Oocyte diameter varied from 1 1 7-327 p.ni (mean 1 78 \^.m). but those with a gelatinous coat reached a diameter of 385 p-in. Fertilization and Development During December 1996, October and November 1997, a total of 10 trials of artificial spawning were successfully completed (Table 2); these were of three types: (a) use of gamete suspensions obtained by dissecting the gonads (OT) of 10 F. picta individuals; (b) oocytes from dissected ovaries together with sperm from in- duced spawning (01) of 10 animals; and (c) oocytes and sperm both obtained from spontaneous spawning (SS) in 10 specimen. The spawned oocytes of F. picta frequently required several seconds before complete hydration and expansion of the albumen membrane. During this time eggs were observed to be surrounded by large numbers of sperm, and occasionally the sperm head could be seen within the micropyle. At 17.5"C. fifteen to twenty minutes after fertilization the first polar body was visible, having the appearance of a small, translu- cent, refringent granule (Fig. 3A). The polar body was located immediately adjacent to the vitelline membrane within the ex- traembryonic fluid of the albumen membrane of the egg. After 90 minutes the first two holoblastic segmentation divi- sions had given rise to four blastomeres of equal size (Fig. 3B,Ci. The third oblique plane of division resulted in the fonnation o 188 Bahamondes-Rojas and Bretos TABLE 2. Fissurella picla. Stages of development obtained in laboratory at 17.5 ± rc and 29-30 9,, salinity. Diameter Fertilization stages Time (fim) OT Egg First minutes 180 OT First polar body 15-20 min. ISO OT 2 Blastomeres <60 min. 180 OT 4 Blastomeres 80-90 min. 180 01. s.s 4-8 Blastomeres 80-90 min. 180 OI Blastula 4—7 hours 180 ss Trochophore 15 hours 180 ss Early veligers 21 hours 180 ss Hatched veligers 40 hours 200 OT = Dissected ovary and testicle 01 = Dissected ovary plus sperm from induced spawnings SS = Spontaneous spawning in females and males. eight blastomeres (Fig. .^D). of which four were very large inac- romeres. located at the vegetative pole of the embryo and contain- ing yolk reserves, and the remaining four were small micromeres located at the animal pole. After 4 to 7 hours successive divisions of the blastomeres had given rise to a blastula, a compact, multicellular sphere that gently rotated within the membrane, suggesting the presence of cilia on the e,\ternal surfaces of the blastomeres. Embryonic development was highly synchronized, but became less so during the larval stages. Fertilized eggs obtained by OT and 01 (Table 2 1 developed only until blastula stage. The trochophore larva, exhibiting a ring of prototroch cilia, appeared 15 hours after fertilization, and was completely envel- oped by the albumen membrane (Fig. 4A). Survivorship was about 5iV'c of fertilized eggs in SS trials (Table 2). Larvae became an early veliger. turning round within the al- bumen membrane. 21 hours after fertilization. Forty hours after fertilization, the albumen membrane had disappeared and the ve- ligers had emerged (Fig. 4B) to spend a short period moving through the water column. The pretorsional larva exhibited the protoconch, an expanded velum, the foot, and the rudimentary P s B 50 (jm 50 pm mac 50 Mm Figure .V Kmbryonic development stages of Fissurella pichi within the albumen membrane. A. Fertilized egg where a polar body (pb» is visible. B. Two-blastomere stage. Arrow shows remnants of micropyle in the albumen membrane. C. Kour-blastomere embryo. D. Embry o « ilh unequal eight blastomeres. View from animal pole, mac: macromeres; mi: micromeres. Spawning and Early Development of F. picta 189 Figure 4. Larval development stages in Fissiirella picta. A. Tro- cliopliore larva. Arrov^ shows ring of prototrochal cilia. B. Pretor- sional hatched veliger larva.f: foot; pr: protoconch; rrm: rudimtntarv retractor muscle; v: velum; vm: vitelline membrane. retractor muscle. The visceral mass was well developed and the yolk reserves and velar cilia diminished. About a 30^0% of embryos obtained by SS trials survived to veliger stage. Development did not continue, and larvae ultimately died. DISCUSSION tion methods tested (thermal, electric, and osmotic shock) by Gonzalez et al. (1999) in F. picta did not produce positive results; this report suggests that a combination of various inductive stimuli should be required to obtain positive responses, as has been shown in the present study. Potassium chloride injections, either alone or combined with other stimuli (exposure to air, gonad maceration), and hydrogen peroxide induced spawning in F. picta. Potassium chloride acts at the cellular level and has been extensively used to induce meta- morphosis in marine invertebrate larvae (Yool et al. 1986; Baha- mondes-Rojas & Tardy 1988; Bahamondes-Rojas 1990) as well as for the induction of spawning. Hydrogen peroxide induces spawning in gravid male and fe- male abalones Haliotis rufescens and Nordotis gigantea. the mechanism possibly being direct stimulation of the enzymatic syn- thesis of prostaglandin endoperoxidase (Morse et al. 1977; Tanaka 1979; Hahn 1989). Our data support those of Vega et al. (1996). who successfully induced spawning in male Fissiirella cumingi with low concentrations of hydrogen peroxide (3 mM when added to the medium, and 5 niM when combined with gonad macera- tion). It is also known that seawater irradiated with ultraviolet light induces spawning in many mollusks (Kikuchi & Uki 1974; Ka- gawa & Nagahama 1981). but our data are not in agreement. Nevertheless, we found ultraviolet irradiation to be effective when combined with PG or dopamine, presumably as a result of syner- gistic action. Moss et al. (1995) induced spawning in Haliotis iris with two agents, hydrogen peroxide and seawater irradiated with ultraviolet light, the former being more effective. The role of prostaglandin remains unclear, although its pres- ence and biosynthesis have been established (Ogata et al. 1978; Nomura & Ogata 1976). We found PG to be a successful agent for inducing spawning in male F. picta, but in females the mechanism may be different. Thus Martinez et al. ( 1996) suggested that in the hermaphrodite scallop Argopecten purpiiiatits. dopamine and PG may be involved in the release of oocytes. The fact that most of the trials were successful only in males may be attributable to different mechanisms or maturation rates in male and female gonads. Our microscopic observations demon- strated that gonad maturation was almost continuous in males, whereas in females there was a latent period during the vitello- genic phase. One outcome from this experiment should nevertheless be em- phasized. Spawning inducers such as KCl and H^O-, are inexpen- sive, easy to use and are widely used for the control of reproduc- tion in molluscs such as fissurellids. Induction of Spawning Sex and Gametes The spawning stimuli used in this study were similar to those used by other authors (Morse et al. 1977; Morse & Morse 1984; Uki & Kikuchi 1984; Pechenik 1986; Martinez et al. 1996) to obtain gainetes from various marine invertebrates. In the case of F. picta. the maximum maturity condition in the gonads detected in this study during the spring (September to December) would fa- cilitate the spontaneous gamete release to get fertilization and de- velopment in a more successful way. Our results suggest that induction of spawning in F. picta was successful, but more effective with males than with females. Simi- lar data have been obtained in previous studies on F. maxima and F. cumingi (Vega & Osorio 1995). The artificial spawning induc- Fissiirella picta is an archaeogastropod which, like all fissurel- lids, does not exhibit external sexual dimorphism (McLean 1984). There is no evidence for hermaphroditism or sex reversal (Bretos et al. 1983). The technique we used for determining the sex of each specimen was non-invasive, successful and simple. There have been few studies of the life cycle (e.g.. spawning, fertilization, and age of sexual maturation) in fissurellids. Ward (1966) described the reproductive cycle of F. barbadensis, and demonstrated the presence of oocytes of 80-180 |j.m in diameter (including gelatinous coat). This species is known to have a pe- lagic phase of two to three days duration (Lewis 1954; Lewis 1960). In Diodora aspeia spawned eggs are 160-198 \xm in di 190 Bahamondes-Rojas and Bretos ameler. williout the gelatinous coat (Hadtleld & Strathiiiann 1996). In /•'. ni(i.\iiiiti (Bretos et al. 1983) the oocyte diameter lies between 120 and 2S0 (xni. and that of F. crassa is approximately 300 |xm. excluding the gelatinous coal (Huaqui'n et al. 1998). Our values for oocyte diameter in F. piclii are similar to the maximum values recorded for other Chilean species. Nevertheless, differences ob- served from tropical little sized species such as F. barhadensis and Diodoni apertitm may be related to differences in reproductive strategy e.g., time to reach sexual maturity. In Diodoni aspeni the external gelatinous coat which envelops the oocyte appears to break open only in response to a mechanical action (Hadfield & Strathmann 1996), whereas the internal mem- brane disappears as a result of the action of enzymes produced by the larva. In Patella (Fretter & Graham 1962) the gelatinous coat of the oocyte disappears a short time after spawning, as in F. picki (this study). The presence of a canal, the micropyle, in the gelatinous coat of the oocyte, and the presence of sperm cells within it. has been observed in F. crj.vwj (Huaqui'n et al. 1998). Diodoni aspeni (Had- lield & Strathmann 1996) and F. picta (Fig. 2B of this article). Fertilizution and Dcveliipiiwiil Knowledge of larval development of Chilean fissurellids is limited mainly to a few unpublished observations; all of them obtained by spontaneous gamete release and fertilization at the laboratory. Vega and Osorio (1993). and Vega et al. (1996) deter- mined that the duration of the pelagic larval phase in F. cwningi is 3-5 days under controlled conditions ( 16-19. 8°C), whereas in F. latiimirginata at 13°C the veliger stage and metamorphosed larva occur 6-7 days after fertilization (Pereira & Quezada 1996). According to Gonzalez et al. (1999), who also got spontaneous gamete release and lertili/ation, the initial trochophore stage was observed in F. picia at 72 hours, and swimming trochophore hatched at 96 hours at IOC. In the present study at 17.5°C. tro- chophores of F. picta were obtained \fi hours after fertilization within the albumen coat, and veligers hatched out of this coat after 40 hours. Our data are not consistent with those of Gonzalez et al. (1999). probably owing to the difference in temperature. It has been described for Hatiotis and other species, that the teinperature can hasten or delay development (Hahn 1989). and this has prob- ably happened in F. picla. The veligers which were liberated in this study showed mor- phogenetic movements associated with torsion before settlement and metamorphosis took place. In this regard, our observations agree with those of Page (1997) for the archaeogastropod Hatiotis kamtscliatkami. demonstrating cephalo-pedal and viscero-pallial rotation in pretorsional larvae. The fact that development of F. picta took place in the labo- ratory during this study suggests the potential for future cultivation of this species. Nevertheless, it will be necessary to elucidate the principal biological factors and ontogenetic mechanisms required to improve survival rates and to identify the conditions required for settlement and growth of a large number of larvae. ACKNOWLEDGMENTS This research was supported by Canadian International Devel- opment Agency, and Memorial University of Newfoundland awards, and by the Direccion de Investigacion y Desarrollo, Uni- versidad Austral de Chile (Grant F-96/0I ). We thank Dr. Ray Thompson for translating this manuscript. Dr. Oscar Chaparro for his comments on the manu.script. and Jaime Oyarzo for assistance and help with photography. LITERATI' Bahamondes-Rojas. 1. 1990. Mecanisnies d'lnduclion niiturelle et artifi- cielle des metamorphoses des Mollusques: Eiihrancluis doriae (Trinchese, 1879) Gasteropode Nudihranche et Rudilapes philippi- narum (Adams et Reeve. 1850) Bivalve Veneride. La Rochelle: These de Doctoral. Universite de Poitiers. 119 pp. Bahamondes-Rojas, I. & J. Tardy. 1988. Induction a la metamorphose chez Eiibrcmclnis doriae (Trinchese. 1879) (Mollusque: Nudihranche) par diverses substances bioactives. Haliotis. 18:121-130. Bretos. M.. I. Tesorieri & L. Alvarez. 1983. The biology of Fissiirella maxima Sowerby (Mollusca: Archaeogastropoda) in Northern Chile. 2. Notes on its reproduction. Binl. Bull. 165:559-568. Brelos. M. 1988. Pesqueria de lapas en Chile. Medio Ambienle. 9(2):7-l2. Bretos, M., J. Gutierrez & Z. Espinoza. 1988. 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Regulation of maturation and spawning of an abalone. Haliotis (Gastropoda) by external environmental factors. Aqiiaculture. 39:247-261. Vega. G. & C. Osorio. 1995. Evaluacion del potencial de cultivo de Fis- surella mu-xima y Fissiirella ciiiningi. .XV Jornadas Ciencias del Mar, Universidad Catolica del Norte. Sede Coquimbo. p. 137. Vega. G.. C. Osorio, J. Remonsellez & A. von Marees. 1996. Avances en el cultivo de Fissiirella ciimingi (MoUusca: Archaeogastropoda). IX Congreso Latinoamericano de Acuicultura. 215-219. Ward. J. 1966. The breeding cycle of the keyhole limpet Fissiirella Inir- badensis Gmelin. Bull. Mar Sci. 16(4):685-695. Yool. A.J., S. M. Grau, M. G. Hadfield, R. A. Jensen, D. A. Markell & D. E. Morse, 1986. Excess potassium induces larval metamorphosis in four marine invertebrate species. Biol. Bull. 170:255-266. Journal of Shellfish Reseanh. Vol. 21. No. 1. 193-200. 2002. TYRIAN PURPLE FROM MARINE MURICIDS, ESPECIALLY FROM PLICOPURPURA PANSA (GOULD, 1853) LUDWIG C. A. NAEGEL' AND CHRIS J. COOKSEY" ^Ct'iitro Interdisciplinario de Ciencias Marinas, lustitittoPoUtecnico Nacional La Paz. B.C.S. 23000 Mexico; '59 Swiss Avenue. Watford, Herts WD] 8 7LL. United Kingdom ABSTRACT A review of the literature disclose.s that most inarine snails of the family Muricidae produce in the hypobranchial gland a viscous secretion containing, besides mucus and biologically active compounds, minute amounts of chromogens. These chromogens develop enzymatically and under the influence of light and o.xygen into a purple pigment known as "Tyrian Purple". "Royal Purple" or shellfish purple. In the hypobranchial gland the enzyme purpurase is kept apart from the chromogens. so that no pigments are formed under normal conditions. Different species of muricids produce different pigments, depending on the number and concentration of different chromogens and on the varying light intensity and oxygen availability during pigment formation. The main pigments obtained from the hypobranchial gland from muricids are indigoids. The pigment of P. pansa is mainly 6.6'-dibromindigo with smaller amounts of 6-bromoindigo and 6,6'-dibromoindirubin. similar to that of A7»/r.v hrandaris. KEY WORDS: "Tynan Purple", gastropoda, muricidae. Plicopurpuia pansa. hypobranchial gland INTRODUCTION A review of the hypobranchial gland of muricids. its secretions, including "Tyrian purple", seemed justified in view of the growing interest in natural dyes and marine products with pharmacological properties. The majority of purple producing marine snails belong to the family of Muricidae and most, if not all. produce a colorless se- cretion in the hypobranchial gland, which turns purple on exposure to air and light (Fretter & Graham 1994). In antiquity, the purple from the muricids Miire.x tninculits, M. brandaris. and Purpura haemastoma was produced in the eastern Mediterranean countries - now called, Crete, Lebanon, and Israel. Through the Phoenicians the art of purple production was spread from the Mediterranean to West Africa and Ireland (Jackson 1917). Purple dyes were used extensively by Egyptians and sub- sequently by Greeks and Romans. In view of the enormous quan- tity of marine snails needed to produce a minute amount of the dye, the scarcity of the animals, and the high costs of production. Tyr- ian purple was at that time a most expensive luxury article. In addition there was the symbolic importance of purple as a sign of royalty, power and wealth, and the belief that it could possess magic and supernatural powers (Reinhold 1970). At that time it was the only known fast vat dye. other than indigo. With the Arab conquest of Palestine in 638 A.D., and finally with the fall of Constantinople in 1453 A.D. the use of Tyrian purple became, with a few exceptions, extinct in the Old World (Herzog 1919; Bom 1936b; Clark et al. 1993). Through archaeological studies it was confirmed that during the Middle Ages on the west coast of France the muricid Nucella lapillus was used as a source for purple (Gruet 1993). From the I6th to the 18th century the artisanal use of purple for marking linen was widespread in Ireland. South Wales and Cornwall, as well as in Scotland, France, Norway and other parts of Europe (Cole 1685; Jackson 1917). In Japan, the muricid Rapana bezoar was of importance in ancient dyeing processes (Baker 1974). On the Japanese peninsula Shima. professional seafood collectors stained their diving suits, made of cotton, with the purple from marine snails believing that it contained supernatural powers (Yoshioka 1974). Corresponding author. Ludwig C. A. Naegel. E-mail: lnaegel@cibnor.mx The use of muricids for dyeing on the pacific coast of the Americas dates at least from pre-Columbian times. In the same way as the now extinct Mediterranean purple industry the exploi- tation of the dye of marine snails led also on the pacific coast of the Americas to a product of high economic value. Today, however, there is not much general interest existing in Tyrian purple derived from marine snails, since similar pigments can be obtained from synthetic substitutes at much less cost (Bom 1936c). However, two remarkable exceptions have to be men- tioned: (a) the dark violet-blue tekhelet color, which is relevant to Jewish religious rituals derived from the Mediterranean muricids Murex erinaceus and M. trunculus. and b) on the Pacific coast from Peru to Mexico, the hypobranchial secretion of the muricid Plicopurpura pansa (Gould 1853) has been exploited since pre- Columbian times by Indians for dyeing cotton yam, which until now is subsequently woven into traditional dresses (Martens v. 1874. 1898; Schunck 1980a; Nuttall 1909; Jackson 1917; Bom 1936c; Gerhard 1964; Turok et al. 1988; Yoshioka 1974; Thomp- son 1994; Garay 1996; Sandberg 1997). v. Martens (1898) pointed out that the use of the pigments from P. pansa for dyeing in Central America must have had a very long and pre-Columbian tradition and were not brought by the Spanish conquistadors from Europe to the New World. Its presence in archaeological textiles and pictures confimied his finding. The camivorous muricid Plicopurpura pansa (Gould 1853). according to Kool ( 1993) conspecific with Purpura pansa (Gould 1853). inhabits intertidal rocks exposed to the open sea with high impact waves. The range of P. pansa extends at the Pacific from the north-west coast of Mexico (Baja California Sur) (Clench 1947; Keen 1971) to northem Pern (Pena 1970; Paredes et al. 1999). Hypobranchial or Mucous Gland Since the mid 1 8th century the hypobranchial gland of muricids has attracted the interest of natural scientists, investigating its func- tional role, and the astonishing production of Tyrian purple. Fretter and Graham (1994) consider the main function of the hypobran- chial gland to be a secretor of mucus for trapping and cementing particulate matter sucked into the mantle cavity with the respira- tory water current, prior to its expulsion. 193 194 Naegel and Cooksey The hypobranchial or niuciis gland is an elongated epithelial struclLire located in the dorsal mantle cavity between the gills and recto-genital organs, immediately below the shell. In the gland three distinct anatomical and functional areas have been described: two lateral regions composed of eight distinct cell types, among them many active secretory cells (mucocytes). ciliated on the ven- tral surface, and possessing pores through which the mucus and other secretory products are released into the mantle cavity, and a central area where the formation of the '"purple" precursors takes place and where secretory products accumulate prior to their re- lease from the snail (Bolognani Fantin & Ottaviani 1981; Roller et al. 1995). The purple precursors (tyrindoxyl sulphate) and the en- zyme (arvl sulfatase = purpurase) that induce the transformation of the purple precursors into pigments are only localized in the median zone of the hypobranchial gland (Erspamer 1946) and are kept separate, so that no reaction occurs. Mollusk purple as such does not occur in the live animal, but it is formed during a se- quence of chemical reactions from the secretions produced by the animal. When the animal contracts vigorously the cells are mas- sively liberated, burst open by mechanical or osmotic pressure, and their contents dispersed into the mucus (Lacaze-Duthiers 1859). These observations were later refined by the histological work of Bernard (1890). who found a well-developed innervation in the gland, suggesting a role in perception (Verhecken 1989). The pharmacological action by extracts of the hypobranchial gland was discovered by Dubois (1909), and he described for the first time their toxic and paralyzing action in both wann- and cold-blooded species. The secretion of the hypobranchial gland from a large number of muricids contains, besides mucus, the precursors of the purple dye, proteins (aryl sulfatase, purpurase), and toxins and narcotizing agents, like serotonin (5- hydroxytryptamine), murexine (urocanylcholine,), choline ester and biogenic amines (Erspamer 1952: Erspamer & Benati 1953; Whittaker 1960; Malaszkiewicz 1967; Huang & Mir 1971; Roseg- hini et al. 1996; Shiomi et al. 1998). The secretion from the hypobranchial gland of P. pcinsa can be obtained by "milking"' without harming the annuals. It is a milky- white liquid, which turns on exposure to air and light, at first yellow, then greenish, bluish and finally purple ("Tyrian purple"). During personal field observations (unpublished) we observed that P. pcinsa uses the secretion to immobilize prey {Nerira sp.. Lii- lorina sp.) in the intertidal zone, and does not resort to drilling through the shells of other snails. Additionally interesting to notice is the fact that during the predation no purple color is formed on the prey, despite the presence of oxygen and intense light radia- tion. The chromogens containing the hypobrancial secretions seem to be purely incidental, and their functional role, if any, is presently unknown (Clench 1947). The volume of secretion obtainable from P. puusi.1 depends not only on the size and sex of the animals, the time interval between the each "'milking", but also on the season. Its production and use may be in proportion to the type of food the snails feed on. From small animals of less than 2 cm shell length can be obtained about 0.5 ml of secretion, from 5-6 cm large animals up to 4 ml (Rios-Jara et al. 1994). It has to be kept in mind however, that in this volume only a minute proportion consists in the dye precursors. Chemistry of Tyrian Purple Formation Several preliminary studies on the chemical composition of the pigments of P. pansa are available. The comparison between the chemical composition of the hypobranchial secretion of other mu- ricids will lead to a better understanding of the metabolic pathways that lead to the final production of Tyrian purple. Since the re-discovery by Cole (1685) of "Tyrian Purple" from Nidfllii iPnrpiini} lapillus a number of researchers have worked on the determination of the chemical composition of the secretion of the hypobranchial gland. Most remarkable, considering the lim- ited knowledge of organic chemistry at the beginning of the 19th century, is the analytical work by Bartolomeo Bizio about the origin and properties of Tyrian purple from the Meditenancan muricids Murex tniiiciiliis and M. hniiuUiris (Ghiretti 1994). When collecting the glandular secretion of the snails he made the impor- tant observations, first, that as soon as the coloriess fluid is ex- posed to light and air it becomes immediately yellow and greenish, and soon afterwards it turns into deep emerald green, blue, deep blue and finally reaches the purple color. Next, that during the production of the purple dye, a highly odorous compound is re- leased. In comparing the color differences between the purple from Murex tninciihis and M. bnmdaris he discovered that they are species specific. Bizio also determined that Tyrian purple is a substance with chemical properties similar to indigo. Schunck (1879) isolated and crystallized the pigment from the ""ink" of Nucflhi (Purpura} lapillus. and determined the chemical proper- ties. He called the pigment punicin. To obtain 7 nig of punicin he extracted the hypobranchial gland of 400 animals, after which he reports "'my patience was exhausted". Friedlander (1909) isolated 1.4 g of the pure pigment from 12.000 hypobranchial glands from Murex hrandaris. and showed that it was 6.6'- dibronioindigo. Recently, using ad\anced analytical methods, Fouquet (1970). Baker and Duke (1973), Michel et al. (1992) and Koren (1994, 1995) among others, have confirmed that the major pigment from all studied muricids is 6,6'-dibromoindigo. Different species of muricids produce different color qualities of the dye, depending mainly on the number and concentration of the different chromogens. Fouquet (1970) found four differ- ent chromogens in the hypobranchial gland of M. trunculus: I) indoxyl sulfate, II) 2-methylthio-indoxyl sulfate. III) 6-bromoin- doxyl sulfate, and IV) 6-bromo-2-methylsulfonyl-indoxyl sul- fate, and he described the chemical pathway leading to Tyrian ptirple: The first step in the purple production is hydrolysis of the sulfate group with purpurase (aryl sulfatase). Indoxyl sulfate (I) and 6-bromoindoxyl sulfate (III) are then oxidized by oxygen to give indigo and 6,6'-dibromoindigo, respectively. With 2-me- thylthio-indoxyl sulfate (II) and 6-bromo-2-niethylsulfonyl- indoxyl sulfate (IV) oxidation is followed by dimerisation and the dimer is photolysed in light to give indigo and 6.6'-dibromoin- digo respectively together with methanethiol or dimethyl disul- fide. These reactions as described by Foquet (1970) are shown in Figure 1 . At the time of Fouquet's studies the possibility of cross- coupling of the indoxyls which accounts for the large percentage of 6-bromoindigo in the pigment of M. rruiuuluswdn was un- known. The composition of the chromogens of other muricids is less complicated. Thais clavigera. T. hronnii. Dicathais orbita. M. brandaris and N. lapillus contain 6-bromo-2-methylthio-indoxyl sulfate (IV); M. erimiceus contains a single different chromogen and Purpura luwmasuiuui and Rapana bezoar contain two other different chromogens, but the chemical structures are not known (Baker 1974; Hiyoshi & Fujise 1992). The reaction pathways of Tyrian Purple 195 (i) (ii) (I) —^ indoxyl -> indigo r (Hi) (ii) 2- <— dimer 1 <— methylthio- indoxyl l/ 6-bromo- (iii) dimer 3 <^ -^^""^^ J\ indigo <- <] ni (i) (II) (i) ^ (ii) 6,6'- (iii) dimer 2 (ii) 6-bromo-2- (i) (III) -^ 6-bromo- -> dibromo- <— <— methylsulfonyl- <— (IV) indoxyl indigo indoxyl Reagents: (i) aryl sulfatase, (ii) oxygen, (iii) light. oso. X=H, Y=H, (I) indoxyl sulfate X=H, Y=SCH3. (II) 2-methylthio-indoxyl sulfate X=Br, Y=H, (III) e-bromandoxyl sulfate X=Br. Y=S02CH3, (IV) 6-brofno-2-methylsulfonyl-indoxyl sulfate Figure 1. The chroniogtns from Miirex iriiiiculiis and their reactions to give indigoid pigments (Kouquet, 1970). 6-bromo-2-methylthio-indoxyl sulfate (tyrindoxyl) to give indi- goid pigments are shown in Figure 2. The composition of the different chromogens is not only de- pendent on the species of muricids. but also environmental, and physiological condition of the animals. The light intensity and oxygen availability also play a role during pigment formation. According to historical reports the best seasons to exploit the purple snails in the Mediterranean are autumn and winter. During summer the animals are hidden and in spring they lay eggs, at which time the hypobranchial secretion presumably is losing its coloring power and is not suited for color production (Bom 1936: Cardon & du Chatenet 1990). Fouquet (1970) cites Schaefer (1941 "Neuere Ansichten iiber den antiken Purpur" Chemiker Zeitung. 273) and O. von Fiirth (1903 "Vergleichende chemische Physiolo- gie der niederen Tiere". Verlag G. Fischer. Jena, page 377) who stated that there are seasonal changes in the chromogens of snails from the "trunculus" and "brandaris" types, due to age. gender, and food. Chemistry of Tyrian Purple from P. pansa For more than a hundred years the chemical composition of "Tyrian Purple" from P. paiisa has attracted the interest of chem- ists. Edward Schunck (I88()a) obtained a sample of cotton yarn dyed on the west coast of Nicaragua with the extract of Purpura patuhi (now P. pansa). From 24 g of dyed material he obtained 99 mg of pure crystalline pigment with all the properties of punicin. which he had earlier obtained from Purpura capillus (Nucella lapillus) (Schunck 1879). Thirty years later it was shown by Fried- lander (1909). that Schunck's punicin was 6.6'-dibromindigo. In 1922 Friedlander obtained from Mexico a sample of yarn dyed with the excretion of P. aperta (the zoological description has to be P. pansa. since P. aperta does not occur on the Pacific coast of the Americas). The analysis of the dye showed no differences in solu- bility, color, and absorption spectrum between the dye from M. brandaris. which he had analyzed before and for this reason he concluded with some certainty the dye of P. pansa consists mainly of 6.6'-dibromoindigo (Friedlander 1922). Saltzman ( 1992) showed that the reflectance spectrum of cloth dyed with the "ink" of P. pansa had a maximum absorption at 520 nm. Very similar results obtained Withnall et al. (1993) and Clark et al. ( 1993) for synthetic 6,6'-dibromoindigo. Mass spectrometry was used by McGovern et al. (1991) to confirm that the major colorant of the cotton sample from Saltzman. was 6.6'- dibromoindigo with traces of 6-bromoindigo. It was found that direct introduction of the cotton sample into the mass spectrometer led in addition to the detection of large amounts of 6-bromoindigo also to some indigo. This artifact arose from interaction of the cotton fiber and 6.6'-dibromoindigo at the high injection tempera- tures, leading to debromination and the formation of indigo. The problem can be avoided by extraction of the dye from the fiber with hot quinoline. or with dimethyl sulfoxide (McGovern et al. 1991). Using this extraction methodology, it was observed that apart from the major 6.6'-dibromoindigo component there were also smaller amounts of monobromoindigo and indigo which have been previously obscured by other low molecular weight materials. The analytical technique of choice for the characterization of mixtures of indigoid dyes is the application of the High Perfor- mance Liquid Chiomatography (HPLC). pioneered by Wouters and Verhecken ( 1991 ). This technique allows the characterization of pigments by retention time and absorption spectrum. Koren (1994) applied this technique to a sample of Dr. Saltzman's ma- terial containing the Mexican purple fiom Oaxaca. He could only detect 6.6'-dibromoindigo. A recent chemical study of the pig- ments of P. pan.sa confirmed the finding that the main component of the dye is 6.6'-dibromoindigo (90'7r); with 9% monobromoin- 196 Naegel and Cooksey SCHj SCH, SCH, tyrindoxyl sulfate tynndoxyl tyrindoleninone tynndoleninone I 0 H H 0 tyriverdin 6-bromoisatin i light 0 H tynndoxyl 6.6'-dibromoindigo 6,6'-dibromoindirubin Figure 2. The production of indigoids from t>rindoxyl sulfate in "brandaris-type" nuillusks. digo, and I'X dibromoindirubin (Withnall et al.. unpublished). Re- sults obtained by different authors (Wouters 1992; Cooksey et al. 1992; Keren 1993, Withnall et al. unpublished) using HPLC to determine the chemical composition of the indigoid constituents of the purple dye from various muricids are shown in Table 1. Since the values in Table 1 were obtained using a variety of HPLC protocols, close comparison is not justified, but some trends can be noted: P. pansa belongs to the M. bmndaris group con- taining no indigo in the pigment, some 6,6'-dibromoindirubin, and showing a higher propoilion of 6-bronioindigo than the average. TABLE 1. The composition (in %, obtained through HPLC analysis) of the indigoid constituents of the purple dye from various muricids. 6-Mono 6,6'- 6.6'- Bromo Dibromo nibromo Indigo Indirubin Indigo Indigo Indirubin Reference Murex bmndaris 0 0 3 83 14 Wouters (1992) Thais liaemastoma 0 0 3 91 6 Wouters (1992) Niicella lapillus 0 0 3 88 9 Cooksey et al., (IW2) Nucella lapillus g 0 1 77 14 Withnall et al.. (unpublLshed) P. pansa 0 0 9 90 1 Withnall et al.. (unpublished) P. pansa 0 0 16 77 7 Wouters (pers. com). Murex rruiiiutit.s 55 7 35 3 0 Wouters (1992) Murex irumiiliis 3 0 15 63 T Koren (1995) Tyrian Purple 197 Textile Dyeing with "Tyrian Purple" In using the "ink" for dyeing materials two significant differ- ences have to be mentioned between the Mediterranean muricids and P. pansa: (a) the Mediterranean snails have to be killed to obtain the chromogens, whereas P. pansa can be "milked" to ob- tam the dye withotit harming the animals; and (b) the "milk" from the P. pansa can be applied directly on textiles where the final pigments are formed in the presence of light and oxygen. In textile dyeing, there are two methods for dyeing with niol- lusk purple. The most simple is to have the chromogens react in the presence of light and air to obtain directly the final pigment on the fiber, as is the case with P. pansa. Since the molecular structure of mollusk purple is indigoid, there exists also the possibility of start- ing the dyeing process with the final oxidized purple colorant by reducing it in an aqueous alkaline bath, and applying the highly water soluble, and almost colorless leuco-form as a vat dye. just like indigo. Exposure to air oxidizes the leuco-form back to the indigoid pigment (Verhecken 1993; Clark et al. 1993; Schweppe 1998). In sunlight, the brominated leuco-indigoids can be photo- debrominated, leading to 6-bromoindigo or indigo after aerial oxi- dation and changing the purple color to blue. This chemistry of reduction and photodebromination of 6.6'-dibromoindigo, was first described by Driessen (1944), as shown in Figure 3. DISCUSSION Different species of muricids produce different color qualities of the ink (Born 1936a), depending on the number and concentra- tion of different chromogens. According to Verhecken ( 1 993 ) de- pending on the precursors and chemical reactions for the formation of the pigments, two groups of dyes from marine muricids can be distinguished: the "trunculus type" where light is not necessary, and the "brandaris type" requiring both light and oxygen. Since for the formation of the dye of P. pansa light and oxygen is necessary, the dye of P. pansa according to this definition belongs to the "brandaris type". Another fact supports this characterization: the pigments of the "brandaris type" contain mainly 6,6'- dibromoindigo. The pigments of P. pansa contain 90% 6,6'- dibromindigo, confirming that the snails are forming part of the "brandaris type". This is in contrast to the purple pigment derived from M. tnmcnhis, which is exceptional in containing non- reduce oxidise 6,6'-dibromoindigo (purple) leuco-6,6'-dlbromoindlgo light 0 H reduce oxidise H 0 6-bromoindlgo (purple/blue) leuco-6-bromoindigo light reduce oxidise '"*90(blue) leucoindigo Figure 3. The reduction and photodebromination of 6.6'-dibromoindigo. 198 Naegel and Cooksey brominaled precursors, leading to widely varying mixtures of in- digoid pigments, including indigo and indiruhin (Malaszkiewicz 1967). The biosynthesis of the chromogens in the hypobranchial gland of muricids originates from tryptophan, an essential amino acid for animals, which is enzymatically split into indole and pyruxate. After a sequence of chemical reactions different intermediates are formed from indole, which lead finally to the colorless precursor of indigoid pigments tyrindoxyl sulfate, and subsequently through an enzymatic reaction with aryl sulfatase to give the yellowish inter- mediate tyrindoxyl (Fouquet 1970). In the presence of oxygen the red tyrindoleninone is fomied. which reacts together with tyrin- doxyl to give the greenish tyriverdin. In the presence of light tyriverdin is photolysed to give dimethyl disulfide and the purple, insoluble pigment 6.6'-dibromoindigo (Verhecken 1989). Addi- tionally, from photolysis of tyrindoxyl or tyriverdin in the presence of oxygen. 6-bromoisatin can be formed which reacts with tyrin- doxyl to 6.6'-dibromoindirubin (Withnall et al.. unpublished). Oxygen and the light intensity during the chemical reactions of the intennediate substances determine the final composition of the pigments. The higher content of 6-bromoindigo than average in the pigments of P. pansa may result from the development of the dye under conditions of high light intensity. Under these conditions, any leuco-6,6'-dibromoindigo would be photodebrominated to give leuco-6-bromindigo. which on aerial oxidation would give 6-bromindigo. Under high intensity light conditions, the photo- elimination of dimethyl disulfide from tyriverdin to yield 6.6'- dibromindigo will proceed rapidly, leading to a low concentration of tyriverdin. Consequently, oxidative cleavage of tyriverdin, a bimolecular reaction, to give 6-broinoisatin is a minor reaction pathway, leading to a low concentration of 6.6'-dibroinonidirubin in the purple pigments of P. pansa (Withnall et al.. unpublished). The importance of the chromogens in the metabolism of the pui-ple snails is unclear. The presence of the enzyme aryl sulfatase. which presumably occurs in all muricids (Erspamer 1946). sup- ports the hypothesis that the chromogens could serve as a storage for the highly unstable indoxyls. which are formed enzymatically by the aryl sulfatase from sulfate esters (Fouquet 1970). Additional attention needs the question about the biological function of the indo.xyls and their substituted bromo and methylthio analogs. It could be possible that these bromo and thio substituted indoxyls. like the iodine derivatives of tyrosine could act as hormones in the metabolism of the snails. Since the chromogens. besides mucus and bioactive substances, have their origin in a specialized area of the hypobranchial gland, it is feasible that the gland could ha\e additionally inner secretory acti\ities (Fouquet 1970). We obser\ed during field work that P. pansa. above sea lex el uses the secretion to immobilize their prey, without the formation of purple pigments (unpublished personal observations). This sup- ports the finding that under normal circumstances the enzyme purpurase is kept apart from the chromogens. and therefore no pigments are formed, despite the presence of oxygen and light (Verhecken 1989). Additionally, in preliminary, yet unpublished personal studies, we could show, that the secretion from P. pansa is toxic to nauplii of Artemia, and has gram negative and gram positive antibacterial properties. From snails of less than 2 cm shell length can be obtained about 0..^ ml of secretion and from large animals 5-6 cm up to 4 ml (Rios Jara et al. 1994). About one liter of secretion is required to dye about 200 g of cotton (Acevedo Garcia et al. 1993; Michel Morfin 20()()). Since the average size of P. pansa is about 3 cm and a collection of more than 1 ml secretion per animal seems difficult, the enormous number of at least 1 .000 snails has to be "milked" to obtain 1 liter of secretion to dye only 200 g of material. Since too frequent "milking" does harm to the animals it was the right de- cision of the Mexican government to permit only Indian commu- nities the traditional exploitation of P. pansa for its pigments and to declare it a protected species. In contrast with the Mediterranean region, where the use of purple from marine snails has long been forgotten and the craft of dyeing today cannot exactly be reconstructed, in remote Pacific regions of Mexico (in the States of Oaxaca and Michoacan) and with the Indian community of the Borucas in Costa Rica (Turok 1999) its use is continuing now and represents the survival of a knowledge of considerable antiquity. However, as Thompson (1994) observed that this old tradition will be lost in the future. As Thompson (1994) notes "In the eariy 20th century in Mexico shell- fish purple was in much more widespread use than it is now\ The beliefs, languages, and crafts of the Mexican Indians are fast dis- appearing. The progressive "westernization" of rural Mexico has led people in many villages to abandon their traditional textiles and customs, in favor of factory-made cloth and western-style clothes which are readily axailable everywhere. Cultural and social decay is continuing to the point that the demand for traditional textiles has almost vanished. Weavers in a few \ illages fomieriy noted for their excellent textiles ha\e turned to making more 'commercial" articles, for sale to people, such as tourists, outside their culture — a classic manifestation of the "aiiport art' phenomenon."" Acevedo Garcia. J.. M. Turok Wallace & M. A. Escalante Cavazos. 1993. El caracol Purpura. Su uso y manejo en Mexico. Resumen de la III Reunion Nacional para el estudio del caracol P. p. pansa. 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(MOLLUSCA, GASTROPODA) IN RELATION TO SHELL STRUCTURE DOMINIQUE SUD,' * JEAN-MARC PONCET," ARMELLE SAIHI,' JEAN-MARC LEBEL,^ DOMINIQUE DOUMENC,' AND EVE BOUCAUD-CAMOU" ^ Museum Naliunal d'Histoire Naturelle. Laboratoirc de Biologic des Invertebres Mariiis et Malacologie, CNRS ESA - 8044. 57 rue Cuvier. 75 231 Paris Cedex 05, France: -(Jniversite de Caen. Laboratoire de Biologie el Biotechnologies Marines IBBA. Esplanade de la pai.x. 14032 CAEN Cedex. France ABSTRACT The mantle cytology and ultrastructure of the abalone Halioiis mherciikiw were investigated and different structures are detailed in relation to shell structure. The mantle edge of the abalone O-i mm near the shell edge) was chosen because it corresponds to the most active area in shell formation. The mantle is composed of a thin lamella of connective tissue wrapped in monolayered epithelia. the inner epithelium (facing the body cavity) and the outer epithelium (facing the shell). At the edge, the mantle ends with the division of the edge in two folds. The main features of glandular and nonglandular cells were characterized. The inner epithelium, responsible for ion exchanges with the external environment, is composed of three glandular cell types and ciliated or unciliated nonglandular cells. The periostracal groove, where the periostracum originates, shows high metabolic activity, with many secretions inside both from glandular and nonglandular cells. Numerous cilia have been observed in relation to these secretions. The outer epithelium could be divided into two distinct areas: a first short area in the outer part surface of the outer fold at the mantle edge (approximately 250-300 p.m long), called the tubular area, and a larger area which extends on the whole shell surface beyond the tubular zone. These two areas are both folded, hut in the tubular area folds form tubules. Moreover, some alimentary pigments have been observed to be associated with the tubular zone. Both structural characteristics of these two distinct areas as well as features of the cells indicate that they are responsible for the formation of the two distinct layers of the shell: the tubular zone (the calcite prismatic layer) and the outer epithelium (the aragonite nacreous layer), respectively. A£} WORDS: nantle. ultrastructural study, shell formation, marine gastropod. Halioiis INTRODUCTION The shell of the mollusc abalone. Halioiis Uiherculata. is made up of three distinct layers (Nakahara et al. 1982; Wilbur & Saleud- din 1983): the outer layer or periostracum. which is a tanned proteinaceous layer, a middle prismatic layer (calcite). and an in- ner nacreous layer (aragonite). The calcified layers are a tight association of calcium carbonate minerals with organic matri.x. Despite numerous studies of the mineralization processes in mol- luscs (reviewed by Wilbur 1964; Wilbur 1972). the precise mode of foimation of shell is far from clear. The organic matrix plays an important role in mineralization, providing sites for crystal nucle- ation and a template for crystal growth and orientation. Each layer of the shell corresponds to a different composition of this organic matrix. The precursors of this organic matrix are first secreted by the mantle into a small cavity between the mantle and the shell (the extrapallial space). Some studies on mollusc mantle structure show that a regionalization of the mantle exists in relation with the secretion of the three shell layers (Beedham 1958; Kawaguti & Ikemoto 1962b; Wilbur & Saleuddin 1983). Whereas the ultrastructure of the molluscan mantle edge, es- pecially the bivalves, has been studied in great detail, the epithe- lium of the inner (facing the mantle cavity) and the outer (facing the shell) surface of the mantle has received considerably less attention. In molluscs, the structure of the mantle, and more par- ticularly the mantle edge, shows a high degree of variation. In bivalves, the mantle edge is generally composed of three main folds (Jabbour-Zahab et al. 1992; Morrison 1993): an inner mus- cular lobe, a middle lobe believed to be sensory, and an outer lobe (Richardson et al. 1981 ). It is generally held that the periostracum in bivalves is secreted by the cells lining the surface of the outer *CorTesponding author. E-mail: sud@mnhn.fr lobe. In some gastropods, the mantle is also arranged into lobes, however, in numerous species the mantle lobes can be reduced in size and number (e.g.. Biainplialaria glalyrata. Bielefeld et al. 1993a; Bielefeld et al. 1993b). or are virtually lacking (e.g.. Lit- torina littorea. Bevelander & Nakahara. 1970). An additional fea- ture of gastropods is the presence of one or more groups of mar- ginal glands embedded in the connective tissue (Bevelander & Nakahara 1970; Saleuddin 1975). Because no study of abalone mantle has been undertaken to our knowledge, we investigated the histology and ultrastructure of the whole mantle, and more particularly near the shell edge, which is considered to be the more active part of the mantle in the bio- mineralization processes. Then we attempted to relate features of the cells to formation of the different shell layers: the perios- tracum. the calcitic prismatic, and the aragonitic nacreous layers. MATERIALS AND METHODS Source and Maintenance of Animals Adult (6-7 cm in length) and juvenile (mph lacunae are designated b) the black arrows (xl60). (b) Subdivision of the mantle edge into different func- tional areas: 1, inner epithelium: 2, epithelium of the outer face of the inner fold: 3, bottom of the periostracal groove: 4, epithelium of the inner face of the outer fold; 5, tubular area of the outer epithelium; and 6, outer epithelium, which extends on the «hole shell surface bevond area 5. luicrovilli. 2-5 \x.n\ high (Fig. 2a). Cells are interconnected by intercellular junctions comprising an apical desmosome and sep- tate junctions. A distinguishing feature of the inner epithelial cells is the highly convoluted interdigitalions of the plasma membranes below the apical desmosome (Fig. 2b). Under the basal lamina, a thin sheet of collagen fibers is observed (orientated parallel to the basal lamina). Two major cell types can be identified, depending on the secretions of their cytoplasm: epithelial glandular cells (where the cell cytoplasm is filled with secretory granules) and epithelial nonglandular cells. Epithelial nonglandular cells are generally similar in size. Cilia are occasionally observed, interspersed between the microvilli (Fig. 2a). but the general characteristics of these cells remain simi- lar along the luantle IE. These epithelial cells contain a basal nucleus in which luost of the heterochromatin is concentrated in the periphery. A Golgi complex is often observed in the vicinity of the nucleus. Mitochondria are mainly located near the nucleus as well as in the apical part of the cell cytoplasm, even though some have been observed in the whole cytoplasm. Numerous vacuoles are located in the apical part of the cells. Some vacuoles contain some dark-stainiui; contents, which are sometimes heterogeneous Mantle Ultrastructure of a Marine Gastropod 203 Figure 2. Area 1. (a) limer iiuintle epithelium iTEMi with nontjlaii- dular cells. Cilia Ihlack arrowsl are interspersed with niicrntilli (M\ I. Some vesicles with pigments are observed in the apex of the cells (white arrows! 1x4,50(11 lb) Detail (TEMi of highlv convoluted plasma mem- branes below the microvillar border. Cells are separated from each other by intercellular junctions comprising a desmosome (white star) and septate junctions (black arrowheads) (x20.0(l()). aniJ couW be related to some kin(i of pigments. Tonotllament hunehes are observed mainly orientated in the axis of the cell (Fig. 2b). A general feature of the glandular cells (or secretory cells) is their goblet shape. The narrow openings of glandular cells can sometimes be seen. The main features of these cells are the oc- currence of numerous secretory granules in their cytoplasm and a basal small nucleus surrounded by a more or less developed rough endoplasmic reticulum (Figs. 3. 4. and 5). Three glandular cell types could be distinguished depending on their secretions. Type A glandular cells (Fig. 3) contain clear ovoid granules, closely packed and homogenously stained, which tend to coalesce. Type B glandular cells (Fig. 4) contain distinct, darkly staining granules, which have a denser core in some cells. The membrane of type B granules is generally still distinct when they are discharged. Type C glandular cells (Fig. 5) contain some distinct, membrane-bound granules, with rosette-like mucous granules. Type C cells have peculiar secretory granules of heterogeneous stages: in a clear and finely granular matrix an array of complex chains of electron- dense material is found. The basal nucleus is surrounded by a well-developed rough endoplasmic reticulum of enlarged cisternae filled with finely granular material. Between the rough endoplas- mic reticulum and the secretory granules. Golgi stacks occur. The immature granules from the trans Golgi only contain finely granu- lar material, then the dense complex progressivelv condenses and develops. Figure 6 resumes the main features of the IE: a monolayered epithelium which constitutes ciliated (Ci). or nonciliated. nonglan- dular. and glandular cells (A. B. C types) with microvillous border (Mv). I iniue .^. \rea I. Detail of type A glandular cells in the inner mantle epithelium (TEM): The cytoplasm is filled with ovoid granules, clo.sely packed, homogeneously stained. N, nucleus (xl4,(l(IO). Tlw Periostracal Groove: Areas 2a. 2h, .?, 4a, 4h We divided the periostracal groove, which runs parallel to the mantle edge, into three different parts of spatial and equal impor- tance, depending on the structure of the cells. The first part (a) corresponds to the opening of the periostracal groove directly in contact with external seawater: areas (2a) and (4a) are, respec- tively, the epithelium of the inner and outer folds of the periost- racal groove. Principal features of the cells are very similar to those of cells of the IE: in the 4a area, characteristics of the IE are less marked (plasma membrane is not as convoluted as in the 2a area, and pigment granules are less representated than in the 2a area). The second part (b) corresponds to the middle of the peri- ostracal groove: areas (2b) and (4b) are. respectively, the epithe- lium of the inner and outer folds of the periostracal groove. A principal feature of this area is the presence of a group of ciliated cells of the epithelium 4b (outer fold. Fig. 7) and the presence of numerous secretions from nonglandular cells of the epithelium 2b (inner fold. Fig. 7). All cilia are orientated to the opening of the periostracal groove and some secretions are often found associated with the cilia. Principal characteristics of the cells are quite similar to that of cells previously described. In the periostracal groove, the microvillous border is not equivalent in the inner and outer folds. In the epithelium of the outer fold (4a. 4b). microvilli are dense and ananged in a brush border, whereas the cells of the inner fold (2a, 2b) are lined by microvilli irregularly arranged. A lot of material seems to be dis- charged by these cells, as clear vesicles with a fine granular con- tent. We can also observe some secretions from nonglandular cells in the microvilli in area 2b (in the inner fold). 204 Slid et al. Fisure 4. Arta I. Detail dI t\|)t B glandular cells in the inner mantle epithelium (TEM): The cytoplasm is filled with distinct, darkly stain- ing granules, which have a denser core in some cells |x30.0(l()). In areas (a) and (b) of the inner and outer fold, we can notice the presence of numerous B-type glandular cells. A-type glandular cells have been observed in some sections in the epithelium of both outer and inner fold. However, C-type glandular cells have only been found in the epithelium of the inner fold. At the bottom of the periostracal groove (area ?<), the cells become gradually cuboidal. Essentially, nonglandular cells are ob- served. Cilia and cell secretions could not be observed (Fig. 8). The inner and outer folds appear morphologically equivalent. Fine Structure of the Outer Mantle Epithelium: Areas 5 and 6 In H. tiiheniilata. two distinct areas of the outer mantle epi- theliuin can be distinguished: a short area near the periostracal groove (area 5) and the other part of the outer mantle epithelium (area 6). The structure of the cells and the structure of the folds in the OE determine this separation. Area 5 corresponds to a very folded epithelium, which forms some tubules when this area is quite developed. In scanning electron micrographs of the outer mantle epithelium, this area corresponds to an alveolous structure in external view: this area has been called the tubular zone (TA. Fig. 9). In area 6. the OE displays folds that are quite parallel to the mantle margin (Fig. 9). In area 5 the tubular /one is more or less developed, depending on the collected specimens in a same size class. To observe the well-developed tubular zone, some juveniles have been observed by TEM (Fig. 10). The principal features of these cells are their cuboidal shape, a high nucleoplasmic ratio, and numerous round- shaped mitochondria. In Figure 10. tubules are cut transversally. Figure 5. .\rea 1. Detail of type C glandular cells in the inner mantle epithelium (TEM): The cytoplasm is Tdled with distinct, membrane- bound granules, which have a heterogeneous content. N, nucleus (xl4.(H)0). and we can observe in the lumen of each tubule some small mi- crovilli and some secretions. In this area, pigments are found trav- elling from hemolymph lacunae to tubules; they correspond to an obvious red and/or green line along the border of the OE in live specimens. The pigments are carried by "migrating cells" (likely hemocytes). which have a central nucleus and a cytoplasm filled with four or five vacuole sites containing a large central core ot heterogeneous granules (Fig. 1 1 ). Figure 12 summarizes the main features of the tubular zone: a monolayered epithelium (which is very folded) composed of cuboidal cells and tubules (T) containing microxilli (Mv). alimen- tary pigments (Pi) in vacuoles, and soine secretions in the lumen (Lu). In area 6. a different structure than the OE has been observed: cells are low columnar (I5 (xm) (Fig. 14). depending on studied specimens in the same size class. Meanwhile, the OE is always folded. In some specimens with low columnar cells (Fig. 13), numerous "empty" cells have been observed and the other cells have a cytoplasm where few organelles are present, corresponding to low cell activity. In other specimens, the OE is very high and narrow columnar (Fig. 14): numerous mitochondria, Golgi apparatus, and inclusions are found in the cytoplasm of these cells (Fig. 15). In such cells, all or- ganelles, and in particular, all Golgi complexes, are found orien- Mantle Ultrastructure of a Marine Gastropod 205 Figure 6. Area 1. Schematic drawing of the main features of the cells of the inner epithelium. In this area, three types of glandular cells are present: types A. B, and C (encircled A, B, C), with nonglandular cells, with or without cilia (Ci). BL, basal lamina: Ci, cilia; GS, Golgi stacks; M, mitochondria; Mv, microvilli; N, nucleus; RER, rough endoplasmic reticulum; Pi, pigments; TB, tonofilament bunches; V, vesicle (not to scale). tated in the longitudinal axis of the cells (Fig. 15). The microvil- lous border is well developed in very high columnar cells and numerous protruding cell processes are found associated with this microvillous border (Fig. 14). In low columnar cells the microvil- lous border is quite nonexistent excepted in fold of the IE; how- ever, in the cells of the OE, some distensions of the membranes are generally observed and they are often associated with mitochon- dria (Fig. 15). The schematic Figure 16 resumes the main characteristics of the OE far from the periostracal groove: very high columnar cells containing numerous mitochondria (Mi), Golgi apparatus (G). and Figure 7. Area 2b-4b of the periostracal groove (TEM): Nuniirous cilia are interspersed in the microvillar border of the epithelium of the outer fold (4b). The microvillar border of the inner fold (2b) is irregu- lar and some cell protruding processes can be observed (black or white arrowheads). Some secretions of the B type glandular cells are asso- ciated with cilia (*). IE, inner mantle epithelium: OE, outer mantle epithelium (x9,0UO). microvillar border (Mv| with protruding processes, orientated in the longitudinal axis of cells. DISCUSSION Many publications describe shell formation in molluscs and some of them reported the structure of the mantle and its role in shell formation. But the majority of these studies dealt with bi- valves (Kawaguti & Ikemoto 1962a; Kawaguti & Ikemoto 1962b; Neff 1972;Bubel 197.^a, 1973b, 1973d; Garcia-Gasca et al. 1994). Most of the work on gastropods has been done on the terrestrial snail Heli.x and on freshwater gastropods (Zylstra et al. 1978. Bielefeld et al. 1993a, Bielefeld et al. 1993b); only one reference to a marine gastropod was found: Lillorina tittorea (Bevelander & Nakahara. 1970). Although shell production is often assumed to be quite similar among molluscs, there are important morphologic Figure 8. Area 3. lioltiiju of the periostracal groove (TEM). The cells are cuboidal. Only few secretions and no cilia can be observed in this part of the periostracal groove (x4,()00). 206 SUD ET AL. Figure 9. Areas 5 and 6. External structureof the outer nianlle epithelium (SEM). Near the periostracal groove (PG), the tubular area (TA) (area 5) appears as honeycomb lobe at the surface of the mantle (double black arrow I, whereas the outer epithelium (OEl beyond area 5, covering the whole shell surface (area 6) (single black arrow) ha\e some folds parallel to the periostracal groove. This tubular area (area 5) can be distinguished from the outer epithelium covering the remainder of the mantle (area 6| (x35(l). differences of the mantle between bivalves and gastropods due to different shell structure and morphology (Hedegaard, 1497). As Martin et al. (1983) have demonstrated with their study of the gonads in Haliotis ntfescens. two cell types can be defined depending on their cytoplasm content: glandular (or secretory) cells and nonglandular cells. In H. tiihercuhita. ultrastructural stud- ies have allowed us to determine three types of glandular cells with regard to their secretion xesicles content. Type A and B glandular cells are present in the whole epithelium, whereas type C cells only occur in the IE and in the inner fold epithelium of the periostracal groove. Type A glandular cells correspond to a typical mucous cell. Type A and B glandular cells are present in various species (for example: Lymnaea stagnalis, Zylstra et al. 1978). Type C glandular cells are rarely represented in molluscs and have already been described in the gastropod Rhodope sp. (Has/prunar & KCin/ 1996). Glandular cells, and )iotably type B. aie highly represen- tated in the periostracal gi'oove. While in the IE glandular cells play a role in the protection of the mantle (Lemaire-Gony & Bou- dou 1997), various authors repon the presence of glandular cells. which might facilitate the movement of the periostracum out of the groove or protect the site of periostracum formation against invad- ing water (Bielefeld et al. 1993a; Bielefeld. 1993b; Garcia-Ga.sca et al. 1994). Some ulti'astructural studies demonstrated that glan- dular cells play a role in periostracum formation (Tsujii 1968a; Tsujii 1968b; Bevelander & Nakahara 1969a; Bevelander & Na- kahara 1969b; Nakahara & Bevelander 1971): this role is to be assumed in H. litherculala. The role of glandular cells in mineral deposition is controversial in the studies; Tsujii (I960) suggests that in Pincuula manensii. glandular cells in the OE do not par- ticipate in shell forirtation. whereas Beedham (1958) (studying Aiunlonhi cygnea. Mytilus ediilis. and Ostrea edulis) believes that secretion may be incorporated in the organic matri.x of the shell. Figure 10. ,\rea 5. Tubular area of the outer nianlle epithelium (TEMl in a juvenile abalone. .Structure in tubules are shown transversally cut. Cells contain a central nucleus (N) and numerous mitochondria can be observed (arrow heads). In the lumen of the tubule some .secretions are present (*) (x4,000). Mantle Ultrastructlire of a Marine Gastropod 207 Figure 11. Area 5. Tubular area of the outer mantle epithelium (TEM). Alimentary pijjments (AP) are found in \acuole.s of wandering hemocyte-like cells. HI,, hemolymph lacunae; hemocyte (*) (x3,000). Hillmun (1961) suggests that not all glandular cell types could interfere with the biomineralization processes. The organic matrix of the shell mineral layers (which are calcite and aragonite) con- tains some mucopolysaccharides (Garcia-Gasca et al. 1994). As a consequence, the participation of glandular cells in shell formation could not be ruled out in H. niberciilata. and further studies need to be undertaken to determine the nature of each glandular cell and the role of glandular cells in shell formation. In H. tubercidata. the mantle edge ends with a periostracal groove, parallel to the mantle surface, which divides the mantle into two folds: the inner and outer folds. In all molluscs, the periostracum is secreted by the mantle edge, but the mode of ^\i •^ Figure 13. Area 6 of the outer mantle epithelium (TK.Ml. Oetall of one fold: Numerous empty cells can be obser>ed (*) (x3,000). formation and the morphological structure of the mantle edge vary from species to species. Generally, in molluscs, the periostracal groove divides the mantle into folds, although it is virtually lacking in some species such as the gastropod L. litlorea (Bevelander & Nakahara 1970). The mantle edge of bivalves generally consists of three folds compared with the usual two folds among (freshwater or terrestrial) gastropods. In bivalves, the periostracum originates from epithelial cells lining the inner surface of the outer fold of the periostracal groove and/or from a row of basal cells at the bottom of the groove (Kawaguti & Ikemoto 1962a; Dunachie 1963; Neff 1972; Bubel 1973a; Bubel 1973c; Bubel 1973d; Saleuddin 1974; Petit et al. 1979). In some gastropods, such as Helix sp.. the pe- riostracal groove is described as a single layered, flattened Figure 12. Area 5. Schematic drawing of the tubular area of the outer epithelium (OE). The epithelium is folded and when well developed, it forms tubules (T). Some alimentary pigments (AP) are observed near the hemolymph lacunae (HL). H. hemocyte; M, mitochondria: .Mv micro\illi: N, nucleus (not to scale). 208 Figure 14. Area 6 of the outer mantle epithelium (TEM(. \ ery high columnar cells are seen; numerous protruding processes (arrows) can be observed within the microvillar border 1x2,00(1). glandular epithelium called the "periostracal gland" (Beedhani 1958), although it does not form a compact body or have a duct. The cells forming the gland seem to be equivalent to the basal cells described in other bixalves (Saleuddin 1974). In some gastropods (L. suignalis and Biamphalaria pfeifferi). some different types of gland cells are found which have their cell bodies sunken into the underlying connective tissue at the base of the groove (Zylsti'a et al. 1978). In gastropods, the periostracal groove is generally shallow, but it is deeper in H. niherculata and morphologically similar to that in the bivalves: this could be due to the primitive status of this gastropod (Stasek & McWilliams 197.^). Periostracal gland and basal cells, described respectively in gasti'opods and in bivalves (Hillman 1961; Kawaguti & Ikemoto 1962a'. Kawaguti & Ikeinoto 1962b: Bubel 1973a: Bubel 1973b: Petit et al. 1979). have not been Figure 15. Area 6 ol the outer mantle epithelium ( TK.M). Detail ol'one very high columnar cell: Inclusions of different content are present in vesicles (V). GS, Golgi stacks; M, mitochondria (x40,00()). Figure 16. ,Vrea 6. Schematic drawing of an active outer epithelium. Palissadic cells are tall and narrow, tilled with numerous organites arranged in the longitudinal avis of the cells; (iolgi stacks ((iS), mito- chondria (M). rough endoplasmic reticulum (RER), The microvillar border (Mv) is well developed and some protruding processes (black arrows) are observed (not to scale). found in H. tuberculata. Secretions of material have been observed in the outer surface of the inner fold, whereas it is generally ac- cepted that, in bivalves, the cells lining the inner surface of the outer fold contribute to the periostracum (Bubel 1973b). A group of ciliated cells have been observed in the inner surface of the outer fold of the periostracal groove of H. tiiberciikita. Such cilia have been described in some bivalves {Pinctada iiiargariiifeni. Jabboui-Zahab et al. 1992). but are generally located at the bottom of the groove. These cilia may help to conduct and aggregate the periostracum material outside the periostracal groove as well as microvilli in the brush border along the inner surface of the outer fold. A distinct membrane-like lamellar layer has not yet been ob- served in anv preparation or fixation in H. mbercukiui periostracal groove, although such a membrane-like layer is reported for other gastropods and bivalves (Saleuddin 1 979; Saleuddin & Petit 1983). This could be explained by the fact that animals have not been anesthetized and mantle contractions could tear up the perios- tracum. Nevertheless, the peiiostracum has also not been found in luveniles fixed in lolo with their shell. Anesthetized animals with 3-aminobenzoic acid ethyl ester (MS222) or 1% MgCI, could be useful to answer this question. Moreover, no periostracal units described in other bivalves or gastropods (Saleuddin 1976; Bielefeld et al. 1993b; Schaefer & Haszprunar 1997) have been observed in cells of//. tuht'icuUita. It seems that the secretions and the formation of the periostracum differ from those previously described, and a more detailed study of periostracum formation must be undertaken to determine how the periostracum is secreted. In H. mbeiTiilata. two distinct areas have been identified in the OE. These two areas are morphologically different; both epithelia are folded, but in the outer surface of the outer fold, the epithelium can form tubules, whereas beyond this, the OE folds are quite similar and are almost orientated parallel to the periostracal groove side. In the first area (5). located in the outer surface of the outer Mantle Ultrastructure of a Marine Gastropod 209 fold, cells are cuboidal. and numerous mitochondria have been observed. According to Istin and Masoni (1973i. in bivalves, the number of mitochondria underlying the epithelial border of the outer fold indicates a metabolic activity for such cells. This activity is not linked only with calcium movements, but may be linked with matrix components synthesis. In the vicinity of this area, in the connective tissue, and near the hemolymph lacunae, some accu- mulations of partially digested pigments have been found. These pigments originate from the algae diet, giving red or green color of the prismatic layer, depending on the algae consumed as food. In Haliotidae, these pigments are incorporated into the calcitic layer of shell (Leighton 1961 ). Thus, this particular area called here the tubular zone (5), can be related to the secretion of the calcitic layer in H. tubeicuhiia. This area has already been described by Crofts ( 1929) as the "glandular acini." This tubular area is always present in juveniles of H. nihcrciilaia. but it could be less developed or even absent in adult specimens. A lower development of tubular area in adult specimens could be related to a decrease in shell growth rate compared to juveniles. The presence or the absence of this area may be considered as a cyclic shell secretion, whereas the other area (6) of the OE is likely related to the formation of the aragonitic (nacreous) layer. This regionalization of the outer mantle epithelium exists in other molluscs: according to Nakahara and Bevelander (1971 ), in P. radiata. the prismatic layer of the shell is derived exclusively from the tall columnar cells lining the outer surface of the outer mantle fold. Although some authors found a distinction between the secretion of the different shell layers (Beedham 1958, Ostrea ediilis: Kawaguti & Ikemoto 1962b, Mitscidus senlioiisia: Jabbour- Zahab et al. 1992. Pinciada maxima: Schaefer & Haszprunar 1997, Laevipilina antanlica). other authors found this distinction not to be effective (Petit et al. 1980 - Amblema sp.). In H. tuberculuta. this regionalization is in accordance with the differences in the organic matrix components of each shell layer and with the pres- ence of alimentary pigments incorporated into the calcitic layer. In area 6 of the OE in relation with the nacre secretion, two different structures of these epithelia have been found: a tall co- lumnar and a very high, narrow columnar epithelium, depending on the activity of the mantle. These two epithelial structures have been observed, depending on studied specimens. Such morpho- logical differences may be correlated with seasonal and/or indi- vidual variations. In the tall columnar epithelium, organelles are not in great number and numerous "empty" cells have been ob- served, whereas in the very high, narrow columnar epithelium, numerous organelles are found (mitochondria. Golgi complexes, rough endoplasmic reticulum, numerous inclusions of various types). It seems that the last epithelium is much more active than the first. A large number of cytoplasmic vesicles as well as nu- merous mitochondria were found, indicating an involvement in the mechanisms of shell formation attributed to this epithelium (Wil- bur 1964; Istin & Masoni 1973). Moreover, the very high, narrow columnar epithelium is associated with protruding cell processes, which can be related to the secretion of the organic matrix com- ponents of the shell (Wilbur 1964; Jabbour-Zahab et al. 1992). In the very high, narrow OE. some distensions of the intercellular spaces have been found: these could act as ion pumps, particularly for calcium (Zylstra et al. 1978: Richardson et al. 1981). The structure of the mantle margin in H. nihercidata is very peculiar and could not be related morphologically to the mantle of other gastropods and bivalves. A spatial organization of the mantle exists in relation with the secretion of the different shell layers: the periostracal groove in relation with the secretion of periostracum. the tubular area (in the outer surface of the outer fold) in relation with the deposition of the prismatic layer, and the OE (beyond the previous area) in relation with the secretion of the nacreous layer. This spatial organization can be related to a temporal variation, because the OE seems to have different structure depending on the stage of secretory activity of the mantle. This temporal variation could be related to the incremental bands deposited annually, monthly, daily, or during another shorter period (Wilbur 1972). because shell formation is incremental rather than continuous. ACKNOWLEDGMENTS This study was supported by the French Ministry of National Education. Research and Technology (Grant 97 CO 230). LITERATURE CITED Beedham. G. E. 1958. Observations on ttie mantle of the Lamellibranchiu, Quart. J. Microsc. Sci. 99:181-197. Bevelander. G. & H. Nakahara. 1969a. An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs. Calcif. Tissue Res. 3:84-92. Bevelander. G. & H. Nakahara. 1969b. An electron microscope study of the formation of the ligament of Mytilus edulis and Pinciada radiata. Calcif. Tissue Res. 4:101-112. Bevelander. G. & H. Nakahara. 1970. An electron microscope study of the formation and structure of the periostracum of a gastropod. Littoriita littorea. Calcif. Tissue Res. 5:1-12. Bielefeld. U.. K. H. Kbrtje, H. Rahmann & W. Becker. 1993a. The shell- forming mantle epithelium of Biomplialaria glabrata (Pulmonata): ul- trastructure, permeability and cytochemistry. / Moll. Stud. 59:323- 338. Bielefeld. U.. W. Peters & W. Becker 1993b. Ultrastructure and cytochem- istry of periostracum and mantle edge of Biamphalaria glabrata (Gas- tropoda. Basommatophora). Acta Zool. (Stockholiitl 74:181-193. Bubel. A. 1973a. An electron-microscope study of periostracum formation in some marine bivalves. I. The origin of the periostracum. Mar. Biol. 20:213-221. Bubel. A. 1973b. An electron-microscope study of periostracum formation in some marine bivalves. II. The cells lining the periostracal groove. Mar. Biol. 20:222-234. Bubel. A. 1973c. An electron-microscope study of periostracum repair in Mytilus edulis. Mar. Biol. 20:235-244. Bubel. A. 1973d. An electron-microscope investigation of the cells lining the outer surface of the mantle in some marine molluscs. Mar. Biol. 21:245-255. Crofts. D. R. 1929. Haliotis. Liverpool Mar. Biol. Comm. Memoirs on typical British marine plants and animals. Liverpool University Press. 174 pp. Dunachie, J. F. 1963. The periostracum o( Mytilus edulis. Trans. R. Soc. Edinb. 65:38,^-411. Garcia-Gasca, A., R. Ochoa-Baez & M. Betancourt. 1994. Microscopic anatomy of the mantle of the pearl oyster Pinciada inazailanica (Han- ley, 1856). / Shellfish Res. 13:85-91. Haszprunar, G. & E. Kiinz 1996. Ultrastructure and systematic significance of the epidermis and haemocoel of Rhodope (Gastropoda, Nudibran- chia. Doridoidea?). J. Subniicrosc. Cytol. Pathol. 28:485-t97. Hedegaard, C. 1 997. Shell structure of recent Vetigastropoda. / Moll. Siiid. 63:369-377. no SUD ET AL. Hillman, R. E. 1961. Formation ot the perio^tracum in Mcneiuiiia iiwi- cemiria. Science 134:1754-1755. Istin. M. & A. Masoni. 1973. Absoiption et redistribution du calcium dan.s le manteau des lamellibranches en relation avec la structure. Calcif. Tissue Res. 11:151-162. Jabbour-Zahab, R.. D. Chagot, F. Blanc iS: H. Grizel 1992. Mantle histol- ogy, histochemistry and ultrastructure of the pearl oyster Pinctada margarinferu (L.). Aqiial. Living Resinii: 5:287-298. Kawaguti. S. & N. Ikemoto. 1962a. Electron microscopy on the mantle of a bivalve. Fahulina niticliilci. Biol. J. OI 0.05). After 12-h immersion, the retention efficiencies of all biotin- supplemented diets were significantly different and showed a negative correlation with the biotin supplemental content {r = 0.87). By taking into account the leakage rates in the first 3-h immersion and the amount initially encapsulated, the real amounts of biotin delivered to the abalone were about 0, 0.49. 0.87, 2.88. 5.75. 9.37, and 17.96 mg biotin/kg diet for DO. D0.5. Dl. D3. D6. DIG, and D20, respectively. Survival and Growth Survival, specific growth rate (SGR) and daily increment in shell length (DISL) data are shown in Table 2. During the 1 lO-day experimental period, there were no significant differences in sur- g 80 96.1 95.8 94.6 95.3 E]:!h 84. 2 J., T EF^ S3 1 fir 69. 2a ^ in ^ 92. 7 90. 3 81. 4 rii Jl k J, DO. 5 Figure 1. Tht retention efficiency (RE) of dietary biotin in six biotin supplemented diets ut different interval (3, 6 and I2h, respectively) immersed in seawater in triplicate. Error bars are SE. Means with the same letter are not significantly different [P > 0.05). In the first 6h immersion. RE were not significantly dilierent among all diets. After 12h, the values were significantly dilTerenl and show a negative rela- tionship with the biotin supplemental content (/' = 0.87). vival (89.2-96.2%.) with any of dietary treatments (ANOVA. P > 0.05). The SGR of the antibiotic group (0.83%/day) was signifi- cantly lower than those of biotin treatinent groups (1.10-1.28%/ day) (P < 0.05). There were no differences observed in SGR among biotin treatment groups during the experimental period (ANOVA. P> 0.05). Similarly. DISL were significantly higher for dietary biotin treatment groups (60.0-69.1 p.m/d) than for the antibiotic group (50.9 |xm/d) (P < 0.05). Among all dietary biotin treatments, di- etary biotin levels influenced DISL significantly (ANOVA. P < 0.05). DISL increased from 60.0 to 69.1 |a.m/d with dietary biotin levels increasing to I mg/kg. There was a decline in DISL observed in abalone fed diets with biotin supplementation higher than 3 mg/kg. Thus, the broken-line regression model (Robbins et al. 1979) was used in this study to express the relationship between DISL and dietary biotin content. The regression equations are shown in Figure 2. As the break point at 0.42 mg/kg gave the least mean square enor. the adequate amount of dietary biotin for ju- venile abalone is estimated to be 0.42 mg/kg. Carcass Composition and Biotin Concentration As seen in Table 3. all the moisture (75.30-77.91%). crude protein (55.87-58.35%). and crude lipid content (5.16-6.37%) of abalone were not affected by dietary treatments (ANOVA, P > 0.05). However, the visceral biotin concentration (VBC) signifi- cantly responded to dietary treatments (ANOVA, P < 0.05). The differences among the eight groups were all significant. By addi- tion of 0.4% tetracycline hydrochloride in the basal diet, the VBC was significantly lower than those of other groups were. A linear increase in the VBC was observed when the biotin supplementa- tion le\el increased (r = 0.91). Carboxylase Activity The activities of pyruvate carboxylase (EC 6.4.1.1 ) and acetyl CoA carboxylase (EC 6.4.1.2) in abalone fed test diets are ;ire- sented in Table 4. Both enzyme activities showed similar trends lo 214 WU ET AL. TABLE 2. Effect of dietary biotin on survival and grovvtli of abalone. H. discus haiinai Ino. (mean [s.e.), n = 3l. Dietary Initial Shell Biotin Initial Weight Length Final Weight Final Shell Length Survival SGR' DISL- Diet (mg/kg) (nig) (mm) (nig) (mm) C^f ) (%) (um/d) DO (1 171.2 (27. .S) 11.5(0.71 576.9(18.1)"' 18.1 (0.2)''" 90.01 7.5) 1.10(0.15)" 60.0 (5.3)" D().5 0.5 1 173.3(27.5) 11.6(0.4) 599.3 (38.9)'"- 18.5(0.2)-* 95.3(1.4) 1.13(0.13)" 62.7 (4.0)" Dl 0.91 173.4(5.0) 11.8(0.2) 711.8(29.2)'' 19.4(0.4)" 96.2(1.4) 1.28(0.05)" 69.1 (4.0)'" D3 3.04 I7S.6(12.5) 11.6(0.2) 629.0 (85.0)'='' 18.7(0.9)-"' 94.2(5.2) 1.14(0.08)" 64.5(6.5)"' D6 6.03 181.1 (22.5) 11.9(0.6) 613.5(43.4)'^" 18.7(0.9)-'" 90.8(3.8) 1.11 ((.).08)" 61.8(5.6)" D12 10.11 175.0(5.0) 11.7(0.3) 599.0(27.2)"' 18.5(0.5)"" 92.5(2.5) 1.12(0.04)" 61.7(3.2)" D20 19.89 177.3(22.5) 11.7(0.4) 629.0(71.6)' 18.0 {e.g)-"" 89.2 (7.6) 1.15(0.17)" 62.0(1.7)" Antihiolir 0 170.7(15.0) 11.5(0.4) 424.0(39.4)"'' 17.1 (0.3)" 92.5(2.5) 0.83(0.01)-" 50.9 (2.3)" ANOVA F 0.27 0.46 5.70 4.75 2.52 2.74 3.84 P 0.956 0.S51 0.019 0.005 0.059 0.044 0.012 ' Specific growth rate. - Daily increment in shell length. ^ Supplemented with 0.4'7f tetracycline in basal diet. "^ Means in the each column sharing the same letter are not significantly different based on Tukeys' test (P > 0.05). (dietary treatments. Compared to the basal group, two carboxylase activities in viscera wei'e significantly depressed when animals were fed the antibiotic diet (93.8 U/mg protein and 7.31 U/mg protein for the activity of pyruvate carboxylase and acetyl-CoA carboxylase, respectively). Apart from the antibiotic diet, the ac- tivities of pyruvate carboxylase activity ( 155.9 U/mg protein) and acetyl-CoA carboxylase (8.33 U/mg protein) of basal group were the lowest among the biotin treatment. The activities of both en- zymes generally increased with increasing dietary biotin up to 3-mg/kg diet then leveled off. The differences between the lowest group and the groups fed diets with biotin higher than 1 mg/kg diet were significant iP < 0.05). It is obvious that the relationships between both enzyme activities and dietary biotin levels were in accord with the broken-line regression model (Robbins et al. 1979). Based on pyruvate carboxylase and acetyl-CoA carboxylase activities, the regression equations were Y = 8.97 + 0.03 x (X - 0.70) (r = 0.93") and Y = 171.1 +03S x {X-0.61} {r = 0.85). respectively. Therefore, the biotin requirements were estimated to be 0.67-0.70 ma/ka that based on two carboxylase activities. DISCUSSION Abalone are known to be slow feeders or nibblers. They can take seseral hours before consuming one feed flake. Thus, it is necessary to reduce leaching to precisely quantify the requirement of water-soluble micronutrients including water-soluble \ itamins. Microencapsulation is one of the most potential methods to reduce leaching by sheltering encapsulated materials from outer environ- ment (Louis 1970; Gupta & Rao 1985: Shun et al. 1988). Accord- ing to Marchetti et al. (1999). the retention efficiencies of lipid- walled \itamin B, in pelleted food were 87.5* and 72.5'7f for 1 and 2-h immersion, respectively. In the present study, the RE of biotin was up to 90.3-96. 1'/r after 3-h immersion. It is evident that leaching out of biotin froni the experimental diets was reduced in a great degree. A previous study indicated that the guts of most TABLE 3. The effects of dictarv biotin on carcass composition and VBC' of abalone. H. discus haiinai Ino. (mean |s.e.|, n = 3. Protein" Lipid' .\Ioister VBC Diet ('7f) C^f) {'7c) (ng/g) DO 56.30(0.14) 5.74(0.78) 75.30(3.85) 202.2(17.6)" D0.5 56.88(0.10) 6.37 (0.38) 75.51 (0.81) 292.3(21.9)'= Dl 56.73(1.60) 5.64(0.51) 75.73 (0.28) 369.4 (27.4)" D3 58.16(0.59) 6.15(0.64) 77.91 (1.11) 555.2 (40. !)■-■ D6 58.35(1.92) 5.79(0.25) 76.03 (0.42) 936.2(22.2)' D12 56.39(0.83) 5.16(0.44) 76.60(1.22) 1068.4(31.3)^ D20 56.42(0.67) 5..59(0.28) 77.31 (2.77) 1208.7(59.2)" Aniibioric^ 55.87 (0.97) 6.04(0.04) 76..50(0.47) 75.8(7.2)" ANOVA F 2.21 1.22 0.75 566.23 P 0.089 0. 1 35 0.636 0.000 0 5 10 15 Dietary biotiti(mg/)tg) Figure 2. The effect of dietary biotin on daily increment in shell length (DISL) of abalone. Each point represents the mean of three groups o abalone (n = 3), with thirty abalones per group. Based on the broken- line model, the biotin requirement is estimated to be 0.42 mg/kg. ' Viscera biotin concentriition. " Dry weight basis. ' Dry weight basis. ■• Supplemented 0.4<7r tetracycline in basal diet. ' " Means in the each column shanng the same letter are different based on Tukeys' test iP > 0.05). not signiticanllv Dietary Biotin Requirement of Juvenile Abalone 215 TABLE 4. Effect of dietary hintin on carboxylase acti> ities in viscera of abalone H. discus haniiai (mean |s.e.l, ii = i). Pyruvate Carboxylase' Acetvl-CoA Carboxviasc- Diet (U/nij; Protein) (ll/nig Protein) DO 155.9 (4.07 )*" 8.33(0.19)'" DU.5 167.5 {6.63)"'^ 8.80(0.14)'"" Dl 173.2 (5.59f 9.36(0.21)' D3 175.0(7.72)' 9.45(0.331' D6 171.8(8.33)' 9.20(0.29)' D12 174.7(9.25)' 9.32(0.14)' D20 178.3(5.31)' 9.40 (0.24)' Antibiotic^ 93.8 (6.22)" 7.31 (0.21)-' ANOVA F 93.88 41.12 P 0.000 0.000 ' Per unit equal 1 (jimol of H['''C]0, incoiporaled into oxaloacetale per minute at 30°C. " Per unit equal 1 pmol of H|'''C]0, incorporated into nialonyl-CoA per minute at 37°C. ' Supplemented with 0.4% tetracycline in basal diet. ■" Means in the each column sharing the same letter are not significantly different based on Tukeys' test (/> > 0.05). abalone were full of food after 2 to 3-h feeding with premium quality diets (Mai et ul. 1998b). There were no significant differ- ences observed in retention efficiency when all diets immersed in seawater for 6-h (ANOVA, P > 0.05). Thus, in this study, the leaching may not influence the result of biotin requirement for abalone by encapsulating biotin and other water-soluble vitamins. On the other hand, wall-forming material calcium alginate is apt to be digested by abalone as high level of alginase appears in the digestive tract of abalone (Oshitna 1931: Nakado & Sweeny 1967; McLean 1970; Toshio 1985; Hugo & Maria 1998). In comparison to those reported by other authors (Uki et al. 1985; Uki & Wa- tanabe 1992; Mai et al. 1995a; Mai et al.l995b). the satisfactory abalone growth indicates encapsulated vitamins can be efficiently utilized by abalone and supports our conclusions as follows. Until now. the essentiality of dietary biotin for any mollusk species was unknown. Results of this study clearly show that di- etary biotin is necessary for juvenile abalone as the shell growth (DISL) was significantly influenced by dietary biotin. The shell growth of abalone fed lower levels of dietary biotin was depressed implying that biotin probably affects the course of biomineraliza- tion of shell. This is in accord with the findings of Bain et al. ( 1988) that biotin deficiency affects bone growth in broiler chick. During the 110-day experimental period, dietary biotin did not significantly affect SGR. However, there still was an obvious trend that lower biotin levels reduced SGR. As we know, biotin is a type of micronutrient in diets and the signs of biotin deficiency are usually produced under specific conditions. Thus, a 110-day ex- perimental period is probably not long enough to significantly affect SGR in abalone. In juvenile animals, body length growth is usually faster than the body weight gain. This agrees with DISL being a more sensitive parameter to dietary biotin than SGR. and DISL may be a responsive criterion for estimating dietary biotin requirement for other mollusk juveniles. Based on the daily incre- ment in shell length (DISL). the optimum biotin requirement for the maximum growth is estimated to be 0.42 mg/kg diet by the broken-line regression analysis. Compared with other reports, the optimum requirement for juvenile abalone is higher than that of brook trout (0.05 and 0.25 mg/kg) (Poston & McCartney 1974). lake trout (0.1 mg/kg) (Poston 1976). rainbow trout (0.05-0.14 mg/kg) (Woodward & Frigg 1989; Castledine et al. 1978). com- mon carp (0.02-0.03 mg/kg) (Ogino et al. 1970), tilapia (0.06 mg/kg) (Shiau & Chin 1999), but less than the requirements re- ported for the mirror carp (2.0-2.5 mg/kg) (Gijnther & Meyer- Buorgdorff 1990) and grass shrimp (2.0-2.4 mg/kg) (Shiau & Chin 1998). The different biotin requirements probably attribute to the differences of experimental procedures and species. The presence of high dietary fat has been shown to obscure effects of biotin in rats, chicks, brook trout, and rainbow trout (Jacobs et al. 1970; Marson & Donaldson 1972; Poston & Mc- Carteney 1974; Walton et al. 1984). In the present study. 3.68% of lipid was measured in the basal diet. According to Mai et al. (1995a). optimum dietary lipid content was 3-5% to maintain maximum weight gain for abalone. Therefore, 3.68% of lipid is just sufficient to meet requirements of the abalone and lipid effects on biotin requirements can be omitted here. As a coenzyme for several carboxylases, biotin affects the me- tabolism of amino acids, carbohydrates, and lipids. Thus, some studies have demonstrated that the proximate composition of ani- mals usually respond to dietary biotin treatment. Poston (1970) reported the carcass fat content of lake trout was depressed when animals were fed biotin-free diets. The crude ash, fat and protein contents of mirror carp were significantly affected by dietary bi- otin (Giinther & Meyer-Buorgdorff 1990). Similar results were also obtained with common caip (Ogino 1970) and tilapia (Shiau & Chin 1999). However, in the current study, the crude protein and lipid contents in the soft body tissues, which consist of mantle, foot muscle, and viscera that include all inner organs, of juvenile aba- lone remained independent of biotin supplementation. This effect may be manifested by a longer experimental duration. It is also generally believed that carboxylase activities are depressed in bi- otin deficient animals such as mammals, fishes and shriinp (Deodhar & Mistry 1969; Arinze & Mistry 1971; Poston & Mc- Carteney 1974; Castledine et al. 1978; Walton et al. 1984; Shiau & Chin 1998. 1999). In the present study, the pyruvate carboxylase and acetyl-CoA carboxylase activities of abalone visceral tissues positively responded to dietary biotin levels. This implies that abalone did experience biotin deficiency, which would lead to depressed enzyme activities. Thus, both carboxylase activities are useful criteria in estimating dietary biotin requirements for aba- lone. Based on two carboxylase activities, the optimum biotin requirement for the maximum enzyme activities is estimated to be 0.67-0.70 mg/kg diet by the broken-line regression analysis. In the present study, the recommended biotin requirement for maximum carboxylase activities is higher than that for maximum growth (0.42 mg/kg diet). This means that the biotin level resulting in maximum enzyme activities and the level resulting in maximum growth are not necessarily the same. In some species of land animals, such as calves and cows, biotin has been found synthesized in considerable amounts by intestinal bacteria (Victor & Rachel 1945). In the present study, when tetracycline was incorporated into basal diet as an antibiotic group, a deficiency developed. The SGR DISL of antibiotic group were significantly lower than those of the basal group (P < 0.05). Similarly, the visceral biotin concentration (VBC) and the carbox- ylase activities were also obviously depressed by addition of tet- racycline. Sugita et al. ( 1992) examined the biotin-producing abil- ity of intestinal microflora in freshwater fishes and proved (he !16 WU ET AL. requirement of freshwater fishes for hiolin was significantly influ- enced by intestinal microflora. Thus, we believe that the poor growth of the abalone in the treatment with antibiotics could at- tribute to the suppression of intestinal microflora that can probably synthesize biotin. Further investigation is needed to provide direct evidence. Another interesting phenomenon is that VBC increased from 292.3 to 1208.7 ng/g with increasing biotin supplemental levels (r = 0.91). Theses results indicate that biotin can be stored in abalone viscera in amounts corresponding to the dietary supply. Therefore, the VBC can be used to monitor biotin status in aba- lone. To our knowledge, the toxicity of biotin in animals is still unccrtaui. however, depressed growth was observed when abalone were fed high levels of dietary biotin and this would require further studies. ACKNOWLEDGMENTS This study has been supported by grant No. 39925029 from National Science Foundation for Talented Youths of the People's Republic of China and the Excellent Young Teachers Program of MOB of the P. R. China. We thank Dr. Q. F. Ye (Institute of Nuclear Agriculture. University of Zhejiang. China) for his excel- lent technical assistance. LITERATURE CITED AOAC. 1995, In: P. Cunnitf, editor. Official Methods of Analysis, 16th ed. Washington, DC: Association of Official Analytical Cheni. Arlnze, J. C. & S. P. Mfstry, 1971. Activities of some biotin enzymes and certain aspects of gluconeogenesis during hiotin deficiency. Coin/). Biochem. Physiol. 386:285-294. Bain, S. D.. J. W. Newbrey & B. A. Watkins. 19S,S. 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Watanabe. 1992. Review of the nutritional requirements of abalone {Hnliotis spp.) and development of more efficient artificial diets. In: S. A. Shepherd. M. J. Tegner & S. A. Guzman del Proo, editors. Abalone of the world: Biology. Fisheries and Culture. United Kingdom: Fishing News Books.Oxford. pp. 504-517. Viana. M. T., L. M. Lopez & A. Salas. 1993. Diet development for juvenile abalone Halioiis fulgens. Evaluation of two artificial diets and mac- roalgae. Aquacullure 117:149-156. Victor. A. N. & B. Rachel. 1945. The synthesis of B \ itamin by intestinal bacterial. In: R. S. Harris & K. V. Thimann. editors. Vitamins and Hormones. Vol 111. New York: Academic Press, pp. 23^8. Walton. M. J.. C. B. Cowey & J. W. Adron. 1984. Effects of biotin deficiency in ranibow trout (Salmo gairdncri) fed diets of different lipid and carbohydrate content. Aqiiaciihure 37:21-38. Woodward. B. & M. Frigg. 1989. Dietary biotin requirements of young rainbow trout iSalmii .iiainliwri) determined by weight gain, hepatic biotin concentration and maximal biotin-depended enzyme activities in liver and white muscle. J. Nmr. 1 19:54-60. Zempleni. J.. T. A. Trusty & D. M. Mock. 1997. Lipoic acid reduced the activities of biotin-depended carboxylases in rat liver. J. Niilr. 127: 1776-1781. Joiinwl of Shellfish Rfsciirch. Vol. 21. No. 1. 219-226, 2002. IDENTIFICATION OF SOUTHERN HEMISPHERE ABALONE (HALIOTIS) SPECIES BY PCR-RFLP ANALYSIS OF MITOCHONDRIAL DNA NICHOLAS G. ELLIOTT,' JASON BARTLETT,' BRAD EVANS," AND NEVILLE A. SWEIJD^ ^CSIRO Marine Research. GPO Box 1538, Hobart. Tasmania 7001. Australia: 'Department of Zoology, University of Cape Town. Private Bag. Rondesboscli. 7701. Cape Town. South Africa. Current address: BENEFIT Secretariat. Box 912 Swakopmund. Namibia: School of Zoology. University of Tasmania. GPO Box 252-05. Hobart. Tasmania 7001, Australia. Current address: School of Marine Biology & Aquaculture. James Cook University. Townsville. Queensland 481 1 , Australia ABSTRACT Illegal fishing and species-substitution of abalone (genus Haliotis). a highly valuable marine gastropod, are of world- wide concern. A mitochondrial DNA PCR-RFLP analysis of fragments of the cytochrome oxidase I (mtCOI) and II (mtCOII) genes was developed for the identification of 1 1 Southern Hemisphere species of abalone. These included five temperate and one tropical species from Australian waters, three temperate species from New Zealand and two temperate species from South Africa. All species, with the exception of the Haliolis ruhralH. conicopora complex, can be unequivocally identified using the combined profiles from four individual restriction enzyme digests {Ddel. Hhal, HinFI and Hpall) on a 1 93 base pair fragment of mtCOI. Six species each displayed a unique profile for a single restriction enzyme. A one hundred and fifty-nine base pair fragment of mtCOlI allowed individual identification of six of the species using the combined profiles from five individual restriction enzyme digests {Dilc/. EcoRV, Hhal. Hpall. and R.sal). These primers failed to amplify in H. iris. Again H. rubra and H. conicopora could not be separated, and neither could H. australis and H. spadicea. No DNA sequence variation in either fragment was observed between H. rubra and H. conicopora; the latter may be a subspecies of//, rubra. The use of both fragments and a minimum of two restriction enzymes is recommended for species differentiation. DNA was successfully extracted, PCR amplified and identified from canned tissue and mucous samples of //. rubra. A conformational mutation in the mtCOI fragment was observed in H. miilac. but in no other species nor in the mtCOII fragment. KEY WORDS; Haliotis. abalone, mitochondrial DNA, identification INTRODUCTION Abalone. genus Haliotis Linnaeus, are a highly valuable com- mercial marine univalve niollusk. There are over 55 recognized species worldwide (Geiger 1998). of which nearly half are ex- ploited by commercial or recreational divers. Abalone generally inhabit rocky reefs to depths of 65 m, but more are usually found in shallower waters to 30 m. The foot muscle of abalone attracts high prices in Asian markets, with species differential. Once the distinguishing shell and mantle tissue have been removed, it is very difficult to differentiate the commercial product of one spe- cies from another. The high price, market demand, ease of harvest and similarity of processed product between species makes aba- lone very suitable targets for illegal marketing and both highly organized and small scale poaching. Abalone poaching and species-substitution of abalone products is of concern to many countries, including the USA (Daniels & Floren 1998), Mexico (Ponce-Di'az et al. 1998). South Africa (Sweijd et al. 1998) and Australia. The value of the illegal trade is difficult to quantify. Conservative estimates in Australia alone are over $US25M annually. The legal Australian abalone fisheries. dominated by Haliotis rubra, account for about half the annual world abalone harvest of ca. 10,500 mt (FAO 2000) and is worth around $US80M per year. The South African abalone fishery (H. midae ca. 500 mt/yearl is worth approximately $US 1 5M with legal sales of confiscated (poached) abalone from just one area fetching over $USlm (Sweijd et al. 1998). In New Zealand the main com- mercial species is H. iris and the illegal harvest is estimated at Corresponding author. Nicholas G. Elliou, CSIRO Marine Research, GPO Box 1538. Hobart. Tasmania 7001. Australia. E-mail: nick.elliott@ marine.csiro.au about 33% of the annual commercial catch of 1 ,300 mt (Roberts et al, 1999), The high but unknown level of illegal harvesting of abalone creates major problems for fishery managers endeavoring to maintain viable and economic fisheries. Whilst H. rubra is the dominant commercial species within temperate Australian waters, both H. laevigata and H. roei are subject to significant levels of commercial fishing under indepen- dent quota systems, and a H. scalaris fishery is under consider- ation, A problem for fisheries enforcement is the overlapping ranges of these and non-commercial species. Such species richness is common with abalone (Geiger 1999). and after removal of the characteristic shell and mantle species identification of the foot muscle is obscure. The need exists for a definitive means of iden- tifying tissue and by-products (e.g., mucous in instances of sus- pected poaching when tissue has been disposed) of individual aba- lone species. Identification of plant and animal species when morphological characters have been removed is possible using either protein or DNA-based methods (Palumbi & Cipriano 1998; Toro 1998; Jo- hannesson & Stenlid 1999; Hare et al. 2000; Sweijd et al. 2000). The protein based methods are very dependent on tissue quality; generally requiring fresh or frozen material. Often identification for commercial needs may require analysis of processed (dried or canned) tissue or degraded tissue. DNA-based methods are rela- tively independent of tissue quality, and those that rely on ampli- fication of small DNA fragments are less likely to be affected by degradation (Mackie et al. 1999). A number of techniques are available for species identification including; random amplifica- tion of polymorphic DNA (RAPD) (Martinez & Malmheden Yman 1998). restriction fragment length polymorphism (RF-LP) analysis (Innes et al. 1998; Wolf et al. 2000). direct DNA sequenc- ing (Quinteiro et al, 1998) and single-stranded conformation poly- 219 220 Elliott et al. morphism (SSCP) (Mackie et i\\. 1999) of PCR (pdlymerase chain reaction) amplified fragments. A PCR-RFLP analysis of a 1,300 base-pair (bp) fragment of the nuclear lysin gene was devised for identification of two South African abalone species. Haliotis midae and H. spadicea (Sweijd et al. 1998). Generic PCR primers that amplify across the intron differentiated between species based on the size of the intron. Preliminary analyses found that the size of the lysin intron varied greatly between other Haliolis species (generally 500 to 1.100 bp). but the intron in the Australian greenlip abalone H. laevigata was over 4,000 bp (unpublished data). Products of such size are not ideal for species identification tests with the likelihood of unreli- able PCR products due to tissue and DNA degradation. To differentiate between the more common Southern Hemi- sphere abalone species a PCR-RFLP method was developed using short fragments (less than 200 bp) of the mitochondrial DNA (mtDNA) molecule. To satisfy potential legal scenarios in Austra- lia and South Africa, 11 species were included. Within species variation and potential non-Haliotis amplification of our designed primers were examined in addition to testing the primers with canned abalone tissue and abalone mucous samples. MATERIALS AND METHODS Sample Collection and DNA Extraction Whole individuals (live or frozen) or alcohol preserved tissues were obtained for 1 1 purported Haliotis species: Haliotis asinimil Linnaeus, Queensland, .^ustralia (30 individuals); Haliotis aiistra- lis Ginelin, New Zealand (10); Haliotis conicopora Peron, West- em Australia, Australia (11): Haliotis iris Gmelin, New Zealand (10); Haliotis laevigata Donovan, Tasmania & Victoria, Australia (62): Haliotis midae Linnaeus, South Africa (10); Haliotis roei Gray, Western Australia, Australia (10); Haliotis rubra Leach. Tasmania, Victoria & New South Wales, Australia (50); Haliotis scalaris Leach, Tasmania & Western Australia, Australia (22); Haliotis spadicea Donovan, South Africa (10); Haliotis virgiiiea Gmelin. New Zealand (10). Total genomic DNA was extracted from ca. 25 mg of foot muscle or gill tissues using a modified CTAB (hexadecyltrimeth- lammoniumbromide) protocol (Grewe et al. 1993). Tissue was incubated overnight at 50' C instead of 30 to 60 min at 60X. To verify the use of our PCR primers on processed product. DNA was extracted from commercially canned H. rubra. Approxi- mately 0.5 g tissue was digested for 30 min at 65°C in 5 mL digestion buffer ( 100 mM Tris. 50 mM EDTA. 400 niM NaCl, 1% SDS). 50 (iL proteinase K ( 10 mg/mL) was then added and the solution was incubated overnight at 55''-C. 150 \x.L NaCl (5 M) and 520 jjiL of 10% CTAB were added and the solution incubated at 65°C for 1 h with regular mixing. Samples were then extracted once with equal volumes of chloroform/isoamyl alcohol (24: 1 ) and precipitated with 2 volumes of 100% ethanol. Precipitated DNA was washed twice with 70% ethanol. once with 100% ethanol, air-dried and re-suspended in 200 |xL TE. PCR amplification was also tested using DNA extracted from H. rubra mucous. Two mucous samples were obtained by placing individual freshly captured H. rubra in separate plastic bags for approximately 2 h, removing the abalone and placing the bag and fluid contents at 4°C, Tissue samples were taken from the indi- vidual abalone as positive controls for DNA extraction. DNA was extracted from mucous swabs taken from the sides of the bags and from the control tissue samples using the modified CTAB protocol described above. In addition, a 600 |iL sample of fluid (mixture of seawater and mucous) from the bottom of each plastic bag was taken, incubated overnight in 20 jjlL proteinase K (10 mg/niL) and 5% SDS, and then genomic DNA extracted using the same modi- fied CTAB protocol. Genus specificity of the PCR amplification was tested on total genomic DNA extracts (using above CTAB protocol) from a va- riety of marine organisms. These consisted of an alga (unidentified red alga), an anemone (unidentified), a crustacean (Antarctic krill Euphausia superha). mollusks (unidentified chiton and Pacific oyster Crassostrea gigas) and teleosts (bigeye tuna Thunnus obe- sus. southern bluefin tuna T. maccoyii, pink ling Cenypterus bla- codes. Patagonian toothfish Dissostichus elegiiioides. school shark Galeorhinus galeits and gummy shark Musteleus antarcticus). PCR Primers and Amplification Generic PCR primers were designed for the mitochondrial cy- tochrome r oxidase subunit I gene (mtCOI) by alignment of either our own unpublished or published Haliolis sequences (Metz et al. 1998). DNA sequences used for the design of the mitochondrial cytochrome c oxidase subunit II gene (mtCOII) were either our own or other unpublished sequences (Sandy Degnan. University of Queensland). The primers designed to amplify a 193 bp fragment of the mtCOI gene were designated HALCOi-NGI (5'-ClGACATRGCITTYC- CICGACT-3') aTid HALC01-NG2 (5'- CCGGCTARGTGIAGIGA- RAAAAT-3'). Those designed for a 159 bp fragment of the intCOII aene were designated HALC02GENA (5'-CAATYTGAACYAT- TCTMCCAGc''-3') and HALC02GENB (5'-CCTTAAARTCT- GAGTATTCGTAGCC-3'). (Degenerate nucleotide lUB codes; 1. Inosine = A, C. G or T; M, aMino = A or C; R, puRine = A or G; Y. pyrimidine = C or T). PCR reactions consisted of 50 to 100 ng of total genomic DNA, 2.5 mM MgCU, 200 |xM each dNTP, 10 pmoles of each primer, and 0.55 U Taci DNA polymerase (Biotech) in a buffer supplied by the manufacturer. PCR amplifications were carried out in a 50 ^.L final volume using a Perkin Elmer GeneAmp® System 9600 with hotlid. The cycling parameters were as follows: denaturation at 95°C for 3 min, 10 initial amplification cycles (94°C for 30 s, 60-55''C for 30 s, 72°C for I min. with a decrease in the annealing temperature of 0.5°C per cycle), a further 25 amplification cycles (94°C for 30 s, 55°C for 30 s, 72"C for 1 min) and final extension at 72°C for 5 min. Negative controls, without DNA template, were prepared for each series of amplifications to exclude the possibility that PCR reagents and buffers were contaminated with template DNA. Amplification products were examined by electrophoresis through a 2% agarose gel (GIBCOBRL) made up in 1 X TBE. Gels were stained in ethidium bromide at a concentration of 0.5 (j.g/mL and visualized under UV light. A one-hundred bp lad- der (GIBCOBRL) was run concurrently to facilitate sizing of am- plification products. DNA Sequencing PCR products were sequenced to confirm variation in restric- tion fragments and sizes, and to improve PCR primer design. PCR products were purified using Wizard™ PCR purification columns (Promega) according to manufacturers instructions, and sequenced using an ABl Prism '^' BigDyeT" Terminator Cycle Sequencing Read\ Reaction Kit (Perkin Elmer). Cycle sequencing reactions Southern Hemisphere Abalone Identification were electrophoresed on an ABI377 automated DNA sequencer (Perkin Elmer) and analyzed using ABl Prism '^' Sequencing Analysis Version 3.3 (Perkin Elmer). RFLP Analysis For each individual of the I I species, four separate restriction digestions of the mtCOI fragment were performed using the four enzymes Ddcl, Hhal. HiiiFI and Hpall (New England Biolabs. Genesearch). For the mtCOII fragments five separate restriction digestions were performed for each species individual using the enzymes Dclel. EcoRV. Hhal, Hpall and Rsal (New England Biolabs, Genesearch). Restriction digestions were carried out in a 15 (jlL total volume consisting of 5 jjlL of PCR product, 1.5 p,L digestion buffer supplied by the manufacturer, 0.5 [xL enzyme, and 8 (jlL ddHoO for all enzymes except Hlial. Digestions for Hlial were carried out in a 15 \i.L total volume consisting of 5 p-L of PCR product. 1 .5 |j.L digestion buffer supplied by the manufac- turer, 0.5 p-L enzyme, 1.5 |j.L 10 X Bovine serum albumin (BSA) and 6,5 |xL ddH.O. Mitochondrial haplolypes were scored by electrophoresis of 10 |xL of digested PCR product in a 3% agarose gel made up in 1 X TBE at lOOV for 3 h, stained in ethidium bromide (0.5p,g/mL) and visualized under UV light. Electrophoresis of restriction digestions was also performed on 12% polyacrylamide (Austral Scientific) gels made up in 1 ,\ TBE and run for 2 h at 100 V. RESULTS DNA Exlraclioii and PCR Amplification DNA e.xtraclions from fresh, alcohol preserved and canned tissue, resulted in high yields of high molecular weight total ge- nomic DNA. Amplification of these extracts consistently produced high quality PCR products. Extractions from mucous scrapings and fluid samples from plastic bags produced a small amount of high molecular weight genomic DNA. PCR amplification of these extracts failed at times to yield a product when undiluted, however when diluted 10 fold, produced a strong PCR product in all samples (Fig. 1). PCR amplification of non-Haliotis DNA with the designed primers was only observed in the tuna samples. Both tuna species amplified ( 160 bp fragment) with the mtCOl primers. Sequencing of the tuna mtCOl products confirmed that the observed product was not contamination from abalone DNA, While nucleotide dif- ferences and RFLP cut site differences existed to separate these teleost products from abalone products, high levels of nucleotide sequence conservation suggests that the amplified product was part of the tuna COI gene. Restriction Digests intCOI The expected 193 bp fragment was generated in each abalone species following PCR amplification with the HALCOl-NGl/ HALC01-NG2 primers. Comparison of the DNA sequences indi- cated suitable restriction sites for discrimination between species using four restriction enzymes (Fig. 2). Intraspecies restriction digest polymorphisms were observed in four species, but in each case for a single individual for only one enzyme (Table 1 ). Two of the observed polymorphisms were the result of a loss of a restriction site and two the result of a gain. All individuals showing a different restriction fragment profile for the species were sequenced to confirm the profile. All other digestions 10 11 12 13 14 15 16 M Figure 1. Electrophoretic analysis of undiluted (lanes 1 to 61 and di- luted (xlO. lanes 9 to 141 193 bp mtCOl (upper image) and 159 bp mtCOII (lower image) PCR fragments for H. rubra tissue and mucous samples. M = UK) bp ladder. Samples in lanes are as follows: 1, 2, 9 and 10 muscle tissue: 3. 4, 1 1, and 12 fluid sample from plastic bag: 5, 6, 13, and 14 mucous swab from plastic bag; 7 and 15 positive H. rubra DNA (x20 dilution) control: 8 and 16 negative H,0 control. returned a single restriction pattern in all individuals examined for each species. At this 193 bp fragment, six of the eleven species had a unique species-specific restriction pattern for at least one enzyme, and so could be individually identified (Table 1 ). With the exception of the H. rubra and H. conicopora pairing, all species are discernible from each other using the four restriction enzymes, regardless of all but one observed polymorphism. The exception polymorphism was a single H. nihni individual that had gained a Dilel cut site, and therefore had a profile similar to H. scalaris. The restriction profiles for the canned tissue, mucous and fluid samples all matched that expected for H. rubra. One purported H. scalaris individual returned a different pro- file at three enzymes to all other H. scalaris individuals. This particular individual displayed the expected cut pattern for H. lae- vigata for all four enzymes; three of which are diagnostic between the two species for all other specimens analyzed. Laboratory con- tamination was ruled out and the results confirmed with repeated tissue sampling. DNA extraction and PCR amplification for this one individual. The observed fragment lengths produced in this study were all examined on agarose and (non-denaturing) polyacrylamide gels and confirmed by sequence analysis. A fragment mobility change was observed in the mtCOI fragment for H.inidae when run on a polyacrylamide gel (Fig. 3). This assumed conformation-induced mutation was only observed in H. midae. Restriction Digests mtCOII The expected 159 bp fragment was generated in each abalone species following PCR amplification with the HALCOIGENA/ 222 Elliott et al. H. rubra 1 CTGACATGGC TTTTCCTCGA CTAAATAATA TAAQATTCTG HinFI 50 ACTACTCCCA CCCTCACTAA CCCTTCTATT 100 AACATCGGGT GCTGTAGAAA GTGGTGrrqr; Hpall .C. .C. . . . .A. . C. C. .C. .c. .c. .A C. , .c. .c. HmFI G T . .T T . GT . . . . G . . .A C. Hpall H. scalaris HmFI -G T . .T T .G . . .A H.asinina HinFI -G HinFI linFI . . .T. . . .T. C , .T. .C , , .A. .C. .C. , .T . .A. .TT.G. . -T. . .T. . . .T. .T. .TT .T. .T, . A . . TC . .GT. . . . .A. .T. . r.A. .C. . . . ,r G . . . . Hpall . . .A . . .A. .G . . . . .c. .G Hpall . . .A. . H.midae .A A. . .0 .A A. . .A. .T .G CIGACATRGC A. C. G. .c. .c. .N. 'TYC T. HinFI .C ...„ ^^. HinFI .G T. "CICGA CT - c. c. c. .c. .c. .c. . . . HinFI I Hpall . . .A. . HinFI -G , . H.australis H.iris H. virginea HALCOl NGl HmFI T. . T. . T. . G . .T. . .T, .T. .C . .T. .C , T . . .A. .T. .C. . .A. .C. . . . . .G. .CT. . . TC . CC . . A . . TC . G. . C. . ^ — .. Hhal . . .A. .A . .0. . . .A. .G . .C. . . . .c Hpall . . . .C A. . .C..A.. Hhal HALC01-NG2 H . rubra 101 GACAGGATGA ACAGTCTACC CCCCACTATC CAGCAACCTA 150 GCCCATGCCG Gf^QQATCAGT Hpall Hhal AGACTTGGCA ATTTTTTCAC TTCACCTAG£ CGG Hpall 200 H. laevigata H. scalaris Hpall Hhal .T Hhal .T .A G. . .C A Hpall Hpall . . .T Hpall H.asinina H. roei T. . . . .G. . . C. . A G. . c G .!.. Ddel T. .T C . . .T T. .G C T. .T T T. .T T . . .T T - Hpall G.__ , Hpall Ddel ICC. . T A ..CC. .A.. c Hpall Ddel Hpall T A. . , A. . , A. . , A. . . . . .C. . . , . .C. .G , . .G. . . . . .C. . . . . .T. .A. . . . .T. .G. . A. . T T. . T. . . . .T. . . . .T. . TT. . . . TT . G . . C .A. . .A. . T Hpall Hpall C. .T. , . . .A. . Ddel Hhal Hpall Hhal G C. .A. . . Hpall H.iris .T. . ...A. , C. .G. ^ • * ■ c C T C C A T Hpall H . virginea Hhal ,.,... .r. . .CT NO C Hpall HALCOl-NGl HALC01-NG2 Hhal Hpall TAAAARAGIG AIGTGRATCG GCC Figure 2. Stquence alignment ol the 193 bp mtCOI fragment for eleven abalone species. Primer sequences and cut sites for the four restriction enzymes Ddel, Hhal. HinFI, and HpaH are included. (N = sequence data unclear whether (.' or T). TABLE 1. Expected restriction fragment lengths for eleven Haliulis species for the 193 hp mfCOI fragment when cut with restriction enzymes Ddel. Hhal. HinFI and Hpall. The number in parenthesis represents the total number of individuals examined for each species that displays the given restriction pattern. Unique restriction profiles are shown in bold. H. laevigata numbers include the misidentified H. scalaris individual. COI Restriction Digestion Patterns Ddel Hhal HinFI HpaH H. Lisimna H. aiislrulis H. t onictipora H. in.s H. hii'\'igiita H. midae H. roei H. ruliro H. .sdiltiri.', H. spudicea H. virginea 7.5I).I37(29) 7,187 (X) 193(10) 193(11) 193(10) 193(63) 193(10) 29,164(10) 193(49) 29.164(1) 29,164(21) 193(10) 193(10) 193(30) 79,114(10) 42.151 (ID 42.1.51 (10) 193 (62) 94,99(1) 42.151 (10) 193(10) 42.15! (50) 42.151 |21) 42,151 (10) 42,57,94(9) 42.151 (1) -M.159(.Wl 193(10) 34,159(11) 19,174(10) 34,159(62) 193(1) .U.159(10) 6,34,97(10) 34.159(49) 193(1) 34,159(21) 15,19,159(10) 193(10) 3.42.72.76(30) 3,93,97(10) 3,42.51.97(11) 3,190(10) 3,42,148(63) 3,42.148(10) 45.51.97(10) 3.42.5 L97 (50) 3.42,51.97(21) 3.190(10) 3.190(10) Southern Hemisphere Abalone Identification 223 Ddel 1 2 3 Hhal 2 3 Hpall 1 2 3 Figure 3. RFLP patterns on a non-denaturiny polvacrylamide gel of the 193 bp nitCOI fragment for three abalone species produced with four restriction enzymes. Species \ - H. midiie. species 2 - H. rubra, species 3 - H. laevigata. M - 100 bp DNA ladder. Reduced mobilty in H. midae fragments suspected to be due to a conformational mutation. HALC02GENB primers, except H. iris that failed to amplify for all ten individuals examined. Comparison of the DNA se- quences for the other species indicated suitable restriction sites for discrimination between species using five restriction enzymes (Fig. 4). All restriction digestions for the five enzymes resulted in a single restriction pattern for each species, except for two enzymes for H. rubra (Table 2). The two polymorphisms were each ob- served in two different individuals, all were sequenced to confirm the observed RFLP. None of these four individuals was respon- sible for the polymorphisms observed at the mtCOI fragment, and the individual H. nihra with a mtCOI profile similar to H. scalaris was clearly identified as H. rubra at this fragment. The restriction profiles for the canned tissue, mucous and tluid samples all matched that expected for H. rubra. As with the mtCOI RFLP analysis, an unusual species profile was observed for three enzymes with a single H. scalaris indi- vidual (the same individual), and again all three profiles match that recorded for//, laevigata. Sequence data showed a 100% similar- ity to H. laevigata across the 159 bp fragment, while three other //. scalaris samples each differed at 9 nucleotides from the H. laevi- gata sequence. At this 159 bp fragment, three of the ten species (excluding H. iris that did not amplify) had a unique restriction pattern for the enzyme Ddel and so could be individually identified (Table 2). In addition to the //. ruhra/H. coiiicopora complex it was not possible to separate H. australis and H. spadicea using the five enzymes on this fragment. All remaining species combinations were separable from each other using one to five of the enzymes (Table 2). Fragment mobilities on polyacrylamide gels were all consistent with known fragment lengths; no conformation induced mutations were observed in the mtCOIl fragment. DISCUSSION The ability to identify abalone species from tissue samples and/or mucous is important to the continued survival of significant abalone fisheries. The tests described in this article will provide one more tool in the fight against illegal fishing, which has the potential, along with commercial over-fishing and environmental variables (Davis et al. 1998, Shepherd et al. 1998), to lead to the decline and collapse of fisheries. The methods are straightforward H. rubra H.conicopora H. laevigata 1 CAATTTGAAC CATTCTACCA 50 GCCATTATCC TTATTTTCCT CGCCCTACCA TCCTTGCGGC Hhal TCCTTTACCT ACTAGACGAA GTCGGTATAT 100 CGTGCCTTCT c -C.C. Hhal .T. .A .T .CI Ddel CT. ....... . .A. . .A. .G EcoRV .A. . . . _._C C. T . . . .c. . . c C. .c. ... .G. -C. . . . . A. . . Ddel ... A . .G. . . . .T. . . . .C. . Hhal A. . . . . .TCC. .A. . . . .A . . . .C Ddel . .T. . .A. .G EcoRV _._T CT. T. C. . . . .c. . . c. . . . .T. . . C. .C. A. .0. .C C. . H. midae H. spadicea H. australis H.Virginia HALC02GENA HALC02GENB H. rubra H. conicopora H. laevigata . .0. . . . . TC A . . A . . . . .C C . .G. . . . .G. .A. .T. .CT. CAATYTGAAC 1 2. . . . .C YATTCTMCCA C C . .TT .A A. .C. A. . . ■.,1... ^.■ Ddel . .G. .G ..C.C . . .CA. .A. . . AC T . . A . .T C T. . . T T. .G .A .A CT . .A A. . 101 AACAATCAAG GCAACTGGTA ArrAGTGATA CTGAGG Ddel CTAC GAAl 150 ^ACTCAG Ddel 159 ACTTTAAGG . . .G. .T. T .A . . .G. . . . . . .A. . . . ...C..A. Hpall . . .C. . . . .T. . . . .T. .A. ... .A. Ddel -G Rsal Ddel -G G. . Ddel DdeJ Rsal G. . Ddel .T. .A. ... .A. . G G . . Ddel . . .G. T Hpall Rsal Ddel .T. .T. .A -A .A . . .r . .c. Hpall ...C..A. Hpall ...C.c. Hpall ... .A. ... .A. ... .A. Ddel Ddel Ddel T Ddel Ddel Ddel HALC02GENA HALC02GENB DdeJ -- -- -- CCGATG CTTATGAGTC TRAAATTCC Figure 4. Sequence alignment of the 159 bp mtCOII fragment for ten abalone species. This fragment did not amplify for H. iris. Primer sequences and cut sites for the five restriction en/ymes [)del. EcoRV, Hhal. Hpall, and Rsal are included. (1 = sequence data unclear whether C or T: 2 = sequence data unclear whether C or .A). 224 Elliott et al. TABLE 2. Expected restriction fragment lengths for 10 Haliotis species for the 159 bp mtCOII fragment when cut with restriction enzymes Ddel. KcoRV, Hhal. Hpall, and Rsal. H. iri\ did not amplify with these primers. The number in parenthesis represents the total number of individuals examined for each species that displays the given restriction pattern. I nique restriction profiles are shown in bold. H. luevigala numbers include the misidentified H. scalaris individual. con Restriction Digestion Patterns Ddel EcoRV Hhal Hpall Rsal H. asinina 13.3H,108(30) 71.88(30) 159(30) 44.115(30) 159(30) H. australis 13.15.131 (10) 159(10) 159(10) 44.115(10) 159(10) H. coiiicopora 13.15.131 (11) 1 59 ( 1 1 ) 58.101 (11) 159(11) 159(11) H. iris H. Icievigahi 13,15,60,71 (63) 71.88(63) 58.101 (63) 159(63) 30.129(63) H. midae 13.15,131 (10) 159(10) 159(10) 159(10) 159(10) H. roei 13,146(10) 159(10) 159(10) 44.115(10) 30.129(10) H. rubra 13.15.131 (50) 159(50) 58.101 (48) 159(2) 159(48) 44.115(2) 159(50) H. scalaris 13.71,75(21) 159(21) 1 59 1 2 1 1 159(21) 30.129(21) H. spadicea 13.15.13! (10) 159(10) 159(10) 44.115(10) 159(10) H. virginea 13.38.108(10) 159(10) 159(10) 44.115(10) 159(10) and suitable for use in any laboratory with basic DNA analytical equipment. The PCR-RFLP tests utilize short DNA fragnients that can be amplified from processed products and slightly degraded material, and therefore are of potential forensic use. Care has been taken in this study to include e.xantination of intraspecies variation as well as possible non-Hiiliolis amplifica- tion with our PCR primers. Samples from different geographic locations were examined for the two main Australian commei'cial species (H. rubra, five locations and H. laevigata, three locations). While not exhaustive, the results suggest that what limited in- traspecies variation exists can be accounted for using the two fragments and multiple restriction enzymes. Both PCR primer sets devised for the test are relatively degenerate and so cross genus amplification was not unexpected. However, of the groups we have exaniined only DNA from the tunas {TIntnnus spp.) anipli- fied, and it was possible to easily differentiate these from Haliotis species. The restriction patterns produced by Dciel for the intCOIl frag- ment would discriminate three of the species, while five other species would be differentiated by a single restriction pattern at the mtCOI fragment. Such species-specific patterns are useful, how- ever as rare polymorphisms may exist it would be wise to confirm identification with multiple enzyiT)es and/or both short fragments. None of the rare polymorphisiT)s observed occurred in niore than a single individual, and no individual displayed (nore than one varia- tion. With the exception of the H. riihra/H. coiiicopora pairing, all other combinations of the 1 1 abalone species can be differentiated from each other using two or more of the restriction profiles shown in this study. We therefore reconimend using both fragments and at least two of the restriction enzymes included here to differen- tiate species. The ability of our test to differentiate between species was inadvertently put to the test during the intraspecies examinations. Of 20 purported H. scalaris individuals, one was found to display a different restriction profile at six of the nine profiles examined. The combined profile of this individual matched completely the expected profile for H. laevigata: and was confirmed by DNA sequence analyses. Although occupying different niicrohabilats, these two species have overlapping distributions and co-occur in the saiT)e area (Shepherd 1973). Shell and mantle morphology did not separate the abeirant individual from other H. scalaris indi- viduals. This individual is either a H. laevigata and morphological characters between the two species are more plastic than currently recognized, or a hybrid between the two species. Naturally occurring hybrids between abalone species with overlapping ranges, although relatively rare, have been reported (e.g., Talmadge 1977; Sasaki et al. 1980: Aral et al. 1982; Messier & Stewart 1994). The two Australian species H. rubra and H. laevigata, also show evidence of backcrossing and introgression (Brown 1995). There are no records of hybrids between H. laevi- gata and H. scalaris. but H. laevigata is more closely related to H. scalaris than to H. rubra (Brown & Murray 1992), and hybrids would not be unexpected. Allozynie analysis of the abenant indi- vidual could not confirm nor refute its putative hybrid status as there are no known diagnostic loci between the two species (Brown 1991). The possible existence of hybrids, albeit at low frequencies, does not minimize the validity of our mtDNA-based test for aba- lone. However, the possibility of hybridization and backcrossing between species does question the legal "species identity" of an individual. If hybrids were infertile and only Fl hybrids were possible, then a single diagnostic nuclear DNA marker would con- firm the individual as a hybrid, and the mtDN.A marker would confirm the maternal species. Such individuals could be legally classed as hybrids. However at least some abalone hybrids appear to be fertile and backcrossing occurs (Brown 1995), and identify- ing the "nuclear lineage" of a potential backcross offspring would require multiple nuclear DNA markers. Even then it could never be proved that an individual was not the offspring of a backcrossing event, except based on probability. A suite of nuclear DNA mark- ers could never disprove a claim of backcrossing, although making it impi'obable. On the other hand using a mtDNA-based test, the maternal lineage of the individual can always be validated. We suggest that for legal purposes where hybrid backcrossing may exist between abalone species that the genetic "species identity" of an individual be classified as its maternal lineage, which can be confirmed from its mtDNA. Hybrid individuals (those with nitDNA of one species and nuclear DNA wholly or partly of Southern Hemisphere Abalone Identification 225 another species) while biologically acknowledged should not be legally recognized as the existence of backcross hybrids cannot be disproved except by probability based on a large number of diag- nostic nuclear DNA markers. Mitochondrial DNA in abalone as in most organisms appears to be only maternally inherited (Conod 2000). The aberrant individual in our study therefore is classed as H. laevigata. The advantage of the tests described here to previous studies (Sweijd et al. 1998) for abalone is the smaller size of the DNA fragment; an advantage when examining processed or slightly de- graded material (Mackie et al. 1999). The lysin gene protocol described by Sweijd et al. (1998) did aim for fragments less than 300 bp. but the presence of an intron increased this at least three times, and for H. laevigata by about ten fold (unpublished data). The authors did however successfully use PCR primers for a smaller 146 bp fragment to discriminate between canned H. luidae and H. rubra products. PCR inhibition was observed when testing our primers on the mucous samples of H. rubra. Dilution ( 10 fold) to a lower con- centration did not have the same inhibitory effect. Similar PCR inhibition due to high levels of polysaccharides is common in plant tissue extracts (Fang et al. 1992), and inhibition due to muco- polysaccharides in the abalone mucous may have caused the ob- served PCR failure. No DNA sequence variation was observed between H. rubra and H. conicopora in either short mtDNA fragment examined in this study. In an assessment of all recent taxa in the family Hali- otidae, Geiger (1998) concluded that there was some justification for sub-species recognition of conicopora under H. rubra. Allo- zyme data suggested conspecifity but shell and geographic distri- butions indicated distinct taxa. Fifteen of 22 DNA microsatellite primers developed for use in H. rubra amplified a similar product in H. conicopora (Evans et al. 2001). This compares to the con- servation of only 1 2 of the 22 markers in other temperate Austra- lian species (H laevigata. H.scalaris and H. roci). Our short DNA sequences lend some support to the possibility of sub-species sta- tus for conicopora. however further research is required to resolve the issue. The altered mobility of the H. midiu' mtCOI fragment run on polacrylamide gels is most likely due to a conformation change. Conformational mutations attributed to sequence-specific varia- tions are restricted to polyacrylamide gels and not seen on agarose gels (Singh et al. 1987). The location and conservation of this conformation variant requires further investigation. Its presence, however, raises a note of caution when using RFLPs as mobility variation of fragments seen on polyacrylamide gels may be mis- leading as they can be length or conformation polymorphisms. It is therefore recommended that species differentiation using the RFLP tests described here be run only on agarose gels. The test described here fulfills the aim of our study to provide a relatively straightforward and cost-effective means for identify- ing several abalone species of commercial importance to Australia. Costs for any DNA-based analyses are not insignificant, but the PCR-RFLP technique is generally considered more cost-effective for routine species identification than alternatives such as direct DNA sequencing of the PCR product (e.g.. Asensio et al. 2000). The opportunity to sequence a PCR product is of course still avail- able for differentiation of individuals if problems arise following PCR-RFLP analysis. To increase the potential value of this study to the sustainability and protection of abalone fisheries worldwide, additional species, particularly from Northern Hemisphere waters, need to be incor- porated either into this test or a modified one. so that a single test is available for discriinination of all abalone species. ACKNOWLEDGMENTS This study was funded in part by the Australian Fisheries Re- search and Development Corporation (Project 1999/164), with ad- ditional financial assistance from the Tasmanian Abalone Council and the Tasmanian Marine Police. The authors are grateful for their support as well as that provided by the South African Police Services. Phil and Audrey Critchlow. Sandy Degnan. Greg Magu- ire, Elizabeth O'Brien, Rodney Roberts and Tasmanian Seafoods Pty Ltd. Sharon Appleyard, Malcolm Haddon and Bob Ward pro- vided useful comments on an earlier version of this paper. LITERATURE CITED Aral. K.. H. Tsuhaki. Y. Ishilani & K. Fujino. 1982. Chromosomes of Haliotis discus lumnai Inn and H. discus Reeve. Bull Jap Soc Sci Fish. 48:1689-1691. Asensio, L.. I. Gonzalez. A. Fernandez. A. Cespedes. P.E. Hernandez, T. Garcia & R. Martin. 2000. Identification of Nile perch iLciies niloricus). grouper [Epinephehis guazu). and wreck fish {Polyprion americaims) hy polymerase chain reaction-restriction fragment length polymor- phism of a I2S rRNA gene fragment. J Food Protection. 63:1248- 1252. Brown. L. D. 1991. Evolutionary genetics and population structure In abalone (genus Haliotis). Doctor of Philosophy Thesis. 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C.P.: 97UU0 ABSTRACT In order to study the distribution and abundance of Strombus costulus. larvae and adults, samples were collected bimonthly from August 1997 to July 1998 at six sites at Banco Chinchorro. To collect larvae, replicate surface plankton tows were made with a conical net. Larval density ranged from 0.0018 ± 0.026 veligers/10 m' to 4.77 ± 3.50 veligers/10 m'. Presence of all larval stages suggested a complete development of veligers from egg stage to metamorphic competence in the reef lagoon; 8.529}- of the larvae corresponded to stage II veligers. 18% to stage III and 72.65'7f to stage IV. At the bottom, all conch found within three 100-m- replicate areas were counted, shell lengths were measured and egg masses recorded. Conch density varied from 0.0025 to 0.22 conch/m- with a dominance of juveniles. The total population in Banco Chinchorro was estimated at 1.3 x 10* conch, but only 8% were estimated to be of legal size. Larvae and adults were more abundant in Cayo Centro, the principal distribution site. Egg masses were abundant from May to October with a greater abundance in May. Banco Chinchorro is an important source of S. costatus veligers and sustains an adult and juvenile population within the reef lagoon. Nevertheless, this S. costatus population is not large enough to support a commercial fishery. It is necessary to protect the reproduction sites to maintain larval supply downstream. KEY WORDS: Caribbean, distribution, larvae. Strombus costatus INTRODUCTION In 1997. Mexico harvested 140.021 tons of mollusks. with a total value of US$39.79 million. This harvest represents 10.06% of the country's total fish and invertebrate catch and couesponds to 4.15% of the total economic value (SEMARNAP. 1998). In the Yucatan Peninsula, the mollusk fishery is niultispecific; catches principally consist of i|ueen conch iSlrdiiihiis fiigas Lin- naeus. 1758). and on a minor scale, milk conch (SiroiiihK.'i antaliis Gmelin, 1791), and the fighting conch (S. piigilis Linnaeus. 1758). Other gastropods are also caught, such as the red conch. (Pleu- roploca gigantea, Kiener, 1840) and the black conch. (Xancus angulata. Lightfoot. 1786) (Sosa-Cordero et al. 1993; Perez 1997). The milk conch (5. costatus) is widespread in the Caribbean, but it is commercially caught only off the Yucatan (Stoner 1997) where it has the same market value and demand as the queen conch (Aldana-Aranda & Patiiio-.Suarez 1998). In the South of Quintana Roo fishing regulations only allow harvesting mollusks in Banco Chinchorro. The queen conch is the targeted species and the milk conch are not harvested. The decline of 5. gigax. between 1985 and 1990 in the Yucatan Peninsula, led to the closure of the conch fishery off Yucatan State and a ban in some areas off Campeche State and northern Quintana Roo State. In addition, a system of capture quotas was established in sourthern Quintana Roo. With the descend of the queen conch fishery, alternative sources such us milk conch could be caught to meet the growing demand for conch meat earmarked for the local markets and the Costa Maya's tourism industry in southern Quintana Roo. The potential for the non-exploited resource milk conch. S. costatus, needs to be evaluated. There are only a few studies on milk conch in the Caribbean. Percharde ( 1968) studied the distribution of the genus Strombus in Trinidad and Tobago. Brownell ( 1977) repotted that S. gigas was the most important fishery resource in Los Roques. Venezuela, but E-mail: alberto@ecoosur-qroo,mx that other mollusks such as S. co-ttatus and S. piigilis also contrib- uted to the fishery, Appeldoorn (1985) studied the growth, mor- tality and dispersion of laboratory reared S. gigas and S. costatus in Puerto Rico, and Berg et al. ( 1989) described the abundance and distribution of S. costatus in Bermuda. In Mexico. Aldana-Aranda et al. (1989) studied the effect of temperature and algal food on larval growth of milk conch. Recently. Aldana-Aranda and Patiho- Suarez ( 1998) reviewed algal diets used in larviculture. of several Strombid species, including the milk conch, and Shawl et al. (in press) reared S. costatus juvenUcs raised from egg masses laid in captivity. There are no studies related to the milk conch at Banco Chin- chorro, Consequently, the objective of this work is to describe the distribution and abundance of 5. costatus adults, determine their reproduction sites, and evaluate larval abundance. This study tests the following hypotheses: ( I ) the lagoon reef is an important site for reproduction and distribution of milk conch; and (2) there is complete larval development (egg to metamorphosis competence) of this specie in Banco Chinchorro, MATERIALS AND METHODS Study Area Biosphere Reserve Banco Chinchorro is a false atoll on Quin- tana Roo's South coast, within the Mexican Exclusive Economic Zone (18°23'; I8°47'N. 87°14'; 87°27'W) (Jordan and Martin 1987) (Fig. I ). Chinchorro is 46 km long and 19 km at its maxi- mum width, with a reef lagoon area of 560 knr. The reef lagoon has an extensive sand bottom with patches of sea grass. In the North these species are most abundant. Thalassia testiidiiuun Banks ex Koning. Halodule wrigthii Ascherson. and Syringodium fdifonne Kiitzing. Reef patches are common in the South region of the lagoon. Banco Chinchorrt) has four keys; in the north there are two small keys known as Cayo Norte. In the central area, there is Cayo Centro, which is the largest, and in the southern area. Cayo Lobos, which is the smallest (Fig. 1). 227 "* '8 DE Jesus Navarrete Kisure 1. Map of Banco Chinchorro showing six sampling sites within the reef lagoon. The dry season extends Ironi March to June, while the rainy season is from July to October, and the cold season, characterized by strong northerly winds ("nortes"). is from November to Feb- ruary. Sampling Methods Samples were collected bimonthly from August 1997 to July 1998, within the reef lagoon, in six traditional queen conch fishing sites: Cayo Lobos (I8°23'45, O'N, 87°2r 20.9'W), Isla Che (18°30' 12.3'N. 87°26'13. I'W). Cayo Centro {18°33' 32.7'N. 87°18' 24..'^'W), Cayo Centro Oeste (18°33'24, I'N. 87°24'; 56.6'W). Penelope (18°42' 47.6'N. 87°14'55.5'W). and Cayo Norte (18°45'28. I'N. 87''47'()1. 1 "W) (Fig. 1). At each site, tem- perature (°C) and dissolved oxygen (mg/1) were measured at sur- face level using an oxygen meter (YSI model 58). Salinity was measured with a thermoconductivity meter (OHALUS model 50). Replicate surface plankton tows were made at each site using a conical net. 0.50-m dia. with a 202-jjLm mesh size. Plankton tows were conducted for 15 min at a velocity of -1.0 m/min. Water \oluiTie was measured with a flow meter (General Oceanic model 203 IH) attached to the net. Plankton were fixed with a mixture of neutralized 5% sea water-formalin (Stoner & Davis 1997a). Tows were conducted diurnally and additional night collections were made at Cayo Centro and Cayo Lobos. Gastropod larvae were sorted from other plankton using a dis- secting microscope (x20). Identification and developmental stages (I to IV) were assigned following the descriptions of Davis et al. ( 1993). and larvae were counted and measured for maximum shell length. Total larval abundance was standardized to larvae/10 m\ To determine the abundance of juvenile and adult conch, bi- monthly samples were conducted at each site. All conch found in each of the three circular unit replicates (100 nr) were counted. The shell length and lip width were measured to the nearest mm using calipers, after measurements all conch were returned to the sea bottom. Egg masses within the unit samples were counted. Conch abundance was compared among months and sites sampled using a two-way Analysis of Variance ( ANOVA). on Log (x -I- I) transformed data. The procedure considered independence between sites and months. RESULTS Maximum mean temperatures were recorded during August and October, 28.9 ± 0.55 and 29.3 ± 0.28°C (/! = 6), respectively. whereas lowest temperature occurred in December (26.3 ± 0.94 n = 6). Dissolved oxygen varied between 5.87 ± 0.37 mg/1 in July and 7.01 ± 0.56 mg/1 (/; = 6) in August. Salinity ranged from 35.9 ± 0.12% in October to 37.0 ± 0.9%f {n = 6) in March. A total of 245 larvae were collected over the 1 2 months. Most larvae were collected in July during night tows (158 veligers at Cayo Lobos and 75 veligers at Cayo Centro). Diunial tows con- tained a very low number of veligers. Six larvae were collected in October; one at Isla Che. one at Cayo Centro. and four at Centro Oeste. In May, six larvae were collected at Penelope. No veligers were found in August. December, and March. Larvae were cat- egorized into the following stages, 8.53% of larvae were stage II (451-750 |jim). 18.77% corresponded to stage III (751-950 jxm) and 72.65% were stage IV at Cayo Centro in October, to 4.77 ± 3.50 veligers/10 m" at Cayo Lobos in July (Table 1 ). A total of 280 juvenile and adult conch were collected during the sampling period. Conch abundance was significantly different between sites [P = 0.0036), Cayo Centro had the highest density and Isla Che had the lowest (Tables 2 and 3). A total 184 indi- viduals were found at Cayo Centro; 89 conch were collected in TABLE 1. Total number of larvae and density (veligers/KI m') of Strombiis coslalus (all stages) collected at Banco Chinchorro. August 1997 through July 1998. No ^eligers were found in August. December, and March. Cayo Lobos Isla Che Cavo Centro Counts Density Counts Density Counts Density October May July+ l.'^S 4.77 ± 3..M) 1 ().()]8±0.03 1 ().()26 + 0.0.^ 7.S 1.62 ±1.07 i Night ccillections C. Centro Oeste Counts Density U.ll ±0.15 Penelope Counts Density 0.17 ±0.24 S. cosTATUs Larvae and Adults at Banco Chinchorro 229 TABLE 2. Milk conch adults density (no. conch/ni'^) at Banco Chinchorro. Cayo Lobos Isia Che Cayo Centro C. Centro Oeste Cayo Norte August 0.0075 0 0.0075 0.0025 0.0100 October 0.0050 0.0025 0.0825 0.0025 0.0200 December 0.0125 0.0025 0.0025 0 0 March 0.0725 0 0.2225 0.0125 0.0125 May 0 0 0.1400 0 0.0025 July 0.0125 0.0025 0.0050 0.0050 0.0550 March, 56 in May. .^.^ in October, 3 in August, 2 in July, and 1 in December. Juvenile and adult density varied from 0.0025 conch/ m~ to 0.222 conch/ni". Despite significant differences between sites (Table 3), a uniform conch distribution was used to calculate the population size at Banco Chinchorro. Based on the lowest conch density (0.0025 ind/m"^), and a lagoon area of 560 km" for Banco Chinchorro, a total of 1.3 x 10** conch were estimated. However, only 100,000 conch were estimated to be harvest size (8%). This low population size does not appear sufficient to sup- port a commercial fishery. The highest numbers of conch found in Banco Chinchorro were collected in March, with a total of 128 organisms. Fifty-seven conch were collected in May. 45 in October. 32 in July, 1 1 in August, and 7 in December. The size-frequency distribution varied from 30 to 1 80 mm shell length with 92% of total sample of conch in the range of 81 to 160 mm shell length (Fig. 2). Reproductive activity, copulation, and the presence of egg masses in medium sands, were observed from March to Deceinber. A total of 1 13 egg masses were counted with the majority at Cayo Centro (39 egg masses in October, 58 in May, and 1 in December). Fifteen egg masses were counted at Cayo Lobos in March. DISCUSSION Temperature has a direct influence on the beginning and ces- sation of conch reproductive activity, egg-hatching time, and on the duration of the larval phase (Berg et al. 1989; Appeldoorn et al. 1983; Stoner et al. 1992; Stoner & Davis 1997a; Pechenik 1999). At Banco Chinchorro, egg masses of S. cosiatiis were first observed in March and were found until December when bottom water temperature decreased to 26.3"C. In Banco ChinchoiTO. the reproductive season of S. costatiis is during a ten-month period, with a peak in copulation and egg laying in May. This period is longer than in Trinidad and Tobago where mating occurs from November until June (Perchard 1968) and longer than in Venezu- ela, where reproduction occurs from November to May (Brownell 1977). In comparison, the reproductive season for queen conch, S. gigas. has great variation. It varies from 5 to 12 months, with the shortest season duration in Bermuda and Florida and the longest at Banco Chinchorro (Cruz 1984; Corral & Ogawa 1987; Stoner et al. 1992). A difference in reproductive cycles of S, gigas and S. cos- tatiis as a function of temperature was mentioned by Brownell (1977). He observed that S. costatus reproductive season began when the season for S. gigas usually ended. Brownell (1977) as- sociated this with a one-centigrade degree decline in mean water temperature. However, in this study S. aisluttis reproduction sea- son was from March to December and coincided with S. gigas reproduction period at Banco Chinchorro. In Banco Chinchorro. the number of S. costatus egg masses exceeded that of S. gigas (113 vs. 19 egg masses) for the same study period (de Jesiis-Navarrete 1999). This could be an effect of fishing activities, because only S. gigas is collected not S. costatus. Females constitute a high percentage (-65%) of the S. gigas catch, (pers. obs.) and that might also explain why there were higher number of S. costatus egg masses, juvenile and adults than S. gigas. in some areas, like Cayo Centro. Fishing practices are known to modify the structure of popu- lations by reducing the overall biomass (Alcala, 1988; Roberts, 1995), decreasing age and size at sexual maturity (Harnielin et al. 1995), and altering sex ratios and genetic structure (Ryman et al. 1995). The displacement of target species due to the effect of fishing pressure and a consequently higher abundance of non- target species was mentioned by Seijo et al. ( 1997). This may be happening in Banco Chinchorro, as the 5. gigas population de- clines there may be an increase in the 5. costatus density, due to habitat and food resources becoming available. A greater quantity of 5. costatus larvae was found in July when mean water temperature was at its maximum. It is possible that this is related to higher egg mass production, and increase in food availability for the larvae (Davis 1998). Local water circulation might also influence number of larvae retained inside of Banco ChinchoiTO. Larvae were collected from nearly all stages (II, III and IV) indicating a complete larval growth process within the reef lagoon. Although the current circulation pattern of Banco Chin- choiTO is unknown, it is probably influenced by trade winds, and TABLE 3. Results of two way ANOVA of S. costatus abundance in Banco Chinchorro Quintana Roo, Mexico. Source of Variation Sum of Squares D.F. Mean Square F-ratio p-level Sites Months Residual Total 20.5434 8.24020 21.7742 50.5578 5 5 25 35 4.|{J86 1 .6480 0.8709 4.7170 1.8229 0.0036* 0.1316 230 DE Jesus Navarrete August O c o cr 0 '-4— • 40 30 ^ 20 10 0 40 30 - 20 1 0 0 40 30 20 1 0 - G 4 0 30 20 I 0 0 40 30 - 20 1 0 - 0 40 30 -\ 20 10 H «=11 ■Ijjjij October ■ ill. ■ «=45 December M arch ,»=128 lllli- May «=57 111. July iIlo* «=32 Siphonal length (mm) Figure 2. Relative frequency (%) and size distribution of juvenile and adults of Siniiiihiis costatiis at Chincliorro Bank. this ciaulation would transport larvae tov\ard the iiUerlor of the reef lagoon causing local retention. In Florida. Stoner et al. ( 1997) found that larvae of 5. costatiis were very abundant in early stages and rare in advanced development stages. They noted that the abundance of later larval stages was associated with water flow produced by the wind, which suggests local recruitment from spawning populations that inhabit outer reefs. The same effect could occur in Banco Chinchorro. Evaluating populations outside iif the reef lagoon might explain why 6S9f of the larvae were collected at Cayo Lobes, the southern site of Chinchorro. The high number of larvae may indicate a contribution from deep-water S. costatiis populations or populations external to Banco Chinchorro. The density of .S'. cosiouis larvae found ui this study (4,77 ± 3.50 veligers/IO m') is higher than the values reported from other sites in the Caribbean. In Florida, densities of 0.04 to 1 .40 veiigers/ 10 m' have been found with maximum abundance peaks in June (Stoner et al. 1997). In the Bahamas. Stoner and Smith (1998) reported 5. costatiis densities ranging from 0.026 to 0.069 veiigers/ 10 m\ In Banco Chinchono. the higher percentage of S. costatiis larvae m night tows (9.'ip). may be due to lack of intense surges and wind action that were prevalent during the day. Stoner and Davis (1997b) documented vertical movement of S. gigas larvae toward deeper zones due to wind and surge effect, and Stoner and Smith ( 1998) noted that wind and surge also produce horizontal trans- portation of larvae. Density of S. costatiis adults and juveniles found in Banco Chinchorro is higher (0.22 conchs/m") than other values reported in the Caribbean. In Bermuda. S. costatiis density was highest in the basins, (0.00299 conchs/ni"). than on the platform, (1.9 x lO"'* conchs/ni") (Berg et al. 1989). In Puerto Rico, the density of 5. costatiis varied from 0.18 x lO""* conchs/nr in reef patches to 0.0032 conchs/m' in coarse sands to 0.0049 conchs/m" in rubble areas (Appeldoorn 198.5). Although the densities found in Banco Chinchon-o exceed these values, 92<7f of the population consisted of individuals smaller than 170-mm shell length, which cannot support commercial harvest. In Yucatan State. 5. costatiis shell sizes varied from 65 to 225 mm shell length, and legal fishing size (180 mm) represented over 65% of the relative abundance of conch catches, (Perez 1997). In Banco Chinchorro, the large size was 180-mm shell length and this represented only 8% or an estimated population of 100,000 conch. Therefore commercial catch of this species is not recommended. Banco Chinchorro was designated as a Biosphere reserve in 1996. The main goals were to conserve biodiversity, protection, and enhance overexploited species, such as queen conch (S. gigas) and spiny lobster [Paiuilints argiis). It is now clear that other species should be considered in the management plan, including the milk conch, S. costatiis. Many marine reserves like Banco Chinchon'o, have small nucleus areas to protect fishing species. At Banco Chinchorro, only 3.2'7f of the total lagoon area comprises of protected zones. It is likely that these areas do not contain the full habitats needed to protect or enhance species during their entire life cycle. Therefore, the role of these nucleus areas in protecting species and connecting different habitats together cannot be totally ascertained (Appel- doorn & Lindeman, in press). In the establishment of management plans, data on distribution and abundance of adults and larvae, reproductive sites, egg mass numbers, and recruitment dynamics at a regional level are required. For example, the data from this study suggests that critical areas like Cayo Centro with its high distrib- tuion of milk conch adult, juveniles, egg masses and larvae needs to become a conch protected area within the Biosphere Reserve Banco Chinchorro. In general, marine reserves help to protect some fishing spe- cies, and can be effective in the recovery of commercial exploited species (Alcala 1988; Roberts & Polunin 1991; Roberts 1995; Allison et al. 1998). However, to assure conservaton of the species it is essential to design reserves with an understanding of the ontogenetic requirements of target and non-target species. Re- serves need to be placed in strategic locations to protect larval production sites and settlement sites. This information on source sites and sink sites will support metapopulation d\ nanucs. and help S. cosTATus Larvae and Adults at Banco Chinchorro 231 in optimize fisliery benefits for many marine invertebrates species like S. costatiis that have a pelagic larvae. In the Bahamas, a high density of S. fiii^a.s larvae and adults ha\c been reported in a protected area in Bahamas (Stoner & Ray 1996). It is possible that both S. gigiis and 5. coshiln.s populations in Banco Chinchorro can be maintained for conservation and fisheries if reserve areas are expanded and management regulations to be practiced. ACKNOWLEDGMENTS El Consejo Nacional de Ciencia y Tecnologi'a (CONACyT) grant 420P-N93()6 supported this research. The authors thank Jose Oliva and A. Medina for help in the field. The comments of M. Davis, E. Sosa, and S. Monks and one anonymous reviewer im- proved the manuscript. LITERATURE CITED Alcala, A. C. 1988. Effects of marine reserves on coral fish ahiiiKlance>. mk\ yields of Philippine coral reefs. Amhio. 17; 194- 199. Aldana-Aranda D., A. Lucas. T. Brule. E. Salguero & F. Rendtin. 1989. Effect of temperature, algal food, feeding rate and density on larval growth milk conch iSlminhiis cosuilus) in Mexico. Aquaculture 7fi: .^6 1 -.371. Aldana-Aranda. D. & V. Patino Suare/. 1498. Overview of diets used in larviculture of three Caribbean Conchs: Queen Conch Strombns f>ii>iis. Milk Conch Srnmihiis c(i\kilu.'. and Fighting Conch Siroinlnis piigilis. Aquaculture 167:163-178. Allison. G. W.. J. Lubchenco & M. H. Carr. 1998. Marine reserves are necessary but not sufficient for marine con.servalion. Ecol. Appl. 8(Supl I ):S79-S92. Appeldoorn. R. S. & K. C. Lindeman. In Press. A Caribbean wide survey of non take Marine Reserves: Spatial coverage, administrative attrib- utes and effectiveness. Proc. Gulf and Caribb. Fish. Inst. 54. Appeldoorn. R. S. 1985. Growth, mortality and dispersion of juvenile, laboratory-reared conchs. Srivmhus gifjas and Slroiuhus cosralus. re- leased at an offshore site. Bull. Mar. Sci. 37(3):785-793. Appeldoorn. R. S.. D. L. Ballantine & P. E. Chanley. 1983. Observations on the growth and survival of laboratory reared juvenile conchs. Strnin- hu.s gigiis and S. coslatu.s. J. Shellfish Re.s. 3:82. Berg. C. J. Ward. B. Luckhursi. K. Nisbet & F. Cuuper. 1989. Observa- tions of breednig aggregations of Queen conch. Slrombus gigai in Bermuda. Proc. Gulf and Caribb. Fish. Inst. 42:161-171. Brownell. W.N. 1977. Reproduction, laboratory culture and growth of Stroiiihus giga.<:, S. ciKtUihis and S. pugili.s in Los Roques. Venezuela. Bull. Mar. Sci. 27(4):668-68(J. Cruz. R. S. 1984. Avances en la experimentaciiin de la poiducclcin masiva de caracol en Q. Roo. Mexico. Proc. Gulf ami Carihh. Fish. Iiisi. 37:12-20. Corral. J. L. & J. Ogawa. 1987. Cultivo masivo del caracol Slrombus gigas en estanques de concreto. Proc. Gulf and Caribb. Pish. lust. 38:344- 35 1 . Davis. M. 1998. The effects on natural foods, temperature and salinny on the length of larval life for the tropical gastropod veligers of Strouibus gigds. PhD Dissertation. Florida Institute of Technology, Melbourne. Florida. 136 pp. Davis. M.. C. Bolton. & A. W. Stoner. 1493. A comparison of larval development growth, and shell morphology in three Caribbean Strom- bus species. The Veliger 36(3):236-244. de Jesus-Navarrete. A. 1999. Distribucion y abundancia de larvas velfgeras del caracol rosado {Slrombus gigas Linne. 1758) en Banco Chinchorro Quintana Roo. Mexico. Tesis Doctoral. CINVESTAV-Merida. 223 pp. Harmelin. J. G.. F. Bachet & F. Garcia. 1995. Mediterranean marine re- serves: fish indices as a test of protection efficiency. Marine Ecology- Pubbliczioni Delia Stazione. Zoology 16:233-250. Jordan. D. E. & E. Martin. 1987. Chinchorro: morphology and composition of a Caribbean atoll. Atoll, Res. Bull. 310, 27 pp. Pechenik, J. A. 1999. On the advantages and disadvantages of larval stages in benthic maruic invertebrate life cycles. Mar. Ecol. Progr. Ser. 177: 269-297. Percharde, P. L. 1968. Notes on distribution and underwater observations on the molluscan genus Strouibus as loiiiul iii the waters of Trinidad and Tobago. Caribb. ./. Sci. 8:47-55. Perez. M. 1997. Distribucion. abundancia y mortometria e Indice de factor de Condicidn del recurso caracol en la costa oriente de Yucatan. Tesis de Maestri'a. CINVESTAV. Merida. 96 pp. Roberts. C. M. 1995. Rapid build-up offish biomass in a Caribbean maruie reserve. Cons. Biol. 9:815-826. Roberts. C. M. & N. V. C. Polunin. 1991. Are marine reserves effective in management reef fisheries? Rer. Fish Biol. Fish. 1:65-91. Ryman, N.. F. Utter & L. Laikre. 1995. Protection of intraspecific biodi- versity of exploited fishes. Rev. Fish Biol, ami Fish 5:417—146. SEMARNAP. 1998. Anuario estadistico de Pesca 1997. SEMARNAP. Mexico. D.F. 455 pp. Seijo. J. C. O. Defeo. & S. Salas. 1997. Bioeconomia Pesquera. Icoria modelacion y manejo. FAO Documento Tecnico de Pesca (368). 175 pp. Shawl. A. L.. M. Davis & J. Corsaut. In press. Captive breeding for the gastropod conch {Strouibus spp.). Proc. Gulf Caribb. Fish. Insl. 54. Sosa-Cordero. E.. A. Medina Quej. A. Ramirez-Gonzalez. M. Dominguez- Viveros & W. Aguilar. 1493. Invertebrados marinos explolados en Quintana Roo pp. 709-734. In: S. Salazar-Vallejo & N. E. Gonzalez, editors. Biodiversidad Marina y Costera de Mexico. CONABIO- CIQRO. 865 pp. Stoner. A. W. & N. P. Smith. 1998. Across-shelf transport of gastropod larvae in the Central Bahamas: rapid responses to local wind condi- tions. J. Plaiik. Res. 20(1): 1-16. Stoner. A. W. 1997. The status of Queen Conch. Strouibus gigas. Research in the Caribbean. Mar. Fish. Rev. 59(3): 14-22. Stoner. A. W. & M. Davis. 1997a. Abundance and distribution of Queen Conch veligers {Slrombus gigas Linne) in Central Bahamas: I. Hori- zontal patterns in relation to reproductive and nursery grounds. ,/. Shell- fish Res: I6(l):7-18. Stoner. A. W. & M. Davis. 1997b. Abundance and distribution of Queen Conch veligers {Strouibus gigas Linne) in Central Bahamas: II. Vertical patterns in nearshoie and deep-water habitats. ./. Shellfish Res. 16(1): I y-29. Stoner. A. W.. N. Metha & T, N. Lee. 1997. Recruitment of Strouibus gigas veligers to the Florida keys Reef tract: Relation to hydrographic events. J. Shellfish Res. l6(l):l-6. Stoner. A. W. & M. Ray, 1996. Queen conch, Slrombus gigas. in fished and unfished locations of the Bahamas: effects of a marine fishery reserve on adults, juveniles and larval production. Fish. Bull. 94:551- 565. Stoner, A. W., V. J. Sandt & I. F. Boidron-Metairon. 1992. Seasonality in reproductive activity and larval abundance of queen conch Slrombus gigas. Fish. Bull. 90:161-170. Joiinml ofShfUflsh Re.secinh. Vol. 21, No. 1, 233-237, 2002. VELAR CHARACTERISTICS AND FEEDING CAPACITY OF ENCAPSULATED AND PELAGIC LARVAE OF CREPIDULA FECUNDA GALLARDO, 1979 (GASTROPODA, CALYPTRAEIDAE) O. R. CHAPARRO,' A. E. SOTO,' AND C. E. BERTRAN' 'lii.stituto de Biologiu Marina Dr. J. E. Winter. Univcrsulad Austral dc Chile. Casilla 567, Valdivia. Chile; 'Institiito de Zoologi'a, Universidad Austral de Chile. Casilla 567. Valdivia. Chile ABSTRACT Veligers of Ciepiclulu fcciiiidii develop the cap;icity lor ingesting particukitc material during early stages of their development within the egg capsules. The potential feeding rate of encapsulated larvae is low compared with that of hatched larvae and pelagic larvae of other mollusks. Once the veligers emerge from the capsules, they increase the capacity for particle retention. This situation could result from the increase in the velar area, length of the ciliated border, length of the preoral cilia, potential filtering area and the width of the feeding canal, or to some combination of these, compared with encapsulated larvae of the same shell length. Extra-capsular development of the velum is an adaptive feature of the species which promotes efficient feeding and active locomotion during the pelagic phase, m preparation for settlement and metamorphosis on inshore rocky substrates. KEY WORDS: Crcpuiula fecunda. veliger larvae, velar morphology, particle clearance rate INTRODUCTION The presence of a lobed velum is a common characteristic in veliger larvae of the Mollusca (Strathmann & Leise 1979), where in free living species the velar lobes carry out functions of swim- ming and feeding (Hadfield & laea 1989). Veligers have external borders on the velar lobes that include preoral and postoral ciliary bands (Strathmann et al. 1972; Strathmann & Leise 1979). The pre-oral band is formed of long cilia that produce water currents used in locomotion and feeding. The postoral band consists of shorter cilia that beat toward the preoral band. The combined activity of these ciliary bands promotes capture and retention of food particles. Between the two ciliary bands lies the food canal covered with very small cilia that transport food particles to the larval mouth. In suspension-feeding niollusks the pailicle clearance rate (CR) may be used to calculate ingestion rate when food concentration is known (Sprung 1 984). Clearance rate in veliger larvae is partially determined by the length of the velar margin and the length of the preoral cilia (Strathmann et al. 1972). Strathmann and Leise (1979) observed that although longer preoral cilia were related to higher rates of water flow, they were less efficient in capturing particles in the smaller size ranges. The latter may be compensated for by an increase in total filtering area that increases the larval clearance rate. In addition to ciliary size, the length of the velar margin is important in larval feeding. Increase in ciliated area is accoinpa- nied by an increase in CR (Strathmann et al. 1972). The rate of clearance is not only influenced by the factors cited, but also by the ability of the organism to transport captured particles away from the primary sites of capture to the mouth. Thus, the rate at which the cilia in the food canal transport particles to the mouth directly affects the overall CR. Crepiditla fecunda. a sedentary filter-feeding gastropod, inhab- its intertidal and shallow subtidal zones on the Chilean coast. It is a protandric hermaphrodite whose reproductive mechanism in- cludes the deposition of egg capsules on rocky substrates, followed by parental brooding (Gallardo 1976. 1977. 1979). Most eggs in Corresponding author. O. R. Chaparro, Instituto de Biologia Marina Dr. J. E. Winter. Universidad Austral de Chile. Casilla 567. Valdivia. Chile. Phone: +56-63-221791; Fax: +56-63-221455; E-mail: ochaparr^mercuriii. uach.cl the capsules develop, and veliger larvae of approximately .'iOO p.m in shell length are released from the capsules (Gallardo 1976. 1977. 1979) to complete their development in the inshore plankton prior to settlement. Encapsulated larvae have no apparent source of food, and it is not known whether they are capable of suspension-feeding prior to eclosion. Development of the ciliated velum within the capsule (Gallardo 1977, 1979; Chaparro et al.. in press) suggests that these larvae are capable of feeding and swimming before hatching, and that these properties would improve upon eclosion. since the pe- lagic larvae must actively feed and swim in preparation for settle- ment and metamorphosis (Gallardo 1977. 1979. 1989). We hy- pothesize that the ability of C. fetiiuda larvae to remove particles from suspension is initiated during the intracapsular phase and further developed during the pelagic phase, including increases in the size of the cilia, filtration area, and capacity for particle trans- port \Mthin the larval food groove, MATERIALS AND METHODS Stacks of adult specimens of C. fecunda were collected from the intertidal at Yaldad Bay, Chiloe (43'08'S; 73"44'W). In the laboratory, individuals were removed from the substrate in order to expose the egg capsules from which embryos and/or larvae were obtained for experimentation. Before each experiment, the cap- sules were observed under a stereomicroscope to identify the de- velopmental stage of the embryos. Developmental stages (egg, morula, bhistula, trochophore, and veliger) were identified by their morphology. Fifteen to 20 indi- viduals of each group of larvae were observed microscopically and videotaped as described below. The shell lengths of the larvae were determined from images captured on videotape (see later). Clearance Rale Clearance rate (CR) was quantified in several encapsulated stages and in pelagic veligers using the method of Coughlan (1969). Several thousands of einbryos (egg. morula, trochophore) and veliger larvae frotn different spawning masses were manually removed from capsules and placed in individual 1-L glass aquaria containing 500 ml 0.45 p,m filtered seawater (salinity 30 %c. tem- perature 17°C). Embryos used in each aquarium came from the same egg muss. Laboratory cultured Isochrysis galhana was added to each experimental system (final concentration 3 x 10'' cells/ml^ 233 234 Chaparro ht al. Aquaria were stirred to ensure iidequiite mixiiii;. At intersals be- tween one and two h. the concentrations of algae remaining in the aquaria were determined using an ELZONE 180 XY particle counter. Control aquaria were run during each experimental periiid under the same experimental conditions but without lar\ ae. At the end of each experiment, five samples of 1 .5 nil each were obtained from each experimental system to determine the number of embryos or larvae in each aquarium. For measurement of shell length, larvae from each experiment were fixed in 5% formaldehyde and stored in Eppendorf tubes to await analysis. They were measured as previously described. Larval cultures were maintained to determine the CR of pelagic larvae of C. fccunda. Naturally hatched larvae were collected on a 100 (jil nylon screen as they emerged from the capsules and placed in 100-L aquaria containing filtered seawater as described above, with gentle aeration. Each day the larvae were fed ad libitum with /. galluina. The seawater was replaced every two days. For deter- mination of clearance rate, a known number of larvae were placed in experimental aquaria as described above for the encapsulated larvae, and the same procedures used to determine CR. Groups of larvae from these experiments were also set aside for measurement of shell length. Velar Morphology Larvae obtained from capsules as well as pelagic larvae were videotaped by placing them in a plankton decantation chamber with seawater under an inverted microscope fitted with a video camera. Selected images were captured for subsequent processing on a computer equipped with an ATI Corp. "All in Wonder" video card and Scion Image 3.0 PC software. We obtained the velar area (extended velar lobe), length of the ciliated boi-der, length of the preoral cilia, and shell length. The width of the food canal was also determined. The potential feeding area was calculated by multi- plying the length of the ciliated border by the length ( = height) of the cilia in the preoral band. Velocity of Particle Transport A suspension of red plastic particles 2-10 |xm m diameter was offered to larvae in a plankton settling chamber as previously described by ChapaiTO et al. (1993). Trajectories of these particles along the food groove were videotaped as described above for observing larvae, and the distances traversed by the particles were calculated using the Scion Image PC program. Simultaneously, the time taken by particles to traverse measured distances was deter- mined (Ward 1996J for calculations of transport velocity. These ♦ Encapsulated larvae o Pelagic larvae E M T 200 400 Stage-Larva shell length (nm) Figure 1. Clearance rate in different enibr>()nic stages, encapsulated ill = 281 and pelagic (/; = 10) larvae of C. feciiiida. E = egg (» = 5l, M = morula (;i = 5| ,T = trochophore in = 5). determinations were cairied out on larvae obtained from capsules and on pelagic (culttired) larvae. RESULTS Clearance Rate No uptake of particles was observed in early developmental stages, including the egg. morula, blastula and trochophore (Fig. 1 ). The clearance rate (CR) of encapsulated veligers of C.fcciiiula increased with the size of the individuals. The initial CR was 0.31 jjil h'' larva"' in larvae with shells 20S p-iii in length, increasing to 5.13 |j.l h"' larva"' in pre-hatched larvae 353 ixm in shell length (CR = 0.071e '""=i-'^^-'' -''■'" ''-■"-'^ r^ = 0.4564. n = 28) Figure 1. Recently hatched veliger larvae showed much higher values for CR than those of the same size that remained encapsulated (Table 1). CR values ranged from 45.3tJLl h"' larva"' in larvae 337 p.m in shell length at one day post-eclosion to 464 p,l h"' larva"' in larvae 442 p-m in shell length at 13 days post eclosion (CR = 1.0952 \elar Morphology Velar Area The velar area of encapsulated larvae showed linear growth during the developmental period, fluctuating between 0.012 mnr TABLE L Crepidala fecunda. Comparison of clearance rate iCRl, area of a velar lobe (VA). length of ciliated velar border (LCVB). length of preoral cilia (CL), potential filtering area (I'FA). and width of food groove (VVF(;) in encapsulated and pelagic larvae. \ alues represent indi\iduals of 340 urn in shell length and were obtained from appropriate regression equations. C^r I = percent increase in \ariable when comparing pelagic stage (recently eclosed) with encapsulated larvae having the same shell length. Stage Shell length (Mm) (Ml CR h"' • larv" VA (mm") LCVB (mnrl CL (Mm) PFA ( mm' ) WFG (pm) Encapsulate Pelagic <7c .WO 340 2.28 74.21 0.037 688 66 0.034 19 0.049 739 83 0.061 21 32.4 7.4 25.8 79.4 10.5 Velar Characteristics and Feeding of Crepiduu Fecunda 235 in larvae 188 yim in shell length and 0.056 nmr in larvae 372 |j.m in shell length (velar area = 0.0002 x larval shell length - 0.0312. r = 0.8755. n = 52) Figure 2. In recently liberated pelagic larvae of C. fecunda the velar area was 32.4 % greater than in encapsulated larvae having the same shell length (Table 1). The velar area was 0.054 mm" in recently eclosed larvae 340 p-m in shell length, reaching 0.198 mnr in larvae ready for metamorphosis at 16 days post-eclosion at 650 |xm shell length (velar lobe area = (6 x 10"^) x (larval shell length)' "-"-'. r- = 0.9586, )i = 5) Figure 2. Length of Ciliated Velar Border The length of the ciliated border in encapsulated larvae showed gradual growth with development. The initial length. 287 p,ni in larvae 205 p.m in shell length, increased to 755 p.m in larvae 372 |xm in shell length (length of ciliated velar border = 858.36 x In (larval shell length) - 4315.5 , r" = 0.9724, n = 19) Figure 3. The length of the ciliated velar border in recently liberated pelagic larvae of C. fecunda was 7,4% greater than that of encap- sulated larvae having the same shell length (Table 1 ). The length of the border varied from 772 ixm in larvae of 340 (jtm shell length, to 1548 |xm in larvae of 650 p.m shell length (ciliated length of velar border = 1.7826 x (larval shell length)' '"■", r" = 0.9307. /I = 5) Figure 3. Length of Preoral Cilia These cilia showed rapid growth during intracapsular develop- ment, reaching lengths of 80 (j.m in larvae with shell length 400 (xm (ciliary length = -0,0023 (larval shell length)" -i- (1,7307 x larval shell length) -256.29, r^ = 0.8954. n = 29) Figure 4. The preoral cilia of recently hatched pelagic larvae were ap- proximately 25,8'7f longer than the cilia of encapsulated larvae having the same shell length (Table 1 ). The length of these cilia in recently eclosed larvae was about 82 p,m. This increased to 1 11 p.ni in those larvae at 15 days post eclosion that were ready to undergo ^ 1 * Encapsulated larvae " 1 _ 1500 E a. 0 Pelagic larvae J- c ^^*f f I2U0 . r ^"-"'^'^ " ea E J ^ X b. ^ WO ^^ y=l.7826x ■u ^i^^ R" = 0.9307 n -. _P^ ■3 M)U tM^ 0 iT c 30(1 ■ 0 - ^g;t ^ y= 858.36Ln(x) - 43 1 5.5 R" - 0.9724 Larva shell length (fim) Figure 3. Length of ciliated velar margin (periphery of an extended velar lobe) in relation to larval size in encapsulated (/( = 19) and pelagic in = S) larvae of C. fecunda. When the SD bar is not shown, it is smaller than the symbol size. Each data point represents the mean for at least 15 individuals for encapsulated larvae and 25 individuals for pelagic larvae. metamorphosis (ciliary length = 61.316e «*i="-vai sheii iengih_ r- = 0.9041. (1 = 5) Figure 4. Potential Filtering Area The potential filtering area increased during larval development from 0.002 mnr in larvae of 205 [xm shell length to 0.054 mm' in larvae of 372 |jLin shell length [potential filtering area = (-5 x I0~^) X (larval shell length)" + (0.0006 x larval shell length) - 0.1 121, r- = 0.9417. n = \6] Figure 5. In recently eclosed pelagic larvae, the potential filtering area was about 79.4'7f greater than that of encapsulated larvae of com- parable .shell size (Table 1 ), This value was 0.06 mm" in one-day- old larvae, increasing to 0.18 mm" in larvae at 15 days post eclo- ♦ Encapsulated larvae O Pelagic larvae < y = 0.0002x-0 03l2 R- = 0.8755 ^-^ ^■^^ y = (6'lo-')x"^-' II ai*^^ R' = 0.9586 ui^^^ — 1 1 1 1 1 Larva shell length (fim) Figure 2. Area of an extended velar lobe in relation to size in encap- sulated (n = 52) and pelagic (/; = 5) larvae ni C. fecunda. When the .SI) bar is not shown, it is smaller than the symbol size. Each data point represents the mean for at least 10 individuals for encapsulated larvae and 25 individuals for pelagic larvae. 120 . ♦ Encapsulated larvae T too - o Pelagic laj-vae _. ^— --"^ — ^ .2 m - h^^^"""^^ y = 61.316e°»«»^ O ^=^W- R-= 0.9041 0£ e -J 60 . 40 - A I y = -0.0023x' + 1.7307X - 256.29 20 - jfii_ r' = 0.8954 0 . 150 250 350 450 550 650 750 Larva shell length (^m) Figure 4. Length of preoral cilia in relation to larval size in encapsu- lated (H = 29) and pelagic (h = 5) larvae of C. fecunda. When the SD bar is not shown, it is smaller than the symbol size. Each data point rep- resents the mean for at least 15 individuals for encapsulated larvae and 25 individuals for pelagic larvae. 236 Chaparro et al. ♦ Encapsulated larvae s « 1 o 'C 0 16 . 012 - 0 08 . o Pelagic larvae , „r.T^T 0 003x / v = O0221e / ' R= = 0 9505 y^ y o 1 B U 1 0t)4 - 0 - JT^ y = (-5'10 >'+00006x-() 1121 JC R' = 09417 ^ , , 3 20(1 !^ ISO 1_ 0. ♦ Encapsulated larvae ^ Pelagic larvae - y = 0.02X ' °" R- = 0.3562 Larva shell length (fim) Figure S. Potential filtering area in relation to larval size in encapsu- lated (n = 16) and pelagic (/; = 5) larvae of C.feciinda. Kach data point was calculated by multiplying the mean length of the ciliated velar edge by the mean length of the preoral cilia. .- I c-i^ • /\ mil , nnn3*larvi)l shell length ^2 _ sion (potential filtering area = ().()221c '^ . r - 0.9505. /? = 5) Figure 5. Width of Food Groove The width of the larval food groove increased with the size of the individual. The groove of encapsulated larvae 205 (Am in shell length was 6.22 [xm wide, increasing to 34.98 \x.m in pelagic larvae of 650 (jLin shell length (Fig. 6). In recently eclosed larvae, the width of the food groove was 10.5% greater than that of encap- sulated larvae of the same shell length (Table I ). Velocity of Particle Transport in the Food Groove Particle transport velocity increased throughout the larval en- capsulation period, with minimum values of 86 |j.m s"' in larvae 225 |ji,m in shell length, increasing to 270 [Lva s~' in larvae .?00 jxm in shell length (approaching eclosion) [particle velocity = 0.02 x (shell length)' "". r' = 0.3562. n = 16] Figure 7. 4(.) - ♦ Encapsulated larvae - 35 - o Pelagic larvae , ) 30 - - 25 - 20 - 1 15 - 1 10 - s _ 11 - ♦ I Larva shell length (fim) Figure h. Width of larval feeding groove in relation to individual size in ('. fcciiiida. Vertical bar = standard deviation. Larva shell length (^nl) Figure 7. Transport velocity of particles in the larval food groove in relation to individual size in encapsulated in = 16) and pelagic in = 5) larvae of C. fecitiida. When the SI) bar is not sho\\n. it is smaller than the symbol size. Kach data point represents the mean for at least HI individuals for encapsulated larvae and 20 individuals for pelagic lar- vae. In pelagic larvae the velocity of the particles remained rela- tively constant with values near 250 jo-m s"' (Fig. 7). DISCUSSION All encapsulated stages of C. fecunda veligers are able to ingest particulate material. This ability is related to the development of the ciliated velum (Gallardo. 1977, 1979), and particulariy to the presence of opposed ciliary hands (Chaparro el al.. in press). In the pelagic phase of the life cycle, development of the velum is of vital importance to swimming and feeding prior to settlement in this species, thus a large and well developed velum appears to be related to the mixed developmental strategy used by C. fecunda (Gallardo, 1979). Encapsulated larvae of C. fecunda develop the ability to ingest particles before their transition to the planktotrophic habit. Chap- arro et al. (in press) have observed particle capture and ingestion by pre-eclosed larvae which were fed immediately after artificial excapsulation, suggesting readiness for the planktt)trophic phase. The low CR observed in veligers removed from capsules is probably attributable to the early stage of development, as ob- served in larvae of other molluscan species (Hawkins et al. 1984; Welborn & Manahan 1990). Values for particle clearance by C. fecunda veligers obtained from capsules are lower in all cases than values reported in the literature for pelagic molluscan larvae (Mac- Donald 1988; .Sprung 1984; Bayne 1965). although CR in C. fe- cunda pelagic larvae is well above the rates cited by these authors. The rapid growth of the velum in encapsulated larvae of C. fecunda may be related to the mode of nutrition and the necessity for efficient swimming in the pelagic stage. Once the larva is free from the capsule, it is advantageous to have as large a velum as possible (Fretter & Graham 1962). The increase in the length of the ciliated border oi the velum in C. fecunda is a result of enlargement of the bilobed velum, not the formation of new lobes. The length of the preoral cilia in encap- sulated and pelagic larvae of this species falls within the size range reported by Strathmann ( 1987). i.e.. 30 to more than 100 |xm. and Velar Characteristics and Feeding of Crkpidula Fecunda 237 also agrees with the 50 fj.m value given by Riedel ( 1992) for larvae of the gastropod Cuheslana spcnfilcri (200 fxm shell length), which also exhibits mixed development. The potential filtering area of recently eciosed pelagic larvae is nearly 80% larger than that of encapsulated larvae of the same size (370 p.m shell length). This is due to the elongation of the ciliated border of the velum and growth of preoral cilia, and provides the larvae with a large surface area for particle capture, as suggested by the CR of pelagic larvae in this study. This is consistent with observations on the planktotrophic character of these larvae (Gal- lardo. 1977. 1979. 1989). The transport velocity of particles within the food groove of encapsulated larvae of C. fecunda increases with developinental stage to values near 250 jjim s"'. In pelagic larvae this velocity is more or less constant but the width of the food canal continues to increase during pelagic development, sug- gesting that pelagic larvae are able to capture and handle several particles at the same time, or increasingly larger particles (or par- ticle aggregates) as they develop. Together with the increase in potential filtering area, this may explain the high values for CR in pelagic larvae of C. feciiinla. The greater capacity for particle clearance in pelagic larvae compared with encapsulated larvae in C. fcciiiula may be related to increases in the velar area, cilated length of the border of the velum, length of the preoral cilia, potential filtering area, and the width of the food groove, or to some combination of these vari- ables. These changes are obviously adaptive as the larvae are released into the environment, where they must be able to swim and feed before settling. Some larvae remain in the capsule, yet have a shell size similar to that of newly released larvae, suggest- ing a differential rate of development between shell and velum, the latter requiring more time to develop before eclosion. ACKNOWLEDGMENTS Financial assistance was provided by an operating grant to ORC from the Fondo Nacional de Investigacion Cientifica y Tec- nologica (FONDECYT 1980984) and the Direccion de Investiga- cion of the Universidad Austral de Chile (S 200114). We also thank the Canadian International Development Agency (CIDA) for support during the preparation of the manuscript. LITERATURE CITED Bayne. B. L. 1965. Grovvtii and delay of nielanicirphosis (if the lar\ae of Mylilii.s ediilis (L.). O/^lH-lla 2:1—17. Chaparro. O. R.. R. J. Thompson & J. E. Ward. 199.'^. //) r/ivi observations of larval brooding in the Chilean oyster. Ostrea citilensis Philippi. 1845. Biol. Bull. 185:365-372. ChapaiTO. O. R., J. L. Charpentier & R. Collin. Embryonic velar structure and function of two sibling species of Crepidulu with different modes of development. In press. Coughlan. J. 1969. The estimation of filtering rate from the clearance of suspensions. Mar. Biol. 2:356-358. Fretter, V. & A. Graham. 1962. Larval forms. In: British Prosobranch Molluscs. London: Ray Soc. pp. 448^76. Gallardo, C. S. 1976. Natural history and reproduction of Crepidulu dilatata Lamarck in a Mehuin Bay population (Valdivia province. Chile). Medio Ambiente. 2:44-50. Gallardo. C. S. 1977. Two modes of development in the inorphospecies Crepidulu dUatata (Gastropoda: Calyptraeidae) from Southern Chile. Mar. Biol. 39:241-251. Gallardo, C. S. 1979. Twin species of Crepidulu genus (Gastropoda. Ca- lyptraeidae) in the Chilean coast; a redescription of C. dilatata Lamarck and description of C. fecundu n. sp. Stud. Neotropical Fauna Environ. 14:215-226. Gallardo, C. S. 1989. Reproduction patterns and vital cycle of bentic ma- rine mollusks. an ecological and evolutive approach. Medio Ambiente 10:25-35. Hadfield. M. G. & D. K. laea. 1989. Velum of encapsulated veligers of Petulocoiuinis (Gastropoda), and the problem of re-evolution of plank- totrophic larvae. Bull. Mar Sc. 45:377-386. Hawkins. A. J. S.. P. N. Salked. B. L. Bayne. E, Gnaiger& M. Lowe. 19X4. Feeding and resource allocation in Mytilus edulis: evidence for time averaged optimization. Mar. Ecol. Prog. Ser. 20:273-287. MacDonald. B. A. 1988. Physiological energetics of Japanese scallop Pu- tinopecten yessoensis larvae. / £v/). Mar. Biol. Ecol. 120:155-170. Riedel, F. 1992. A re-evalualion of the ontogeny of Cuhestana spengleri (Perry. 1811) (Gastropoda: Tonnoidea: Ranellidae). Veliger 35:117- 121. Sprung. M. 1984. Physiological energetics of mussel larvae (Mytilus edu- lis). II. Food uptake. Mur. Ecol. Prog. Ser. 17:295-305. Strathmann. R. R. 19X7. Larval feeding. In: A. C. Giese. J. S. Pearse & V. B. Pearse. editors. Reproduction of Marine Invertebrates. Volume IX. General Aspects: Seeking Unity in Diversity. Blackwell Scientific Pub- lications & The Boxwood Press. Pacific Grove. CA. pp. 465-550 Strathmann. R. R. & E. Leise. 1979. On feeding mechanisms and clearance rates of moUuscan veligers. Biol. Bull. 157:524-535. Strathmann, R. R., T. L. Jahn & J. R. C. Fonseca. 1972. Suspension feeding by marine invertebrate larvae: clearance of particles by ciliated bands of a rotifer, pluteus. and trochophore. Biol. Bull. 142:505-519. Ward. J. E. 1996. Biodynamics of suspension-feeding in adult bivalve molluscs: particle capture, processing, and fate. Inv. Biol. 1 15:218-231. Welbom, J. R. & D. T. Manahan. 1990. Direct measurements of sugar uptake from seawater into moUuscan larvae. Mur. Ecol. Prog. Ser. 65:233-239. Jounwl iif Slu'llfish Rfxcinrh. Vol. 21. No. 1, 2.^4-241, 2002. DISTRIBUTION, DENSITY AND LENGTH-WEIGHT RELATIONSHIP OF CHITON ARTICULATUS SO WERE Y, 1832 ( MOLLUSC A-POLYPLACOPHORA) ON ISLA SOCORRO, REVILLAGIGEDO ARCHIPELAGO, MEXICO OSCAR EFRAIN HOLGUIN QUINONES AND JESUS EMILIO MICHEL-MORFIN Centra Intenlisclplinario de Ciencias Marinas Insfttiito Politecnico. Nacional Apartado Postal 592. La Paz. B.C.S. 23096. Mexico ABSTRACT Chiton articidciuis is an abundant mollusk species on the Isla Socorro. In March 1992 C. articulatus populations were sampled at four sites on the southwestern and northern coast of the island. Average densities of C arliculatus ranged between 3.7 to 8.5 ind/nr among sites. The length-weight relationship for all sampled nidividuals (N = 385) is W(g) = 0.00022 L"''. KEY WORDS: Cliiimi uriiciilalus. mollusk, distribution, density. Isla Socorro INTRODUCTION Chitons are polyplacophoran niollusks that are common grazers of intertidal epilithic and endolithic algae. Since their first appear- ance in the late Cambrian, chitons have modified hard marine substrates using their strong raduia (Glynn 1070; Rasmussen & Frankenberg 1990). Cliilon articulatus is a species that was recently very abundant along the Pacific tropical coast of Mexico, today C. articulatus is uncommon because of over exploitation by fishermen. The foot is commercially utilized and sought in subsistence fisheries. In the Revillagigedo Archipelago (Fig. 1 ) it is a potential resource that has integral usage possibilities, it is still an important member of the malacofauna since it has not been fished commercially (Vil- lalobos 1960: as C. laevigatus. Keen 1971: Ferreira 1983: Holguin et al. 1992: Holguin 1994: Bautista-Romero et al. 1994: Mille- Pagaza et al. 1994; Emerson 1995). Only one study has been conducted for the Mexican Pacific continental shore population of C. articulatus. in the Acapulco area (Rojas-Heirera 1988). Little is known about the Revillagigedo Archipelago chiton populations, because of its distance from the Mexican mainland. This article describes the distribution, density and length-weight relationship of C. articulatus sampled on Isla Socorro, Revillagigedo in March 1992, MATERIALS AND METHODS The Revillagigedo Archipelago (Fig. 1) is located approxi- mately 350-650 km, southeast of the Baja California Peninsula and 580 km west of the Colima coast (Llinas-Gutierrez et al. 1993). The Archipelago is comprised of four oceanic islands of volcanic origin. Socorro, Clarion. San Benedicto and Roca Partida. The surface area of Isla Socorro, the largest island, is 1 67 km" with a maximum diameter of 16 km in a NW-SW direction. Geo- graphically Isla Socorro is located at 18°4r57"N and 1 10^56'33"W (Troyo-Dieguez & Pedrfn 1994). The island is com- posed primarily of braced igneous rock, with the presence of smooth stones and sandy or stone-sandy beaches (Holguin 1994). During March 1992, C articulatus were sampled in the follow- ing rocky areas, from south to north (Fig. I): Bahia Braithv\aite (La Braulia). Bahia Binners, Bahi'a Grayson (Palmasola), and Bahi'a Academia (Playa Noite). Some of the areas are more acces- sible than other areas where there is a greater abundance of or- ganisms. At each one of the four satnpling locations, we took samples along a transect of 50 m~ in the intertidal zone. We laid out transects parallel to the coast line, using 25 m cord which delineated a 2 m wide sampling swath. The entire sampling was accomplished during ebb tides. The density of C. articulatus in TABLE L Mean densities for Chiton arliculatus at four sites on Isla Socorro, Revillagigedo. Mexico, March 1992. Figure L Map of the sampling sites on Isla Socorro, Revillagigedo, March 1992. Maximum Number Area No. Total Relative Chitons Locality (m-) Organism Density (m') Grouped Bahia Binners 50 186 3.72 22 Baliia Braithwaite 50 424 8.48 35 Bahia Grayson 50 193 3,86 33 Bahia Academia 50 249 4.38 41 239 240 QUINONES AND MlCHEL-MORFIN 20 K 30 35 40 45 50 95 90 95 100 105 Length (mm) Figure 2. Length frequency distribution of Chiton aiiiiiilaliis sampled on Isla Socorro, Revillagigedo, March 1992. each rocky shore are;i was determined from the coiinl of all Iimiisj organisms present m the transect. The maxiniiim number of the chitons grouped was determined. A sample of 30-40% of chitons (N = 385) was taken from the transects in order to be measured and weighed. The length and weight measurements from selected chitons were recorded. The length was measured using a vernier caliper, taking the major distance between the front and the posterior parts of the body. Weight, including the shell, was obtained with a three-arm scale having an accuracy of ±0.5 g. We liberated all the animals in the rocky intertidal zone after measuring them. RESULTS AND DISCUSSION 90 • / 80 70 y = 0CI002x"' r' = 0 86 n=385 09/ 0/ ~ 60 % 50 5 40 o o 0 '^.^ cb o 30 20 o > F' 10 ,^ ^ 60 Length (mm) o Braithwaite -Binners -> Grayson AAcademia Figure 3. Length-weight relationship of Chiton articulatus sampled on Isla Socorro, Revillagigedo, March 1992. B Binners y = 6E-05x^" R^ = 0 96 n=55 On Isla Socono all rocky coasts exposed to strong waves are populated by Chiton articiikiltis. The species is discontinuously distributed along the reef line; in some areas adult animals over 4 cm length were found crowded within a narrow belt along the coast, while in adjacent areas animals bigger than 3 cm were not found at all. According to Rojas-HeiTcra (19X8) the size of the mollusk in its first sexual maturity is 40 mm. We observed juvenile C. arliculotiis «4 cm) in Isla Socorro, mostly in the rock fissures and in the cracks of the intertidal belts occupied by the sea urchin Echiiiomt'lni vaiilvunii. We observed that for C. anicidutiis the TABLE 2. Mean lengths and weights for Chiton articulatus at four sites on Isla Socorro, Revillagigedo, Mexico, March 1992. ;i Length (mm) Weight (g) Locality min max mean s.d. min max mean s.d. Bahia Binners 5S 42 1(38.0 67.9 13.9 4.3 S6.3 23.3 19.1 Bahi'a Biaithvvaile \m 24 Q.^i..^ 66.3 124 2.0 73-4 26.6 14.2 Bahia Grayson 1^ 44 102.0 73.6 10.6 9.0 S-^.3 33.3 14.9 Bahi'a Academia 86 49 90.0 71.9 8.9 8.,^ 49.0 27.4 9.3 TOTAL .W.-S 29 lOS.O 69.6 12.5 2.0 S6.3 27.9 14.3 B Grayson 0/ y = 6E-05x>" 0 y r' = 090 n=75 ,^ > 0 B Academia y = 0CI002x^" 0=86 Length (mm) Figure 4. Length-weight relationship of Chiton articulatus for each sampling site on Isla Socorro, Revillagigedo. March 1992. Distribution, Density and Length-weight Relationship of C. articulatus 241 major feeding activity was nocturnal and food was generally com- posed of crusty filamentous algae and diatoms. Several other species of invertebrates coexist with C. aiiiciila- nis. Some are chiton predators such as the gastropods Plicopitr- pura pansa and Thais spp., as well as the cephalopod Octopus sp. Other coexisting species are Littorina aspera, L. modesta and L puUata. Nerita finuciilata, Diodnva iiuiequalis, Chama sqiianiulii;- era. ColiseUa spp. and Fissiirella spp. Several crustacean species can also be found in those areas, such as the decapod crab Grapsus grapsus, the cirriped Tetraclita sp. and the isopod Ligia exotica. We counted a total of 1,052 C. articulatus occurring in the sampling transects (Table 1 ). The highest relative density average (8.5 animals/m^) was found in Bahia Braithwaite and the lowest in Bahi'a Binners (3.7 animals/m") with an average of 5.3 animals/ni" for the four sampling sites combined. We observed the maximum densities of up to 41 chitons/m" in small sections of the transect (Bahi'a Academia. Table 1 ). The abundance of chitons on Isla Socorro is greater than mi Acapulco seaside (4.2 animals/m". Rojas-Herrera 1988). The high density of the mollusk. that we observed in Bahi'a Academia, is probably due to the fact that the bay is far away from any popu- lated center. Bahia Binners which is most accessible to the navy village, shows lower densities because of fishery activity. The length frequency distribution of the 385 chitons collected on Isla Socorro, ransed between 29 and 108 mm with a mode of 70 mm (Fig. 2). The smallest chiton sampled weighed 2.0 grams, while the longest weighed 86.5 grams (Table 2). The average values are of 69.6 mm and 27.9 g for length and weight respec- tively (Table 2). The length- weight relationship W = 0.0022 L" '' where W is total weight (g) and L= total length (mm) was derived from all 385 C. articulatus measured in March 1992 on Isla So- corro (Fig. 3 and Fig. 4). There are no significant differences between length-weight re- lationship in the sampling areas. The major length weight per unit was found in Bahi'a Braithwaite and the minor weight was found in Bahi'a Grayson. It is important to point out that the predominance of Chiton bigger than 6 cm. in the rocky intertida zone of the Isla Socorro, is due mainly to its geographic location, this part of the island is almost inaccessible by land or sea. The population of this species in general, is unaltered and its partial exploitation under strict protection measures is possible. ACKNOWLEDGMENTS We thank Direccion de Estudios de Posgrado e Investigacion del Institute) Politecnico Nacional and Consejo Nacional de Cien- cia y Tecnologia for funding this work. Thanks to the Comision de Operacion y Fomento de Actividades Academicas. Thanks to Sil- via Mille P. Alicia Perez and Ma. de Jesus Parra for their help alone the field work. LITERATURE CITED Bautista-Romero. J.. H. Reyes-Bonilla. D. B. LIuch-Cota & S. E. Lliich- Cota. 1994. Aspectos generales sobre la Fauna Marina. In: R. Ortega & V. Castellanos. editors. La Isla Socorro. Reserva de la Biosfera. Ar- chipielago de Revillagigedo. Mexico. Puhl. No. S. C/BNor. S.C. Mexico, pp. 247-275. Emerson. W. K. 1995. A Zoogeographic Summary of the Marine Mollusks of the Revillagigedo Island (Tropical Eastern Pacific Ocean). The Ff.v- tivus, American Mu.ieum of Natural History. 27(1);3-18. Ferreira, A. J. 1983. The Chiton fauna of the Revillagigedo Archipielago. Mexico. The Veliger. 25(4):307-322. Glynn, P. W. 1970. On the ecology of the Caribbean chitons.- Acanihoft- leura granulata Gmelin and Chiton tubercnUitus Linne; density, mor- tality, feeding, reproduction and growth. Smithsonian Contr. Zool. No. 66. 21 pp. Holguin. Q. O.. S. Mille-Pagaza & A. Perez-Chi. 1992. Resultado de las campanasde muestreo de 1991 para el estudio del bentos marino de isla Socorro. Revillagigedo. Colima, Mexico. Zool. Inf. 24:1-20. Holguin. Q. O. 1994. Comunidades marinas bentonicas. In: R, Ortega & V. Castellanos, editors. La Isla Socorro, Reserva de la Biosfera, Archi- pielago de Revillagigedo, Mexico. Publ. No. 8. CIBNor. S.C. Mexico, pp 225-245. Keen, M. A. 1971. Sea Shells of Tropical West America. Marine Mollusks from Baja California to Peru, 2nd Ed. Stanford: Stanford Univ. Press. 1065 pp. Llinas-Gutierrez. J., D. LIuch. A. Castellanos. 1993. Isla Socorro. Revil- lagigedo. In: S. I. Salazar-Vallejo & N. E. Gonzalez, editors. Biodi- versidad Marina y Costera de Mexico. Mexico: CONABIO-CIQRO. pp. 520-534. Mille-Pagaza, S.. A. Perez-Chi & Q. O. Holguin. 1994. Fauna malaco- logica bentonica del litoral de la Isla Socorro, Revillagigedo, Mexico. Cienc. Mar. 20(4):467^86. Rasmussen, K. A. & E. W. Frankenberg. 1990. Intcnidal bioerosion by the chiton Acanthopleura granulata: San Salvador. Bahamas. Bull. Mar. Sci. 47(3):680-695. Rojas-Herrera. A. A. 1988. Analisis Biologico-Pesquero de la cucaraclia de mar (Chiton articulatus. Sowerby 18321 de Acapulco, Gro.. Mexico. In: Memorias IX Congre.so Nacional de Zoologi'a, Mexico. Univ. Jua- rez Aut. Tab. & Soc. Mex. Zool. Villahermosa, Tab. Mex.. pp. 151- 156. Troyo-Dieguez. E. & S. Pedn'n. 1994. Aspectos hidro-fisiogrdficos y geo- logicos. pp. 43-53. In: R. Ortega & V. Castellanos. editors. La Isla Socorro, Reserva de la Biosfera, Archipielago de Revillagigedo, S.C. Mexico: Mexico. Puhl. No. 8. CIBNor. Villalobos, A. 1960. Notas acerca del aspecto hidrobiologico de la parte sur de la isla. In: A. E. Cobo, et al. editors. La Isla Socorro. Archipielago de las Revillagigedo. Monog. Inst. Geofis. UNAM. 2 pp. Journal of Shellfish Research. Vol. 21. No. 1. 243-248, 2002. CELLULAR IMMUNOLOGICAL PARAMETERS OF THE OCTOPUS, OCTOPUS VULGARIS BEATRIZ NOVOA, CAROLINA TAFALLA, ANGEL GUERRA, AND ANTONIO FIGUERAS Institiito de Investigackmes Marinas. CSIC. Eduardo Cahello. 6. 36208 Vigo, Spain ABSTRACT The white body is the main hematopoietic organ of cephalopods. In this study, we have investigated the capacity of the octopus (Octopus vulgaris) white body cells to petlbmi common cellular defense parameters known to be done by heniocytes of other mollusks such as phagocytosis of zymosan particles, respiratory burst activity and nitric oxide (NO) production. White body cells were capable of respiratory burst and NO production, however, they exhibited a low phagocytic response. Similar capabilities were observed in hemocytes withdrawn from the hemolymph. We have studied the effects of in vitro incubation with bacterial lipopolysacharide (LPS) or zymosan for 24 hours on these two functions. Incubation of the white body cells with zymosan, hut not with LPS. resulted in a significantly increased respiratory burst activity and NO production. We have also investigated the capacity of circulating hemocytes and white body cells to increase their thymidine uptake (indicative of DNA synthesis) in response to LPS and phytohemaglutinin (PHA). In some animals, both mitogens induced a significant increase in thymidine uptake. If this thymidine uptake correlates with cell proliferation, this will be the first report of any proliferation of hemocytes in mollusks. In the hemolymph, we observed two different morphologies under the electron microscope, however, we cannot conclude that they correspond to two distinct cell types. Among white body cells different morphologies that may correspond to intermediate stages were observed. All these findings represent a baseline for future studies to elucidate mechanisms of host defense in this mollusk. KEY WORDS: Octopus vulgaris, hemocytes, white body, respiratory burst, thymidine uptake, nitric oxide (NO), phagocytosis INTRODUCTION Due to the decrease in Spanish cephalopod fishei'ies, the inter- est in the commercial culture of cepahalopod species has gained increasing attention. Diseases are one of the major obstacles in achieving this goal. Several pathogens have been identified in wild and aquarium-kept octopuses, including viruses, bacteria and fungi, Farley (1978) described the presence of viruses in the muscle of the octopus. Octopus vulgaris. Bacteria have been re- ported to cause several disease outbreaks in laboratory reared oc- topuses (Hanlon & Forsythe 1990; Hanlon et al. 1984). Parasites have been blamed for several pathogenic problems. Hochberg ( 1990) described a flagellated parasite in cultured octopuses. Fungi have been also described as causing problems in cephalopods maintained in captivity (Polglase 1980; Polglase et al. 1984). De- spite the threat these pathogens may cause to octopus populations, elucidation of defense mechanisms in these species have received little attention. Humoral defense factors have been identified and are reviewed by Ford (1992). Rogener et al. (1985) and Fisher and Dinuzzo (1991) described hemaglutinating activity and aglutinnins in cell free hemolymph of several molluskan species, including the octo- pus. Octopus vulgaris. In this species, an antiprotease of the a-macroglobulin family was also detected in the hemolymph (Thogersen et al, 1992), Malham et al. (1998) reported lysozyme and antiprotease activity in hemocytes and hemolymph of the lesser octopus Eledone cirhhosa. Like in other mollusks. cepha- lopod hemocytes are believed to play a role in host defense mecha- nisms, however, the function of the hemocytes has been poorly studied. Cowden and Curtis (1981) estimated that the phagocytic capacity of octopus hemocytes was low while high phagocytosis of carbon particles has been described in Eledone cirrhosa (Stuart 1968). Bayne (1983) reported a clearance of Serratia niarcescens by hemocytes of the octopus. Octopus dofleini. Corresponding author. Antonio Figueras, Instituto de Investigaciones Ma- rinas. CSIC. Eduardo Cabello. 6. 36208. Vigo. Spain. E-mail: patol@ nautilus.iim.csic.es The generation of hemocytes of cephalopods is believed to take place in an organ situated around the optic nerve called the white body, gland of Hensen or gland of Faussek (Stuart 1968; Cowden 1983; Bolognari et al. 1980). Hence, the aim of this study was to determine whether white body cells from Octopus vulgaris are capable of performing certain functions believed to be of relevance to defense mechanisms like phagocytosis of zymosan, respiratory burst and nitric oxide (NO) production and to compare their func- tionality to that of circulating hemocytes. We have also examined the ability of two mitogens, bacterial lipopolysacharide (LPS) and phytohemaglutinin (PHA). to stimulate thymidine uptake, an in- dicative of DNA synthesis. In the case of the respiratory burst and NO production, we have also determined the effect of in vitro activation with bacterial lipopolysacharide (LPS) and zymosan. MATERIALS AND METHODS Sampling Adult octopuses (Octopus vulgaris) of both sexes, weighing 2-3 kg were caught from a raft in the Ri'a de Vigo (Spain ) and after a day of adaptation under laboratory conditions, were anesthetized with excess MS-222. When animals were fully anesthetized (iden- tified by muscle relaxation and absence of movement), the visceral cavity was dissected. Once the heart and its associated vessels were reached, around 1 ml of hemolymph was drawn using a 27 gauge needle from the artery and subsequently from the heart, placed in an eppendorf and kept on ice until used. The white body was removed and kept on ice until used. In order to obtain single cell suspensions, individual white bodies were passed through a 100 |jLm nylon mesh using Leibovitz medium (L-15, Gibco) supplemented with penicillin { 100 lU/mL). streptomycin (100 |xg/mL) and 2% fetal calf serum (FCS). The resulting cell suspension was centrifuged (500 x g for 15 min at 4°C) and the cells were resuspended in L-15 supplemented with penicillin, streptomycin and 2% (FCS). Cell viability was deter- mined by Trypan blue exclusion. Cells were resuspended in L-15 supplemented with penicillin, streptomycin and 0.1 "^r FCS at a density of 1x10" cells/mL. 243 244 NOVOA ET AL. Hemocytes were obtained by centrifuging octopus heiiiolyniph (500 X g for 15 min at 4'C) and hemocytes were resuspended in L-15 supplemented with penicillin, streptomycin and 29f FCS. The ceil viability was determined by Trypan blue exclusion test. Cells were resuspended in L-15 supplemented with penicillin, strepto- mycin and 0.1% FCS at a concentration of 1 x 10" cells/mL. Electron Microscopy Pelleted cells (from white body and hemolymph) were fixed for one hour in 1% osmium tetroxide in cacodilate buffer 0.1 M (pH 7.3). In some cases, a previous step of fixation with \':i glutaral- dehyde in cacodilate buffer 0.1 M (pH 7.3) for 15 minutes was performed. Following three washes in 0.1 M cacodilate. the cells were dehydrated with increasing pei-centages of ethanol and em- bedded in Araldite/Poly Bed (Polyscience). Ultrathin sections (50- 70 nm) were stained with uranyl acetate and lead citrate and ex- amined using a Phillips electron microscope CM 100. Phagocytosis Assay To measure the phagocytic ability of octopus cells. 200 |J.L of cell suspensions (derived from white body or hemolymph of six animals) were incubated in chamber slides (Nunc) for 2 h at 18"C in moist incubation chambers to allow the cells to adhere. Medium was removed and the adherent cell layer was washed twice with L-15. Zymosan A (Sigma) resuspended in sterile phosphate buf- fered saline, PBS, at a concentration of 1 mg/mL. was heated at 100°C for 30 min, washed twice, and resuspended in the same PBS volume. Zymosan was added to the cells at a final concentration of 250 jxg/mL. and the same volume of L-15 was added to the con- trols. Slides were incubated in a moist chamber for one hour at room temperature or 18°C to allow phagocytosis. Some slides were kept up to three hours. Slides were then washed twice in PBS, fixed in absolute ethanol, stained with Hemacolor, and mounted with DePex. Two replicas were made for each octopus and at least 150 cells were observed in each replica. Respiratory Burst Activity Respiratory burst activity of octopus cells was assayed by the reduction of ferricytochrome C (Cit C, Sigma) by released super- oxide anion (O^-). following stimulation of the cells with phorbol myristate acetate (PMA, Sigma) (Secombes 1990) in 4 octopuses. White body adherent cells and circulating hemocytes were ob- tained as described above, resuspended in L-15 supplemented with penicillin, streptomycin and 0.1% FCS and dispensed into 96-well tissue culture plates (Iwaki) at a concentration of 1 x 10*" cells/mL (100 jjlL per well). In a preliminary experiment, we determined the specificity of the respiratory burst by assaying the response of the cells to PMA. After 24 hours of incubation at I8°C, octopus cell monolayers were washed twice in phenol red-free Hank's balanced salt solu- tion (HBSS, Gibco). One hundred (jlL of HBSS containing Cit C (2 mg/mL) and PMA (1 ng/mL) were added to each well. As a control for specificity, 300 lU/mL superoxide dismutase (SOD, Sigma) was added to some wells. The optical density (O.D.) was measured at 550 nm after 30 min in a multiscan spectrophotometer (Labsystems). Triplicate wells were used in all the experiments for each octopus and the mean ± SD was calculated. Once the responsiveness of octopus cells to PMA had been deterinined, the effects of stimulation with zymosan or Escherichia coli serotype 0111: 84 lipopolysacharide (LPS) on the respiratory burst of octopus cells triggered by PMA was also studied. After three hours incubation of the cell monolayers at 18"C, LPS and zymosan were added to a final concentration of 50 and 250 p-g/mL respectively. After an additional 24 hours of incubation at 18"C with these substances, the respiratory burst activity was measured by adding 100 p.L of HBSS containing Cit C (2 mg/mL) and PMA ( 1 |j.g/mL) were added to each well. The O.D. at 550 nm was then detemiined as described above. NO Production The ability of octopus cells to produce NO in response to LPS and zymosan was also determined in 4 animals. Cells resuspended in L-15 with 0.1% FCS were disposed into 96-well plates at a concentration of 1 x 10'' cells/mL. After 3 h of incubation at 18°C. LPS and zymosan were added at a final concentration 50 and 250 |j.g/mL respectively. After additional 24 h of incubation at 18°C. the NO concentration present in the cell supernatants was assayed through the Griess reaction (Green et al. 1982) that quantifies the nitrite content of the cell supernatants. since NO is an unstable molecule and degrades to nitrite and nitrate. Fifty (jlL of hemocyte supernatants were reiuoved from individual wells and placed in a separate 96-well plate. One hundred microliters of 1% sulfanil- amide (Sigma) in 2.5% phosphoric acid were added to each well, followed by the addition of 100 |a.L of 0.1% N-naphthyl- ethylenediamine (Sigma) in 2.5% phosphoric acid. Optical density at 540 nm was determined using a multiscan spectrophotometer. The molar concentration of nitrite in the sample was determined from standard curves generated using known concentrations of sodium nitrite (100. 10. 5. 2.5. 1. 0.5. 0.25. and 0.1 |jlM). Effect of Mitogens on (^H)-Thyinidine L'ptake by Octopus Cells The thymidine uptake by octopus cells was assayed following a modification of the method described by Marsden et al. ( 1994) in six octopuses. Briefly, hemocytes derived from the hemolymph or the white body were adjusted to a density of 5 x 10"' cells/mL in RPMI 1640 medium (Gibco) supplemented with penicillin (100 lU/mL), streptomycin (100 |jLg/mL), 25 mM NaHCO, and 1 x 10"^ M 2-P-iuercaptoethanol. Aliquots of 100 |xL of cell suspensions were added to wells of 96-well plates containing 100 |jiL of LPS or PHA dilutions (Sigma) to make final concentrations of 50. 25 and 12.5 |j.g/mL or 5, 2.5, and 1.25 |jig/mL, respectively. Controls without mitogens were also included. After 3 h of incubation at 18°C, FCS was added to the wells to give a final concentration of 10%. Following 48 h of incubation, the cells were pulsed with 0.5 (id of ('H)-thymidine (Amersham). After additional 24 h incu- bation at 18"C. DNA v\as harvested onto glass filter mats. One niL Xylofluor scintillant (Packard) was added to dried filter circles in vials and counts per minute (cpm) were recorded using a Packard liquid scintillation counter. Triplicate cultures were used in all cases. Statistics The data were compared using a Student's t test. Results are expressed as the mean ± standard deviation and differences were considered statistically significant al P < 0.05. RESULTS Cell Morphology After two hours of incubation of the cell suspensions at 18°C, the white bod\ adherent cells were adhered to the bottom of the Octopus Cellular Immunology 245 wells, tlutten and spread over the surface by extending pseudopo- dia. Circulating hemocytes presented the same aspect. When visualized under the electron microscope (Fig. I), what seems as two different morphologies, that were distinct under the inverted microscope, were observed among circulating hemocytes. No differences were found in the quality of fixation when the previous glutaraldehyde step was omitted and therefore the cells were always directly fixed in osmium tetroxide. We identified in the first cell type a kidney-shaped nucleus that occupied about 2/3 of the cell volume with a well-defined nucleolus and abundant heterochromatin in peripheral positions. Their cytoplasm was rich in vacuoles and electron-dense granules of various sizes. The sec- ond cell type had a nucleus with faint chromatin, a round nucleus that occupied about 1/3 of the cell volume. The cytoplasm was rich in vacuoles, but had only scarce granules. However, these results are not conclusive and we cannot assure that what looks as two different morphologies corresponds to two distinct cell types. In the case of white body cells, we were able of identifying cells showing other morphologies that may correspond to interme- diate stages between the two cell types found in the hemolymph. Phagocytosis Activity of White Body Adherent Cells and Circulating Hemocytes The phagocytic activity detected after incubation of octopus hemocytes and white body cells with zymosan was low. The per- centage of phagocytosis observed in circulating hemocytes was 19.3% (SD = 14), while only 9.3% (SD = 8) of white body cells contained zymosan particles in their cytoplasm. In all cases, varia- tions among individuals were high, since in some samples no phagocytosis was observed (0% of phagocytosis). The same results were obtained with the two incubation temperatures (18°C or room temperature) in both circulating and white body cells. No differ- ences were observed when the hemocytes were incubated in their own hemolymph (data not shown). Respiratory Burst Activity Octopus white body cells showed a significant increase in the release of superoxide anion after stimulation with PMA compared with controls, as depicted in Figure 2A. The specificity of the Figure 1. Under the electron microscope, two distinct morphologies were identified among circulating hemocytes. The first cell type (I) is characterized by its kidney-shaped nucleus and the high number of electron-dense granules. The second cell type (III possessed a round nucleus with a few electron den.se granules. Bar = 5 fini. A - T "■i:W' - . T , -1- Cit PMA PMA+SOD Treatmenl Conlml LPS ZyimtiJii Ta-.itinenl c * - T - i T T 1 X c Control LPS Z\niosaii Treatment Figure 2. Respiratory burst activity of octopus white body cells. First, the capacity of PMA to stimulate the respiratory burst activity and the specificity of the reaction was assayed (A) (N = 2). Once, this was determined, the effects of LPS and zymosan on the respiratory burst activity of white body hemocytes (B) (N = 4) and circulating hemocytes (Cl were studied through the reduction of Cit C, stimulating the cells with PMA. Data are shown as the mean O.I), at 550 nm. *Respiratory burst significantly higher than the respiratory burst obtained in con- trols. P < 0.05. reaction was demonstrated since SOD completely inhibited the respiratory burst response of octopus cells in all cases. This re- sponse was also observed with circulating hemocytes. The respi- ratory burst activity of hemocytes incubated directly in their own hemolymph was also assayed. In these conditions, some octopuses did not respond to PMA and did not elicit a respiratory burst response. Figure 2B shows the effect of incubation for 24 hours with LPS or zymosan on the respiratory burst triggered by PMA of white body cells, compared to the respiratory burst observed in cells that had been incubated with L-15 only. The pre-incubation of the cells with zymosan, but not LPS. significantly increased the respiratory burst of white body cells in response to PMA. The same response was observed in cells obtained from hemolymph. showing a higher respiratory burst after zymosan treatment than controls (Fig. 2C). NO Production The NO production of white body cells in response to LPS and zymosan is shown in Figure 3. Zymosan significantly stimulated the cells for NO production in all individuals, however, as in the case of the respiratory burst activity, LPS failed to stimulate the cells. In the case of hemolymph cells, a similar response was ob- served. The NO production in the cultures treated with zyinosan (S.4; SD = 0.8) was higher than the response observed in controls (6.7; SD = 0.3). 246 NOVOA ET AL. Control LPS Zymosan Treatment Figure 3. NO production of cells from the wliile bodj after incubation witli LPS or zymosan. Data are presented as the mean nitrite concen- tration obtained with 4 octopus. 'Nitrite concentration si}>niricantl> hifjher than the one observed in controls onlj treated with 1,-15. P < 0.05. ('H)-Thymidiiie Uptake Not all animals studied showed a significant response. Only two out of six individuals assayed responded with a significant increase of the thymidine uptake in response to mitogens. The individual responses observed in these two animals are shown in Table I . When means were compared, concerning white body cells, a maximum thymidine uptake was observed with the higher LPS dose (687.3: SD = 6.7). In this case, the PHA dose that significantly stimulated the thymidine uptake was 2.5 p.g/ml (495; SD = 20) in comparison to controls (.'^78.3; SD = 2.5). In these two responsive animals, hemocyte thymidine uptake was also significantly affected by the mitogens. All concentrations of LPS significantly increased ('H)-thyniidine uptake compared to non-stimulated controls, although the higher response was ob- served with the highest LPS dose. However, hemocyte thymidine uptake was only significantly increased with the higher dose of PHA. TABLE 1. Thymidine uptake by circulating hcmocytes and white body cells in two responsive animals. Data are presented as the mean cpni obtained in the replicas (N = 3l ± SD. Octopus 1 Octopus 2 White White Circulating Body Circulating Bodv Mitogens Hemocytes Cells Hemocytes Cells LPS 50 |xg/ml 630.6 ± 242 694 ± 35 1 515.3 + 37 680.6 ± 1 04 LPS 25 (jLg/ml 340 ± 1 1 S .%6± 144 457 ±217 370 ± 70 LPS 12.5 jjig/ml 458 ± 98 761 +418 427 ±41 524 ± 142 PHA 5 Jig/ml 340 ± 92 241 ± 171 615 ±128 253 ± 58 PHA 2.5 |xg/m] 23S± 12 475 ± 147 141 ±32 515 ± 154 PHA 1.125 |j.g/ml 178 + 52 313 + 79 222 ± 42 290 ± 55 Control 204 ± 64 381 ± 105 294 ± 94 376 ± 1 2 DISCUSSION The findings in this study suggest that both octopus white body cells and circulating hemocytes are capable of performing func- tions associated with host defense mechanisms. This is particularly important to determine since scanty data is available on the im- mune response of this octopus species. This is the first work in which reagents and techniques usually used in vertebrate immu- nology have been successfully applied to study cellular responses of cephalopods. Under the electron microscope, two distinct morphologies among circulating hemocytes were identified, although it had been described as only one cell type of the hemolymph of Oclopus vulgaris (Bidder et al. 1989). Our results are not conclusive and more work should be done to determine whether these two mor- phologies correspond to different states of activation or they con- stitute two different cell types. Previous studies in bivalve mol- lusks have identified two main hemocyte types in the hemolymph (Fisher 1986; Lopez et al. 1997) that have been subdivided (Auf- fret 1988; Nakayama et al. 1997). In the white body, it is well known that there are cells, refened to as hemocytoblasts. with a large cytoplasmic volume and abundant rough endoplasmic reticu- lum and nucleoli (Ford 1992). Hemocytoblasts transform to leu- koblasts by reducing its cytoplasmic volume and decreasing its nuclear size, whereas secondary leukoblasts (mature hemocytes) are cells with a larger size and a folded nucleus. Both under the light and electron microscope, we were able to identify different morphologies that may correspond to these intermediate stages among white body cells. As well, the two different morphologies that we describe in this work among circulating hemocytes seem to correspond to these two cell types (hemocytoblasts and leuko- blasts). It may be possible that in these individuals maturation ends in the hemolymph or as will be discussed later, that these two cell types are functionally different. The first cell type may correspond to what has been called granulocyes in bivalves (Auffret 1988). These cells possess a kid- ney-shaped nucleus that resembles those of vertebrate granulo- cytes. The second cell type had a round nucleus and a cytoplasm that was also rich in vacuoles, but had only scarce granules. This cell type may correspond to what in bivalves has been called hyalinocytes (agranular hemocytes) (Auffret 1988). Results concerning in vitro phagocytosis by octopus hemocytes are in accordance with those previously reported (Crowden & Curtis 1981) where it was suggested that gill tissue or phagocytes from gill tissue could be responsible for clearing foreign sub- stances and that circulating hemocytes had low activity (Crowden & Curtis 1981: Bayne 1983). However, studies in other cephalo- pod species, like Eledone cirrhosa reported bacterial phagocytic rates of even 40% (Malham et al. 1997). The low phagocytic activity that is observed in the octopus. Octopus vulgaris, circu- lating hemocytes also contrasts with the high phagocytic rate de- tected in bivalves (Mortensen & Glette 1996; Ordas et al. 1999). animals in which phagocytosis is a critical defense mechanism. Many factors can affect phagocytic rates in mollusks such as tem- perature (Carballal et al. 1997), time and pH (Abdul-Salam & Michelson 1980), size and nature of the particle presented for phagocytosis (Bayne 1983). In some cephalopod species, phago- cytosis is not possible in the absence of hemolymph (Stuart 1968). It may be possible that on the contrary to what happens with the respiratory burst (that is inhibited by the presence of hemolymph). Octopus Cellular Immunology' 247 phagocytosis in this species needs humoral factors present in the hemolymph. Octopus hemocytes, however, were able to respond to PMA and release significant amounts of superoxide anion. Respiratory burst activity is an interesting non-specific defense mechanism that vertebrate macrophages use to avoid bacterial and parasitic infec- tions due to the potent microbiocidal effects of oxygen radicals. The reason why the respiratory burst activity was not detected in some of the octopuses while incubated in their own hemolymph is unknown. The immune system is regulated by multiple signals that can up or down modulate the animal responses. Maybe regulating factors, affecting this immune response, are present in the hemolymph of these animals, in the same way that other humoral factors such as lysozyme or antiproteases have been detected in cephalopod hemolymph (Malham et al. 1998). We have also demonstrated that octopus cells deri\ed from hemolymph and white body produced NO. In \'ertebrates. NO mediates many functions including neurotransmission, vasodilata- tion, as well as several immune functions. It is known that this molecule when secreted by macrophages is microbicidal against viruses, parasites and bacteria (Nathan & Hibbs 1991; Tafallaet al. 1999). In the octopus, NO production was significantly increased when the cells were incubated with zymosan for 24 hours. How- ever, incubation with LPS did not have an effect on the NO se- cretion. This is the first report on NO production by cells of any cephalopod. NO production in other mollusks has been shown, in which NO production was demonstrated by indirect methods (Maninez 1995: Otaviani & Franchini 1995). Octopus hemocytes were also able to increase their thymidine uptake after stimulation with LPS and PHA. although this response was not found in all indi\iduals. maybe because this immune function is more related to a specific response not known to be present in cephalopods. Thymidine uptake implies DNA synthesis. so this increase may mean that the hemocytes are proliferating. If so. this will be the first report of any proliferation in mollusks. outside the hematopoietic organ. More work must be done to de- termine if this thymidine uptake detected coirelates with cell pro- liferation. In higher vertebrates, these two mitogens correlate to B and T lymphocyte mitogens, respectively. Since we observed ul- trastructurally two morphologies among hemocytes in responsive animals, it may be possible that LPS and PHA-induced prolifera- tion are being supported by different hemocyte populations, and therefore imply a certain grade of heterogeneity, as in vertebrate immune cells. In conclusion, we have effectively applied immunological tech- niques designed for vertebrates to evaluate cephalopod hemocyte functions. Among mollusks, cephalopods are highly evolved ani- mals with a body design, nervous system and sense organs that often reach vertebrate standards (Budelmann et al. 1997). It seems possible that immune system may also be more developed than other mollusks in which modulation of immune responses by pathological agents or other factors has been reported (Ordas et al. 1999). We have set up different in vitro techniques, such us de- termination of respiratory burst activity, NO production and cell proliferation, which will let us further evaluate the octopus im- mune response against infections or the influence of environmental conditions. This latter aspect is especially relevant because of in- creasing interest that cephalopod aquaculture has had in the past years (Osako & Murata 1983; Guerra & Rocha 1994; Guerra et al. 1994). More work must be done to clarify the regulation of these immune functions in cephalopods and determine their role in the defense against pathogens. ACKNOWLEDGMENTS The authors thank Dr. Guerra"s team. Begona Villaverde and Jose Ramon Caldas for their technical assistance and Dr. M. Faisal (Virginia Institute of Marine Science. USA) for reading and re- viewing this article. LITERATURE CITED Abdul-Salam, J. M. & E. H. Michelson. 1980. Biomphalaria glahrata amoeboeytes: assay of factors influencing in vitro phagocytosis. / Imenebr. Pathol. 36:52-59. Auffret. M. 1988. Bivalve hemocyte morphology. Am Fisheries Society Special Publication 18:169-177. Bayne, C. J. 1983. Molluscan imniunobiology. In: A. S. M. Saleuddin & K, M. Willbur, editors. The Mollusca. Vol. 5. Physiology, part 2. London: Academic Press, pp. 407-486. Bidder, A. M.. M. V. Boletzky. S. V. Boletzky. F. G. Hochberg. K. Mangold, H.-J. Marthy. A. Portmann, C. Teichert & J. Worms. 1989. In: P-P Grasse, editor. Traite de zoologie. Anatomie, systemalique. biologie. Tome V. Cephalopodes. Masson. p. 411. Bolognari, A.. S. Fasulo & A. Licata. 1980. A preliminary comparison of the leukocyte series of the cephalopod Todannles saginams and the granulocyte series in the bone marrow of the rat Rattus rattus. Bull. Zoo/. 41:221-225. Budelmann. B. U., R. Schipp & S. von Boletzky. 1997. Cephalopoda. In: Microscopic Anatomy of Invertebrates. Volume 6A: Mollusca II. Wiley-Liss, Inc. pp. 1 19^14. Carballal. M. J.. C. Lopez, C. Azevedo &. A. Villalba. 1997. In vitro study of phagocytic ability of MyliUts galloproviiiL talis Link, haemocytes. Fish Shellfish Immunol. 7:403-416. Cowden. R. R. 1 972. Some cytological and cytochemical observations on the leukopoietic organs, the "white bodies" of Octopus vulgaris. J. Imenebr. Pathol. 19:113-119. Cowden. R. R. & S. K. Cunis. 1981. Cephalopods. In: N. A. Ratcliffe & A. F. Rowley, editors. Invertebrate blood cells. New York: Academic Press, pp. 301-323. Farley, C. A. 1978. Viruses and virus like lesions in marine mollusks. Mar. Fish. Rev. 40: 1 8-20. Fisher, W. S, 1986. Structure and functions of oyster hemocytes. In: M, Brehelin, editor. Immunity in invertebrates. Berlin Heidelberg: Springer-Verlag. pp. 25-35. Fisher, W. S. & A. R. Dinuzzo. 1 99 1 . 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New Jersey: SOS Publications, pp. 137-154. Stuan, A. E. 1968. The reticulo-endothelial apparatus of the lesser octopus. Eledone cirrho.ui. J. Pathol. Bacterial. 96:401— 112. Tafalla. C. A. Figueras & B. Novoa. 1999. Role of nitric oxide on the replication of viral haemorrhagic septicemia virus (VHSV), a fish rhab- dovirus. \'ci Immuitol. Immunopathol. 72:249-256. Thogersen. J. B.. G. Salvesen. H. Brucato, S. V. Pizzo & J. J. Enghild. 1992, Purification and characterization of an a-macroglobulin protein- ase inhibitor from the mollusc Octopus vulgaris. Bioch. J. 285:521- 527. Journal nf Shellfish Rt-seanh. Vol. 21, No. 1, 249-253. 2002 ESTIMATING GONADO-SOMATIC INDICES IN BIVALVES WITH FUSED GONADS H.-JORG URBAN'* AND JOSE M. RIASCOS V.- Alfred Wegener Institute for Polar and Marine Research, Section for Comparative Ecosystem Researcli, Postfacli 12 01 61, 27515 Bremerhaven. Germany: 'Mnseo de Ciencias Naturales "Federico C. Lehmann " A. A. 5660, Cali - Coloml^ia ABSTRACT A new method was developed to estimate the gonado-somatic index in bivalves, taking into account that in most bivalves gonad and non reproductive tissues are fused. As examples, four tropical bivalve species ^Dollux demifer. Cardita ajfinis. Pmctada imbricara and Pteria colymhus) from the Colombian Pacific and Caribbean were used. The fused gonad regions were replaced by geometric bodies, thus the gonad volumes were estimated with geometric equations (corresponding to geometric bodies) and linear measures obtained directly on frozen soft-body samples. The very good correlation between the gonado-somatic index and the gametogenetic cycle of the stage "ripe", leads to the conclusion that this method is a suitable tool to estimate gonad indices of bivalves with fused gonads. KEY WORDS: method, gonado-somatic index, bixalves. Doiiax demifer. Cardihi affhus. Pmctada imhricaia. Pteria cohmbi(s INTRODUCTION A standard procedure in marine biological reproductive .studies is to determine gametogenetic stages (Guillou et al. 1990) or lo estimate indices (Moore 1934; Giese 1967; Pearse 196.'5). Among many existing indices the so called gonado-somatic indices, cal- culated from the relationship between a variable depending di- rectly on reproductive processes and a variable independent from reproductive processes, are most frequent used. Examples for the variables used in gonado-somatic indices are the gonad volutne and the total volume. Such a gonado-somatic index, followed pe- riodically over a year, is a valuable tool because it is related directly to reproductive activities, such as spawning events. Among marine species, the estimation of gonado-somatic indices in bivalves often presents a problem because most species of this group have fused gonads, such that reproductive and non- reproductive tissues cannot — or are difficult to be separated. Based on four tropical bivalve species, the objective of this article is to present and explain a new method, that permits the estimation of gonado-somatic indices in bivalves with fused go- nads. In order to evaluate the new method, the gonado-somatic index is compared with gametogenetic stages obtained from smear samples. MATERIALS & METHODS Investigations were carried out in three areas: the Tayrona Na- tional Nature Pari:, close to the city of Santa Marta. Caribbean of Colombia (1 1°20'N. 74°10'W, Fig. la), the "Cal?o de la Vela", a Peninsula located in the Guajira province. Caribbean of Colombia (I2°I0'N. 72'20'W. Fig. lb) and the "Bahia Malaga', a bay close to the harbor town of Buenaventura, Pacific Coast of Co- lombia O'-Se'N. 77°19'W. Fig. Ic). In Tayrona Park the pearl oyster Pteria colyitibus. which lives attached to octocorals is a dominant species, while in Cabo de la Vela a characteristic Thalas- sia testudinnm community gives a suitable substrate for a variety of bivalve species, especially a dense PiiictaJa imhricaia popula- *Corresponding author. H.-Jorg Urban. Alfred Wegener Institute for Polar and Marine Research. Section for Comparative Ecosystem Research. Post- fach 12 01 61. 27515 Bremerhaven. Germany. Phone: -H49-471 4831-1315; Fax: ■H49-471-4831-1119; E-mail: jurbanCgawi-bremerhaven.de Figure 1. Study areas a) Cabo de la Vela, Colombian Caribbean; 'i Tayrona Park: Colombian Caribbean; and c) Bahi'a Malaga. Colom- bian Pacific 249 250 Urban and Riascos Diinax denlijer 12 3 4 5 6 Curiiifa ajpnis 12 3 4 5 6 Figure 2a shows the sotl parls (mantle, siphons and gills removed) on the left shell of D. dcnlifcr and C. uffhn.s demonstrating the fused character of somatic and gonad tissues. Figure 2b shows the six transversal sections. In D. dentifer the visceral mass is located in the middle-dorsal body part, partially enclosed by the gonad region. The pedal muscle occupies the major part of the total body volume extending in anterior-posterior direction and enclosing the gonad region. Contrary, in C. affinis the visceral mass is located in the posterior-dorsal part, decreasing towards the anterior end. The gonad region is located in the posterior- ventral zone, increasing its volume towards the middle part (section 3) and then extending 3) Donax dentifer Cardila a/finis Figure 2. a) Morphological description of body tissues (on left valve, mantle, siphons and gills remo\ed) of Donax dentifer and Cardita af- finis. Location where transversal sections were taken is indicated. b» Transversal section according to Figure 2a. Gonadal tissue is given in black, visceral tissue in gray and muscle tissue in white. tion. Finally, at Bahki Malaga the infaunal Doiiax dentifer found in the intertidal of sandy beaches and the borer clam Cardita ajfinis from rocky shores were studied. Sampling for Dmiax dentifer was earned out in 1997/98 as well as in 1999/00, while for the latter three species the study period lasted one year, but at different dates (Pteria colyinhtis: 1994/95. Pinctada iinbricata: 1997/98, Cardita affinis: 2000/01). At monthly intervals, samples were taken and the shell length (ante- rior-posterior axis) of all specimens was recorded with vernier callipeis. Two subsamples of 30 specimens per species each were taken. One subsample was used for length-weight relationships: soft parts were removed and dried at 70°C to constant weight to determine shell free dry weight (SFDW). Monthly length weight relationships were used to estimate a shell free dry weight ( = con- dition) cycle of a standard individual ( = 667f of maximum length) based on Equation ( I ); SL = a- SFDW (I) where SL is the shell length |mm| and SFDW is the shell free dry weight [g]. The second subsample was deep frozen and used to obtain linear measures of gonad regions with a vernier callipers in order to estimate the gonado-somatic index (details are given un- der "Results" later). Finally, after taking the linear measures, ga- metogenetic stages of smear samples were determined according to a microscopic scale (Urban 2000): "developing 1 (= early de- veloping)", "developing 2 ( = late developing)", "ripe" and "spent". RESULTS Using frozen samples, six transversal sections (thick slices) were taken at different locations of the body: these were used to identify the gonad legion geometrically. As an example only the morphological sections o'i two of the four species are presented. m -if-ii"" b) Cardita affinis 's-K triangle prism ^^ ^ a ■be -\- a-b-c 2 4 area C) d) cylinder y.r, = " e) Pinctada imbricata & Pteria colymbus pyramid 1 fe-t Figure 3. Geometric bodies defined according to the morphological examinations of the transversal sections (Fig. 2bl. The linear measures necessary to calculate the volume as well as the formulas are given (see text), a) Ellipsoid of Ihmax dentifer. h) Two fused triangle prisms of Cardita affinis. c, d. and e) Fused cylinder and pyramid of Pinctada imbricata and Pteria colynihns. Simple small letter correspond to linear measures of somatic and reproductive tissues, while gonadal tissues are given as small letters with "'" (e.g. a'). Estimating Gonado-Somatic Indices in Bivalves 251 a) Donax dentifer b) Cardita affinis c) Pinctada imbricata & Pteria colymbus Figure 4. Body tissues (upper row ) and geometric bodies replacing gonad region and or body tissues (lower row), a) Donax dentifer. b) Cardita affinis and c) Pinctada imbricata and Pteria colymbus towards the anterior-dorsal part where its volume decreases gradu- ally. The pedal muscle is reduced, corresponding to a small ex- tension of the posterior-ventral end. The volume of the abductor muscle increases from the middle ventral zone, extending towards the anterior end. Based on the morphological examination of the gonad region (Fig. 2) for all species three-dimensional bodies of the gonad re- gion were defined. In other words, the gonad region was replaced with geometric bodies. The volumes of these bodies and thus the gonad volume, can be estimated using the following geometric equations and linear measures [mm] taken directly on the bivalves from frozen samples. Based on macroscopic criteria such as tex- ture, color, etc.. gonad tissue could be easily distinguished from somatic tissue. Thus, for each species during the study period the gonad volumes |ml) of approximately 30 specimens were obtained at monthly intervals. Donax dentifer The gonad morphology corresponds to an ellipsoid half (Figs, 3a & 4a). located between the foot and the visceral mass. The equation below Figure 3a corresponds to the volume of an ellip- soid half. It is clear, however, that a small visceral region is located in the dorsal part of the "gonadal ellipsoid" (Fig. 2a). On average, this visceral tissue occupied 10% (max: 19%, min: 6%) of the total ellipsoid volume. Thus, in order to yield the gonad volume, the formula was corrected multiplying with 0.9 (Eq. 2): V„ a b TT • - • - • c I /2 ■0.9 (2) Measures are indicated in Figures 3a & 4a; "a" is the gonad breadth (lateral to the body axis), "b" is the gonad length (anterior- posterior body axis) and "c" is the gonad height (dorsal-ventral body axis at transversal section number 3. Cardita affinis The geometry of the gonad region suggests the form of two fused triangle prisms (Fig. 3b & Fig. 4b). Thus, the formula of a single triangle prism has to be multiplied by 2 (Eq. 3): V = [A, base "prismJ ■" a-h,, (3) Where A^,^„^. is the area of the base, which corresponds to a triangle with "a" being one side and "h " the heiehl of the triangle corre- sponding to "a", finally "h the height of the prism. The gonad volume was calculated as the sum of these two prisms (Eq. 4): a-b-c] Tab-c (4) Where "a" is the gonad length (anterior-posterior body axis), "b" is the gonad height (transversal to gonad length corresponding lo transversal section number 3. Fig. 2) and "c" is the gonad breadth at the fusion point of both prisms. (Note: "a" corresponds to the length of both fused prisms, therefore for the two bodies "1/2 a" has to be used yielding Eq. 4.) Pinctada imbricata and Pteria colymbus These two species of the family Pteridae are different from the latter two because the gonad tissue covers the somatic tissues. In D. dentifer and C. affinis the total body volume was estimated with the water displacement method and gonad volume corresponds directly to the geometric abstractions, while in P. imbricata and P. colymlnis first the total body volume (visceral mass and foot in- cluding gonads) and then in a second step the "covering" gonad volume was estimated. The total body volume can be defined by a cylinder (Fig. 3c & Fig. 4c. Eq. 5a) fused with a pyramid (Fig. 3d & Fig. 4c. Eq. 5b). The cylinder corresponds to the visceral mass 252 Urban and Riascos Fig. 5a & c — •— OSl ■ - G- - npe Fig. 5b & d — • — dry weiglil D developing I D developing 2 ■ npe ■ spent Figure 5. a) Overlay of gonado-somatic index and gametogenetic .stage "ripe" cycle, b) Overlay of shell free dry weight cycle and distribution of gametogenetic stages of OoHa.v deiuifer from Bahia Malaga, Colom- bian Pacific, c & d) Cardila affinis from Bahi'a Malaga, Colombian Pacific. and the pyramid to the basal region of the foot. These two bodies have the general formula: a) V,.„, = -IT • r" • h. b)V„ = 3A, (3) Where "r" is the radius, h^.^,, is the height of the cylinder (Fig. 3c). "Abase" 's 'hs ^'■'S^ of the base of the pyramid and "hpy/" is the height of the pyramid (Fig. 3d). So the total body volume of these two species coiresponds to the sum of the \ olume of a cylinder and a pyramid (Fig. 3e. Eq. 6 & 7): 'body.tulpi^r.j^j ■ ^v, .yl pyr b + c\- (6) Where "a" is the length, "b" is the height and "c" is the breadth of the visceral mass (including gonad region) and "d" is the length. "e" is the height and "f" is the breadth of the foot (including gonad region). In the second step the somatic volume was estimated using the satiie equation (Eq. 6). but with linear measures where gonad regions were excluded. Gonad volume was estimated subtracting the somatic volume from the total volume (Eq. 7); V = V, gonptcniliK- liudy 1 „" ^b,.Jy ..,„„,., (b + cy- ■n ■ I 4 ) -J + L e ■ t — 55 ■ Pwria tolymhiiy ■S 50- \ ■' '• a 45 ■ \ ■' ' i 40. \'^"''-. ■■«■■ ■"'■ 1 "■ »—,■.,■'/>, ■' y^\ A ' S 30 ^* V/ \ /' V' g No^ ■ — ~ 80"o- st 5 60%- .a e it .(. . \ 1 f! t A^ V =«^ 1 1 L 1 II 'fi 50 S. X 40 I 30 ! 20 = u 10 s 0 5 ,if 0.5). For the correlation between GSI and SFDW only for Pterin colyinhiis and Donax clciitifer high \alues can be observed. This result is confirmed in Figure 5a & c and Figure 6a & c where GSI and ripe cycles are plotted in overlay graphs. In all species the pattern of GSI and ripe cycle is very similar. Figure 5b & d and Figure 6 b & d show the SFDW cycles together with the distribution of gametogenetic stages. DISCUSSION In order to evaluate a new method it is most convenient to compare the results with alternative information obtained com- pletely independent. This can easily be accomplished for the GSI. It can be assumed thai the GSI is positively correlated with the gametogenetic stage "ripe". GSI gives the mean gonad volume/ total volume relationship per month. In other words if a large part of the population has voluminous gonads, the GSI is high. On the other hand the gametogenic stage "ripe" gives the percentage of specimens with ripe gonads (shortly before spawning). In this developinent stage, egg and sperm size is large, resulting in volu- minous gonads. The results clearly confirm this assumption and thus prove the applicability of the GSI method. Graphically (Fig. 5a and c and Fig. 6a and c) as well as statistically (Table 1 ) a high correlation between the GSI and the gametogenic stage "ripe" exists. It should also be noted that GSI and stage "ripe" are com- pletely independent measures; The GSI is a quantitative index based on linear measures, whereas the "ripe" cycle is a semi- quantitative measure obtained from microscopic observations of smear sample. Together with the GSI and the "ripe" cycle, the shell free dry weight (SFDW = condition) cycle as well as the distribution of all gametogenetic stages is presented (Fig. 5b and d and Fig. 6b and d). Here, the typical pattern of tropical species with a very long or permanent spawning season and continuous reproduction strategy is demonstrated. All four species studied have ripe ovaries through out the year with no resting or inactive gonads found. This char- acteristic pattern is in contrast to cold temperate species. As an example. Urban and Campos (1994) studied the gonad cycles of the infaunal clam Gari solida from Chile at 37°S. In this species during 9 months of the year, developing I and/or spent stages dominate (85'7f ). Only during the latter 3 months did ripe and/or developing 2 stages doininate indicating a short spawning period. Contrasting to the "ripe" cycle only for Pteiia colymhus and Donax dentifer. a high correlation between GSI and SFDW was observed. However, a high correlation between these two variables cannot be expected generally in a tropical environment with little annual variability of biotic and abiotic factors such as temperature, nutrients or phytoplankton. Thus, under rather continuous feeding conditions a condition cycle might be independent from the gonad activities, contrasting to cold temperate latitudes with typical spring/summer phytoplankton blooms. ACKNOWLEDGMENTS Data were obtained in the context of two larger projects: "Bi- valve Culture in the Colombian Caribbean. 01/94-04/99" and "Ef- fects of El Nino on Biological Resources of Latin America 1999- 2001". The first author thanks the following students for their collaboration in the field, as well as working on the samples: Angela Maria Perez, Maria Cristina Murillo, Juan Pablo Assmus and F. Marcos Ablanque. LITERATURE CITED Giese. A. C. 1967. Some methods for study of the biochemical constitution of marine invertebrates. Oceanogi: Mar. Biol. Ann. Rev. 5:159-186. Guillou, J., G. Bachelet. M. Desprez, J. P. Ducrotoy, I. Madani. H. Rybar- czyk. P. G. Sauriau. B. Sylvand, B. Eikaim. & M. Glemarec. 1990. Les modalites de la reproduction de la coque (Cerastodenna edule) ser le littoral franfais de la Manche et de I'Atlantique. Aquulic Living Re- sources 3:29—41. Moore, H. B. 1934. A comparison of the biology of Echinus esciilentiis in different habitats. Part I. / Mar Biol. Ass. U. K. 19:869-885. Pearse. J. S. 1965. Reproductive periodicities in several contrasting popu- lations of Odontasler validiis Koehler. a common Antarctic asteroid. Anwrcric. Res. Ser. 5:39-85. Urban. H.-J. 2000. Aquaculture potential of the Caribbean Pearl Oyster Pinclada imbricaki I. Gametogenic activity, growth, mortality and pro- duction of a natural population. Aquaculture 189:361-373. Urban. H.-J. & B. Campos. 1994. Population dynamics of the bivalves Gari solida. Scmele solida and Protothaca tluua from a small bay in Chile at 36'S. Mar. Ecol. Prog. Ser. 1 15:93-102. .loiimal ,'f Shellfish Research. Vol. 2 I . No. I. 255-2?8, 2002. COMPARATIVE FECUNDITY OF THREE PROCAMBARUS SPECIES ARNOLD G. EVERSOLE* AND YAVUZ MAZLUM Department i reproductive strategy is consistent with exis- tence in a nutrient-abundant, predictable environment. P. zonan- gulus also appears to be more conservative in mobilizing nutrients during starvation than is P. clarkii (Powell 2001). Other compari- sons include length-weight relationships, production characteris- tics, and population studies in aquaculture systems (Romaire & Lutz 1989; Deng et al. 1995; Huner 1994). P. clarkii was introduced into South Carolina for aquaculture in 1978 (Pomeroy & Kahl 1987). Although P. clarkii is the inainstay of the South Carolina aquaculture industry (Whetstone, pers. comm.), there are concerns about its and other crayfish introduc- tions (Taylor et al. 1996). Comparative research on P. clarkii and P. a. acutus has been limited to investigations of the two species in experimental culture ponds. Denson and Eversole (1996) re- ported a dramatic shift in species composition from P. clarkii- to P. a. rtfKrii.v-dominated culture ponds over a relatively short pe- riod. Follow-up 2 years of supplemental stockings (59-1 14 kg/ha) of P. clarkii failed to increase its long-term production in these P. a. «<:7(f((i-doniinated ponds (Eversole et al. 1999). Although re- tlooding of drained culture ponds in September increased P. clarkii percentage in the harvest in comparison to October and November reflooding dates, it did not improve production (Mazlum & Ever- sole 2000). A series of investigations have been designed to gain a better understanding of P. clarkii and P. a. acutus interactions. The focus of this study was to provide comparative reproductive data for P. a. acutus. P. zonangulus. and P. clarkii. Specific objectives were to determine the number of extruded eggs, egg size and composi- tion, egg development lime, and instar size for the three species. MATERIALS AND METHODS Experimental Animals Adult female P. a. acutus and P. clarkii were collected from harvests at two locations in South Carolina; Clemson University Aquaculture Research Facility at Clemson and a commercial farm in Fountain Inn on May 24 and June I. 1999, respectively. Female P. zimangulus were collected from Ben Hur Farm. Louisiana State University. Baton Rouge. Louisiana, air shipped, and arrived at Clemson on May 19. 1999. Individuals Ui = 76) of each species with a complete set of appendages were either placed in simulated burrows (?i = 64) or sacrificed for hepatopancreas moisture de- terminations in = 12). Hepatopancreas Moisture The total length (TL) of 12 individuals of each species was measured to the nearest millirneter. The hepatopancreas was ex- cised, weighed to the nearest 0.01 g after blotting (//„„,) and dried in a convection oven (80°C) to a constant weight (Hj^). Moisture content of the hepatopancreas (HM) was calculated using the fol- lowing formula; HM = - H„ H,, X 100 *Corresponding author. E-mail: aevrsl@clemson.cdu Technical Contribution No. 4728 of the South Carolina Agricultural Ex- periment Station, Clemson University, Clemson, South Carolina. Reproductive Success Individual crayfish {n = 64) were measured (TL) and placed in 1-L plastic containers (simulated burrows). Although these con- 255 256 EVERSOLE AND MaZLUM tainers were not intended ti) duplicate conditions in earthen bur- rows, they do represent a technique to compare fecundities across species under more uniform test conditions. The simulated bur- rows were suspended in restaurant glass racks held in four race- ways receiving dechlorinated aerated water. Each raceway con- tained a rack of 16 individuals of each species. Holes drilled in the bottom and along the side of the container allowed water exchange and maintained 5-cm water depth. Crayfish were held at ambient water temperatures and not fed over the 160-day experimental period. Crayfish were checked 5 days/wk and every fifth day the water was exchanged in the raceway. Water temperature and dis- solved oxygen were checked by meter (Model .S?. Y.Sl). Survival and the presence of extruded eggs were noted by date. A propor- tion of the ovigerous females were removed from burrows, usually within 24 h of oviposition. and the extruded eggs removed v\ ith forceps, counted, and fixed in 10% neutral formalin. Eggs on the other ovigerous females were allowed to develop and hatch. De- velopment time was recorded and the TL of third instars measured. Egg Characteristics Egg subsamples (/i = 4-5) from 12 individuals of each species dried at 80'C were weighed to the nearest 0.001 mg to estimate mean egg dry weight. Following drying, the nitrogen content of egg samples was determined by the Dumas method using a Carlo- Erba NA 1800 (Series 2) nitrogen analyzer. Nitrogen content (per- cent) in an egg sample was multiplied by 6.16 to estimate the percentage protein per egg on a dry weight basis (Noblitt & Payne 1995). Lipid content was determined on duplicate subsamples of 10 eggs each from six ovigerous females per species. Egg lipid content (percent) was determined as described by Mann and Gal- lager (1985). Egg lipid content (percent) was calculated on a dry weight basis as a mean of the duplicate subsamples. Statistical Analysis Data were analyzed with the analysis of variance (ANOVA). analysis of covariance (ANCOVA), and linear regression using Micro-SAS Statistical Software System Version 8 (SAS 1999). A split-block design with trays representing blocks was used to ana- lyze the effects of raceways on the reproductive performance of the three species. The general linear model for ANOVA was used to compare species differences in initial TL, instar TL, hepatopan- creas moisture content, time to ovipositing, hatching time, and egg size (dry weight, lipid, and protein content). The differences be- tween ovipositing and nonovipositing female crayfish within spe- cies were compared by ANOVA. Linear regression was used to determine the relationship between crayfish TL and number of extruded eggs per ovipositing female. The ANCOVA procedure was used to compare slopes and intercepts of the regressions and eggs/female. Differences in means were detected with least- significant difference (LSD) test (Ott 1993). Level of significance was set at P < 0.05. RESULTS Experimental Animals The TL of the species stocked in simulated burrows were sig- nificantly different; P. a. acinus was the longe.st, P. zoiuingiiliis the shortest, whereas P. clurkii was intermediate in TL (Table 1 ). P. zonaiifiiiliis hepatopancreas contained the least amount of moisture and was assumed to be in better conditions than the other two TABLE I. Mean (±SD) and range of tola! length ( TLl of the females used in the experiment, ovipositing and n(>n 0.05). species (Huner et al. 1985; Huner et al. 1990). Mortality in the simulated bunxiws was low; only two individuals each ot P. zi>- iHiii.tiKliis and P. clarkii died over the 16()-day experimental period. Mean (±SD) water temperature and dissolved oxygen was 24.5 ± 2.2 T'C and 4.0 ± I. .38 mg/L. respecti\ely. o\er the period. Percentage of crayfish ovipositing was similar among the four raceways. Because of the lack of difference among the raceways, blocks (raceways) were pooled in subsequent statistical analysis of reproductive performance measures among species. Reproductive Success A significantly higher proportion of P. a. acutus (953%) ovi- posited eggs than P. zoiuiiiguhis (74.2%). and the proportion of ovipositing P. clarkii (3SJ9c} was significantly less than the other two species. In contrast. P. clarkii was the first species to oviposit eggs, more than .30 days earlier than the other two species (Table I ). No crayfish were observed ovipositing eggs within the last 4 weeks of the experiment; however, glair gland development was not assessed in these crayfish. Individuals of P. a. acutus entire size range (90-127 mm TL) oviposited eggs, whereas some smaller individuals of the other two species did not oviposit (Table 1 ). Although the ovipositing indi- viduals of each species were larger than the nonovipositing indi- viduals, the onl\ significant difference in TL was observed with P. Z(>iiaui;iilus. Number of extruded eggs ranged from 189 on a 85-mm TL P. zonangulus to 764 on a 1 14-mm TL P. clarkii (Table 1 ). Extruded eggs per female varied significantly with increased TL in the three species (Fig. 1 ). These linear relationships explained 80%-90% of the variation among individuals of the species. Using TL as a covariant. ANCOVA indicated significant differences in extruded egg counts among the three species, with P. clarkii being most Comparative Procambarid Feci'ndity 257 fecund (Table 1 1. The slopes and intercepts of the regressions were also significantly different among species. Egg Charuclcristics Eggs of P. a. acutiis and P. zonangulus were similar in weight and significantly heavier than P. clarkii eggs (Table 2). The eggs of these two species also contained a significantly greater propor- tion of lipid and protein than did those of P. clarkii. The smaller eggs of P. clarkii hatched approximately 3 days earlier than those of the other two species; however, the TL of the third instars were significantly shorter than the instars of P. a. aciilus and P. zoiuiih gulus (Table 2). DISCUSSION P. a. aciilu.s and P. zoiuingnliis share many morphological, ecological, and leproductive characteristics. Both species belong to the subgenus Ortmannicus. diagnosed by the terminal element of the first pleopod (Hobbs 1972; Hobbs & Hobbs 1990). Although this characteristic clearly distinguishes P. zonangulus as a species and distinct from P. a. acutus. these two species have similarly shaped chelipeds. carapace, and overall body proportions (Hobbs 1981; Hobbs & Hobbs 1990, pers. observations). Crayfish with similar body forms frequently occur in similar habitats (Hobbs 1975; Holdich & Reeve 1988). P. a. aciilus and P. zonangulus occur in sluggish streams with aquatic vegetation and lentic habi- tats, including those with fluctuating water levels such as borrow pits, roadside ditches, sloughs, and farm ponds (Hobbs 1981; Hobbs & Hobbs 1990; Deng et al. 1993). Adults of both species retreat to burrows in drying habitats and also to oviposit eggs, which is characteristic of tertiary burrowers (Hobbs 1981). P. a. acutus and P. zonangulus shared several of this study's measured reproductive characteristics with the exception of hepatopancreas moisture content and the number oi extruded eggs per female (Table 1 ). Crayfish store large amounts of energy (lipid) in the hepatopancreas for oocyte development and survival while in the buiTow (Huner 1989). For this reason, relative mois- P. clarkii Eggs/Female = -1498.9t19,94TL r'- 0.8997 n= 12 / P. zonangulus Eggs/Female = .426.34*7-60TL r'= 0 7938 n= 24 A p. a, aculus Eggs/Female = -962.92+1 2,1 2TL r'= 0,8033 n=40 80 90 100 110 120 130 Total Length (mm) Figure 1. Relationships between total length (TL, mm) and number of extruded eggs per female for Pnicainhanis clarkii. (closed circles), P. a. acutus (triangles), and P. zonangulus (open circles). TABLE 2. Mean (-fSD) and range of egg dry weight, lipid, protein, hatching time from ovipositing dale to the appearance of instars and the total length (TL) of instars. Variable* No. P. a. aculus P. zonangulus P. clarkii Dry weight 12 (mg/egg) Egg lipid (%) 6 Egg protein O ) 12 Hatching time 9 (days) Instar TL (mm) 200 3.49 ± 0.379' (2.83-4.20) 26.0+ 1.16' (23.6-28.2) 58.9 ± 3,67-' (47.4-60. 1 ) 21.1 ±0.60-' (20-22) 9. .5 ± 0.55-' (9-11) 3.44 ± 0.443' (2.64-+.29) 26.6 ± 2.06-' (22.0-28.6) 57.6 ± 4.37" (43.4-58.8) 21.6 ±0.88" (20-23) 9.6 ± O.SS" (9-11) 1 .94 ± 0.245'" (1.46-2.27) 17.6 ± 1.75*' (19.2-20.7) 45.4 ±3.21" (40.5-50.9) 18.6 ±0.73" (18-20) 7.6 ±0.51" (6-8) * Those means within rows sharing lowercase superscripts are not signifi- cantly different (/'>0.05). ture content of the hepatopancreas has been suggested as an indi- cator of crayfish condition (Huner et al. 1985; Huner et al. 1990). It was expected, based on hepatopancreas moisture content, that P. zonangulus fecundity would be greater than a similar-sized P. a. acutus. which was the case. The "better conditioned" P. zonangu- lus oviposited more lipid as eggs (349.9 mg on a dry weight basis) than a comparable-sized P. a. acutus (271.6 mg lipid). The reproductive characteristics of P. a. aculus and P. zonan- gulus contrasted that of P. clarkii (Tables 1 and 2). For example, P. clarkii fecundity was higher and the extruded egg size was smaller than that observed for P. a. acutus and P. zonangulus. Also, the hatching time and TL of hatchlings (instars) was shorter for P. clarkii than for the other two species. Our observations are consistent with the hypotheses of Noblitt et al. (1995) in that the reproductive characteristics of P. zonangulus and P. a. acutus follow a prudent strategy where more resources (energy) are in- vested in each egg, improving the survival or fitness of offspring in less predictable environments. Instar fitness is more dependent on size in this reproductive strategy scenario, and those species with bigger eggs yield larger instars. The larger instar P. a. acutus (10 mm TL) in allopatric and sympatric competition experiments survived at a higher rate than the smaller P. clarkii instars (8 mm TL) (Mazlum & Eversole. unpubl.). The fraction of dry weight, protein, and lipid extruded in eggs were computed from linear regressions for whole-body female P. a. acutus dry weight, protein and lipid levels (calculated from data of Turker 1997) and for our fecundity estimates in terms of each of these parameters. A standard-sized P. a. acutus (104.6 mm TL) with an average fecundity of 249.4 eggs oviposited 1,065 mg dry weight, 573.7 mg protein, and 271.6 mg lipid. The fraction of the whole-body dry weight, protein, and lipid used for extruded eggs was 10.2%. 13.6'7r. and 22.2%. respectively. The average repro- ductive lipid output (extruded eggs) of 22.3% total-body lipid computed for 3 1 ovipositing P. a. aculus (Eversole et al. 2000) was almost identical to the estimate derived in this study. Similarities in the proportion of lipid devoted to reproduction are explicable in terms of fecundity, egg size, and the total-body content. Because of the differences in systematics and reproductive strategies among the species, it might be expected that the allocation of energy (lipid) to reproduction differs as well. Unfortunately, this compari- son will have to wait until compatible data are available for tho species or reproductive strategies. 258 EVERSOLE AND MAZLUM ACKNOWLEDGMENTS Funding was provided by the S.C. AqLuiciilliire Reseaixh Ini- tiative. S.C. Agricultural Experiment Station, and the Turkish Min- istry of Education. Dr. L. W. Grimes provided statistical advice. Dr. Robert Romaire provided the P. zoiiaiifiulKS. and Ms. J. Richard- son suffered through multiple drafts of this manuscript. We also thank .Shane M. Welch and Danny R. Jones for reviewing the manuscript. LITERATURE CITED Deng, X., D. L. Beehler & K. R. Lee. 1993. Comparative life history studies of two sympatric Prncamharu.s crawfishes. ./. Shellfish Res. 2:343-350. Denson. M. J. & A. G. Eversole. 1996. Effects of tornuilated feed on crayfish production in South Carolina. Freshwawr Cniyfish 11:550- 565. Eversole, A. G. & W. R. McClain. 2000. Crawfish culture. In: R. Stickney, editor. Encyclopedia of aquaculture. New York: John Wiley, pp. 188- Eversole, A. G., Y. Mazluni & C. Erkebay. 1999. Effects of stocking Procambants clarkii (Girard) in culture ponds dominated by Procam- barus acutus acunis (Girard). Freshwater Crayfish 12:584-592. Eversole, A. G., Y. Mazlum. Q. C. Fontenot & H. Turker. 2000. Evaluation of a non-invasive technique for predicting reproductive success in white river crayfish. Int. Assoc. Astacology Syinp. 13:(abstract). Hobbs. H. H. Jr. 1972. Crayfishes (Astacidae) of North and Middle America. Biota of freshwater ecosystems. Identification Manual No. 6. Cincinnati, Ohio: Environmental Protection Agency. 173 pp. Hobbs, H. H. Jr. 1975. Adaptations and convergence in North American crayfishes. Freshwater Crayfish 2:451-551. Hobbs, H. H. Jr. 1981. The crayfishes of Georgia. SmithsniL Coiiirib. Znnl. 318:1-549. Hobbs. H. H. Jr. & H. H. Hobbs III. 1990. A new crayfish (Decapoda: Cambaridae) from southwestern Texas. Proceed. Biol. Soc. Wash. 103: 608-613. Holdich, D. M. & I. D. Reeve. 1988. Functional morphology and anatomy. In: D. M. Holdich & R. S. Lowery. editors. Freshwater crayfish: biol- ogy, management and exploitation. Portland. Oregon: Timber P^e^s. pp. 11-51. Huner. J. V. 1989. Survival of red swamp and white n\er crawfishes under simulated burrow conditions. Crawfish Tales 8:29. Huner. J. V. 1994. Ecological observations of red swamp crayfish. Pro- cainbarus clarkii (Girard, 1 852) and white river crayfish. Procaiiibariis zonangulus Hobbs & Hobbs 1990, as regards their cultnation in earthen ponds. Freshwater Crayfish 10:456-468. Huner, J. V.. H. Kononen & O. V. Lindqvist. 1990. Variation in body composition and exoskeleton mineralization as functions of the molt and reproductive cycles of the noble crayfish. .Astaciis iist{ieiis L. (De- capoda, Astacidae) from a pond in Central Finland. Comp. Biochem. Physiol. 96A:235-240. Huner. J. V., O. V. Lindqvist & H. Kononen. 1985. Responses of intermolt noble crayfish, Astuciis astaciis (Decapoda. Astacidae), to short-term and long-term holding conditions at low lemperatures. Aciiiaciillure 47:213-221. Mann. R. & S. M. Gallager. 1985. Physiological and biochemical energet- ics of larvae of Teredo navolis L. and Baiikia goiddi (Bartsch) (Bi- valvia: Teredinidae). J. E.xp. Mar. Biol. Eeol. 85:211-228. Mazlum. Y. & A. G. Eversole. 2000. Effects of Hooding dates on popu- lation dynamics in South Carolina ponds. J. Appl. .Ai/iiueiiltiire 10:17- 28. Noblitt. S. B. & J. F. Payne. 1995. A comparative study of selected chemi- cal aspects of the eggs of the crayfish Procambanis clarkii (Girard, 1852) and P. zonangulus Hobbs & Hobbs. 1990 (Decapoda: Cam- baridae). Crustaceana 68:695-704. Noblitt. S. B.. J. F. Payne & M. Delong. 1995. A comparative study of selected physical aspects of the eggs of the crayfish Procambarus clarkii (Girard. 1852) and P. zonangulus Hobbs & Hobbs. 1990 (De- capoda, Cambaridae). Crustaceana 68:575-582. Ott. R. L. 1993. An introduction to stadstical methods and data analysis. Belmont. California: Duxbury Press. 1051 pp. Pomeroy. R. S. & K. H. Kahl. 1987. Crayfish in South Carolina: devel- opment and current status of the industry. Clemson University. Clem- son, South Carolina: South Carolina Agricultural Experiment Station Bullclm 661. Powell. M. L. 2001. The effects of temperature and nutrient level on energy metabolism in two sympatric crayfish species. Ph.D. Dissertation. Bir- mingham, Alabama: University of Alabama at Birmingham. 153 pp. Romaire. R. P. & C. G. Lutz. 1989. Population dynamics of Procambanis clarkii (Girard) and Procambarus acutus acutus (Girard) (Decapoda: Cambaridae) in commercial ponds. Aquaculture 81:253-274. SAS Institute. Inc. 1999. SAS guide for personal computers. Version 8.0 edition. Cary. North Carolina: SAS Institute, Inc. 452 pp. Taylor. C. A.. M. L. Warren Jr.. J. F. Fitzpatrick Jr.. H. Hobbs III. R. F. Jezerinac, W. F. Pfleiger & H. W. Robison. 1996. Conservation status of crayfishes of the United States and Canada. Fisheries 21:25-38. Turker. H. 1997. Evaluation of a nondestructive method for determining body composition of white river crayfish. M.S. Thesis. Clemson. South Carolina: Clemson University. 24 pp. Journal of Shellt'ish Reseunh. Vol. 21. No. 1, 259-265. 2002. CONFIRMATION OF TWO COMMON MUD CRAB SPECIES (GENUS SCYLLA) IN THE MANGROVE ECOSYSTEM OF THE MEKONG DELTA. VIETNAM D. J. MACINTOSH/* J. L. OVERTON,' AND H. V. T. THU" ^ Centre for Tropical Ecosystems Research. Department of Ecology and Genetics. Universir\- of Aarhiis. Aarhits. Denmark 'Department of Biotechnology and Aquacultitre. Institute of Oceanography. Nhatrcmg. Vietnam ABSTRACT Adult and iiivenile mud crahs (genus Scxlla) were collected from two sites in the Mekong Delta. Vietnam. The specimens were examined morphologically and analyzed by starch gel electrophoresis to confirm the species present based on a recently published revision of the genus Scylla that recognizes four species from adult morphological characters and electrophoresis. The Mekong Delta samples contained two species. S. olivucea ("red crab") and S. paianuunosiiin ("green crab"). Ratios derived from measurements of the carapace width and the frontal spines, which are species diagnostic in adult mud crabs, did not separate juvenile S. panwmmosain and S. olivacea reliably. However, in combination, differences in the shape of the frontal spines, the spination on the cheliped. and cheliped shape and color, make it possible to distinguish juveniles of these two species down to a size of about 1 .5 mm carapace width. This is the modal body size of new recruits entering the mangroves of the Lower Mekong Delta. The diagnostic morphological features of adult and juvenile S. olivacea and S. paramamosain from the Mekong Delta are based on the architecture of the frontal lobe spines and the number of dorso anterior propodal spines and ventral carpal spines. These are described, together with features relating to their coloration and habitat preferences. The recognition of two named species of Scylla within the mangrove populations of mud crab in the delta is important to fisheries management and aquacullure development, as both adult and juvenile mud crabs are fished heavily for marketing and for stocking in aquaculture systems, respectively. KEY WORDS: Scylla. mud crab, species identification, mangrove. Mekong Delta INTRODUCTION Mud crabs of the genus Scrlla are the only swimming crabs (Family Portuiudae) that are found habitually in tropical and sub- tropical mangrove forest ecosystems; their habitat extends to man- grove estuaries, embayments and surrounding coastal waters (Macnae 1968). As well as providing shelter from predation and desiccation, especially during moulting, mangroves also provide mud crabs with a plentiful supply of food in the form of other crustaceans and molluscs (Arriola 1940; Hill 1979). Mud crabs have significant economic importance to artisanal fisheries throughout the Indo West Pacific region. They are also contributing increasingly to aquaculture production in several countries, most notably Vietnam and the Philippines (Johnston & Keenan 1999; Fortes 1999). Their rapid growth and high market value, coupled with easy post-harvest handling, make them an attractive alternative to farming shrimp within coastal areas (Over- ton & Macintosh 1997). The growing importance of mud crab fanning is also supported by a rapidly expanding demand for crab products, both regionally and internationally. The mangrove forests of Vietnam originally covered an area of up to 400.000 ha (Maraund 1943), of which about 250.000 ha flourished in the Mekong Delta. The greatest concentration of mangrove ( 150.000 ha) was in the Minh Hai Peninsula, which is now divided into Ca Mau and Bac Lieu provinces (Fig. 1 ). Despite heavy exploitation in recent decades, mangroves are still the dom- inant habitat in the extensive saltwater-influenced regions of the delta. Within the coastal provinces of the Lower Mekong Delta (LMD), rural communities are heavily dependent on mangrove- based fisheries and aquaculture to support their livelihoods. These include crab fishing and crab culture as a secondary activity to ^Corresponding author. D. J. Macintosh. Institute of Aquaculture. Univer- sity of Sliding. Stirling FKI4 7EB. Scotland. United Kingdom. Phone: +44-1786-467785; Fax: +44-1786-472133: E-mail: d.j.macintoshta stir.ac.uk shrimp farming within the mangrove forest ecosystem. Average mud crab production in the LMD ranges from 504 to 839 kg/ha/y (World Bank/Danida 2000). Nearly all the available brackishwater areas of the delta have been converted to extensive aquaculture ponds for shrimp and/or crab production integrated to varying degrees with the mangrove forest (these fanning systems are de- scribed by Binh et al. 1997; and Johnson et al. 1999). To date, there are no commercial hatcheries in Vietnam to support mud crab culture and, consequently, the natural recruitment of juvenile mud crabs into the mangrove ecosystem provides the only source of seed available to crab farmers. A previous study using morphonietric analysis confirmed the existence of two common moiphs of Scylla within the South China Sea region (Overton et al. 1997; Overton 2000), while Keenan et al. ( 1998) have proposed a total of four species of mud crab based on genetic data from a wider geographical range. They also de- scribe morphological characters to distinguish the four species. Although Keenan et al. (1998) attribute tv\'o species to Vietnam, only one species is mentioned from the Mekong Delta, called Scylla paramamosain Estampador. Moreover, the study is based entirely on large adult specimens (carapace width greater than 95 mm), because the distinguishing characteristics of each species are more clearly defined in the adult stages. Until now, no species identifications have been reported for the juvenile stages. The people who catch or rear juvenile mud crabs in the Mekong Delta recognize two morphs of Scylla on the basis of color, using the local names "cua xanh" (green crab, due to its greenish carapace) and "cua lua" (red crab, due to dark red coloration on the lower chelae). These color differences are not distinct in the smaller juveniles, however, especially after capture. In the present study, adult and juvenile mud crabs were col- lected from the Lower Mekong Delta. They were then examined morphologically and analyzed using electrophoresis to determine which species are recruiting into the mangrove crab fishery. The main objective was to produce scientific descriptions of both ju- veniles and adults of the species identified, which confirm Iheir 259 260 Macintosh et al. 10°N g^N 60 Km 105 °E 106 °E 107 "E Figure 1. The Mekong Delta, Vietnam sliovving the location of the sampHng sites in Bac Lieu and Kien Giang provinces. local identities based on color and behavior. This knowledge is vital for mud-crab stock assessment and for the future develop- ment of mud crab culture using selected, known species of Scylla. Recruitment and growth studies on juvenile mud crabs are par- ticularly needed in this regard. MATERIALS AND METHODS Crab Collection Study sites were chosen within two of the most southerly coastal provinces of the Mekong Delta; Bac Lieu (9°00'N: 105°14'E) and Kien Giang (9°22'N; 104°26'E) (see Fig. 1). The crabs were collected from mud crab fishers or traders. They were interviewed to confirm that the crabs obtained were all fished from the selected locality. In the fiist investigation. 2.^ adult crabs. 84 to 125 mm carapace width (CW), including both common morphs of Scylla. were sampled from the commercial mud crab fishery in Bac Lieu Prov- ince. Their species identities were confirmed by comparing their morphology and allozyme mobility with those published by Keenan et al. (1998). In the second investigation, juvenile mud crabs (CW 45-62 mm) from Bac Lieu and Kien Giang provinces (62 and 58 individuals, respectively) were studied. The majority of specimens from Kien Giang were the "red" morph, whereas all those collected from Bac Lieu in the second sample were typical of the "green" morph. In addition, a larger number of smaller juve- niles (CW 10—4^5 mm) were available for examination from the commercial crab seed fishery in Ca Mau. Crab Measurement Three physical measurements were made on each sampled mud crab using digital calipers, namely Frontal Lobe Width (FLW). Internal Carapace Width (ICW) and Frontal Median Spine Height (FMSH) (Fig. 2). Measurements were recorded to the nearest 0.1 mm. From these measurements, two ratios described by Keenan et ICW Figure 2. Illustration of the three measurements forming the ratios used to discriminate hetween the two Scylla species. FL\\ : Frontal l.ohe Width: FMSH: Frontal Median Spine Height; ICW: Internal Carapace Width. al. (1998) as discriminating between the four known species of Scxila were used to compare the red and green morphs of Scylla from the LMD. These ratios were FLW/ICW and FMSH/FLW. Allozyme Electrophoresis Use of genetic markers, including allozyme electrophoresis, are the most reliable in identifying Scylla species (Keenan et al. 1998; Overton 2000). Other mud crab specimens representing the four Scylla species proposed by Keenan et al. (1998). which had been identified earlier (Overton 2000). were used as reference material to help interpret the results from electrophoresis using the mud crabs obtained from the Mekong Delta. Muscle tissue was extracted from each crab specimen and stored at -80°C. The allozymes extracted from the muscle tissue were separated using starch gel electrophoresis. The electropho- retic techniques used followed those described by Hanis and Hop- kinson (1976). Shaklee and Keenan (1986) and Pasteur et al. ( 1988). Four allozymes. using two buffer systems that distinguish the four species of Scylla identified by Keenan et al. ( 1998). were used to identify the two common species collected from the LMD. These enzymes were alanine aminotransferase (ALAT), argenine kinase (ARGK). mannose phosphate isomerase (MPI) and phos- phoglucomutase (PGM). They were visualized using standard staining procedures (Shaw & Prasad 1970; Harris & Hopkinson 1976; Shaklee & Keenan 1986; Pasteur et al. 1988). RESULTS The results of the allozyme electrophoresis confirmed that the two coinmon species o( Scylla in the Mekong Delta are S. olivacea Herbst (red morph) and S. panuiumiosain Estampador (green Mud Crab Species in the Mekong Delta 261 morph). as described by Keenan et al. (1998). Figure 3 and Ta- ble 1 illustrates the fixed mobility observed between the LMD specimens and the typed samples of the four species (also based on Keenan et al.). The frontal spine morphology represented by the ratios FLW/ ICW and FMSH/FLW did not prove to be conclusive in distin- guishing the two species identified from the allozyme data. Both adults and juveniles of the green morph iS. puramamosain) have narrower frontal lobe widths (compared to their internal carapace width) and taller frontal lobe spines, than crabs of the red morph (5. olivacea). This conforms with the spinal architecture of sharp frontal spines on the green morph and smooth, obtuse frontal lobe spines on the red morph (Fig. 4). However, the ranges obtained for each of the ratios show that there is a large overlap between the two species and that it is not possible to identify an individual crab solely from either of these ratios. Although both juveniles and adults of the two mud crab species from the Mekong Delta could be distinguished from each other using the above ratios, it is less clear which species they represent when they are compared to the equivalent ratios for the four ScylUi species proposed by Keenan et al. ( 1998) (Table 2). Mean values of FLW/ICW for the juvenile red and green morphs of Scylla lie out with the ratios reported by these authors. The mean values for the adult specimens from Bac Lieu are within the published ranges for the four Scylla species recognized by Keenan et al. 1998. but they lie between the values of 0.037/0.038 for S. serratalS. para- mamosain and 0.412/0.415 for 5. tntnquebarica IS. olivacea (Table 2). Mean values for the ratio FMSH/FLW for adults and juveniles of both Vietnamese morphs also fall within the ranges set by Keenan et al. 1998 for the four Scylla species they recognize (Table 2). FMSH/FLW ratios positively identified the adult green morph from Bac Lieu as S. paramamosain and the juvenile red morph as S. olivacea. but the other two sample groups cannot be classified reliably on the basis of this ratio. This is not surprising as there is also considerable overlap in the ranges of the FMSH/ FLW values for each species as reported by Keenan et al. 1 998. DISCUSSION The results from allozyme electrophoresis were conclusive in identifying the two common species of Scylla in the coastal man- grove ecosystem of the Mekong Delta. Based on the recent review of the genus Scylla by Keenan et al. (1998). the morphs recognized by fishers and crab farmers as "red crab" and "green crab" are S. olivacea and 5. paramamosain. respectively. These confirmed identifications help to overcome the long-standing confusion about the identity of mud crab species when their fisheries biology, or use in aquaculture is described. As noted by Fortes (1999). several authors have assumed a single species. Scylla serrata. in aquacul- ture. despite evidence that this mud crab is actually rare in the Southeast Asian region compared to the other three species of Scylla now recognized (Keenan et al. 1998; Overton 2000). It is not surprising that the morphometric ratios based on cara- pace measurements (FLW/ICW and FMSH/FLW) were inconclu- sive for the identification of juvenile mud crabs, or that the values obtained do not lie within the equivalent ranges for adult speci- mens. Developmental changes in exoskeletal dimensions between juvenile, sub-adult and adult crabs are well documented (Hartnoll 1982). Indeed the relative allometic changes in the dimensions of the body are often used to define the molt of maturity in crustacean species (e.g.. Somerton 1980; Paul & Paul 1995). However, the same ratios obtained from the adult mud crabs sampled also did not conform to the equivalent values reported by Keenan et al. ( 1998). This apparent discrepancy may be explained by (a) opera- tor differences when taking carapace measurements with calipers; or (b) natural variation in the morphology of these closely related species. It is notable that there is a high degree of overlap between the ratios recorded by Keenan et al. (1998). and clearly morpho- metries alone cannot be used to identify individual mud crabs reliably. The morphological characters used to identify the species in adult samples can also be extended to juvenile crabs down to a size of 1 .5 cm carapace w idth. the modal size of new recruits into the Mekong Delta. In particular, the architecture of the frontal lobe spines and the number of spines on the carpus are reliable char- acters which can be used to distinguish juveniles of S. olivacea and S. paramamosain, even in the field. The presence of two species in the Mekong Delta mud crab fisheries is important from a fisheries management and aquacul- ture development perspective, as natural recruitment of juvenile mud crabs into the mangrove ecosystem is the only source of seed available to crab farmers in the delta. It will now be possible to investigate and compare ecological differences between these two species, especially their potential in aquaculture. The yield of mud crab in Ca Mau Province was 5000 tonnes in 1999, of which 1000 tonnes came from aquaculture production (Xuan 2001). In the ten-year fisheries development plan for Ca Mau Province (2000 to 2010), the production of cultured mud crab is expected to increase to 3,500 tonnes per annum. This increased emphasis on aquaculture has important implications regarding the sustainable level of exploitation of the mud crab seed recruiting into the coastal mangroves. From observations of the crab seed fishery in Ca Mau, it is clear that there are significant seasonal fluctuations in the supply and species composition of juvenile mud crabs caught by local fishers. It also appears that the recruitment dynamics of S. paramamosain and S. olivacea vary from province to province in the delta (Macintosh: personal observation). By being able to identify these two common species of Scylla down to the early juvenile stages entering the mangroves, a more accurate assessment of this important fisheries resource can now be at- tempted. Scylla paramamosain and S. olivacea are farmed throughout the brackish water regions of the Mekong Delta, usually in earthen ponds constructed within the mangrove forests. The farmers gen- erally stock juveniles of both species purchased from fishermen or crab dealers. Their general opinion is that S. olivacea is the more aggressive species. It also likes to burrow, which weakens the pond structure and making harvesting very difficult. In contrast, Scxlla paramamosain is regarded as more suited to pond culture as it seldom burrows. However, female S. olivacea are very popular as "egg crab" (crabs with mature ovaries) because they develop large ovaries at a comparatively small body size. Differences in size at maturity and gonosomatic index have also been recorded in sympatric populations of S. olivacea and S. paramamosain in tlie Gulf of Thailand (Overton 2000). By confirming their species identity at the juvenile stage, it will now be possible to research the 262 Macintosh et al. 107 100 Origin Genotype 100 95 Ongin 100/100 SP./S-S./S.T. Genotype Genotype Figure 3. Illustration of allozyme mobilites for Scylla species using a) mannose-6-phospiiate isomerast (MPI): h) pliosplioglucomutase (PGM); c) argenine kinase (ARGK); and d) alanine aminotransferase (ALAT, using ultra violet stain); from Overton tunpublished). Note: S.O. = Scylla olivacea; S.S = S. serrata; S.T. = S. Iranquebarica and S.P. = S. paramamosain. Mud Crab Species in the Mekong Delta 263 TABLE 1. Results of mobility for four allozymes used to compare two Scylla morplis from the Lower Mekong Delta with typed specimens of the four species of Scylla recognized by Keenan et al. ( 19981 used as markers (from Overton, 20(10). Adults Juveniles Specimens from Overton (2000) Bac Lieu Bac Lieu Bac Lieu Kien Giang Scvlla Scylla Sc\lla ,Sfv//fl Locus Green Red Green Red serrala traiiquebahca paramamosain olivacea Numbers of indi\iduals 11 i: s S 6 5 5 b ALAT 100 95 nd nd 100 100 100 95 ARGK 75 75 nd nd 100 75 75 75 MPI 100 95 100 95 100 100 KJO 95 PGM 100 85 100 85 100 107 100 85 biological basis for the farmers' observations about red crab (5. olivacea) and green crab (5. paramamosain) in aquaculture. Stud- ies of their comparative tolerance to different combinations of salinity and temperature, their comparative growth rates and matu- ration under different pond conditions, and the possible affect of interspecific interaction between S. olivacea and S. paramamosain in mixed culture, could yield valuable information to improve mud crab farming in the Mekong Delta. Diagnoses of the Two Common Species of Scylla in the Mekong Delta Scylla olivacea Herbst Dorsal carapace brown to dark green. Dorsal chelae and pereio- pods range from dark brown to green, no reticulation present. Ventral propodus orange/red. Carapace rounded with blunt, obtuse frontal lobe teeth all equal in size, with shallow interspaces. Short, Ictn 1cm Figure 4. Drawings of the frontal carapace spination and left cheliped of adult Scylla paramamosain (a and b) and Scylla olivacea (c and d) based on specimens collected in the Mekong Delta, Vietnam. 264 Macintosh et al. TABLE 2. Means, standard deviations (S.D.) and ranges for three morphonu-tric characters (measured in mm I and two rati.ms used to discriminate between four species of mud crab, Scjila described by keenan et al. ( 1998). Adults Juveniles Keenan et al. (1998) Bac Lieu Bac Lieu Bac Lieu Kien Giang Scylla Scylla Scylla Scylla Character Green Red (Jreen Red serrata puramamosmii trunquehurica olivacea Numbers of individuals II 12 62 58 68 9 25 66 ICW (mm) Mean S.D. Rantie 104.84 10.24 84.39-34.24 107.64 13.67 85.51-1333.54 51.07 3.81 45.00-61.30 55.71 3.87 47.11-61.65 138.40 23.20 95.50-191,70 114.70 9.00 104.80-134.10 113.70 11.40 97.10-137.80 107.50 10.10 95.00-133.90 FLW (mm) Mean 4(1.S8 42.82 22.62 25.30 nd nd nd nd S.D. 3.57 3.99 1.94 1 .65 Range 34.24-15.68 38.84-48.41 I9.8(J-3I.70 21,70-27.85 FMSH (mm) Mean 2.37 1.75 0.90 0.58 nd nd nd nd S.D. 0.44 0.2S 0.24 0,17 Range 1.72-2.92 1.16-2.05 0.40-1.40 0.40-1.20 FLW/ICW Mean S.D. Range 0,39 0.01 0.38-0.41 0.40 0.02 0.36-0.42 0.44 0.03 0,41-0.63 0.45 0.01 0.42-0.48 0,37 0.02 0.34-0.41 0.38 0.01 0.36-0.39 0.41 0.02 0.38-0.44 0.42 0.02 0.37-0.45 FMSH/FLW Mean S.D. Range 0.06 0.01 ().()4-0.()7 0.04 0.01 0.03-0.05 0.04 0.01 0.(11-0.05 0.02 0.01 0,02-0,05 0.06 0.01 0.04-0.10 0.06 0.01 0,04-0,08 0.04 0.01 0.03-0.05 0.03 0.01 0.02-0.04 obtuse anterolateral spines uniform in size and shape. None to two vestigial spines present on dorsal propodus of the chelae. Pro- nounced nodule present on inner lateral propodus. Spination on ventral carpus of chelae ranges from two vestigial, nodule-like spines to a total absence of spines. Abdominal flap of mature females dark brown to purple with a pale lateral stripe on each abdominal segment. Coloration of juveniles similar to that of the adults. Chelae are elongate, with a distinctive dark red flash on the outer propodus and fixed dactyl. Chelae bear blunt, obtuse frontal lobe teeth that are equal in size, with shallow interspaces. Two sharp spines on dorsal propodus. and one pronounced nodule on inner lateral pro- podus. Single spine present on ventral carpus. Scylla paramamosain Estampador Adult carapace pale olive green. Dorsal chelae and pereiopods pale green with prominent dark green/brown reticulation. Reticu- lation breaks up and forms brown dots on outer upper half of propodus. Lower half of outer lateral propodus pale yellow to cream with orange to red flashes on tips of propodus and dactylus. A black patch is present on ventral surface of caipus. Carapace flattened dorso-ventrally, with short, triangular fron- tal lobal teeth with angular interspaces. Central pair of frontal lobe teeth slightly protruded in some individuals. All anterolateral spines uniform in morphology, and compressed with small spaces between spines. Two sharp spines on dorsal propodus with ridges following posteriorly behind spines. One small spine present on inner lateral propodus. Two sharp spines on ventral carpus, on some individuals the anterior spine is vestigial, or missing. Juveniles have similar body coloration to adult form. Carapace bears short frontal lobal teeth, sharp and triangular with angular interspaces. Two pronounced spines on the dorsal propodus. No nodule on inner lateral propodus. Ventral carpus usually bears two spines, but 6% of crabs examined have only one spine. Hahilat Scvlla olivacea is associated with mangrove forests, in particu- lar estuarine and lower salinity areas associated with the river systems well within the mangrove zone or deltas where there is a high amount of freshwater runoff. S. olivacea prefers to burrow in the soft embankments during low tide. S. olivacea has an extensive distribution, populations of this species having been identified from the coastlines fringing the western Indian Ocean to northern and western Australia and islands of the Pacific Ocean (Keenan et al. 1998). Another species. Scylla inmqnelnirica. is often associ- ated with S. olivacea. These two mud crabs occupy similar niches in the mangroves of East Malaysia and the Philippines (Macintosh, personal observation). Scvlla paramamosain inhabits a more subtidal environment than Scxila olivacea. This species is found in the mouth of estua- rine areas and shallow subtidal mudflats and along the coastal fringes of mangrove areas. Keenan et al. 1998 also remarks that the habitat of this species extends to coral reefs areas, as in Singapore. Scvlla paramamosain is widely distributed along the coastlines of the South China Sea particularly along the coast of Vietnam and the eastern seaboard of the Thai-Malay peninsula (Overton 2000). ACKNOWLEDGMENTS The authors thank the staff of the Aquaculture and Fisheries Sciences Institute of Can Tho University. Vietnam for providing facilities and manpower support for this study. Financial assistance Mud Crab Species in the Mekong Delta 265 was provided by Daiiida through the Can Tho University - Aarhus University Linl< in Environmental Sciences (CAULES) project. and ill the form of a Danida scholarship awarded to Mr H.V.T. Thu. LITERATURE CITED Arriola, F. J. 1940. A preliminary study of the life history of Scyllii sciratu (Forskal). Philippine J. Science 73:437-246. Binh. C. T., M. J. Phillips & H. Demaine. 1997. Integrated shrimp- mangrove farming systems in the Mekong Delta of Vietnam. Aquae. Res. 28(8):599-610. Fortes. R. D. 1999. Mud crab research and development in the Philippines: an overview, pp. 27-32. In: C. P. Keenan & A. Blackshaw, editors. Mud Crab Aquaculture and Biology. Proceedings of an International Scientific Forum held in Darwin. Australia, April 21 to 24. 1997. ACIARproc. no. 78. 216 pp. Harris. H. & D. A. Hopkinson. 1976. Handbook of En/yme Electropho- resis in Human Genetics. North Holland Publishing Company. Amster- dam. The Netherlands. Hartnoll. R. 1982. Growth. In: D. E. Bliss & L. E. Abele. editors The Biology of Crustacea: Embryology, Morphology and Genetics (volume 2). London: Academic press, pp. 1 1 1-I85. Hill. B. J. 1979. Aspects of the feeding strategy of the predatory crab,Scv//(( sen-am. Mar. Bint. 53:209-214. Johnston, D. & C. P. Keenan. 1999. Mud crab Culture in Minh Hai Prov- ince. South Vietnam. In: C. P. Keenan & A. Blackshaw. editors. Mud Crab Aquaculture and Biology. Proceedings of an International Scien- tific Forum held in Darwin. Australia. April 21 to 24. 1997. ACIAR proc. no. 78. 216 pp. Johnston. D.. B. Clough. T. X. Tran, & M. Phillips. 1999. Mixed shrimp- mangrove forestry farming systems in Ca Mau Province. Vietnam. Aquae. Asia 4:6-12. Keenan, C. P., P. J. F. Davie & D. L. Mann. 1998. A revision of the genus Seylhi de Haan, 1833 (Crustacea: Decapoda: Brachyura: Pornmidae). Raffles Bull. Zool. 46:217-245. Maraund, P. 1943. L'Indochine forestiere. Insl. Reck. .\gro. Indochine: 185-194. Overton. J. L. 2000, Morphonietric. genetic and reproductive characteris- tics of mud crabs (genus Scylla de Haan 1833) from Southeast Asia. PhD Thesis. Stirling. Scotland: University of Stirling. 406 pp. Overton. J. L. & D. J. Macintosh. 1997. Mud crab culture: prospects for the small-scale farmer. INFOFISH International 5:26-32. Overton. J. L.. D. J. Macintosh & R. S. Thorpe. 1997. Multivariate analysis of the mud crab Scylla serrata {Brachyura: Portunidae) from hnir locations in Southeast Asia. Mar. Biol. 128:55-62. Pasteur. N.. G. Pasteur. F. Bonhomme, J. Catalan & J. Britton-Davidian. 1988. Practical Isozyme Genetics. Chichester. UK: Ellis Horwood Ltd. 215 pp. Paul. A. J. & J. M. Paul. 1995. Changes in chela heights and carapace lengths in mature male red king crabs Paraliihoides camthschaiicus after moulting in the laboratory. Alask. Fish. Res. Bull. 2:164-167. Shaklee. J. B. & C. P. Keenan. 1986. A practical laboratory guide to the techniques and methodology of electrophoresis and its application to fish fillet identification. Melbourne. Australia: CSIRO Marine Labo- ratories Publication. 177 pp. Shaw. C. R. & R. Prasad. 1970. Starch gel electrophoresis of enzymes — a compilation of recipes. Biochem. Gen. 4:297-330. Somerton. D. A. 1980. A computer technique for estimating the size of sexual maturity in crabs. Can. J. Fish. Aquat. Sci. 37:1488-1494. World Bank/Danida. 2000. Environmental indicators pilot study: technical appendix. Prepared by Aarhus University, Denmark/Can Tho Llniver- sity, Vietnam. 88 pp. Xuan, T. T. 2001. The status and the prospect of mud crab culture in Ca Mau Province, Vietnam. In: 2001 Workshop on Mud Crab Rearing. Ecology and Fisheries January 8 to 10. 2001. Vietnam: Can Tho Uni- versity. 28 pp. Joiinuil ,if Shellfish Research, Vol. 21. No. 1. 267-272. 2002. PHYSICO-CHEMICAL CHANGES IN ACID SULFATE SOIL DURING SEMI INTENSIVE CULTURE OF PENAEUS MONODON FABRICIUS, IN CLEARED MANGROVE AREAS OF THE CHAKARIA SUNDARBANS, BANGLADESH BIPLOB DAS,' YUSUF SHARIF AHMED KHAN,' KHAN TOWHID OSMAN," PRANAB DAS,' AND MD. NURUL AMIN' Institute of Marine Sciences, University of Cbittagong, Chittagong-4331 . Bangladesh: 'Department of Botany, University of Chittagong, Chittagong-4331, Bangladesli ABSTRACT The present study analyzed physico-chemical properties of pond soil during Penaeus monodon culture in a semi- intensive shrimp farm in Cox's Bazar district. Bangladesh during winter 1996 to 1997. Soils were acidic with high concentrations of SOj-S. extractahle Fe and AI. Soluble salt concentration were in order Na>Mg>Ca>K. The physico-chemical properties changed with flooding both under simple inundation and inundation with shrimp culture. The most noticeable change was soil P". SOj-S and extractable Al. Extractable A! decreased a minimum value at 145 days after preparation of the culture ponds. Extractable Na and K were increased gradually while, Ca and Mg remained fairly constant throughout the entire period. The extent of change was different for the culture and the inundated ponds. Results of the present study indicated a positive effect on the improvement of acid sulphate soil in the cleared mangrove forest area because of shrimp culture. KEY WORDS: Peiuieti.s moiuiJon. shrimp pond, culture INTRODUCTION Pond soil plays an important role in the balance of an aquacul- tural system and consequently on the growth and survival of aquatic organisms. Sediment/soil can function as a buffer. It pro- vides water nutrients and serves as a biological filter through the adsoiption of organic residues (Ray & Chien 1992). Soil thus playing a vital role in aquaculture practices is considered the "Chemical Laboratory'" of the pond (Felix 1988). In ponds there is an intense interchange of organic and mineral compounds between the soil and the water (Wrobel 1983). Accumulation and decom- position of organic matter take place on the pond floor. As a result macro elements are eliminated from tlie pond water while the decomposition of organic matter releases inorganic substances and acids, and dissolutes minerals. These go into solution with water and influence aquatic biota. A number of conditions are conducive to the formation of acid sulfate soils (Pons & Van Breemen 1982). Vast tracts of acid sulfate soils abound in Asia and Africa (Singh 1980; Brinkman & Singh 1982). In Bangladesh about 0.23 M ha of land constitute acid sulfate soils (FAO 1988). However, these are considered problem soils everywhere resulting in severe limitations for agri- cultural use (Nhung & Ponnamperuma 1982; Van Breeinen & Pons 1978; Ponnamperuma & Solivas 1982; Hechanova 1983; Simpson et al. 1983). Severe acidification of pond water in acid sulfate areas has been reported by many workers (Webber & Webber 1978; Lin 1986; Boyd 1989). The low P" exerts its effects through the in- fluence on most aspects of pond water chemistry. The main prob- lems arising after construction or deeper excavation of fish ponds in acid sulfate soils comprise insufficient growth of algae, poor condition and consequent slow giowth of phosphate fertilizers (Camacho 1977; Bay Ion 1981; Brinkman & Singh 1982; Poer- nomo & Singh 1982). Low P" of the acid sulfate soils causes dainage to gill tissues of fish (Ferguson 1988) and influences the impact of toxins (Alabaster & Lloyd 1980; Colt & Armstrong 1981) and heavy metals (Boyd 1989). In acid waters crustaceans and fish may experience impaired ionic regulation. P" <4.8 is Corresponding author. Yusuf Sharif Ahmed Khan, Institute of Marine Sciences, University of Chittagong, Chittagong-4331, Bangladesh lethal for the Penaeids (Tsai 1990). Additionally, a high organic matter content in the bottoin soil depletes O, of water (Wrobel 1983). Characteristics of acid sulfate soils in some areas of Cox's Bazar have been reported by Rahman (1990), Rahinan et al. (1993), and Mahmood and Saikat (1995), But no study has .so far been done on the changes of pond bottom acid sulfate soil that take place by the amendments done for shrimp culture. Keeping these views in mind, this work was undertaken in a semi-intensive shrimp farm at Cox's Bazar. Ponds constructed on acid sulfate soils revealed physico-chemical changes in soils of shrimp {Pe- naeus monodon) culture ponds as well as inundated ponds. MATERIALS AND METHODS Location of the Farm The seini-intensive shrimp farm namely "Chakaria Chingri Khamar" is situated on the western bank of the Matamuhuri River, about 5 km upstream from the estuary and about 65 km south of the Chittagong City. Sampling P. monodon was cultured in 14 ponds during winter of 1996 to 1997. Among those, three ponds (P-1, P-2 and P-3) were selected randomly on the basis of the previous years production data and soil P" values to accommodate the maximum variability for the present study. From the rest of the fallow ponds (temied as inun- dated ponds), three others (P-4. P-5 and P-6) were selected where pond preparation was done similarly to the culture ponds but stocking was not. Soil samples from 0-15 cm and 15-30 cin depths of pond bottom were taken from three spots diagonally of each pond (cul- tured and inundated), so that one spot was in the center and two were in the corners. Samples were drawn underwater with a spe- cially designed sampler. Soil sampling was done 30 days before (DBP) and 7, 50. 95 days after completion of pond preparation (DAP). Soil samples were also taken at 145 DAP from the culture ponds. Analysis of Soil Samples Soil texture was determined according to Bouyouco's Hydrom- eter method (Piper 1950), P" from 1:2 soil water suspensions and 267 268 Das et al. TABLE 1. Textural classes of different soil samples collected from culture ponds and inundated ponds. Pond Type Pond No. Depth (cm) Sand ( f/r I Silt ( % 1 Clay ( % ) Textural Class Culture Ponds Inundated Ponds p = 1 00- 1 3 L'^-30 P = 2 00-15 15-30 P = 3 00-15 15-30 P = 4 00-15 15-30 P = 5 00-15 15-30 P = 6 00-15 15-30 65 49 67 52 48 66 70 45 65 55 69 14 13 25 12 23 24 13 14 28 13 19 12 34 Sandy Clay 22 Sandy Clay Loam 26 Clay Loam 21 Sandy Loam 25 Sandy Clay Loam 28 Clav Loam 21 Sandy Clay Loam 16 Sandy Loam 27 Clav Loam 22 Sandy Clay Loam 26 Sandv Clav Loam 19 Sandv Loam organiL- matter I'roiii loss ol ignition. Total nitrogen was deter- mined by micro-Kjeldhal digestion and distillation procedures. Available phosphorus was extracted with Bray and Kurtz No. 2 extractant (0.03 N NHjF in 0.10 N HCl) followed by Spectropho- tomelric determination according to SnCl, reduced molybdophos- phoric blue color method (Jacl\son 195SI. Extraclable Ca. Mg. Na. K and Mn were determined by 1 N NH^OAC. P" 7.0 ± 0.1 satu- ration and FE by 1 N NHjOAC. P" 4.6 saturation followed by Atomic Absorption Spectrophotometry (Allen et al. I9S6). RESULTS AND DISCUSSION Physico-chemical parameters of the culture ponds and inun- dated ponds are tabulated in Table 2 and Table 3 respectively. The textural classes of the soil samples are shown in the Table 1 . Soil samples of the present study were sandy clay to clay loam in texture. The texture has been inherited from the deposition of the sediments. Such compositions of acid sulfate soils are also met with the saline soils of Chakaria Sundarbans (Rahman et al. 1990). The textural variation could have been created by the sedimenta- tion processes during past mangrove swamp systems. The soils were found rather poor in total nitrogen in compari- son to the organic matter content resulting in high C/N ratio, that may inhibit the decomposition of organic matter. This feature shows the characteristics of peat layers. A high organic matter poor in nitrogen is also conducive to reduction processes. Similar total nitrogen values were reported earlier from other acid sulfate soils of the area in association with low organic matter (Rahman et al. 1993). Values of soil P" remained neutral. After submergence, the P" of acid sulfate soils gradually increases due to reduction and in- activation of SOj-S through microbial activities (Singh 1982). This reduction rate depends on the presence of sulfur reducing bacteria, P" of the system, energy source (organic matter), etc. (Van Bree- men 1976; Pons et al. 1982). In the present investigation, the P" of soils was found to be reduced by aii-drying with the average values ranging from 3.68 to 6.2. Further reduction could have been achieved by slow oxidation with prolonged drying. Additionally, there were significant differences in P" values of the soils of the same pond at different spots. It might be due to the fact that the sulfidic horizon had not been uniformly parallel to the land surface and during excavation residual sulfidic materials were left. Evi- dence indicates that the pond soils had ucid sulfate characters. In TABLE 2. Phsico-chemical parameters of different soil samples collected from culture ponds. Pond Deptll pH I EC Salinit> OM N, P., S, s„ Na., K Ca„ Mg. Fe, Mn, \l No. Parameters (cml .Mi>ts( Dry (ds/m) (ppll 1 percent) (percent) (ppml ippti ippt) (ppl) (ppt) (ppt) (ppti (ppni) (ppnil (ppni) P-1 BP n-l.s 5.72 5.16 8,92 11 42 7 90 0.16 62.98 I 40 1 ()4 7.78 1 (6 0.92 (57 88.20 58.87 66.30 I.S-30 6.64 5.08 16.93 21.67 10.42 0.13 5.^.50 1,26 3.80 8.30 0.8( 0.84 2,()( 115.87 45.00 128.(3 AP 0-15 6.06 5.59 1 1 34 14..57 9.70 0.16 63,20 0,57 5.23 6.88 1.05 0.89 (..50 128.44 48.70 43.37 l-^i-.TO 6.43 5.39 16.55 21.17 12.68 0.17 58,73 0.79 12.29 8.97 (.23 1.06 2.(2 176,15 68.72 83.59 P-2 BP tn.'; ft.7S 4.79 12.88 16.48 10.51 0 19 69.30 0,92 3.97 6.33 0.48 0.89 (.94 124,33 73.33 99.40 l-'i-.IO 6.13 4.36 20.92 26.69 12.73 0.17 53.(5 2,07 S..39 8.7S 0.76 OSS 2.24 169,4(1 73.33 2(5,43 AP 0-15 6.65 5.72 14.59 18.69 10.44 0 18 60,93 0.73 5.98 8.24 (.16 1.02 1.58 133,75 50,83 3 (.04 15-30 6.38 5.14 18.65 23.86 12.26 0.15 55.04 0,89 1141 9.77 1.04 0.94 1.99 172.13 73.08 58,47 P-3 BP 0-15 6.87 5.28 9.64 12.33 8.87 0.18 58.77 0,68 3.51 5.82 1.18 (..^6 2.05 105,33 93.80 65.46 L5-30 6.54 4.57 13.69 17.52 10.84 0.17 48.95 0.58 6.58 6.95 0,79 0.70 2.04 122.46 52.00 (.W.63 AP 0-15 6.50 5.53 14.82 18.98 10.53 0.17 62.3 ( 0.78 6.42 8.27 1,37 0,97 1.78 1.54.42 .50.48 30.33 1.5-30 6.41 5.19 19,96 25.54 13.35 0.16 57.(1 1.07 12.54 10.67 1,31 0,96 2.21 192.24 71.88 69.87 EC = E(eclrica( Cdnduciivity: OM = Organic Matter; N, = Tota( Nilrogcn; P^ = Avai(ab(e Phosptioms; S^ = Su(phate Su(fur; S„ = Oxidizab(e Su(fur: Na„ = Extraclab(e Sodium; K,. = Extractab(e Poias.sium; Ca.. = Extractable Calcium; Mg.. = Exlractable Magnesium; Fe.. = Extraciable Iron; Mn.. = Extraclable Manganese; A!., = Extractable Aluminum; BP = 30 days Before Production & AP = Mean value of 5 days, 50 days. 95 and 145 days Adcr production. Physico-Chemical Changes in Acid Sulfate Soil 269 TABLE 3. Phsico-chemical parameters of different soil samples collected from inundated ponds. Pond No. Parameters Depth (cm) PH Moist Dry FX (ds/ml .Salinit> ippti OM (percent) N, (percent) P., ippm) (ppt) (ppt) Na, (ppt) (ppt) Ca, (ppt) Ms., (ppt) Fe, (ppm) Mn„ (ppm) Ale (ppm) P-4 BP 0-15 5.6<) 4..13 19.50 15--TO 4.79 i.bH 31.28 AP 0-15 5.82 5.29 19.73 15-30 5.67 4.86 29.30 P-5 BP 0-15 5.93 4.77 14.77 15-30 6.75 4.27 37.51 AP 0-15 5.78 5.77 13.56 15-30 6.12 5.U 20.22 P-6 BP 0-15 5,81 4,67 12,05 LS-.W 6,44 4 48 20,42 AP 0-15 5,92 5,51 14.07 15-30 5.98 4.87 18.76 24.96 15.75 0,22 36.66 2,50 5 52 1 1 10 0.52 0 66 2 :s 4U.04 19.56 0.09 32.28 2,93 12.42 14.06 1.76 0.79 2 25 25.25 16.08 0.13 29.77 1.78 7.64 9,29 0.78 1,28 1 70 37.22 16.62 0.12 26.31 1.83 13.87 12,71 1.14 1.61 2 56 18.90 15.60 0.19 40.88 1.27 5.54 7,26 0.43 0.73 1 96 48.00 12.99 0.15 41.05 1.53 11.57 7,778 0.57 0.62 2 23 14,59 10.02 0.17 57.48 1.07 3.75 7.88 1.15 1.17 1 68 25.89 14.99 O.IS 50.23 1 27 13.67 9.75 0.92 1.11 2 20 15.43 11.00 0.16 39.30 1 .07 4,51 6.09 0,54 060 1 66 26.14 12.87 0 19 41.23 1.55 6,65 6.81 0.55 0.56 1 94 18.01 11.38 0 17 49.37 0,96 549 7.31 1.11 1.07 : 56 24.00 13.19 0.16 49,41 1.43 11.59 8.27 0.66 0.87 1 9S 177.53 71.13 219.50 326.00 92.60 602,03 143,29 49.19 101.29 212.58 87.39 216.69 185.00 127.93 148.87 178.54 107.33 262.33 110.33 68.33 37.25 225,18 87,04 92,00 190,73 71.40 141.67 252.46 59.73 216.37 126 57 63.27 47.96 21004 93.18 126.48 EC = Electncal Conductivity; OM = Organic Matter; N, = Total Nitrogen; P^ = Available Phosphorus; S, = Sulphate Sulfur; S„ = Oxidizable Sulfur; Na^. = Extractable Sodium; K,. = Extractable Potassium; Ca, = Extractable Calcium; Mg^ = Extractable Magnesium; Fe^ = Extractable Iron; Mn^ = Extractable Manganese; Al^ = Extractahle Aluminum; BP = 30 days Before Production & AP = Mean value of 5 days, 50 days, and 95 days After production. TABLE 4. Correlation and regression co-efficient between chemical parameters of soil In culture ponds. Independent Dependent St. Variahle Variable No. > ^ 1 P"(airdiy) EC 2 pH Salinity 3 pH Organic Matter 4 pH Total-N 5 pH Availahle-P 6 pH Sulfate-S 7 pH Oxidizable-S 8 pH Extractable Na 9 pH Extractable K 10 pH Extractable Ca 11 pH Extractable Mg 12 pH Extractable Fe 13 pH Extractable Mn 14 pH Extractable Al 15 Organic Matter Total-N 16 Organic Matter Available-P 17 Organic Matter Sulfate-S 18 Organic Matter Oxidizable-S 19 Organic Matter Extractable Ca 20 Organic Matter Extractable Mg 21 Organic Matter Extractable Fe 22 Organic Matter Extractable Al 23 Organic Matter Extractable Mg 24 Extractable Ca Extractable K 25 Extractable Ca Extractable Fe 26 Extractable Ca Extractable Fe 27 Extractable Ca Extractable Mn 28 Extractable Ca Extractable Al 29 Extractable Ca Total-N Correlation Co-efficient Regression Co-efficient a b 33.07123 3.34.353 44.82607 -4.68567 12.35626 -0.14819 0.17374 -0.00101 29.36178 5.57392 253.2928 -38.70149 10.10835 -0.37784 12.-36769 -0.73409 2.02837 -0.16984 0.66337 0.05491 3.41039 0.28362 162.7490 -2.09753 119.7637 -10.91958 389.0405 -60.73047 0.1595 0.(.)0I415 66.77517 -0.62038 295.15233 -21.40631 -4.2054 1 .06366 -3.3018 1.061 10 0.94343 0.00102 1.24631 0.05671 50.22998 8.76288 42.89656 2.00984 1.67808 0.23581 0.87904 0.06789 132.4095 20.08978 23.6488 39.83474 131.01246 -67.90904 0.62448 1 .96549 -0.34109"''^ 0.38810* -0.02294'^^ -0.02367"^^ 0.35322"^=^ -0.06742'"''' -0.04 140^^ -0.19885"^^ -0.11505"^^ 0.16318'^= 0.48109** -0.02689^^ -0.38776'"^ -0.55449** 0.21277"^^ -0.25392"^^ -0.24085'^^ 0.75288*** 0.76297*** 0,01974^^ 0.62131** 0.71889** 0.11847'^'* 0.13485'"'^ 0.29724^^ 0.08586"^^ 0.41462* -0.20863^^ 0.25 100^'^ NS: Not Significant * Significant at 57c leyel ** Significant at \'i level *** Highly Significant at 5'* level 270 Das et al. TABLE 5. Correlation and regression co-efficient Ijelwten chemical parameters ol soil in inundated ponds. Independent Dependent Correlation Regression Co-efficient St. \ ariable \ ariable Co-efficient No. X ^ r a b 1 P" (air dry) EC -0.51448* 50.91311 -6.11398 2 P" Salinity -0.57717** 71.19709 -9.09594 3 pH Organic Matter -0.45927* 25.03221 -2.19786 4 pH Total-N 0.23651^^ 0.08123 0.01552 5 pH Available-P 0.47452* -1.68086 8.69177 6 pH Sullale-S -0.76008** 5.12290 -0.72512 7 pH Oxidizable-S -0.273 le'^'* 18.66437 -1.92903 8 pH Extractable Na -0.25399^^^ 14.18430 -1.0025 9 pH Extractable K -0.19285^^ 0.17974 0.14421 10 pH Extractable Ca 0.23543'^^ 0.13667 0.18206 11 pH Extractable Mg 0.44329* 3.26257 0.24920 12 pH Extractable Fe -0.55978** 473.3843 -57.52583 13 pH Extractable Mn -0.37716''^ 143.671 -12.96930 14 pH Extractable Al 0.81063*** 1040.365 -177.6317 15 Organic Matter Total-N -0.39531^^ 0.23594 -0.00544 16 Organic Matter Avaikible-P -0.71448** 80.31563 -2.7357 17 Organic Matter Sulfate-S 0.58190** -0.15350 0.12600 18 Organic Matter Oxidizable-S 0.64261** -4.29046 0.94829 19 Organic Matter Extractable Ca 0.70217** -4.57551 1.07604 20 Organic Matter Extractable Mg 0.18387''^ 0.64117 0.02791 21 Organic Matter Extractable Fe 0.78295** 0.72273 0.09197 22 Organic Matter Extractable Al 0.60919** 0.71976 13.08166 23 Organic Matter Extractable Mg 0.59527** -236.0092 27.2572 24 Extractable Ca Extractable K 0.29912^^^ 1.82904 0.16691 25 Extractable Ca Extractable Fe 0.41430* 0.66661 0.42843 26 Extractable Ca Extractable Fe -0.29640"^^ 224.666 -39.3892 27 Extractable Ca Extractable Mn -0.19698"^^ 87.4361 -8.7412 28 Extractable Ca Extractable Al -0.26591^^ 223.517 -75.3507 29 Extractable Ca Total-N -0.34532'"^ 1.70459 -4.t)679 NS: Not Significant * Significant at 5% level ** Significant at 1% level *** Highly Signi ificant at 5'7r level the Chakana Sundarbaiis zone, dry P" of surface soil was found as low as 3.15, which increased gradually with depth (Mahmood & Saikat 1995). The wet soil P" in the surface soil of the present study was less than the sub-soil. Excavating a depth greater than 1 m for pond construction might have exposed the less acid sub- soil. Aeration of water might have caused some oxidation. The higher reduction in P" of the sub-soil due to drying might, on the other hand, be responsible for downward leaching of sulfate. Air dry soil P" was found to range from 2.6 to 4.3 in some acid sulfate soils of Cox's Bazar and Chakaria (Rahman et al. 1993). Liming during the past cropping might have also caused an increase in P" value. Electrical Conductivity (EC) values of the presently studied pond soils are quite high. ECe values greater than 80 ds/m were reported from Senegambia (Vieillefon 1977). Some of the present values (ECe = EC 1:2 x 4 approximately) appear to be still higher and soil salinity may be responsible for the salinity of inundated water because of the similar trend of variation with ponds. In the present findings, available P concentration was found frequently above 24 ppm. In acid sulfate soils, low available P is a result of solubility in acid reaction, insolubiliz.ation of fixation by Fe. Al and Mn. and low release from organic matter (Banerjea & Ghosh 1970; Andriesse et al. 1973; Subosa & Bautista 1991). Addition of phosphate fertilizer to acid sulfate soil is ineffective because of fixation of added P in the form of irreversible iron and aluminum phosphates. In alkaline conditions, colloidal materials in mud and organic matter may be inactive P (Tisdale & Nelson 1975; Singh 1982; Poernomo & Singh 1982). Available P in pond soil might have increased due to application of 25 kg TSP/ha. The problems of extreme acidity of soils in agriculture and aquaculture arise from toxicities of H,S, SO4, Fe. Al, and Mn (IFP 1974: Singh 1982). In the present study SO4-S values ranged from 0.26 to 2.93 ppt. oxidizable S from 1.04 to 15.41 ppt. extractable Fe from 88.2 to 326.0 ppm. extractable Al from 0 to 602.03 ppm and extractable Mn from 37.2 to 127.93 ppm. Extractable Ca was found to be low and the concentration followed the sequence Na>Mg>Ca>K. Another study of Rahman (1990) on acid sulfate soils of Cox's Bazar and Chakaria, the ranges of soluble Fe. Al and Mn were found to be fiom 52 to 75 ppm. from 3 to 260 ppm and from 2 to 22 ppm respectively (Rahman 1990). There was. how- ever, a basic difference between the nature of the two studies and soil type. Concentrations of iron and aluminum in the present study were found lesser than the acid sulfate soils of many other coun- tries (Baylon 1981; Hechanova 1983; Andriesse et al. 1973). The extractable Mn values in the present study were found correspond- Physico-Chemical Changes in Acid Sulfate Soil 271 ing to the active Mn concentrations reported from the Chakaria Sundarbans (Rahman 1990). In acid sulfate soils of Thailand, Viet- nam, and Philippines active Mn ranges from 5 to 400 ppm (Pon- namperuma 1972), Actual acid sulfate soils have smaller amounts of active Mn (Tanaka & Yoshida 1970; Van Breemen 1976). Con- centration of Mn in the present study were found higher than the flooded acid sulfate soil (Rahman 1990; Attanandana 1971 ). From the present study it was observed that during shrimp culture or keeping the ponds inundated without stocking, soil P" increased gradually while SO4-S and extractabie Al decreased rap- idly. Such phenomena are characteristic of acid sulfate soils under flooding and may be exploited in their reclamation and utilization. Extractabie Al was found negatively correlated (Table 4 and .'i) with the soil P", which was also observed by some other workers (Van Breemen 1973. 1976; Baylon 1981; Rahman 1990), On the other hand, oxidizable S and extractabie Fe were positively cor- related with the organic matter. It appears that transformation of aluminum was P" dependent and transformation of .S and Fe in submerged systems were organic matter dependent. Both S and Fe transformations are microbial processes, the agents of which may utilize organic matter as energy source. So. submergence of many acid sulfate soils may eliminate two most important problems, low P" and high Al. but the removal of S seems to be inadequate if the soil/sediment contains high organic matter. Therefore, flooding these soils is a temporary relief. Together with liming, this might keep the soils more tolerable. To remove pyrite. drying of pond bottom with subsequent repeated flushing has been advised (Felix 1988) but prolonged drying, or intense pyrite oxidation may render the soils more acidic, CONCLUSION The extents of increase in P" and decrease in extractabie Al and SO4-S were higher in the culture ponds than those under simple inundation. This suggests that shrimp culture had a positive effect on the improvement of acid sulfate soils under submergence. This could be due to fish fed. mechanical aeration of water, frequent water exchange, shrimp excretion, mixing of surface soil by bur- rowing, leaving Ca rich in shells during molting, etc. In the light of the above discussion, it seems that clearance of mangrove for- ests would lead to destruction of environmental balance. In cleared areas, however, cultivation of shrimp under careful management should be preferred to other forms of land use because of the better scope of soil improvement. 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PHYSICAL, CHEMICAL AND BIOLOGICAL VARIATION AMONG FRESHWATER CRAYFISH (CHERAX ALBIDUS CLARK, 1936) RESEARCH PONDS CRAIG S. LAWRENCE,' - NOEL M. MORRISSY,' PHILIP E. VERCOE," IAN H. WILLIAMS," AND YUK W. CHENG' ^Department of Fisheries, WA Marine Research Laboratories, North Beach. WA 6020. Western Australia: 'Animal Science Group. Faculty- of Agriculture. The University of Western Australia. Nedlands. WA6907. Western Australia ABSTRACT Previous workers pertbrming classical agricultural field e.Kperiments and aquaculture pond trials have emphasized that in order for results from experiments to be analyzed and evaluated, it is important to either demonstrate that the experimental units are homogeneous or quantify any variation between plots, ponds or blocks of these experimental units. Moreover to ensure that results of aquaculture experiments are applicable to industry, research ponds must have similar characteristics to those of industry. Physical, chemical and biological characteristics of 24 research ponds were recorded at the Avondale Research Station. Western Australia. This demonstrated that: (a) The ponds behaved in a manner smiilar to farm dams typical of the Western Australian wheat belt; (b) The water chemistry and turbidity profiles of the ponds were within the range recorded for wheat belt farm dams; (c) The average % organic matter of the sediments was 3.49'7r. which was within the range recorded for farm dams in Western Australia (d). The coefficient of variation for growth of yabbies [Cherax albidus) among the ponds at the Avondale Research Station was 9.26%. The comparatively low level of variation among the ponds was attributed to a number of factors including the age and design of the facility, and the homogeneous water supply for all ponds. Power analysis has been applied to determine the number of replicates required for experiments in these research ponds. A'£l' WORDS: variation, ponds, crayfish. Clienix albidus. power analyses INTRODUCTION Agricultural plot experiments on field research stations provide a bridge between glass house experiments and industry paddocks (Fisher 1958; Fisher 1960). Similarly, aquaculture research pond experiments provide both a bridge between aquarium studies and commercial ponds, and a more realistic estimate of production than laboratory aquarium or tank studies that tend to underestimate yields (Shell 1983). For the results of aquaculture experiments to be applicable to industry it is essential to be able to measure production character- istics both accurately and in a system that mimics the natural environment in ponds as closely as possible. Aquarium studies provide an opportunity to measure single characters accurately in a tightly controlled environment. However, they do not simulate production in ponds well because they do not provide the same natural biota, or algal and clay turbidity. Consequently, long-term growth performance and nutritional health of freshwater crayfish, such as yabbies {Clwra.x albidus Clark 1936). is usually so poor in clean aquaria that experimental comparisons are compromised (Morrissy 1984a). Generally, aquaria studies tend to underestimate yields (Shell 1983). Furthermore, laboratory tank experimentation is impractical at industry densities of 1-5 yabbies/m". Since den- sity and growth of freshwater crayfish are related inversely (Mor- rissy 1992; Mills & McCloud 1983; Brown et al. 1995; McClain 1995a; Morrissy et al. 1995), tank experiments in small containers at high densities are unrealistic and give poor growth and survival ( Ackefors et al. 1 989; Verhoef & Austin 1 999a; Verhoef & Austin 1999b). Another alternative is to estimate production characteristics of yabbies (C albidus) directly from commercial ponds. In contrast Corresponding author. Craig S. Lawrence. Department of Fisheries. WA Marine Research Laboratories. PO Box 20, North Beach. WA 6020. Aus- tralia. E-mail: clawTence& fish. wa. gov. au to the smaller Eastern Australian yabby (C destructor) industry that relies on wild caught and pond reared animals, the much larger farmed Western Australian yabby (C albidus) industry is based on large "commercial ponds" that are argillotrophic. clay-based, pad- dock catchment dams filled by rainfall runoff to provide drinking water for sheep (Lawrence 1998; Lawrence & Jones 2001). How- ever, it is difficult to obtain useful infonnation from the large ponds and farm dams used for rearing yabbies (C. albidus) because of the variability in physical and biological characteristics (Mor- rissy 1974; Lawrence, et al. 1998). In addition there are other limitations of farm dams for efficient research. Management prac- tices differ between dams, they are difficult to drain and hence measure the total population, and they generally contain so many yabbies (C albidus) that the labor required to measure production traits like growth and population size is excessive. Research station experiments include most of the advantages of true ecological studies, where the population of animals is influ- enced by many uncontrolled natural factors, as is experienced in commercial ponds, and classical experimentation, usually single factor, under highly controlled conditions in the laboratory. To carry out yabby (C albidus) experiments a research facility, consisting of 25 ponds supplied by a homogeneous water supply from a header dam. was built near Beverley Western Australia (32°7'S. 116°55'E). This was capable of supporting replicated, randomized and reproducible experiments, with controls, in an environment that simulated farm dams. Large variability among ponds in aquaculture experiments of- ten leads to imprecise estimates of treatment effects. Previous workers in classical agricultural field experiments such as at Roth- amsted (Fisher 1958; Fisher 1960) and aquaculture pond trials at Auburn (Shell 1983) emphasized that in order for results from experiments to be analyzed and evaluated, it is important first to: (a) demonstrate that the experimental units are relatively homoge- neous; or (b) to quantify any variation between plots, ponds or blocks of these experimental units. Cross-over (Change-over) designs, which make comparisons 273 274 Lawrence et al. directly w ithin the same pond instead of between ponds, have been proposed for use in aquaculture experiments to eliminate the varia- tion between ponds (Smart et al. 1997). The construction of an appropriate cross-over design is challenging (Cheng 1996; Cheng & Street 1997) as: (a) due to environmental factors most species in aquaculture respond significantly differently according to seasonal variations over a year; and (b) the interaction between seasonal and other treatment effects are unknown. It is likely that seasonal varia- tion and the effect of the interaction between seasons and other treatments in each pond within a year may be greater than the variation among ponds within the same time frame. Consequently cross-over designs require a longer experimental period and the analysis may involve a more complex statistical model. To in- crease the degree of precision for estimation of treatment effects, the most efficient way to account for between pond variation is to minimize the variability between ponds and quantify that level of variation. By measuring the level of variation between ponds prior to commencing a field trial program it was possible to (a) take varia- tion between experimental units into account when planning the randomization and replication of treatments in future experiments. and (b) detennine whether results recorded from future experi- ments will be due to the application of treatments or merely a result of naturally occurring variability between ponds. This study aimed at testing the homogeneity of experimental units, quantifying the variation between these units, determining the number of replicates required when planning experiments and confirming that the ponds had similar characteristics to wheat belt farm dams. MATERIALS AND METHODS The site for ponds was selected according to clay profiles from the region and to ensure that all ponds were placed as close as practicable to each other. All 25 ponds were constructed v\ ithin the same soil type with the same dimensions (10 m x 10 m water surface area, 1.5 m deep and 3:1 side, or batter slopes). The 25 ponds all received water from the same supply dam. The twenty-five 0.01 ha ponds were filled from the water stor- age dam four weeks prior to stocking. Two weeks prior to stocking each pond with yabbies (C. atbidus) for the first experiment, the ponds received the addition of 50 L of sheep manure to condition pond sediment and increase organic matter to the level commonly found in farm dams. Twenty-four of the 25 ponds were divided into 6 blocks ac- cording to possible sources of variation (clay type, location — north, south, east, west, upper row, lower row); a randomized block design was used, each block contained 4 ponds which re- ceived the same four treatments (yabbies 4.5/nr unfed, yabbies 4.5/m- fed lupins at the rate of 2.5 g/m-/week, yabbies l/m" unfed, yabbies 1/nr fed lupins at the rate of 2.5 g/tn-/week). The remain- ing pond received yabbies 4.5/nr fed lupins at the rate of 2.5 g/m~/week. Prior to stocking each yabby (C ulhidus) was weighed, sexed and 10% of animals were tagged by tail punching (Morrissy 1980; Getchell 1987). The ponds were stocked with 7.050 yabbies (C. ulbiclus) (mean weight 19.41 g ± 0.22 SE) at a sex ratio of 1 male: 1 female on the 29th November 1 994 and the trial was harvested between the Xth to the 15th of March 1995. At the commencement and conclusion of the experiment samples were collected for water chemistry analyses. Samples were submitted to the Chemistry Centre of WA for analyses of Ca, N-NO,, N-NO3, CI, Cu. Fe (dissolved), Fe-total (unfiltered sample). HCO,. K. Mn (dissolved). Mn-total (unfiltered sample). Na. P-SR (Phosphorous, soluble reactive). SO4-S (Sulphate, ex- pressed as sulphur). Zn-total (unfiltered sample). CO,. pH. Elec- trical conductixity (25°C) (Econd.). Alkalinity and Hardness. Max-Min thermometers were used to record water temperature in the ponds. At fortnightly intervals subsurface water samples (lO-cm depth) and benthos core samples were collected from each pond. The benthos corer removed a 2-cm diameter core of sediment and clay to a depth of 15 cm. The percentage organic matter of water samples and sediment was determined by drying samples in pre- dried and weighed crucibles and then ashing out. As the loss in weight is due to combustion of organics, percentage organic matter was calculated according to the formula: Organic Matter = [weight of organic matter/di-y weight] x 100% At fortnightly intervals Secchi disk depth was measured in each pond as an index of turbidity. All data in the randomized block design were analyzed using analysis of variance (ANOVA) to determine significant differ- ences among treatment means. Data were considered significantly different at the 0.05 lexel of significance. The coefficient of varia- tion (C.V.) is a measure of variation and was calculated according to the formula: C.V. = standard deviation/mean ( Shell 1983). Power analyses was applied using the methods described by Searcy-Bemal (1994) to determine the number of replicates re- quired for a given number of treatments to measure a difference of 5. 10, and 20% in the growth of yabbies (C albidits) from the research ponds. RESULTS VlV((er Chemistry Water chemistry parameters at the commencement and conclu- sion of the experiment are presented below (Table 1 ). Using the nonparametric Wilcoxon-Mann-Whitney test (Wilcoxon 1945; Mann & Whitney 1947) to examine paired observations, there was no significant difference between the initial chemical parameters and final chemical parameters (P = 0.73). During the experiment water temperature ranged from 13 - 35°C (mean 22'=C ± 0.6 SE). There was no significant difference in minimum (P = 0.51 ) (C.V. = 9.63%) or maximum (P = 0.21) (C.V. = 12.38%) water temperature among blocks of ponds. Turbidity There was no significant difference in turbidity among blocks of ponds at either the commencement (P = 0.67) (C.V. = 4.74%) or conclusion of the experiment iP = 0.73) (C.V. = 11.75%). However, there was a significant decrease in turbidity over the course of the experiment across all ponds (P < 0.0001) by two tailed f-test with paired observations (Fig. 1). As there was no variation in salinity (measured as electrical conductivity Table 1 ), it is probable that the decrease in turbidity was due to a reduction in suspended organic matter (see Fig. 3). Variation Among Crayfish Research Ponds 273 TABLE 1. Water (.hi'iiiistr) parameters of Avondale research ponds at commencement and conclusion of experiment (;i = 25 1. Units Commencement Conclusion Parameter Mean SE Min Max Mean SE Min Max Alkalinity mg/L 186 9 L30 200 172 15 130 200 CO, mg/L II 2 <2 22 13 4 <2 18 Ca mg/L 33 2 26 38 30 3 26 38 CI mg/L 918 49 662 1 1 50 936 103 662 1150 Cii total mg/L 0.05 0.02 <0.02 0.21 0.09 0.04 0.04 0.21 l;ccind mS/m 343 14 259 404 346 31 259 404 I-L- mg/L 0.07 (1.(11 <0.05 0. 1 3 0.08 0.02 <0.05 0.13 He total mg/L 0.8 0.2 0.1 1.4 0.6 0.3 0.1 1.4 HCO3 mg/L 206 10 160 230 185 12 160 210 Hardness mg/L 348 18 270 430 365 35 270 430 K mg/L 7.8 0.5 6.0 10.0 8.8 0.6 7.0 1 0.0 Mn mg/L 0.02 0.00 <0.02 0.02 0.02 0.00 <0.02 0.02 Mn total mg/L 0.02 0.00 <0.02 0.02 0.02 0.00 <0.02 0.02 N-NO, mg/L 0.46 0.24 0.02 2.00 0.52 0.49 0.02 2.00 Na mg/L 593 28 412 678 578 58 412 678 P-SR mg/L 0.05 0.01 0.03 0.08 0.06 0.01 0.04 0,08 SO4-S mg/L 139 6 121 173 146 11 121 173 Zn total mg/L 0.41 0.24 0.03 2.00 0.10 0.05 0.03 0.23 PH 8.4 0.1 7.5 8.8 8.4 0.3 7.5 8.8 Organic Mailer The organic matter of the pond sediment (Fig. 2) was not significantly different among bloclcs of ponds at the commence- ment {P = 0.16) (C.V. = \9M9c). conclusion {P = 0.07) (C.V. = 31.03%) or during the experiment (P = 0.09.) by two tailed r-test. All ponds ranged between 1 .6% and 53% sediment organic matter content. There was no significant difference in the suspended organic matter in water among blocks of ponds at either the commence- ment iP = 0.10) (C.V. = 24.87%) or conclusion (P = 0.13) (C.V. = 21.13%) of the experiment (Fig. 3). However, there was a significant decrease in suspended organic matter o\er the course of the experiment across all ponds (P < 0.0001 ) by two tailed Mest with paired observations (Fig. 3). The low levels of variation in organic matter during this trial may be largely attributed to the low industry based feeding rates (0-2.5 g/m"/week) in this experiment. Yabby (C. albidus) Survival, Growth and Biomass There was no significant block effect on yabby (C. albidus) production determined as either survival (65% ± 2 SE) {P = 0.24), change in biomass of adult yabbies (C. albidus) stocked (P = 0.26), total change in biomass of all yabbies (C albidus) harvested (including juveniles) (P = 0.50) or final mean weight of yabbies (C albidus) (P = 0.47) (Fig. 4). The coefficient of variation for yabby (C albidus) growth (expressed as final mean weight-initial mean weight) between the blocks of research ponds was 9.26% in = 6). These results indicate that there was no trend across the ponds rh * * * * 70 60 'E 50 •S 40 Q. U !S 30 u 8 20 10 0 Bbck Figure I. Turbidity of research ponds (cm ± SE) at the commencement and conclusion of experiment - the higher the Secchi disk depth, the lower the turbidity of the water. 0 U ui u U ^ ^ » u u U ^ U U > 0 z H H H H ^ S K X 8 8 8 8 _] 1 _j _i ,j J J J -1 _i -3 _i -5 m m CQ ca CQ an •s m 03 0 03 0 03 276 Lawrence et al. 2 u 1 E? o 10 9 g 7 6 5 4 3 2 rih n * * * Btock 3^ U. u U ^ Ui o ^^ ^ ^ U! u ^ > o CJ u u O ^ U (J y y o y o Z o O s o R o S _l h^ y o -J ii o ■s m 03 CQ CQ CD ca ■5 03 m oa ca Figure 2. Organic matter (% ± SE) of pond sediment al the commencement and conclusion of experiment. 70-, 60 ? 50 W e 40 CO o 30 -I 1 £? 20 O 10 0 n,n,n,Pl,n^D- ^ - f^ o U U. I 8 8 ■^ _i J -1 -J g CQ 03 oQ oa ^ »/-l 'O ;^ ui u: ui: 8 8 8 8 ^ s — j -1 ^ P3 CQ oa ^ u: :^ U CJ o o o o J -J -1 oa BQ CQ Bbck op Figure 3. Suspended organic matter (% ± SE) of pond water at the commencement and conclusion of experiment. 40 35 30 ^ 25 'So 20 15 10 5 0 BLOCK 1 BL0CK2 B1jOCK3 BLXXX4 BLOCKS BL0CK6 INrrL\L Block Figure 4. Mean ,val)b> (C alhidiis) weight (g ± SE) at commencement of experiment (Initial) and variation in final vabhv (C. alhidus) weights (g ± SE) among pond blocks at conclusion of experiment (Block 1-6). Variation Among Crayfish Research Ponds 277 for change in yabby (C albichis) growth due to inherent pond factors at the site. The low variation in yabby (C. albidiis) growth (C.V. = 9.26%) between individual ponds treated identically showed a highly acceptable homogeneity in the pond site for future experiments. DISCUSSION To ensure that results of aquaculture experiments are applicable to industry, research ponds must have similar characteristics to those of commercial ponds. In addition the variation (C.V.l in yabby (C. alhidus) growth among ponds due to inherent site fac- tors needs to be as small as possible to minimize the replication needed for future experiments to be effective in showing effects due to different treatments. If differences between treatments are smaller than the coefficient of variation then the observed differ- ences are likely to be due to chance variation rather than a treat- ment effect. The coefficient of variation for yabbie growth among the ponds at the Avondale Research Station was 9.26'7f. Experiments within earthen ponds at the Auburn University aquaculture research station in Alabama have produced coeffi- cients of variation for fish production ranging from 4.2-34.6%, with an average of 20%' (Shell 1983). Previous research using adjacent ponds for marron {Cherax tenuiinaniis) experiments at- tributed 1 1% of variation in growth to differences between ponds. (Morrissy 1992; Morrissy et al. 1995). The comparatively low level of variation between the ponds at the Avondale Research Station may be attributed to a number of factors including: ( I ) the age of the facility: (2) the planning and design of the facility to ensure a homogeneous environment: and (3) the homogeneous water supply for all ponds. Comparative trials between ponds, cages and lined tanks have shown that the primary causes of variation are differences in en- vironmental conditions (including water quality, productivity, soil types, and water source) (Shell 1983). This experiment has shown that the pond site was homogeneous and although environmental parameters (i.e.. turbidity and % suspended organic matter which showed an inverse relationship due to the initial algal bloom from pond fertilization declining) change over time, all ponds followed similar patterns of change. The ponds were therefore suitable for testing treatments (such as density, diet, monosex culture etc.) because any observed difference in yabbie growth greater than 9.26% (C.V. ) was likely to be due to the effect of the experimental treatment. For a given number of treatments, power analysis can be used to determine the number of replicates required. Power analyses are therefore considered to be a basic tool in experimental and sam- pling design (Searcy-Bernal 1994). In general, aquaculture experi- ments can only detect relatively large effect sizes with a reasonable power. This is particularly true with pond experiments that are often characterized by high within-treatment variability and low numbers of replicates (Searcy-Bernal 1994; Shell 1983). A power value of 0.80 has been proposed as the minimum desirable to avoid committing a Type II error (i.e.. accepting a false null hypothesis) (Searcy-Bernal 1994). Applying the methods described by Searcy- Bernal (1994) a power table (Table 2) using the data from this experiment shows the miniinum number of replicates (n) required for treatments (k) to measure a difference of .S. 10. and 20% in the growth of yabbies (C alhidus) from the research ponds, using the a = 0.05 level of significance, with a power of 0.8. As would be TABLE 2. Number of replicates (n) required to identify a difference in growth of 5, 10. and 20% with mean 60g and standard deviation 4.5g for a = 0.05 level of significance with a power of 0.8 for (k) treatments. Difference No. Treatments No. Replicates in Growth (k) (n) 5% 3 4 5 20 30 60 70 10% 2 3 4 5 3 3 4 5 20% 2 3 4 5 2 2 2 2 expected from the C.V. of 9.26% an unrealistically high number of replicates (>20) would be required to measure a difference in yabby (C alhidus) growth of 5% between 2 or more treatments (Table 2). In contiast. to record a difference in growth of 10% requires considerably less replicates (i.e.. 3-5 replicates depending on number of treatments under investigation). The condition of the ponds was similar m manner to typical farm dams of the Western Australian wheat belt. The water chem- istry of the ponds was within the range recorded for farm dams in the Western Australian wheat belt (Morrissy 1980; Lawrence et al. 1998; Cheng et al. 2001 ), which is dominated by sea saU ions, (Na and CI), rather than by salts from catchment erosion. (Ca, Mg, SO4). as found in other world freshwaters (Francesconi et al. 1995b). The high salinity recorded 346 mS/m (1885 mg/1), is typi- cal of cleared catchments in the Western Australian wheat belt (Lawrence et al. 1998; Cheng et al. 2001). The values of a number of parameters (i.e.. CI. Na. SO4-S and hardness) were higher than those recorded from most wheat belt farm dams but within the range suitable for yabbies (Moirissy 1980: Lawrence et al. 1998: Cheng et al. 2001) (Table 1). This may be attributed to initial disturbance of the catchment due to dam construction and subse- quent flushing of the catchment. The turbidity of the ponds was within the range recorded for farm dams in the Western Australian wheatbelt (Lawrence et al. 1998). Similarly, the percentage of organic matter in the sediments of the experimental ponds, mean 3.49%. was also within the range recorded for farm dams in the Western Australian wheat belt, which range from 0.27-12.13% organic matter (Lawrence et al. 1998). The research ponds were therefore suitable for conducting ex- periments on yabbie farming and the results were likely to be directly applicable to the farm dam environment. ACKNOWLEDGMENTS This work was supported by funding from FRDC Project No. 94/75. We thank R. Allison and M. Stuckey technical officers. Fisheries WA for assistance in maintaining the experiments. We also thank Dr. G. Maguire. Dr. N. Hall, and Dr. N. Caputi for their coinments. 278 Lawrence et al. LITERATI Ackefors. H.. R. Gydemo & L. Weslin. 1989. Growth and survival of juvenile crayfish. Astacus astacus in relation to food and density. In: N. De PuLiw, E. Jaspers, H. Ackefors & N. Wilkins. editors. Aquaculture - A Biotechnology in Progress. Bredene, Belgium: European Aquacul- ture Society, pp. 365-373. Brown, P. B., K. A. Wilson, J. E. Wetzel & B. Hoene. 1995. Increased densities result in reduced weight gain of crayfish Onoiiectes virilis. J. World Aqiuuult. Soc. 26:165-171. Cheng, Y. W. 1996. Construction of optimal change-over designs. PhD thesis. University of NSW. Cheng, Y. W. & D. J. Street. 1997. Constructions for optimal non-strongly- balanced change-over designs. Comm. Stars. 26:1073-1082. Cheng, Y. W., C. S. Lawrence, N. M. Morrissy & J. E. Bellanger. 2001. The statistical correlations and implied causal relationships between physical, chemical and biological parameters and yabby (C/icra.v alhi- ihis) production in Western Australian farm dams. Freshwater Cray- fish. 13: Fisher, R. A. 1958. Statistical methods for research \v50 mm CL may produce 3^ broods per year. In addition to P. giittcitus. 120 individuals of P. argus. mostly sub-adults (<80 mm CL) were also caught. Size of male and female P. argus was similar, but the size of P. argus was significantly larger than that of P. gunatus. Time at large of recaptured individuals suggests that the reef patches are a temporary habitat for P. argus. but a more permanent residence site for P. giinaius. KEY WORDS: Panulinis gunatus. Pauulirus argus. population dynamics, spiny lobster, abundance, growth, reproduction, Mexico INTRODUCTION Spiny lobsters are ainong the most \ altiahle fishing resources in the world. Population studies provide information on the abun- dance, movements, growth rates, reproductive dynamics, and sur- vival of these species, and are therefore useful to improve their fishery management (Morgan 1980). However, such studies are also important to increase the knowledge on the ecology of the species, whether they support major fisheries or not. In the Carib- bean Sea and adjacent Western Atlantic coasts, two sympatric species of spiny lobsters occur, Panulinis argus (Latreille) and P. gullcitus (Latreille). Pauulirus argus is a migratory, large-sized species that undergoes several ontogenetic changes in habitat. The postlarvae (pueruli) of P. argus settle in shallow, vegetated areas, where they remain throughout the so-called "'algal juvenile"" phase (6-15 mm carapace length (CD). The ""postalgal juveniles" (15—1.5 mm CL) move to crevice-type shelters, also in shallow areas, whereas the "subadults"" (45-80 mm CL) move to coral reef habi- tats. Further on, the adults (>80 mm CL) migrate to deeper, more diverse habitats (Butler & Hermkind 1997), In contrast, P. gunatus IS a small, rather sedentary species. The pueruli of P. gutlatus are believed to settle directly on the coral reef habitat, and remain in this habitat throughout their entire benthic life (Briones-Fourzan & McWilliam 1997; Sharp et al. 1997). Pauulirus argus supports major fisheries throughout its geo- graphic range, but P. gunatus is mostly a by-catch or secondary catch in most areas, with specific fisheries only in Bermuda and the French West Indies (Evans & Lockwood 1994). Consequently, numerous and extensive population studies of P. argus have been conducted in locations such as Florida (e.g., Lyons et al. 1981; Forcucci et al. 1994), Cuba (review in Baisre 2000), Jamaica (Mun- ro 1974), and Mexico (review in Briones-Fourzan & Lozano- Alvarez 2000), In contrast, most population studies on P. gutlatus have been conducted in Martinique (Farrugio 1975, 1976; Farrugio & Saint-Felix 1975; Marfin 1978), and Bermuda (Sutcliffe 1953; Evans & Lockwood 1994; Evans & Evans 1995, 1996; Evans et al. 1995, 1996). but also in Florida (Caillouetetal. l971;Chitty 1973; Sharp et al. 1997) and in Mexico. In the Caribbean coast of Mexico (coast of the state of Quintana Roo), P. guttatus amounts to -6% of the lobster catch (Padilla- Ramos & Briones-Fourzan 1997), and has been the subject of specific studies on size distribution (Briones-Fourzan 1991). movement patterns (Carrasco-Zanini 1985; Lozano-Alvarez et al. unpubl. data), reproductive dynamics (Briones-Fourzan & Con- treras-Ortiz 1999), and the description of its puerulus (Briones- Fourzan & McWilliam 1997). Also, comparative studies have been conducted on biological, ecological and fisheries aspects of P. guttatus and P. argus (Colinas-Sanchez & Briones-Fourzan 1990; Briones-Fourzan 1995; Padilla-Ramos & Briones-Fourzan 1997; Briones et al. 1997), and on the den choice and occupation patterns of shelters by these two sympatric species (Lozano-Alvarez & Briones-Fourzan 2001). The present paper provides information on a field investigation into the population dynamics of adult P. guttatus in a coral reef of northern Quintana Roo. Monthly changes in the population size of adult P. guttatus were explored, by means of capture-recapture techniques, in a group of coral reef patches separated, but not entirely isolated, from adjacent coral patches. Because P. guttatus is a sedentary species, we hypothesized that population additions would be due mostly to recruitment of young adults, and popula- tion losses to predation-induced mortality. We also aimed to de- tennine the growth rates of P. guttatus and to compare them to those reported for P. argus. as well as to obtain direct evidence for repetitive breeding of females throughout the year. Although our study was focused on P. guttatus. our samplings also yielded in- dividuals of P. argus. providing an opportunity to compare the size ranges and time at large of individuals of both lobster species in this reef patch habitat. 279 280 Negrete-Soto et al. MATERIALS AND METHODS Sliidv Area The study was conducted in the coral reef at Puerto Morelos. in northern Quintana Roo (Fig. 1 ). Puerto Morelos is located in the northern portion of a barrier-fringing reef tract that extends from Belize to the Yucatan Strait. Rather than a continuous barrier, the coral reef in Puerto Morelos consists of a series of reef patches, separated from the coast by a reef lagoon 300-1000 m in width. The reef lagoon (<5 m in depth) is covered by seagrass meadows, a habitat where juveniles of P. argus dwell, but where P. guriatKs does not occur (Briones-Fourzan 1995). Along the reef tract, the sloping fore-reef has relatively few high-relief features, but hard coral cover is dense at the reef crest and in the back-reef zone (Ruiz-Renten'a et al. 1998). providing an intricate habitat with numerous crevices and caves where both P. gunanis and P. aigiis occur (Briones-Fourzan I993-. Lozano-Aivare/ & Briones-Fourzan 2001). Lobster Sampling and Tagging The main study site (site 1 ) consisted of a group of several reef patches, close to each other (maximum distance between adjacent patches: 50 ni). but relatively separated (-200 m) from the rest of the reef tract. In addition to their relative isolation, we chose these reef patches because they are not fished for lobsters, owing to their proximity to the navigational channel to the port. The area of the patch complex was 2.5 ha, excluding the areas between patches, which consisted mostly of sand and sparse sea grasses. Average depth around the leef patches was 4 to 5 m. From October 1986 to November 1987, 20 lobster traps (mesh size: 5 x 2.5 cm) were deployed by divers in the underside of ledges or coral formations around these patches. The distance between adjacent traps ranged from -20 to 100 m. The traps remained fixed for 10-13 days every month, during the dark portion of the lunar cycle, after which the traps were recovered and relocated the following month. The divers carefully extracted the lobsters from within the traps every 20° 50' — Figure 1. Location of study sites on the coral reef tract at Toerto Morelos, Mexico. morning during each sampling period, unless impeded by bad weather. Lobsters were tagged with modified Australian spaghetti- type tags (Lozano-Alvarez et al. 1991; Lozano-Alvarez 1992) that were manually assembled in the laboratory as described by Chittle- borough (1974). These tags consist of an individually numbered vinyl "spaghetti" and a small plastic toggle, joined by a thin nylon thread. The toggle is inserted in the dorsolateral muscle of the lobster, between the cephalothorax and abdomen, with a stainless steel applicator. The original toggles measured II x 3 x 0.5 mm, but we shortened them to 9 mm in length with a grinding machine before assembling the tags, in order to reduce their possible del- eterious effects on the relatively small-sized P. guttatus. All lobsters were measured (carapace length. CL, in mm. from between the rostral horns to the posterior margin of the cephalo- thorax) with digital calipers (±0.1 mm), and injuries (i.e. number and type of missing appendages) were recorded. The reproductive stage of females was determined according to the following scale (Briones-Fourzan & Contreras-Ortiz 1999): ( I ) clean carapace, no extruded eggs or traces of spermatophore; ( 2 ) new and intact sper- matophore on sternum; (3) newly extruded eggs (bright orange); (4) dark orange eggs, with eyespots visible; (5) brown eggs, em- bryo and eyes cleariy visible; (6) remnants of empty egg capsules and/or eroded spermatophore. After tagging, the lobsters were re- turned immediately to natural crevices in the coral patches. To reduce tag loss, individuals that were about to molt or recently molted ("soft-shelled") were not tagged. All lobsters were care- fully examined to ascertain whether they had lost a tag, as evi- denced by a distinctive scar in the site of tag application. Additional information on size composition and sex-ratios was obtained from a large, elongated patch (site 2) located 4 km north of site 1, where 13 traps were used in a similar fashion as in site 1. Lobsters from site 2 were not tagged because they were sacri- ficed to study their diet (Colinas-Sanchez & Briones-Fourzan 1990) and the fecundity of females (Briones-Fourzan & Contreras- Ortiz 1999). Size Distribution We compared the overall size distribution of P. argus and P. guttatus; of P. giittatiis between sites I and 2, and of males and females of each species, with Student's /-tests for unequal sample sizes (Zar 1984). When necessary, data were log-transformed to homogenize variances. To explore temporal changes in the size distribution of P. giitlcitus in site 1. monthly data were grouped in 2-mm size classes and analyzed with a one-way repeated measures ANOVA, with time as the repeated factor, followed by a Tukey's test for unequal sample sizes (Winer 1971). Population Size and Survival The population size of P. guttatus was monthly estimated by means of the Fisher-Ford model (Fisher & Ford 1947). which relies on several tagging occasions and several recaptures. We chose this model because the capture-recapture data were rela- tively scarce and the survival rate was fairiy constant (see Results). In these circumstances, the Fisher-Ford model tends to yield more reliable results than other models based on multiple-recapture data (Bishop & Sheppard 1973; Begon 1979; Lozano et al. 1982). while still providing estimates of population losses (death -I- emigration), and population additions (recruitment -i- immigration). The Fisher- Ford model assumes a constant survival rate (ct>), but this assump- tion can be analyzed by means of two goodness-of-fit tests (Begon Population Dynamics of Panul/rus guttatus in Mexico 281 1 979); a test of the period-to-period differences in (observed vs. expected periods survived) (test 1 ). and a test of the independence of 70 mm CL). Only single-molt observations were used to estimate molt increments. The intermolt period (in weeks) was then calculated by dividing the average molt increment (mm CL) by the average growth rate. The results were then sum- mari/ed in a table. Reproductive Aspects The monthly percentage of ovigerous females was obtained from the total sample to determine the main reproductive season. Data from females of P. gnrtatiis that were recaptured in different reproductive stages allowed for a preliminary estimate of the egg- incubation period, and of the duration of a complete breeding cycle. RESULTS In total, 920 lobsters (including recaptures) were obtained. 778 (85%) P. guttatus and 142 (15%) P. argus. Results on the issues explored are given for each species separately. Paniilirus guttatus Size Distribution In all, 551 P. guttatus were caught in site 1 (410 males and 141 females), and 227 in site 2 ( 160 males and 67 females). Mean size of P. guttatus was similar between sites U = 0.782, df = 777, P = 0.495). Overall, male P. guttatus ranged in size from 42.4 to 87.5 mm CL (mean ± SD; 64.5 ± 7.6 mm CL), significantly larger than females (range: 44.5 to 73.5 mm CL; mean ± SD: 59.3 ± 5.2 mm CL) (log-transformed data. ; = 8.748, df = 774, P < O.OOOI ) (Fig. 2A). (a) Panulirus guttatus 20 0 .^ <^^ C? fot. ^ ^t. ^q, ^^ ^- ^^ ^ ^ .. .. ., ,, ^<^, ^ „, ,. ., ,. Carapace length (mm) HFemales(N= 175) BMales (N= 456) (b) Panulirus argus 20 0 (A 0) in o 0) O) ra c 0) u 9) Q. 150 100 <^ .0, q, q, q, q, a ^'^ ^^ ^'^ J^ r?> i-^ <^- «?• - ^^ a'* -l}" <§> q,*" qQ N^ ' <&' \^' ^ <&' 'P' ^' ^' ■" Carapace length (mm) H Females (N= 82) BMales (N= 59) Figure 2. Size distribution of the total sample (including recaptures) of (a) Panulirus guttatus and lb) Panulirus argus caught in traps in coral reef patches at Puerto Morelos, Mexico. Tagged and Recaptured Individuals Individuals of P. guttatus (excluding recaptures) in site 1 were 331 males and 127 females (sex ratio 2.6:1 ). Of these, we tagged 227 males and 90 females, and recaptured 62 males (27.3%) and 12 females (13.3%). Individual males remained at large for 1-50 weeks, and females for 1—43 weeks (Fig. 3a). No significant dif- ferences were found in time at large of males and females (x" = 1.22, df = 5, P > 0.95). Some individuals were recaptured on multiple occasions (from two to seven), yielding a total of 119 recaptures of males and 26 recaptures of females. Fifteen lobsters ( 10.3% of recaptures) lost their tags, but were taken into account when estimating the population size by calculating the "average age" of their tags, based on the average age of the tags of the individuals concurrently lecaptured (Lozano et al. 1982). Of the recaptured individuals, 53% were recaptured within a distance of 20 m from their site of release, 45% at distances from 20 to 50 m, and 2% at distances from 50 to 100 m. Predation-induced mortality within the traps was high (28%^), accounting for 94 males (69 untagged and 25 tagged) and 33 females (28 untagged and five 282 Negrete-Soto et al. (a) Panulirus guttatus 1-7 ^ , ^ I I "^ I , -15 16-23 24-31 32-39 > 40 Total weeks at large I Males (N = 62) HFemales (N = 21) (b) Panulirus argus u> 80 0 -1 ^ 0) ^^H +-» (/) ^ 60,0 - ^H o |fl »^ ^^^ S 40.0 - ^^^ D) ^^BSs TO S 20 0 - ^K^ vv* 40 I Males (N = 10) HFemales (N = 14) Figure 3. Ptrcentage of indhidual lobsters recaptured in different periods at large (In weeks), (a) Panulirus guttatus, (b) Panulirus argus. tagged). Predators found in traps included groupers (Epineplwhis spp.). triggerfish (Batistes vetida). snappers (Liirjiiniis spp.). and moray eels {Cymiuithorax spp.). Population Size and Survival A lower catchability of female P. liiittiilus in traps was reported by Evans and Lockwood ( 1994) in Bermuda. A differential catch- ability of males and females in our traps would contradict one of the principal assumptions of the Fisher-Ford model, namely that all individuals are equally catchable. Therefore, before applying the Fisher-Ford model, we explored whether the biased sex ratio of P. guttatus was (a) the actual sex ratio over the size range of trappable lobsters, caused by a lower number of females in the largest size classes due to a differential growth rate of males and females (Wenner 1972), or (b) an artifact due to a differential catchability of males and females in traps. To test (a), we plotted the percent- age of males in each 5-mm size class of the total catch. In species where males reach significantly greater sizes than females, such as in palinurids (Morgan 1980), male percentages tend to fluctuate around 50 in the small size classes, then decrease as adult females accumulate in one or a few size classes, and further rise as size increases until approaching 1009f in the largest size classes (Wen- ner 1972; Herrnkind & Lipcius 1989). However, our curve showed higher percentages of males in most size classes, indicating that females were undersampled. and suggesting a differential catch- ability of males and feinales. This was further confirmed by a test of difference in catchability between population sub-groups (i.e.. males and females! (Begon 1979), which was highly significant (X" = 28.756, df = 12, P< 0.001). The most likely cause for this differential catchability is a dif- ferent beha\'ior of males and females (Lozano et al. 1982). Ap- parently, the protracted reproductive season of P. guttatus (Farru- gio 1976; Sharp et al. 1997: Briones-Fourzan & Contreras-Ortiz 1999) results in females being overall less active than males, as evidenced by the higher occunence of male P. guttatus in fixed gear, such as nets and traps compared to samples obtained by divers, who search actively for lobsters (Table 1 ). Sex ratios, then, may depend on the sampling method, the location, and the size TABLE 1. Panulirus guttatus. Size-ranges and sex-ratios of individuals sampled with different methods in several locations of its geographic range. Sampling Sample Sex-Ratio Size Range (C L. mm) Location Method Size (M:Fl Males Females Source Florida Divini; S94 1.2:1 32-S5 36-7 1 Caillouet et al. (1971) Florida Divmg 1477 0.7:1 32-S4 26-71 Chitty (1973) Florida Diving U2 (.).5:l 1 X-75 21-63 Sharp etal. (1997) Mexico Diving 212 1.7:1 40-89 44-78 Padilla-Ramos & Briones-Fourzan (1997) Martinique Nets 234 2.8:1 33-70 40-60 Farrugio ( 1975) Martinic|iie Nets 772 1.7:1 36-66 30-66 Farrugio ( 1976) Martinique Nets 1461^ 1 .9: 1 37-74 34-68 Marfin (1978) Bermuda Traps \52 10.0:1 64-88 58-74 Sutcliffe (1953) Bermuda Traps 919 13.0:1 51-81 49-69 Evans & Lockwood (1994) Bermuda Traps 1656 16.0:1 51-83 51-69 Evans etal. (1996) Jamaica Traps 114 2.1:1 43-61 4.3-70 Munro{1974) Mexico Traps 136 1.9:1 55-82 54-65 Carrasco-Zanini (1985) Mexico Traps 631 2.6:1 42-88 45-74 Present paper CL is carapace length: M is males: F is females Population Dynamics of Panulirus guttatus in Mexico 283 range of individuals in the samples. Allowing tor a higher propor- tion of males in the largest size classes, the 1.7:1 sex ratio of the P. i^itttaiiis sampled by Padilla-Ramos and Briones-Fourzan ( 1997) from fisher divers in Puerto Morelos over a fishing season (Table 1 ), appears more realistic than ours. Consequently, we applied the Fisher-Ford model separately to the male and female data, and obtained separate estimates of population sizes and survival for each sex (Begon 1979). The estimated survival rates (4)) were 0.727 for males and 0.867 for females. The two tests for the constancy of 0.10) and of test 2 x" = 6.901 (df = 12, /• > 0.75). For females, the overall result of test 1 was x" = 4.770 (df = \2.P> 0.95) and of test 2 x" = 13.840 (df = 12. P > 0.25). Hence, the assumption of a constant survival rate for both males and females was reasonably supported. Male population size ranged from 97 to 373 individuals, with higher values in November 1986. and from April to September 1987 (Table 2a). The size of the female population varied between 69 and 435, with higher values in April, and from June to Novem- ber 1987 (Table 2b). When adding the male and female population estimates, higher abundances of adult P. giiltaliis occurred in April, and from June to November 1987 (Table 3). In contrast to the more heavily biased sex ratios in our samples, monthly sex ratios in the population varied from 0.3 to 2.8 males: 1 female, with an overall sex ratio of 1.3:1 (Table 3). The monthly population size was converted to density (number of lobsters ha~') and confronted with the monthly mean size of lobsters (Table 4). The mean size of P. guttatus was significantly different in time (F = 2.637, df = 13, 208; P = 0.002), with TABLE 2. Panulinis guttatus. Statistics of the Fisher-Ford model for (a) males: and (b) females in the reef patches. Losses include mortality and emigration: additions include recruitment and immigration. (a) Males Number of Males New Tags in Period Male Population Sampling Period Caught Released Size (N) Losses Additions Oct. 1986 31 28 28 Nov. 42 41 38 219 60 -62.2 Dec. 23 20 13 97 27 43 Jan. 1987 28 19 14 114 31 68 Feb. 46 28 17 Lsn 41 -4.3 Mar. 18 14 9 KI.S 29 218 Apr. 34 23 19 294 80 -47 May 24 14 11 167 46 156 Jun. 33 21 20 278 76 171 Jul. 21 14 12 373 102 -89.5 Aug. 26 15 11 182 50 83 Sep. 18 18 15 215 59 14 Oct. 25 23 19 171 47 -8.8 Nov. 2^ 0 0 115 Total 391 278 226 (b) Females Nui Tiber of Females New Tags Female Population Sampling Period Caught Released in Period Size (N) Losses Additions Oct. lysh 8 6 h Nov. 14 14 14 7S 10 49 Dec. 6 6 6 117 16 -32 Jan. 1987 6 5 5 69 9 20 Feb. 14 11 8 80 11 8 Mar. 10 9 7 77 10 94 Apr. 14 7 7 161 21 -38 May 5 2 2 102 14 45 Jun. 16 10 8 133 18 28 Jul. 7 6 5 143 19 93 Aug. 5 3 3 217 29 -52 Sep. 11 10 8 136 18 60 Oct. 13 12 11 178 24 281 Nov. 9 0 0 435 Total 138 101 90 284 Negrete-Soto et al. TABLE 3. Pamiiinis guttatus. Total population size in the reef patches estimated h\ the Fisher-Ford model, and comparison of sex ratios of individuals sampled in the reef patches «ith those obtained from the model population estimates. Number of Lobsters Sampled (n) Population Size Estimates (N) Males Females Total Sex Ratio Males Females Total Sex Ratio Period (Hm) (n,,) (n.4, + n,.l (M:F) (N\,l (N,.-) (N„ + Nf) (.M:Fl Oct. 1986 .M 9 40 3.4:1 Nov. 46 14 60 3.3:1 219 78 297 2.8:1 Dec. 27 6 33 4.5:1 97 117 214 0.8:1 Jan. 1987 29 6 35 4.8:1 114 69 183 1.7:1 Feb. 51 14 65 3.6:1 150 80 230 1.9:1 Mar. 20 10 30 2.0:1 105 77 182 1.4:1 Apr. 35 14 49 2.5:1 294 161 455 1.8:1 May 25 5 30 5.0: 1 167 102 269 1.6:1 Jun. 33 16 49 2.1:1 278 133 411 2.1:1 Jul. 21 7 28 3.0:1 373 143 516 2.6:1 Aug. 26 6 32 4.3:1 182 217 399 0.8:1 Sep. 19 11 30 1.7:1 215 136 351 1.6:1 Oct. 25 13 38 1.9:1 171 178 349 1.0:1 Nov. 22 9 31 2.4:1 115 435 550 0.3:1 Total 410 140 550 2.9:1 Average 191 148 .?.?9 1.3:1 smaller values from June t(i October 1987 than throughout the remaining months (Table 4). In this period, some of the highest population density values also occurred. The overall average popu- lation density of P. guttatus was 126 indisiduals ha"'. Reproductive Aspects We recorded the reproductive stage of 202 female P. guttatus in the total sample. Of these. 83 (42.19;-) were ovigerous. Oviger- ous females occurred every month, except in August 1987. hi general, the occurrence of ovigerous females was higher (39.3- TABLE 4. PanuUrus guttatus. Population characteristics over the study period. Mean carapace lengths were grouped with a Tukey's test for unequal sample sizes after a repeated-measures ANO\ A. Population density was derived by dividing the total population size (males + females) estimated with the Fisher-Ford model by the surface area of the reef patches (2.5 ha). Populatitm Carapace Den.slt\ Percentage Sampling! Sample I.engtti (mm) Groups ( Lobsters of Ovigerous Period Size (n) (Mean ± SD) of Means ha-') Females Oct. iys6 411 68.5 ± 6.5 c 33,3 Nov. 60 65.1 ±5.8 abc 111) 35.3 Dec. 33 67.1 ±6.4 be 79 42.9 Jan. 1987 35 65.5 ± 6.3 abc 68 54.5 Feb. 66 63.6 ± 8.4 ab 85 52.2 Mar. 30 62.0 ±5.2 ah 67 53.8 Apr. 49 63.1 ±6.8 ah 169 65.4 May 30 61.6±7.9 ah 100 42.9 Jun. 49 60.4 ± 6.8 a 152 39.3 Jul. 28 59.9 ± 6.3 a 191 50,0 Aug. 32 60.6 ± 6.5 a 148 !),() Sep. 30 61.1 ±4.9 a 130 8.3 Oct. 38 61.2 ±5.7 a 129 33.3 Nov. 31 62.3 ± 5.9 ab 204 27.3 Average 63.1 ± 7.4 125 42.1 65.47r) from December 1986 to July 1987. and kiwer (0-359i-) in August to November 1987 (Table 4). Of the 21 recaptured females, three changed from reproductive stage 3 to 6 in 13. 13, and 21 days, indicating an egg incubation period of 2-3 weeks. This agrees with Chitty (1973). who esti- mated the egg-incubation period of P. guttatus in less than 30 days. Evidence for repetitive breeding occurred in six females, which had different broods v\hen captured and when recaptured after 25. 31. 46, 97, 101, and 240 days. Repetitive breeding of female P. guttatus has been reported before (e.g., Chitty 1973; Farrugio 1976: Sharp et al. 1997; Briones-Fourzan & Contreras-Ortiz 1999) and, in Puerto Morelos. large females (>50 mm CL) breed more times in a year than small females (<3() mm CL) (Briones-Fourzan & Contreras-Ortiz 1999). Based on the short incubation period, the large size (>50 mm CL) of all our recaptured females, and the repetitive breeding evidence from recaptured females, we conser- vatively propose an average duration of 90-120 days for a full breeding cycle in large females, i.e., up to 3— f broods per year. (irowth In total, 1 19 recaptures of males (37 uninjured and 62 injured) and 26 recaptures of females (19 uninjured and 7 injured) were considered in the growth analyses. Si/e range at capture of tiiales was 47.8-81.1 mm CL, and of females 53.7-69.0 mm CL. Time between subsequent captures was 1-24 weeks in males, and 2-34 weeks in females. Only 19 males (10 uninjured, 9 injured) and three females (1 uninjured, 2 injured) grew betv\een recaptures (Table 3). In general, itijured individuals had smaller molt incre- ments than uninjured individuals. Small uninjured males showed higher growth rates and molt increments than larger males, but their intermolt periods were similar. The uninjured female had a growth rate of 0.32 mm CL week"'. Thirteen injured males and two injured females molted and regenerated between 1 and 5 ap- pendages in periods of 4 to 1 7 weeks, but showed no increment inCL. Population Dynamics of Panulirus guttatus in Mexico 285 TABLE 5. Panulirus guttatus. Summary of groHth data for males (size range: 47.5-81.0 mm carapace length. CL) and females (size range: 5.^.7-65.3 mm CL) by size class and injury condition at time of release (uninjured: complete individuals: Injured: individuals missing one or more appendages). For each sex and size class, average growth rate (mm CL week ') was estimated by dividing the increase in CL of recaptured individuals by the number of weeks at large. Average molt increments in CL were calculated as in Forcucci et al. ( 1994), by plotting each observation of change in CL against weeks at large and considering only those individuals that underwent one single molt. Interniolt interval was then calculated by dividing the average molt increment by the average growth rate. Number (a) Uninjured Individuals That Did Not Grow That Grev» Size Class Time at Average Intermolt Average Molt Av( erage Growth (CL, mm) Recaptured N l>arge (weeks) N Period (weeks) Increment (CL, mm) Rate ( mm CL week"') Males <60 11 8 5-18 3 10 4.0 0.40 60-65 21 16 1-17 5 8 2.8 0.37 65-70 14 12 1-14 2 12 2.2 0.19 >70 11 11 1-24 0 Total 57 47 10 Females <60 10 9 2-18 1 7 2.1 0.32 >60 9 9 2-34 0 Total 14 18 1 Number (b) Injured Indii .iduals That Did Not Crow That Grew Size Class Time at Average Intermolt Average Molt Av erage Growth (CL, mm) Recaptured N Large (weeks) N Period (weeks) Increment (CL, mm) Rate! mm CL week"') Males <60 9 5 1-16 4 10 2.4 0.16 60-65 20 19 1-24 1 4 1.2 0.30 65-70 IS 16 1-21 2 8 1.1 0.14 >70 15 13 1-12 2 10 1.2 0.12 Total 62 53 9 Females <60 4 3 3-8 1 14 1.6 0.11 >60 3 2 2-7 1 9 1,4 0.17 Total 7 5 1 Panulirus argus The mean size of P. cirgiis (range: 49.0-123.6 mm CL; mean ± SD: 69.8 ± 12.7) (see Fig. 2b) was significantly larger than that of P. i^uttatKs (range: 42.4-87.5 mm CL; mean ± SD; 63.1 + 7.4 mm CL) (log-transformed data; l = 8.055. df = 914. P < 0.0001). We caught 120 P. argus in site 1 (including recaptures) and only 22 in site 2. Overall, there were 48 males (size range; 49.0- 123.6 mm CL) and 72 females (size range; 50.1-98.4 mm CL). Mean size of males and females was similar (mean ± SD of males; 70.3 ± 14.2 mm CL; of females; 69.5 ± 1 1.6 mm CL; r = 0.362, df = 138, P = 0.716). In site I, new individuals of P. argus (excluding recaptures) accounted for 35 males and 41 females (sex ratio 0.85;l). We tagged 33 males and 39 females, and recaptured 9 males {2T^/c ) and 14 females (369f). Most recaptures of P. argus were obtained within 15 weeks; only four individuals remained at large for longer periods (up to 31 weeks) (see Fig. 3b). No significant differences in time at large were observed between males and females (x" = 2.92. df = 3. P > 0.25). Distance of recapture varied from 20 to 150 m. Some individuals were recaptured more than once, yielding a total of 12 recaptures of males and 30 of fernales. Of these, four males (60.8-75.8 tiini CD and seven females (50.1-78.0 mm CL) grew. The average molt increment, intermolt period, and growth rate of males were, respectively, 5.2 mm CL. 8.4 weeks, and 0.67 mm CL week"'; whereas those for females were 4.3 mm CL. 6.5 weeks, and 0.68 mm CL week^'. These data were too scanty to attempt any further analyses on population size or growth, but indicate that individuals of P. argus were less abundant, had higher growth rates, and retiiained in the reef patches for shorter periods than individuals of P. guttatus. Also, in contrast to P. guttatus. there was no evidence of reproductive activity of P. argus in the coral patches; all female P. argus were in reproductive stage I. DISCUSSION In the reef patches surveyed in this study, we caught over five times as many P. guttatus as P. argus. The underlying causes for these disparate results could be interspecific competition, a differ- ential catchability in traps, or a real difference in abundance of both species. Competition is not likely to occur, because P. argus and P. guttatu.s do not seem to compete for food or shelter re- sources (Sharp et al. 1997; Lozano-Alvaiez & Briones-Fourzan 2001 ). Although differences in catchability of both species cannot 286 Negrete-Soto et al. be discarded, we believe that our results reflect a true scarcity of subadult P. argu.s recruiting to the reef habitat. In Puerto Morelos. densities of algal juveniles of P. urtiHs in the shallow vegetated reef lagoon are high (146-263 individuals ha"'. Briones-Fourzan & Lozano-Alvarez 2001a). but the density of the subsequent pos- talgal juveniles is drastically reduced (0-31 individuals ha"'), ow- ing to a lack of crevice-type shelters throughout the reef lagoon (Briones-Fourzan & Lozano-Alvarez 2001b). This would further result in low numbers of subadult P. argiis moving to the reef habitat. Over 80% of the individuals of P. argiis in our sample were subadults (<80 mm CL) as opposed to the individuals of P. guttatus, which were all adults. In addition, each species appears to make a differential use of the reef habitat: subadult P. argus shelter in the reefs during the day. but forage at night in rubble areas or seagrass meadows adjacent to the reefs (Cox et al. 1997). whereas individuals of P. giitlnliis forage on the reef itself (Sharp el al. 1997) and never abandon this habitat completely. Our estimates on population sizes and survival rates of P. gut- tatus were similar to those estimated in the Florida Keys (Sharp et al. 1997). but. although we did acknowledge the tag loss, our estimates may be biased owing to an unquantified tag-induced mortality. When analyzing the effects of the Australian tag on juvenile P. cygiuis (40-75 mm CL). Chittleborough (1974) esti- mated an overall ""tag loss" of 23% over 39 weeks, but he could not separate tag-induced mortality from tag loss. In addition, although the size range of Chittleborough" s P. cygnus and our P. guttatus was similar. Chittleborough's tags had considerably larger toggles ( 14 mm long) than ours (9 mm). Mortality of Hauuirus aiueiicauus (43.5-97.5 mm CL) induced by sphyrion tags (similar to the Aus- tralian tags but with a steel anchor instead of a plastic toggle) was significantly lower (s 5%) in lobsters tagged during intermolt or premolt than in those tagged during postmolt (soft-shelled) (Mori- yasu et al. 1995). a precaution we observed in our study. Lozano- Alvarez (1992), using exactly the same type of tag as ours on a population of juvenile and young adults of P. argus (25-90 mm CL). estimated a tag-induced mortality of 5%. If a similar tag- induced mortality occurred shortly after tagging in our individual P. guttatus. it might have not affected the estimates of survival, but it may have contributed to an underestimation of the population size (Begon 1979, Moriyasu et al. 1995). Therefore, our population size values are probably underesti- mates. However, the estimates of survival rates for males (0.73) and females (0.87), and the average density of adult P. guttatus in our coral patches ( 1 26 lobsters ha" ' ), were comparable to the mean densities of 1 18 and 1 15 adult P. guttatus ha"' estimated by Sharp et al. (1997) in two patch reef areas in a lobster sanctuary in Florida, based on nighttime samplings by divers, with overall sur- vival rates of 0.87 and 0.67 in each area. In contrast, in reef-crest areas of Bermuda, where a specific fishery for P. guttatus exists, Evans and Lockwood (1994) obtained a mean density of 29 ± 7.6 trappable P. guttatus ha"', but with an overall niale:female ratio of 7: 1 . The largest population sizes of adult P. guttatus in our study site were obtained in June to November. Within this period, the lowest percentages of ovigerous females and the smallest mean size of lobsters afso occurred, suggesting a recruitment of small adults to the trappable population during the summer and early autumn. The juveniles of P. guttatus presumably live in the same habitat as adults (Shaip et al. 1997), but we have never observed individuals <34 mm CL during daytime divings in these reefs. However, the coral reef is a very intricate habitat, and the small phases of P. guttatus may hide deep within small recesses and crevices during the day, warranting future extensive surveys by nighttime divings to reveal their occurrence and their contribution to the whole population size. Because P. guttatus is a rather sedentary species, we hypoth- esized that the population additions would be mostly ascribed to the recruitment of small adults. However, we cannot rule out en- tirely the contribution to the population additions and losses of movements of males and females throughout the reef habitat, and from and to our limited study area. Most of our individual P. guttatus were recaptured from distances <50 m, and our reef patches were relatively separated from adjacent reef habitats, but low-relief hard grounds between these and other patches may have been traversed by some individuals. In displacement experiments with tagged adult P. guttatus. Can'asco-Zanini ( 1950 mm CL breed during the four seasons. These authors estimated the index of reproductive potential of P. guttatus by conservatively assuming one brood per year for all size classes. But if females >50 mm CL can breed three to four times per year as suggested by our results, their contribution to the overall egg production may be much higher than that estimated by Briones-Fourzan & Contreras- Ortiz (1999). An effect of repetitive breeding on females is a decrease in their growth rales, due to a reduction in the molt increments and an increase in the intermolt periods (Hunt & Lyons 1986). but our data on female growth were insufficient to explore this assump- tion. Injuries have no clear effect on growth in some lobster spe- cies (Davis 1986; Forcucci et al. 1994). but in others injuries reduce molt increments (Brown & Caputi 1986). In our study, those injured P. guttatus that grew showed, in general, lower molt increments than uninjured individuals. Moreover, other injured individuals molted, as evidenced by their regenerated appendages, but without any increase in CL. Injured lobsters were mostly Population Dynamics of Panulirus guttatus in Mexico 287 males, and the occurrence of detached appendages in our traps suggests that some were injured while in the traps, perhaps through aggressive interactions (Lozano-Alvarez & Briones-Fourzan 200 1 ) or when trying to deter the predators that entered the traps. The real incidence of injuries among the population is undetermined. Our male growth data, although scant and inconclusive, suggest that growth rates of male P. guticitiis decrease as size increases, but few males grew during their time at large. In Martinique, most P. guttatus molt at least twice a year (Marfin 1978). whereas the average intermolt periods of our 10 uninjured males that grew (8- 12 weeks) suggest the possibility of four to six molts per year. Applying the average growth values of uninjured males in Table .'i. it would take a 48 mm CL male about 2.5 years to reach 81) mm CL. However, many individuals did not molt during similar or longer times at large (up to 24 weeks), and if this portion of males is also taken into account in the growth estimates, the results suggest that uninjured adult male P. guttatus may molt at least 2-3 times per year. The average molt increment of males over the entire size range of recaptured individuals was 2.8 mm CL. With these values, a 48- mm CL male P. guttatus would grow to 80 mm CL in about four years. More data are needed to improve these results, which nevertheless indicate that P. guttatus has lower growth rates than P. argus of similar sizes, because it would only take one year for a male P. argus to grow from 48-80 mm (Lo- zano-Alvarez et al. 1991b). Most of the recaptures of P. argus were obtained within 15 weeks, suggesting that these patch reefs are a transitory habitat for these subadults, which would presumably emigrate further on to deeper offshore areas where the large adults dwell (Lozano- Alvarez et al 1991a). In contrast, some individuals of P. guttatus were recaptured over periods up to 50 weeks, suggesting that these patch reefs are a more permanent site of residence for P. guttatus. Our results show that the coral reef patches at Puerto Morelos support a sizeable population of P. guttatus and are also an im- portant habitat to the subadults of P. argus. In the future, more refined studies focused on the population dynamics of P. guttatus should include sampling by nighttime diving, a direct estimation of tag-induced mortality, tagging in a broader area, and increasing the sampling effort to obtain a larger set on grovMh data. ACKNOWLEDGMENTS We thank Felix Colinas, Jorge Siiiionin, Martha Fonseca, David Gutierrez, and Gabriela Contreras for their help in field and/or laboratory activities. This study was funded by Universidad Nacional Autonoma de Mexico. LITERATURE CITED Baisre. J. A. 2000. The Cuban spiny lobster fishery. In: B. F. Phillips & J. Kittaka. editors. Spiny Lobsters: Fisheries ami Ciillure. 2nd edilinn. Oxford: Fishing News Books, pp. 135-152. Begon, M. 1979. Investigating Animal Abundance: Capture- Recapture tor Biologists. London: Edward Arnold. 97 pp. Bishop, J. A. & D. J. Hartley. 1976. The size and age structure of rural populations otRauus ni>negicii.s containing individuals resistant to the anti-coagulant poison warfarin. / Anini. Ecol. 45:623-646. Bishop. J. A. & P. M. Sheppard. 1973. An evaluation of two capture- recapture models using the technique of computer simulations. In: M. S. Bartlett & R. W. Hiorns, editors. The Mathematical Theory of the Dynamics of Biological Populations. 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Panulinis guttatus. in Bermuda, Ecology 34:794-796, Wenner, A. M. 1972. Sex ratio as a function of size in marine Crustacea. .Am. Nat. 106(949):321-350. Winer, B. J. 1971. Statistical Principles in Experimental Design, 2nd edi- tion. Tokyo: McGraw-Hill. 908 pp. Zar. J. H. 1984. Biostatistical Analysis, 2nd edition. New Jersey: Prentice- Hall. 718 pp. Jounuil of Shellfish Rcscanh. Vol. 21. No. 1. 289-294. 2002. RESPONSES OF MIGRATING WESTERN ROCK LOBSTERS PANULIRVS CYGNUS (GEORGE, 1962) TO TWO DIFFERENT TAGGING RELEASE PROCEDURES ROY MELVILLE-SMITH' AND YUK WING CHENG^ ^Western Australian Marine Research Laboratories, P.O. Box 20. North Beach. Western Australia. Australia 6020: 'Western Australia Centre of E.xcellence in Industrial Optimisation. Curtin Universirs' of Technology. GPO Box U1987 Perth. Western Australia. Australia 6845 ABSTRACT In the 1997/1998 season. 3.412 migrating 'white' sub-legal sized western rock lobsters were tagged at Jurien Bay (42—19 m depth), and Cervantes (92-133 m depth) on the Western Australian coast. Most lobsters in = 2.245) were brought ashore to be tagged, held overnight in coffs. and released 1 8-24 hours after capture within 6 km and + 1 0 m depth froiT) where they were taken. A smaller number (n = 1,167) were tagged and released at sea irnmediately after capture. Over three seasons, more animals tagged at sea were recaptured than those tagged ashore (P < 0.01). At both sites, lobsters tagged ashore moved significantly further and faster than those tagged at sea {P < 0.01 ). The correlation between the angle of movement and the speed of movement ranged from 0.25 to 0.42 {P < 0.05 ) for the two tag-and-release procedures and two sites. However, all the lobsters moved in a northerly to north-westerly direction. Growth increments in the first moult after release were unaffected by the different tag and release procedures (P = 0.08); loss of a single limb was. however, likely to result in a 229c decrease in the growth increment. KEY WORDS: Pamilinis cygiuis, tagging, movement, mortality, growth, migration INTRODUCTION Over the last three decades, tens of thousands of western rock lobsters {Paniilinis cxi^niis. George, 1962) ha\e been tagged to estimate movement, growth, natural mortality and discard mortal- ity. Tag and relea.se programs are generally expensive, as they usually require deploying research vessels, chartering commercial fishing vessels, or purchasing part of a cominercial catch. Often only selected size classes are required for tagging, therefore it would be more economical for commercial fishers to retain indi- viduals with the required characteristics and in this way accumu- late large numbers of the animals being targeted for tagging. However, accumulating animals for later tagging would inevi- tably mean displacing theni from their area of capture and previous re,search suggests that this can affect subsequent growth (Brown & Caputi 1983, 1984), recapture rates (Chittleborough 1974: Brown & Caputi 1983) and movenient patterns (Chittleborough 1974) and may lead to nomadism (Hennkind 1980). The object of this investigation was to compare the results of two tag-and-release procedures: one where migrating lobsters were released to the waters where they were caught straight after cap- ture: and the second to bring the lobsters ashore to be tagged, and return them to roughly the saine area the next day. In both cases recapture rates, distance speed, direction of movement and growth increments have been compared for the two release procedures. The lobsters tagged in this study were all migrating animals, known colloquially as 'whites' because they are paler than the dark red resident animals. For most of their lives, adult western rock lobsters show only limited movement: only during the white phase when the lobsters are 4—5 years old, do they move for any distance, migrating from the shallow inshore coastal reefs to the offshore breeding grounds (Morgan 1977: Phillips 1983). The migration takes place each year between late November and January. Tag- ging data have shown that, while most migrating animals move directly offshore, a significant number make more extensive long- Corresponding author: Roy Melville-Smith. Western Australian Marine Research Laboratories. P.O. Box 20. North Beach, Western Australia, Aus- tralia 6020. E-mail: rmsniith@fish.wa.gov.au shore migrations, generally in a north-westerly direction (Phillips 1983: Cheng & Chubb 1998). METHODS Sampling A total of 3,412 migrating sub-legal size ""white" lobsters in the size range 65 mm to 77.9 mm carapace length were tagged and released west of Jurien Bay between 29 December 1997 and 4 January 1998 in 43— +9 in and south-west of Cervantes between 29 December 1997 and 6 Januar> 1998 in 92-133 m depth (Fig. 1. Table I ). The depths at Jurien Bay and Cervantes were chosen because they represented two different stages of the offshore mi- gration, the animals in 43^9 m range being in the process of moving to deeper depths offshore and those at the Cervantes site being at the offshore limit of the migration run. All of the lobsters used for tagging were caught during the course of commercial tlshing. The experimental lobsters (Cer- vantes. ;) = 1.400 and Jurien Bay. /; = 845) were kept alive in tanks with flow-through water circulatory .systems and brought ashore to be tagged. After tagging, they were kept overnight in plastic fishing baskets tied to the side of the fishing boats. The next day. they were placed in tanks, taken to the fishing grounds and released { 18 to 24 hours after capture) together in a single batch on suitable substrate within 6 km and ±10 m depth from the respective sites where they were caught (Table 1 ). GPS details of their release positions were recorded. The control lobsters (/; = 1, 167) were tagged and released at sea within 12 minutes of capture and within 500 m of where they were caught (Table 1 ). All lobsters were tagged ventrally with individually numbered Hallprint type TEA- 1 internal anchor tags, as described by Melville-Smith and Chubb (1997). The tag number, size, sex. number of appendages missing, dates of capture and release, and position and depth of capture and release were recorded for each tagged lobster. Tagging at Cervantes and Jurien Bay was done by different people, both experienced at tagging lobsters: at each site the same person tagged at sea and ashore. Tagged lobsters were generally recaptured by commercial fish- ers, although small numbers were caught by research and recre- 290 Melville-Smith and Cheng 30°00'S 30°20'S 30°40'S 31°00'S INDIAN OCEAN 114''30'E 115"0U'h I15°20'E Figure 1. The Western Australian coast, showing where tagged lob- sters were released in this study. ationul fishers. All tag recaptures were made during the fishing season, which extends from mid-November to 30 June. Fishers were encouraged to provide details of tagged lobsters by offers of a AUD$2 instant lottery ticket for information relating to any tagged animal recaptured. They were asked to record size sex. number of limbs lost. date, position and depth of capture. by their times (one or two and three .seasons) at large and any recaptured within M) days of release were excluded from analyses calculating the mean time at large and distance from release sites, to avoid biasing the results by including migrating individuals. The second and third seasons were combined instead of being treated separately because published and unpublished data show that once western rock lobsters have undertaken their migration and have settled on the deep water spawning grounds, that thereafter their movements are limited. Combining the second and third years increased the power of the statistical tests. Recaptured animals were analyzed for defined time periods at large by student-r tests to estimate the power of the test. General linear modeling was used to model the size of the first growth mcrement after release, with distance moved and loss of limbs treated as covariates; and sex. site of release and release procedure as factors. The uniformity of the directional movement of lobsters tagged at sea and ashore was examined by a Rayleigh test (Zar I99S) and the mean angle of movement of the animals tagged by the two procedures relative to 0° (i.e., True North), was tested by a Wat- son-Williams test (Zar 1998). Angular-linear correlations for lob- sters tagged at sea and ashore were determined by the method described in Mardia (1976) and Johnson and Wehrly (1977). Re- gression methods were used to establish the relationship of the angular movement to the maximum speed of movement. RESULTS Recapture Rate The mean percentage of tagged rock lobsters that were recap- tured from the two release sites and tagging procedures ranged from 7.4% to 12.6% (Table 1). A generalized linear model showed that recapture rates were unrelated to the two release locations (P = 0.38). but that they were significantly correlated with the two tagging procedures (P = 0.03). A binomial test on two proportions (Zar 1998) produced a similar outcome. The overall recapture rate of lobsters tagged at sea was about 4Vr hiaher than those that were tagged ashore. Analysis The percentage of tag recaptures to numbers tagged were com- pared for the two tagging sites and their release sites by a gener- alized linear model with binomial family (logit link). The mean time at large, distance from release sites and speed of movement were calculated. The last two were based on the shortest distance between the point of release and recapture. Animals were grouped Time at Large The time at large before recapture can influence comparisons of different tag-release procedures. For example, one group of lob- sters may move further than another, not because of the experi- mental procedure, but because they were at large for longer. In the first season (to the end of June 1998) after release, animals that were tagged ashore were recaptured significantly later (P < 0.03: TABI.K 1. Western rock lobsters tagged at sea and on shore at Cervantes and .lurien Bay on respectively 29 December 1997 to 6 January 1998 and 29 December 1997 to 4 January I99S, and recaptured by .V) June 20(11. Tagging Procedure Ashore At Sea Release Sites and Depths Release (n) 1 400 S45 224.5 Recaptured (n) Percentage Recaptured Released (n) Recaptured (nl Percentage Recaptured Cervantes (92-133 m) Jurien Bay (43-49 in) Total 103 79 IS2 7.36 9.35 S.ll 564 603 1167 71 72 143 i2.5y 1 1 .94 12.25 Migrating Lobsters and Tag Release Procedures 291 TABLE 2. Days at large (Days) of western rock lobsters released at Cervantes and Jurien Bay in 1997/98 after tagging either at sea (h = 11671 or ashore (;; = 22451. Results are for the sexes combined, recaptured by the end of the first, and second and third fishing seasons at large. Days Before Recapture End of First Season (30 < days <210 After Release) Second and Third Seasons (21)) < days <(210 + 730) Days After Release) Days (mean) SE P Value Days (mean) SE P-value Tagged at sea Tagged on shore .SI ?() 103.12 I 11.70 2.41 2.96 0.0.^ 72 60 414 4.^0,4 9.14 y.24 ().3S conihined sexes) than those that wefe tagged at sea (Table 2). However, in the second and third seasons at large, there was no significant difference between the two groups (P = 0.38: com- bined sexes) (Table 2). Although the data have not been presented here, neither the release sites nor the sex of the lobsters produced different results. Distance Moved Lobsters that were tagged ashore inoved greater distances on the whole than those tagged and released at sea. This difference was significant (P < 0.01) for recaptures at both Cervantes and Jurien Bay made by the end of the first season (Fig. 2). The trend remained clear when only recaptures made in the second and third seasons were considered, but was only statistically significant for the Cervantes data (Fig. 2). The substantial differences in the dis- placement of recaptured lobsters at the two sites and depths have precluded the data being combined in analyses comparing their movement. Depending on whether they had been tagged at sea or ashore, lobsters that were tagged at Cervantes and that were at large for two or three seasons had moved about the same distances as those that had been at large for only one season (means ranging from 38.23-45.53 km for those released ashore, compared to 8.98-17.01 km for those released at sea. Fig. 2). This did not apply to lobsters released at Jurien Bay, where the one-season lobsters had moved shorter distances than the two or more season lobsters (means of 10.87 and 5.03 km for those released ashore and at sea respectively by the end of the first season, compared to 30.06 km and 15.78 km for those released ashore and at sea respectively at the end of the combined second and third seasons at large. Fig. 2). However, the proportional differences moved by the at-sea and ashore release groups at Jurien Bay was similar for those at large for one com- pared to two or more seasons, in all cases those tagged ashore moving two to three times further than those released at sea. Speed As there is little movement by western rock lobsters after their "whites" migration phase which ends by late January, only data for recaptures made from 1 February to 30 June 1998 (i.e., within the first season after release) have been presented in Table 3. Bearing in mind the differences in distance moved between lobsters tagged at sea and ashore (Fig. 2), it is not surprising that the two groups showed significant differences in their speeds of movement at both tagging locations (Table 3). Direction of Movement Compaiisons using a Rayleigh test of uniformity of movement showed that the lobsters did not migrate uniformly in one direc- tion, regardless of whether they were tagged at sea or ashore. A Watson test to compare the mean angles of movement from the two treatments showed no significant difference for either those animals released in the intermediate depths at Jurien Bay (P = 0.2), or those released in the deep water at Cervantes (P = 0.6). In both cases the mean direction of movement was north to nor-nor- westerly. Relationships Between Direction, Distance and Speed of Movement Angular-linear conelations (Mardia 1976; Johnson & Wehrly 1977) used to determine whether distance or speed of nio\ement is correlated with directi(.)n of movement, were determined separately for the two tagging sites and for the animals released at sea and ashore. Some, but not all, of the correlations comparing angle of movement to distance moved were significant (Table 4); however, angle of movement was significantly correlated (P < 0.05) with speed of movement in all cases. A regression analysis comparing speed and angle of movement showed that, while the general direction moved was northerly, recaptured lobsters that had been tagged ashore had a more west- erly component to their movement. Since the correlation between angle and speed of movement was higher for lobsters tagged ashore (0.34, 0.42) than at sea (0.25, 0.33). this indicates that migrating western rock lobsters moved fastest when heading in a north-westerly direction. Growth Increment The results from a general linear model of the growth incre- ments of lobsters that were likely to have moulted once between release and recapture (i.e.. only animals recaptured between Feb- ruary 1998 and July 1998) are shown in Table 5. Distance moved is treated as a covariate (P = 0.15). and sex (P = 0.46) and TABLE 3. Mean speed of movement of recaptured yvestern rock lobsters tagged at Cervantes and Jurien Bay in December 1997 and January 1998 and recaptured between 1 February and 30 June 1998 (i.e., within the first season after release). Release Site and Tagging Recaptu red Speed Depth (m) Procedure (H) (m/day) S.E. P = value Cervantes Ashore 27 392. 1 55.64 92-133 m At sea 26 142.1 30.35 <0.0I Jurien Bay Ashore 23 103.7 28.18 43^9 m At sea s5 -S2..S2 4.50 0.01 292 Melville-Smith and Cheng p<0 01 (a) p<0 01 n=23 n=55 Cervantes Cervantes Ashore At-sea Jurien Bay Jurien Bay Ashore At-sea 50 1 J p<001 40 1 30 20 n=30 n=64 10 0 Cervantes Cervante Ash ore At-sea (b) p=0 32 n=30 i Jurien Bay Jurien Bay Ashore At-sea Figure 2. Mean distance western rock lobsters moved after tagging at Cervantes and Jurien Bay in December I**"}? and January 1998 (a) by the end of the first season excluding the first month after release and (b) in the sec90 m) follows the contours northward, before peeling off inshore along the way to settle on the breeding grounds in depths of around 40-90 m. The distance and direction moved by animals tagged in the depths reported in this study are consistent with the scenario sug- gested above. Those tagged in the middle depths (43—17 m) at Jurien Bay generally moved shorter distances than those in the deepwater (>90 m) at Cervantes. These middle depths (40-90 m) is where the resident breeding stock population occurs. Interest- ingly, lobsters at large for one season at Cervantes, whether tagged at sea or ashore, moved roughly the same distances as those at large for two or three seasons. In contrast, at Jurien Bay. lobsters recaptured in the second and third seasons had moved further than those recaptured in the first season. However, animals tagged at sea and ashore both moved greater distances in the second than in the first. The lack of difference in distances moved once the white migrating lobsters become resident (within the first season) sug- Migrating Lobsters and Tag Release Procedures 293 TABLK 4. Correlations between displacement and angle of moement. and speed and angle of movement of western rock lobsters tagged either ashore or at sea and released at one of two different sites. Correlation Between Correlation Release Tagging Distance Moved and Between Speed and Location Procedure Tagged ashore Angle of Movement P-value Angle of Movement P-valuc Cervantes 0.18 0.3-'i 0.34 0.03 92-133 m Tageed at sea 0.31 0.01 0.2.^; 0.05 Jurien Bay Taased ashore 0.33 0.04 0.42 0.01 43-49 ni Tagged at sea 0.27 0.10 0.33 0.03 gests that the tagaing-release procedure affects distance only in the first season. The differences between the distance travelled by displaced lobsters (shore tagged) and those released where they were cap- tured suggests that movement data from animals tagged during their migration phase be interpreted cautiously. The spatial and temporal displacement resulting from bringing the animals ashore to be tagged and releasing them at a distance from their catch site clearly has an effect. Smaller displacements, such as result from the drifting of tagging platforms or being held overnight in a pot when captured, might also affect the movement of lobsters tagged at sea dunng their migratory phase. Results from this study would therefore suggest that the effects of release procedures on the behavior of migrating lobsters, must be considered before biologi- cal conclusions are drawn. No oceanographic data were collected, either at the tagging locations, or at the anchorages where the animals were held over- night. However, substantial amounts of environmental data have been collected on the western rock lobster grounds in the past, and it can be confidently assumed that differences in the salinity, tem- perature and oxygen concentrations in the coastal waters w here the lobsters were held, compared to the offshore waters where they were caught, would have been well within the seasonal ranges experienced by the species. This does not preclude the possibility that the overnight acclimation of the animals to the changes in water conditions may have contributed to the different behavior of the at-sea and ashore tagged animals. The mean speeds of movement recorded by the inigrating ani- mals tagged in this study are substantially slower than those (1.2 km per day) recorded by Cheng and Chubb (1998). Our speeds have been underestimated because all recaptures over the first season at large were used in the calculation, but western rock lobsters migrate only between late November and late January. The strong correlation between angle of movement, and both dis- TABLE 5. Results of a general linear model of growth increments of lobsters between February 1998 and July 1998 (one moult increment). Name of Variable Estimated P-value Intercept 4.28 0.00 Distance moved 0.00 0.15 Released procedure -0.4.^ 0.08 Released location 0.61 0.03 Sex -0.19 0.46 Single limb loss at tagging -0.94 0.01 tance traveled and speed of movement, is a reflection of the lob- sters that moved furthest (and therefore recorded the fastest speed of movement while at large) generally heading in the northwest- erly direction almost parallel to the coastline, whereas those that moved least (and therefore recorded the slowest speed of move- ment while at large) generally heading in a northerly direction. Single growth increments of lobsters at the end of the first season (after their first moult) did not suggest that migrating ani- mals tagged at sea grew either faster or slower than those held ashore overnight and displaced from their point of capture (P > 0.05). This result differed from that of Brown and Caputi (1984). who found that displaced undersize western rock lobsters of a larger size range than those used in this study, had significantly {P > 0.05) smaller growth increments than nondisplaced animals. Al- though the animals in this study would presumably have been stressed immediately after capture, during tagging and possibly while being held overnight, these affects have previously been shown (Melville-Smith et al. 1997) to be short-term and unlikely to affect the moult increment later in the season. The significant diffeience (P = 0.03) in growth increment between the two tag- ging sites is likely to be due to regional differences in growth, which are well documented in this fishery (Chittleborough 1976; Joll & Phillips 1984). This study investigated the effects of different handling meth- ods and. on the basis of the results, drew some conclusions about the scope for possible cost reduction in tagging programs by hav- ing fishers accumulate selected size-classes of migratory lobsters for tagging ashore. The result has shown that this approach would not be feasible for studying movement patterns or for purposes where recapture rates are critical to the result, such as estimating mortality and/or population sizes. However, growth increments were not significantly affected; if growth measurements were the only requirement from the data, then this cooperative approach between research and industry could result in affordable and reli- able tagging projects. Comparative cost savings of tagging the cumulative catch of a number of fishers ashore, as opposed to a single fisher at sea is not possible, because the result is dependent on catch rates of the animals being selected for tagging and on the number of fishers accumulating that part of their catch. ACKNOWLEDGMENTS We thank Kim Brooks and Doug McCashney for tagging the lobsters in this study, the skippers and their crews for their help and the many fishers and rock lobster processors who returned the recapture details of tagged lobsters. Sonia Anderton ran the tag- ging data base, Mark Rossbach and Jim Christianopoulos did much of the field organization of staff and fishers. Lynda Bellchambers. 294 Mklville-Smith and Cheng Nick Caputi. Vivienne Mawson and Bruce Phillips otTered Liselul suggestions lor improving earlier drafts of this manuscript. The work was part funded by the Fisheries Research and Development Corporation (Project 96/108). Brown. R. S. & N. Caputi. 1983. Factors affecting the recapture of under- size western rock lobster Pamtlinis cygnus George returned by fisher- men to the .sea. Fish. Res. 2:103-128. Brown. R. S. & N. Caputi. 1984. Factors affecting the growth of undersi/e western rock lobster. Painilinis cyginis George, returned by fishermen to the sea. Fish. Bull. 83:567-574. Cheng. Y. W. & C. F. Chubb. 1998. Statistical analysis of the directional movement of the western rock lobster. Proceedings of the Sixth Inter- national Applied Statistics iii Industry Conference. RMIT, Australia, pp. 9-16. Chiuleborough. R. G. 1974. Home range, hommg and dominance m juve- nile western rock lobsters. Aiist. J. Mar. Frcslnicil. Res. 25:227-234. Chittleborough. R. G. 1976. Growth of juvenile Pamilinis limgipes cygnus George on coastal reefs compared with those reared under optimal environmental conditions. Aiisl. ,/. Mar. Fre.shwal. Res. 27:279-295. Gray. H. 1992. The western rock lobster Paimlirus cygnus. Book 1: A natural history. Geraldton: Westralian Books. 112 pp. Hemikind. W. F. 1969. Queuing behavior of spiny lobsters. Science 164: 1425-1427. Hermkind, W. F. 1980. Spiny lobsters: patterns of movement. In: J. S. Cobb & B. F. Phillips, editors. The Biology and Management of Lob- sters. New York: Academic Press, pp. 349—107. Herrnkind. W. F., J. VanderWalker & L. Barr. 1975. Population dynamics, ecology and behavior of spiny lobster. Paniilinis argns. of St. John. U.S. Virgin Islands: Habitation and pattern of movements. Results of the Tektite Program. Vol. 2. Sci. Bull.. Nat. Hi.st. Mus.. Las Angeles Cry. 20:31-45. Johnson. R, A. & T. Wehrly. 1477. Measures and models for angular LITERATURE CITED correlation and angular-linear correlation. J. Ray. Slat. Sac. Series B, 52:183-189. Joll. L. M. & B. F. Phillips. 1984. Natural diet and growth of juvenile western rock lobsters Panulirus cygnus George. J. E.xp. Mar. Bial. Ecol. 75:145-169. Kelly. S.. A. B. MacDiarmid & R. C. Babcock, 1999. Characteristics of spiny lobster, Jasus edwardsii. aggregations m exposed reel and sandy areas. Mar. Freshwat. Res. 50:409-416. Lohmann. K. J.. N. D. Pentchetf. G. A. Neviit. G. D, Stetten. R. K. Zimmer-Faust. H. E. Jarrard & L. C. Boles. 1995. Magnetic orientation of spiny lobsters in the ocean: experiments with undersea coil systems. J. Exp. Biol. 198:2041-2048. Mardia. K. V. 1976. Linear-angular correlation coefficients and rhythmom- etry. Bionielrika 63:403—105. Melville-Smith. R. & C. F. Chubb. 1997. Comparison of dorsal and ventral tag retention in western rock lobsters. Paimlirus cygnus (George). Mar. Fre.slmar. Res. 48:577-580. Melville-Smith. R.. J. B. Jones & R. S. Brown. 1997. Biological tags as moult indicators m Panulirus cvgnus (George). Mar. Freshwar. Res. 48:959-965. Morgan. G. R. 1977. Aspects of the population dynamics of the western rock lobster and their role in management. PhD. Thesis. University of Western Australia. Nedlands. Western Australia. 341 pp. Phillips, B. F. 1983. Migrations of pre-adult western rock lobsters. Panu- lirus cygnus. in Western Australia. Mar. Biol. 76:311-318. Zar. J. H. 1998. Bioslatistical analysis. Upper Saddle River. NJ: Prentice Hall. 663 pp. Journal ,>f Shfllfish Research. Vol. 21. No. I. 295-298, 2002. MUSHROOM TYROSINASE AS A CONTROL MATERIAL FOR PHENOLOXIDASE ASSAYS USED IN THE ASSESSMENT OF CRUSTACEAN 'HEALTH * A. BATTISON.' R. CAWTHORN," B. HORNEY,' AND A. MACKENZIE^ ^Departiiwnt of Pathology ciiul Microhiology, Athiiitic Veterinary College, University of Prime Edward Island. Charlottetown, Prinee Edward Island. Canada: 'Atlantie Veterinary College Lobster Seienee Centre. Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, Prince Edward Island. Canada ABSTRACT Mushroom tyrosina.se (E.C. 1.14.18.1 1 was evaluated as a control material for use in a plasma prophenolo.\idase assay in the American lobster Homanis amerkanits as pan of the assay's validation procedure. Reconstituted, lyophilized tyrosinase solution was stable for 72 h at 2— +°C. Percent recoveries were 94%. 120%, 113%, and 48% for "mid-range"" activity dilutions and 93%, 94%. 87%. and 38% for "low-range'" activity dilutions at 24. 48, 72, and 96 h, respectively. The activity of the reconstituted tyrosinase solution was stable for 4 wk when stored at -80°C, and percentage recoveries, compared to activity of the freshly reconstituted solution, were generally within 15% for both mid-range and "high"-activity dilutions. The between-run coefficients of variation (CVs) were 10,6% for the mid-range and 10.8% for the high-activity dilution over the 4-wk period. Enzyme activity was unstable when stored at -20°C. Mushroom tyrosinase is an acceptable control material for use in assays of phenoloxidase activity. Inclusion of this control material will provide a means to confidently compare results on a day-to-day or run-to-run basis when phenoloxidase assays are used in the assessment of crustacean "health." KEY WORDS: prophenoloxidase assay, control, tyrosinase, crustacean, health INTRODUCTION The prophenoloxidase activating system (PPA.S) and its active en/.yme product, phenoloxidase (o-diphenoliO, oxidoreductase E.C. 1.14.18.1 ) are integral components of the innate defense sys- tem in crustaceans (Soderhiill et al. 1996; Soderhall & Cerenius 1998: Sritunyaiucksana & Soderhall 2000). Dark pigmentation (melaninization) at sites of injury reflects prior generation of phe- noloxidase, followed by the spontaneous formation of tnelanin (Soderhall et al. 1996: Soderhall & Cerenius 1998: Sritunyaiuck- sana & Soderhall 2000). Both the reactive quinone intermediates and the final melanin products fomied during this reaction have antimicrobial and protective properties (Soderhall et al. 1996: Ri- ley 1997). Determination of the activity of phenoloxidase in either whole hemolymph or hetnocyte lysate supernatants has been used to as- sess the "health"' or "'immune" status of crustacean species (Hauton et al. 1997; Sritunyaiucksana et al. 1999; Rodriguez & Le Moullac 2000). Commercial test kits' are available to detect phenoloxidase activity in whole hemolymph samples from shrimp. It is recog- nized that assays used in the assessment of crustacean health should be standardized (Bachere 2000). Evaluation and validation of a laboratory test are required to confirm that an assay is performing within its defined parameters before it can be accepted for use in a diagnostic setting (Bellamy & Olexson 2000). This includes determination of the assay's re- portable range, precision, accuracy, and reagent stability. Control materials are essential to monitor the performance of the assay (Westgard & Klee 1999). Inclusion of a control with each group of samples permits reliable comparison of the results obtained from samples analyzed during different runs. Ideally, control materials are derived from the same biological source as the samples that are being evaluated. When this is not possible, materials with similar properties can be substituted (Westgard & Klee 1999). Most phenoloxidase assays are based on (or are modifications 'Spot On. DiagXotics hic. Wilton. CT 06897. of) the method of Horowitz and Shen (Horowitz & Shen 1952; Aspan & Soderhall 199.5). During development of an assay to detect prophenoloxidase activity in plasma of the American lob- ster, Homarus americanus H. Milne Edwards, a control material was required. A purified source of lobster phenoloxidase having consistent activity was not available. Lyophilized mushroom ty- rosinase was evaluated as a control material because of its simi- larities to phenoloxidase and commercial availability (Aspan & Soderhall 1995). MATERIALS AND METHODS Mushroom tyrosinase (E.C. 1.14.18.1) stock solution was pre- pared by adding 2.5 mL of sodium phosphate buffer (0.2 M Na^POj. pH 7.5) to 13 mg of lyophilized mushroom tyrosinase (Sigma Chemical Company. St. Louis, MO), giving a final con- centration of approximately 10.700 units of tyrosinase activity per niL of solution. Serial dilutions of this reconstituted stock solution were used to detei'mine assay linearity, sensitivity, and within-run precision as determined by calculation of coefficient of variation (^rCV). A second stock solution, calculated to have similar activ- ity per unit volume, was prepared from a different lot of lyophi- lized tyrosinase. This second solution was used to conduct refrig- erated and frozen stability trials. Eight 150-|ji.L aliquots were placed in 0.5-niL polypropylene microcentrifuge tubes (Fisher Sci- entific, Canada). Four aliquots were stored at -20°C, four aliquots were stored at -80"C, and the remaining solution was refrigerated (2^°C), for stability trials. To complete linearity and precision assessments, serial dilu- tions at 1:200, 1:400, 1:800, 1:1600, 1:3200, and 1:6400 of the stock solution were made using a 0.2 M sodium phosphate buffer. pH 7.5. Twenty microliters of each dilution was added to indi- vidual wells of a flat-bottomed tissue culture plate (Falcon Mi- crotest 3072, Becton Dickinson and Company, U.S.A.). There were five replicates per dilution. Wells to be used as "substrate- free blanks", one well for each dilution, also received 20 (xL of the appropriate tyrosinase stock dilution. Twenty microliters of phos- phate buffer was placed in wells used as reagent blanks. Eighty microliters of sterile, distilled water was added to all wells using a 295 296 Battison et al. multichannel pipette. One hundred microhters of the substrate so- lution. 3.8 mM dopamine (3-hydro.xytyramine) (Sigma Chemical Company ). was then added to all wells except for the substrate-free blanks. These wells each received 100 |xL of sterile distilled water. Production of the gold-orange quinone intermediate was moni- tored every 1 I sec (wavelength 470 nm) during a 5-min period in an automated spectrophotometer (SpectraMax. Molecular Devices Corporation. U.S.A.) after a 3-sec mixing cycle. The software package SoftMax^"^' (Molecular Devices Corporation, U.S.A.) was used for calculation of maximum enzyme activity (V,^i„), defined as the rate of maximal change in optical density (OD) of the reaction solution per minute (A mOD/min) over a user-defined time interval. The V^nax used for all further calculations for each dilution was the mean V^nax of the five replicates. Within-run co- etTicients of variation [ %CV = (standard deviation/mean) x 100] were then calculated (Table 1 ). Stability of the refrigerated stock solution was determined by calculating the percentage recovery for two dilutions, identified as having "low" and "mid-range" activities relative to the linear range of the assay, over a period of 4 days. Percent recovery was cal- culated as: [(measured activity at time ".v"/initial activity at recon- stitLition) X 100] at 24. 48. 72. and 96 h (Table 2). Four replicate wells were used for each dilution. Long-term frozen stability of the stock solution, stored at -20°C and -80°C, was evaluated by measuring enzyme activity in serial dilutions of a thawed (room temperature. 20-22°C) aliquot every week for 4 wk. Four replicate wells were used for each dilution. Percent recovery calculations were made for the aliquots stored at -20°C and -SOX (Tables -3a and 3b). Between-run (week to week) %CVs were calculated for each dilution of the aliquots stored at -80°C (Table 4), The refrigerated stability of thawed stock solution, after storage at -80°C, was also evaluated. Enzyme activity was measured at 48 and 72 h post-thawing for two dilutions (mid-range and high ac- tivities). Percentage recoveries were calculated by coniparing the activity after 2 and 3 days of refrigeration to the activity obtained immediately after thawing (Table 5). RESULTS No significant absorbance was detected in the substrate-free blank wells during the initial assays. Substrate-free blanks were not used for the remaining assays. Reagent blanks were used for all assays and also functioned as the plate blanks. The reaction curve TABLE L Sensitivity, linearity, and precision results for reconstituted mushroom tyrosinase. Expected" Activity Observed Activity" cv Dilution (mOD/min) (mOD/minl SD' (%) 1:200 — 41.6 0.53 1.3 1:400 20.8 19.6 0.16 0.8 1:800 10.4 9.3 0.39 5.0 1:1600 5.2 5.0 0.32 6.5 1:3200 2.6 1.4 0.20 14.3 1:6400 1.3 0.4 0.23 60.7 "Calculated from the 1:200 dilution. " Represents the mean of five replicates. ^' Standard deviation. '' Coefficient of variation I^CV = (standard deviation/mean) x 10()|. TABLE 2. Refrigerated (2-4 C) stability of reconstituted mushroom tyrosinase. ... , Percent Reco\er\ (9c)" Initial Activity" ^ (niOD/min) I day 2 days 3 days 4 days Dilution no. I Dilution no. 2 22.4 12,3 94 93 120 94 113 87 48 38 'Percent recovery = (measured activity/uiltial ,ii.ii\itvi x ]()(), ' Represents the mean of four replicates. was linear during minutes 2—1 of the 5-min nnniitoring period, ^niav was determined froin this 2-min period. Initially, a gold- orange product developed in all of the reaction wells containing the dopamine substrate and the mushroom tyrosinase. Subse- quently, a black product also appeared in the wells. At first, only small amounts of the black product were present; however, with time, the gold-orange product was no longer visible and only the black product could be seen. The amount of this second product seemed proportional to the concentration of enzyme solution in the wells. The catechol oxidase activity of mushroom tyrosinase, as mea- sured in this assay system, was linear, and had acceptable precision results, from 5.0 to 41.6 mOD/tnin when evaluated by serial dilu- tion (/?- = 0.99) (Table 1. Fig. 1). Using the activity of the 1:200 dilution as a starting point, the expected and observed (in paren- theses) activities (mOD/min) for each further dilution were 20.8 ( 19.6) for the 1:400 dilution. 10.4 (9.3) for the 1:800 dilution, 5.2 (5.0) for the 1:1600 dilution. 2.6(1.4) for the 1 :3200 dilution, and 1.3 (0.4) for the 1:6400 dilution. The within-run CVs in = 5 replicates), for each dilution were 1.3% for the 1:200 dilution. 0.8% for the 1:400 dilution, 5.0% for the 1:800 dilution, 6.57c for the 1:1600 dilution, 14.3% for the 1:3200 dilution, and 60.77f for the 1:6400 dilution (Table 1). The stability of the freshly reconstituted reagent was acceptable (i.e., generally within 15% of the initial values) for up to 72 h after refrigeration, but decreased to an average of 43% of initial activity by 96 h (Table 2). Percent recoveries for a mid-range dilution at 24. 48. 72. and 96 h were 94%. 120%, 113%. and 48%. respec- tively. Percent recoveries for a low-activity dilution at 24. 48. 72, and 96 h were 93%. 94%. 87%, and 38%, respectively (Table 2). The stock solution was unstable when stored at -20°C. The enzyme activity, calculated as a mean of all dilutions, decreased to 37%, 28%, 27%, and 21% of the initial activity of the freshly re- constituted reagent by weeks 1, 2, 3, and 4, respectively (Table 3a). The stock solution was stable when stored at -80°C. The per- cent recoveries, average of mid-range (dilution no. 2) and high- (dilution no. 1 ) activity dilutions, by week, were 87% for week 1. 99% for week 2. 1 16% for week 3, and 106% for week 4 (Table 3b). The between-run CVs, over the 4-wk period, were 10.6% for the mid-range dilution and 10.8% for the high-activity dilution (Table 4). The refrigerated stability of the thawed stock solution was generally satisfactory for weeks 1-3. but tended to deteriorate by week 4 of storage at -80 C (Table 5). DISCUSSION The value of any assay resides in its ability to consistently provide accurate and precise results. An incorrect result can be Mushroom Tyrosinase: Control for Phenoloxidase Assays 297 TABLE 3a. Frozen stability of mushroom tyrosinase stock solution stored at -20'C for 4 weeks. TABLE 4. Weekly between-run coefficients of variation (CV I for mushroom tyrosinase activity when stored at -80^C. Initial Activity' (mOD/min) Percent RecoM ;ry ( ^r f Mean' ' Activity (mOD/min) Dilution 1 wk 2 wk : 1 wk 4 wk Weeks at -80C Dilution No. 1 Dilution No. 2 No. 1 33.4 37 2,S 26 22 0 33.4 17.0 No. 2 17.0 37 2S 28 1^) 1 28.4 15.1 Mean 37 28 27 21 ~) 31.1 17.7 ° Percent recovery = (measured activity/initial activity) x 100. 3 4 37.2 35.7 20.2 17.9 *■ Represents the mean of tour replicates. Mean SD" 33.1 3.5 17.6 1.9 v\ orse than no result at all. Assay validation include s quality con- CV-C/f) 10,6 10.8 trol steps used to ensure that diagnostic tests pert'orm within pre- determined specifications. Control materials are used to monitor the assay's performance on a run-to-run basis. Ideally, such ma- terials are derived from the same species as the samples to be analyzed; however, this is not always possible. Steps in assay validation usually include (but are not limited tol: determination of the assay's reportable range; evaluation of assay precision by cal- culating both within-run and between-run coefficients of variation C/fCV = standard deviation/mean x I (.XT); and. determining the stability of the assay reagents and the sample under different stor- age conditions (Bellamy & Olexson 2000). Phenoloxidase (d-diphenoliO, oxidoreductase E.C. 1.14.18.1) assays have been used to investigate and assess the health status of a variety of crustaceans in both research and commercial settings (Sritunyalucksana et al. 1999; Rodriguez & Le Moullac 2000). A stable source of enzyme to use as a control material was required in the development of a plasma prophenoloxidase assay for use in H. americanus. A commercial source of purified lobster (//. omeri- caiuis) phenoloxidase is not available. Lobster hemocyte lysale supernatant preparations represented a highly concentrated source of prophenoloxidase; however, these were unsuitable due to their poor long-term storage characteristics and the inherent, inconsis- tent activity between preparations (unpubl. results, ALB). Mushroom tyrosinase (E.C. 1.14.18.1) was evaluated as a po- tential alternative because of its similarity to phenoloxidase. Both enzymes have cresolase (monophenol monooxygenase) and cat- echol oxidase (polyphenol oxidase) activity (Aspan & Soderhall 1995). Determination of the enzyme's copper content and location of conserved histidine residues indicates that arthropod proph- enoloxidase belongs to the tyrosinase group of enzymes found in bacteria, fungi, and plants (Aspan & Sbderhiill 1995). The reported long-term stability of frozen, reconstituted mushroom tyrosinase TABLE 3b. Frozen stability of mushroom tyrosinase stock solution stored at -80 C for 4 weeks. Initial Activity" (mOD/min) Percent Recovery ( % )" Dilution 1 wk 2wk 3 wk 4 wk No. 1 33.4 85 93 112 107 No. 2 17.0 89 105 119 105 Mean 87 99 116 106 ' n = 4 replicates. ^ Standard deviation. " Coefficient of variation (mean/standard deviation) x 100. and its commercial availability made this enzyme an attractive alternative (Kertesz & Zito 1965). Mushroom tyrosinase was readily soluble in the 0.2 M sodium phosphate buffer (pH 7.5) used in this assay system. Other studies have used a sodium cacodylate-based buffer. Sodium cacodylate is listed as a hazardous material and is a recognized carcinogen (Sigma-Aldrich 2001 ). The sodium phosphate buffer used herein presents minimal hazards, is readily available, and was compatible with this assay system. L-DOPA (3.4-dihydroxyphenyl-L- alanine). a commonly used substrate in phenoloxidase assays, was not compatible with the phosphate buffer. Dopamine (3- hydroxytyramine) was chosen as an alternate catechol. Dopamine has been identified as a preferred substrate for phenoloxidase from Mciinltica se.xla (Sugumaran et al. 1999). This may also be true for other invertebrates. Mushroom tyrosinase converted the dopamine substrate to an orange-gold intermediate product, presumably the o-quinone. 4-(2-aminoethyl)-l.2-benzoquinone. This product was eventually replaced by a black precipitate (melanin), which is the expected result of activation of the PPAS /;; vivo (Soderhall et al. 1996; Soderhall & Cerenius 1998; Sritunyalucksana & Soderhall TABLE 5. Refrigerated stability of thawed mushroom tyrosinase stock solution stored at -SOX. Dilution Weeks at -80 C Initial activity'' (mOD/min) Percent recovery ( % )" 2 days 3 days No. 1 No. 2 33.4 28.4 31.1 37.2 36.7 17.0 15.1 17.7 20.2 17.9 nd^ 110 106 106 85 nd 106 86 104 84 nd 135 110 102 75 nd 1.39 103 97 89 'Percent recovery = (measured activity/initial activity) x 100. ' Represents the mean of four replicates. ' Percent recovery = (measured activity/initial activity) x 100. ' Represents the mean of four replicates. ' Not done. 298 Battison et al. > — O c ra t -a Q B O " E a: i, a 0 10 20 30 40 50 Observed activity (mOD/min) Figure 1. Linearity of catechol oxidase activit> of reconstituted mush- room tyrosinase determined by serial dilution. 2000). An identical series of products is observed when either lobster hemocyte lysate supernatant or lobster plasma, after acti- vation with trypsin, is used as the enzyme source for the assay. The reconstituted tyrosinase solution was stable, when refrig- erated, for up to 3 days as indicated by the percentage recovery studies (Table 2). Tyrosinase activity was stable for up to 4 wk when the reconstituted solution was stored at -80°C. Reconstituted mushroom tyrosinase was unstable when stored at -20°C, in con- trast to a previous report (Kertesz & Zito 1965). The discrepant results found in this study may reflect differences in the buffers used for reconstitution. Overall, the refrigerated and frozen (-80°C) stability of reconstituted mushroom tyrosinase was con- sidered acceptable for its use as an assay control material. Ideally, control materials contain a predetermined amount of the enzyme being measured and are incorporated in a material (matrix) similar to the matrix of the sample to be analyzed. In this study, mushroom tyrosinase was reconstituted in a phosphate buffer. This would be the optimal situation when phenoloxidase activity is being measured in hemocyte lysate supernatants where a similar buffer had been used to prepare the lysates. Similarly, incorporation of the mushroom tyrosinase into whole hemolymph or plasma collected from normal, apparently healthy animals would be preferable when phenoloxidase activity is being mea- sured in these types of samples. This was not evaluated in this study. When reconstituted in a sodium phosphate buffer, mush- room tyrosinase is a suitable control material for use in phenoloxi- dase assays, providing assurance that the assay is working cor- rectly and permitting comparison of within-laboratory sample re- sults on a run-to-run or day-to-day basis. Aspan. A. & K. Soderliall. IW5. The prophenoloxidase activating system in invertebrates; Assays of the prophenoloxidase activating enzyme (a serine proteinase) and phenoloxidase. In: J. S. Stolen. T. C. Fletcher. S. A. Smith. J. T. Zelikot'f. S. L. Kaattari. R. S. Anderson. K. Soderhall & B. A. Weeks-Perkins, editors. Techniques in tlsh immunology — 4. Fair Haven, New Jersey; SOS Publications, pp. 161-17(1. Bachere. E. 2000. Introduction; Shrimp immuniiy and disease control. Ai/KMultiire 191;.^-! 1. Bellamy, J. E. C. & D. W. Olexson. 2000. Evaluating laboratory proce- dures. In: J. E. C. Bellamy & D. W. Olexson. editors. Quality assurance handbook for veterinary laboratories. Ames; Iowa State University Press, pp. 61-77. Hauton, C, J. A. Williams & L. E. Hawkins. 1997. The effects of a live iii vivo pathogenic infection on aspects of the immunocompetence of the common shore crab. Cuniim.s nmenas (L.). J. Exp. Mar. Biol. Ecol. 211:115-128. Horowitz, N. H. & S. C. Shen. |y.'i2. Neurosporo tyrosinase. J. Biol. Cliem. 197:549-557. Kertesz, D. & R. Zito. 1965. Mushrooin polyphenol oxidase I. Purification and general properties. Biochim. Biophys. Acta 96:447—162. Riley, P. A. 1997. Molecules in hicus: Melanin. Int. .1. Bioclwrn. Cell Biol. 29:1235-1239. LITERATURE CITED Rodriguez. J. & G. Le Moullac. 2000. State of the an of immunological tools and health control of penaeid shrimp. Acjuaculture 191:109-1 19. Sigma-Aldrich Canada Ltd. 2001. Cacodylic acid sodium salt trihydrate material safety data sheet. Oakville; Sigma-Aldrich Canada Ltd. Soderhall. K & L. Cerenius. 1998. Role of the prophenoloxidase-activating system in invertebrate immunity. Ciin: Opin. Immunol. 10:23-28. Soderhall. K., L. Cerenius & M. W. Johansson. 1996. The prophenoloxi- dase activating system in invertebrates. In: K. Soderhall. S. Iwanaga & G. R. Vasta. editors. New directions in invertebrate immunology. Fair Haven; SOS Publications, pp. 229-253. Sritunyalucksana. K. & K. Soderhall. 2000. The proPO and clotting system in crustaceans. Aquaciiltiiic 191:53-59. Sntunvalucksana. K.. P. Sithisarn. B. Withayachymnarnkul & T. W. Fle- ael. 1999. Activation of prophenoloxidase. agglutinin and antibacterial activity in haemolymph of the black tiger prawn. Penaeiis monodon. by immunostimulants. Fish Shellfish Immunol. 9:21-30. Suguinaran. M.. R. Duggaraju. F. Generozova & S. Ito. 1999. Insect mel- anogenesis. II. Inability of Mandma phenoloxidase to act on 5.6- dihydroxyindole-2-carboxylic acid. Pigment Cell Res. 12:118-125. Westgard. J. O. & G. G. Klee. 1999. Quality management. In: C. A. Burlis & E. R. Ashwood. editors. Tietz textbook of clinical chemistry. 3rd ed. Philadelphia; W.B. Saunders, pp. 384-418. Mmnuil of Shellfish Research. Vol. 21. No. 1. 299. 2U02. ERRATUM ARTIFICIAL ENVIRONMENTAL CONDITIONS CAN AFFECT ALLOZYME GENETIC STRUCTURE OF THE MARINE GASTROPOD PATELLA CAERULEA ANNAMARIA MAURO, NICOLO' PARRINELLO, AND MARCO ARCULEO* Dipartiinemo ili Biologiu Aiilniale via Arcliirafi ]I0 mm) should be planted during April, at the beginning of the growing season, and protected with flexible net- ting (6.4 mm). DESIGN AND CONSTRUCTION OF SUBTIDAL OYSTER REEFS: EVALUATING EFFECTS OF REEF SCALE. Alan .1. Birth, M. W. Luckcnbach, P. G. Ross, R. Ganiniisch, and J. Brubaker, College of William and Mary. Virginia Institute of Marine Science, and J. A. Wesson, Virginia Marine Resources Commission. USA. Restoration of oyster reef habitat in mid-Atlantic estuaries is requisite for restoring oyster fisheries and recovering lost ecologi- cal services. Recent evidence suggests the importance of estab- Shellfish, Restoration, Nuiuiinio B.C., Canada Abstracis, September 2001 307 lishing proper vertical relief and interstitial space in the initial placement of substrate. A third component of reef architecture that has yet to be investigated is the aerial extent or scale of reefs. We are currently evaluating the effects of scale on various components of communities that develop on high relief reef bases ranging in size from 400 m^ to 8000 ". As a result, accurate physical descrip- tions of the constructed reefs are important to meaningfully quan- tify reef scale. We present some preliminary design and ct)iistruction aspects of three dimensional subtidal reefs in the Rappahannock River, a tributary of the Chesapeake Bay. Because of their subtidal nature, describing the reefs physical characteristics becomes difficult. Subsequently, Side-scan sonar mapping, bathymetry surveys and current velocity mapping were conducted during the first year after construction and will be continued in subsequent years as the reefs develop. From a management aspect, where potential substrate is limit- ing and expensive, any ecological advantages or disadvantages derived from allocating resources to many small vs. several large reefs can have direct economic impacts on restoration efforts. Fur- thermore, identification of details of the physical characteristics that may help achieve restoration goals can be extremely important in providing resource managers with the information that can aid future reef design criteria. THE MUCK STOPS HERE A COMMUNITY DECIDES. C. Boldt, Union Bay Liquid Waste Management Committee, P.O. Box 32, Union Bay, British Columbia. Canada, VOR 3B0. What does an ocean side community do when: • residents cannot safely harvest shellfish from its beaches for over 7 years'^ • a quiet, community walk lets you know who is washing clothes, who is having a shower, who has flushed the toilet? • its ditches are unsafe for child's play • its ditches are overgrown with water cress costing $1000"s of dollars to government to clean out? • kids swimming on local beaches can be a health issue? • over 60'^ of septic fields are failing? Well, one community said "Enough is Enough!" While its neighbour were saying NO to a 1 997 valley wide referendum on a solution to liquid waste problems. Union Bay said "Yes". Union Bay has worked hard to involve, inform, educate and enlist residents, local credit union, local businesses, politicians and government agencies from all levels to develop a solution. Working with major funding from Georgia Basin Ecosystem Initiative, Environment Canada and provincial grants. Union Bay has developed a liquid waste treatment solution which will ulti- mately turn waste to wealth, using liquid waste, including a cam- pus for post-secondary institutions to support their environmental program, enhance salmonid habitat, provide an intei"pretive centre. Community process has been the key to significant funding for this project and it has created a climate of acceptance and support for rural grassroots, community-based planning. This process has been accepted by the Regional District of Comox-Strathcona and adopted in at least three other rural communities in the Comox Valley. ACOUSTIC SEABED CLASSIFICATION FOR SHELLFISH HABITAT MAPPING. William T. Collins, Quester Tangent Corporation, Sidney. BC V8L 5Y8, Canada. The amplitude and shape of an acoustic signal reflected from the sea floor is determined mainly by the sea bottom roughness, the density difference between water and the sea floor, and reverbera- tion within the substrate. Remote classification of the sea bottom requires an acoustic data acquisition system and a set of algorithms that analyze the data, determine the seabed type and relate the results of the acoustic classification to the physical properties of the marine sediments. Quester Tangent Corporation has success- fully combined high-speed digital signal processing technology with multivariate statistical analysis to create the QTC VIEW sea- bed classification engine. The QTC VIEW captures and digitizes the seabed echo from a conventional echo sounder, processes, analyses, displays and records acoustic waveform data character- izing the sea floor. The output is a set of point data representing sea floor acoustic diversity. The georeferenced classifications can then be objectively correlated with other data such as sediment prop- erties or directly to fish census. The technology has been applied world wide to map marine sediments for a variety of applications. It has been used for map- ping substrate in support of benthic habitat characterization. The technology can also be configured for use in water depths less than 5 ni. Examples of oyster mapping projects in West Galveston Bay, Texas and Chesapeake Bay, Maryland are highlighted. Strategies for the integration of acoustic and nonacoustic data are included. OYSTER POPULATION RESTORATION IN CARAQUET, N.B.; PHASE II, OPTIMIZING PRODUCTIVITY. Paul Cormier. T. Landry, and J. F. Mallet. Department of Fisheries an Oceans, GFC, P.O. Box 5030, Moncton N.B., EIC 9B6, and Department of Agriculture, Fisheries and Aquaculture, 22 Boul. Saint-Pierre, Caraquet, N.B. EIW IB6, Canada. A decrease in the productivity of oysters in Caraquet Bay, N.B. is generating some interest in restoration projects. This bay repre- sents the most northern location with a sustainable oyster (Cras- sosrrea virginica) population. The first phase of this initiative pro- vided invaluable information on the distribution, abundance and population structure of oyster beds. Over 60% of the oysters found in this bay are pre-recruits to the fishery (35-7.*imm). These oysters 308 Abstracts. September 2001 She sh. Restoration. Nanaimo B.C., Canada are mainly tound in the northern portion of the main bed, which has a relatively low abundance of commercial size oysters (>75mm) and. is locally renown for its "stunted growth oysters"". Samples of oysters from this location where transferred at four stations in the bay and monitored for grov\lh and conditioning. Results are suggesting that the growth limitation of these oysters is associated with the geographical location and even more to the bottom conditions. Management strategies will be developed to optimize the productivity of oyster from this highly successful recruitment location, which offers limited possibility for commer- cial production. can cover large areas in less time, provide accurate measurements of coverage, and simultaneously provide differential GPS coordi- nates. The information acquired from side scan sonar provides a long-term record, which can be used to detect changes due to environmental catastrophes, vessel groundings and harvest pres- sure. A side scan sonar survey was perf"ormed on two cultch plant sites located in the western MS Sound, St. Joe Cultch Plant and Pass Christian Cultch Plant. The St. Joe Cultch Plant site was surveyed before and after cultch planting. On the Pass Christian Cultch Plant only a post side scan sonar survey was conducted. The data was processed and input in a mosaic software program for further analysis. This study will demonstrate the abilities of side scan sonar as a management tool for oyster reef management. ABSTRACT THEME AREA: A. Craig, and C. Downs, EnVir- tue Biotechnologies. Inc. 2255 Ygnacio Valley Rd., Suite H-1. Walnut Creek, CA 94598, USA. Using the tools of proteomics and molecular diagnostics, one of our primary goals is to determine if environmental factors are affecting shellfish susceptibility to infections. This stiategy of re- search can also aid in answering other important issues to shellfish husbandry such as why some shellfish cultivars or strains are more susceptible to infection than others and the cellular mechanisms responsible for disease resistance? Proteomics and functional ge- nomics can be used to discover novel and appropriate molecular biomarkers that can aid in diagnosing the health status of a crop, develop powerful biomarkers to aid in predicting crop yield, and aid in developing genetic markers for advantageous cultivar traits. Finally, understanding the cellular and biochemical mechanisms of shellfish disease may allow for the development of practical mea- sures or treatments to limit disease. SIDE SCAN SONAR AS A MANAGEMENT TOOL IN MIS- SISSIPPI. Dale Diaz, K. Cuevas and W. S. Perret, Office of Marine Fisheries, Mississippi Department of Marine Resources, 1141 Bayview Avenue, Suite 101, Biloxi, MS 39530, USA. Oyster harvest from Mississippi reefs provide jobs for numer- ous fishermen and contribute substantially to the economy. Proper management of these reefs is vital for continued harvest. Enhanced management techniques (i.e. cultch planting) should help to ensure even greater future production. Cultch planting is a key component of management measures conducted by the Mississippi Depart- ment of Marine Resources (MDMR). The use of sounding poles, tongs, dredges or scuba equipment gauged the effectiveness of different cultch planting methods. These methods are not efficient techniques when surveying large areas for coverage rates or dis- tribution of materials. LIsing side scan sonar to monitor cultch plants has a number of advantages over these previously used methods. Side scan sonar CELLULAR-PHYSIOLOGICAL ASSESSMENT OF BI- VALVES AFTER TEN YEARS OF CHRONIC EXPOSURE TO SPILLED EXXON VALDEZ CRUDE OIL USING A NOVAL MOLECULAR DIAGNOSTIC BIOTECHNOL- OGY. Craig A. Downes,' Charles E. Robinson." Arnold Huang.' Gary Shigenaka." and John E. Fauth,' Envirtue Bio- technologies, Inc., 2255 Ygancio Valley Rd., Suite H-l, "Walnut Creek, CA 94598, USA; "Hazardous Materials Response Division, US National Oceanic and Atmospheric Administration, 76(X) Sand Point Way NE, Seattle, WA 981 15, USA; 'Department of Biology, University of Charleston, 66 George Street, Charieston, SC 29422, USA. The objective of this study was to determine the cellular physi- ological status of the bivalves Mya arenaria and Mytilus trossulus in an area impacted by a ten-year chronic exposure of spilled Exxon Valdez crude oil in Prince William Sound. Bivalves were collected from a well characterized mipacted site on Knight Island and from a nonimpacted site north of Bainbridge Island. We used a novel biotechnology known as the Envirtue Molecular Diagnostic System^M (EMDS) to determine if bivalves were (I) physiologically stressed, (2) the nature of the altered physiological state, and (3) whether the organisms were respond- ing specifically to an exposure to polyaromatic hydrocarbons (PAH). Molecular diagnostic analysis indicated that bivalves at the impacted site were stressed, experiencing both an oxidative and xenobiotic stress, resulting in increased protein turnover and chap- erone activity. Further, bivalves from the impacted area were re- sponding specifically to a PAH xenobiotic exposure and accumu- lating DNA-PAH adducts. Finally, species-specific responses were observed that were related to the spatial-habitation characteristics of each species. We conclude that bivalves inhabiting an area impacted by crude oil from the Exxon Valdez spill of 1989 are still adversely affected by the spill's remnants. Shellfish, Restoration, Nanaimo B.C., Canada Abstriicr.s. September 2(101 309 OYSTER AQUACULTURE AS FISH HABITAT IN PA- CIFIC NORTHWEST COASTAL ESTUARIES. Brett Dum- bault,' D. Armstrong.- G. Hosack," and B. Semens," 'Washmg- ton State Department of Fish and Wildlife, Willapa Bay Field Station, P.O. Box 190, Ocean Park, WA 98640, USA; "School of Aquatic and Fishery Sciences, Box 355020, University of Wash- ington, Seattle, WA 98195, USA. Increased pressure on traditionally managed stocks of marine and anadromous fish, calls for protection of essential fish habitat under the Magnuson-Stevens Act. and recent listings of several salmonid stocks under ESA have brought aquaculture activities that take place in coastal estuaries under increased public scrutiny. We initiated a study designed to examine the ecological role that oyster aquaculture plays as habitat in coastal estuaries of the Pacific Northwest. The goal of the project is to identify and quan- tify beneficial and adverse impacts of shellfish farming on eel- grass, juvenile salmonids, and other selected estuarine fauna and flora and to develop farming practices and recommend manage- ment protocols that protect or enhance those resources. We present results of initial fish and invertebrate sampling in selected habitats from Willapa Bay during Spring and Summer 2001 and an experi- ment designed to examine the effects of oyster harvesting on eelgrass habitat. We make a plea to managers to consider oyster aquaculture areas as fish habitat on a broader estuarine scale. DEMONSTRATION AND EVALUATION OF COMMER- CIALLY IMPORTANT BIVALVE CULTURE AND STOCK ENHANCEMENT METHODS IN REHOBOTH AND IN- DIAN RIVER BAY, DELAWARE (1998-2001). John William Ewart,' J. Alderman", and K. Maull," 'Delaware Sea Grant Ma- rine Advisory Service. Graduate College of Marine Studies, Uni- versity of Delaware, 700 Pilottown Road, Lewes. DE 19958. USA: "Delaware Center for the Inland Bays, 467 Highway One, Lewes. DE 19958. USA. Delaware's coastal lagoons, known locally as "inland" bays, have been experiencing the impacts of chronic eutrophication and sediment erosion resultant from several decades of sustained nu- trient input and development from within the sun'ounding water- shed. The Delaware Center for the Inland Bays (CIB) was estab- lished as a nonprofit organization in 1994 to oversee the imple- mentation of the Inland Bays Comprehensive Conservation and Management Plan and to facilitate a long-term approach for the wise use and enhancement of the watershed. The James Farm Ecological Preserve, a 150 acre property with frontage on Indian River Bay, was established in 1998 to serve in part as a demon- stration site for beneficial land use practices and similar watershed based activities such as shoreline stabilization, habitat creation/ protection and shellfish stock enhancement to improve water qual- ity. In 1998. the CIB initiated an ongoing program to evaluate and demonstrate the technical feasibility of using aquaculture methods to maintain or improve populations of commercially viable bi- valves and to create additional habitat for shellfish and other spe- cies. Current field demonstration activities include low density plantings (1-2/square foot) of hard clams (Mercenaria merce- naria). for stock enhancement and recreational fishery improve- ment; monitoring oyster growth, survival and prevalence of MSX and Dermo disease in eastern oysters (Crassostrea viigiuica) held in off-bottom transient gear, submerged trays and Taylor floats; construction and deployment of a Floating Upweller System (FLUPSY) for nursery culture of bivalve seed; and establishment of a 1/4 acre oyster reef to monitor growth, survival, disease preva- lence and habitat development. MOLECULAR DETECTION OF FECAL BACTEROIDES AS SOURCE INDICATORS FOR FECAL POLLUTION IN WATER. K. G. Field, Oregon State University, Department of Microbiology, Corvallis. OR 97331, USA. Fecal contamination of shellfish environments afflicts many regions worldwide, and carries numerous risks to human health. Fecal bacteria, pathogenic protista and viruses may be highly con- centrated in shellfish. Often the problem can't be coiTCCted, be- cause standard methods of measuring fecal pollution in water do not distinguish its source. We have developed a PCR-based alter- native method of fecal source discrimination. The method consists of amplifying I6S rRNA gene fragments of the fecal anaerobic Bciclemides-Prevolella group with specific primers. Because Bacleroides is restricted to gut habitats, its presence in water in- dicates fecal pollution. Molecular detection circumvents the com- plexities of growing anaerobic bacteria. We identified Bacleroides length-heterogeneity PCR (LH-PCR) and terminal restriction frag- ment length polymorphism (T-RFLP) ribosomal DNA markers unique to either cow or human feces, and recovered the same unique fecal markers out of polluted natural waters. We cloned and sequenced the markers and used the sequences to design PCR primers that reliably discriminate human and ruminant sources of fecal contamination. New primers under development will distin- guish elk/deer, horse, pig. chicken, duck/goose, beaver, and harbor seal fecal pollution. With this approach, sensitivity is greater than fecal coliform assays. The method does not require culturing bac- teria, allows for small sample size and easy sample handling, is comparable in complexity to standard tests carried out in food safety and public health diagnostic labs, and lends itself to auto- mation and high-throughput. HABITAT RESTORATION AND SHELLFISH STOCK EN- HANCEMENT INITIATIVES IN NEW JERSEY. Gef Flim- lin,' and Michael P. Stringer," Rutgers Cooperative Extension. 1623 Whitesville Rd.. Toms Ri\'er. NJ 08755. USA; "NY/NJ Bay- keeper, Bldg. 18. Sandy Hook. Highlands, NJ 07732, USA. New Jersey has been a major shellfish production state since colonial times. The Raritan Bay area once boasted a 400-boat oyster fleet from the town of Keyport alone. However, industrial 310 Abslracls. September 2001 Shellfish. Restoration. Nunuimo B.C.. Canada polliilion. water qtiality degradation, and the onset of MSX and Dermo ha\e practically eliminated natural oyster stocks in most areas. Efforts over the past three years have lead to the establishment of two reefs, one on the Liberty Flats near the Statue of Libeity and one off the shore of Keyport. These shellstocked reefs have had cultured oyster seed placed on them and a monitoring program has begun. Another effort is underway in the Mullica River, the last area on the state's Atlantic Coast side to produce oysters, to re- stock the public beds with natural oysters from the seedbeds up- river. This area will be proposed for a larger scale project for a Community Based Restoration Program in the year to come. The Baykeeper and Rutgers Cooperative Extension have been working with the Baynien's Protective Association and the New Jersey Shellfisheries Association, two shellfish industry groups, to propel these initiatives. Primary and secondary school education projects explaining the benefit of oyster restoration and aquacul- ture are being done to garner more support for the projects. It is envisioned that eventually the industry will reap the benefit of these projects v\hen the reefs may be harvested in the future. NEW DIRECTIONS FOR SHELLFISH PROTECTION IN PUGET SOUND. Stuart D. Glasoe, Office of the Governor. Puget Sound Water Quality Action Team. PC Box 40900. Olvin- pia, WA 98504. USA. The Puget Sound Water Quality Management Plan serves as a cornerstone for shellfish protection in Puget Sound. First adopted in 1987, the plan employs a suite of strategies to raise awareness, change behaviors and build and strengthen systems to protect the health of Puget Sound. Although the plan was designed with the dual goal of protect- ing and reopening shellfish beds, early trends were daunting. Be- tween 1987 and 1989 alone, nearly 33,000 acres of commercial beds were downgraded as a result of declining water quality and more intensive monitoring. Trends shifted dramatically in the 1990s as restoration efforts successfully upgraded over 13,000 acres, offsetting nearly identical acreages downgraded during the period. These results show that the strategies are paying off e\en in the face of rapid population growth, but they also reveal an unsustainable emphasis on reactive, restoration techniques. The region must now shift gears and make better use of proven approaches to more effectively and permanently prevent contami- nation of shellfish growing areas. These include better land use planning under the Shoreline Management Act and Growth Man- agement Act to preserve shellfish areas as critical areas and natural resource lands: greater u.se of pollution prevention measures such as low impact development practices and on-site sewage mainte- nance programs; widespread creation of surface water utilities to fund local services and projects: early detection and rapid response to emerging water quality problems: and better communication with all audiences to raise awareness on the impacts and tradeoffs associated wilh pollution and urbanization in shellfish watersheds. SEMIAHMOO BAY WATER QUALITY PRO.IECT: PHASE 1 FINDINGS. Heather Goble, Shellfish Project Coorduiator. Georgia Basin Ecosystem Initiative. Clean Water Action Plan, 224 West Esplanade, North Vancouver, EC, V7M 3H7, Canada. We have now completed Pha.se 1 of a three-year action plan to identify the major sources of contamination to Boundary Bay and propose subsequent actions required to mitigate and remediate the prohibited status of the shellfish growing waters in this area. The objective of Phase 1 of the Semiahmoo Bay Water Quality Project was to characterize non-point source contamination patterns within Semiahmoo Bay. We sampled 19 beach locations along the shore- line as well as 32 natural and manmade storm water drainages into Semiahmoo Bay. Precipitation levels had a positive correlation with bacteriologi- cal counts for both outfall and ocean sampling sites. This indicates contamination in storm water outfalls had a direct impact on water quality along the shoreline of Semiahmoo Bay. Ocean sites dem- onstrated fecal coliform counts that consistently exceeded accept- able standards for shellfish growing waters and occasionally ex- ceeded accepted standards for recreational water quality. The ma- jority of outfalls had fecal coliform counts that occasionally reached values associated with sewage-impacted waters. Boundary Bay sediments and shellfish were tested for common metals and organics that may adversely affect growing areas. The levels of heavy metal residues and organochlorines in shellfish were found to be within acceptable limits. However the tests for lead and arsenic levels were inconclusive and the shellfish will be retested using different parameters. Phase 2 of the Semiahmoo Bay Water Quality Project will conduct contaminant investigations to reduce the impact of con- taminated drainage systems on the shellfish growing waters of Semiahmoo Bay. REMOTE-SET ALTERNATIVE SUBSTRATE ON LEASED GROUND: AN INNOVATIVE APPROACH TO REEF RES- TORATION. William Goldsborough,' Stew Harris,' D. Jack- son,' D. W. Meritt," and S. Tobash,' 'Chesapeake Bay Founda- tion, 6 Herndon Avenue. .Annapolis. MD 21403, USA: University of Maryland, Center for Environmental Science. Box 775. Cam- bridge, MD 21613, USA. Oyster reef restoration in Chesapeake Bay faces a variety of challenges that will require new and innovative approaches. Among the challenges are: identifying sanctuary locations, obtain- ing suitable substrate, establishing oysters on the substrate, and protecting the site from poaching. Thousands of acres of Bay bottom are currently under lease from the states of Maryland and Virginia. Much of this ground is Shellfish. Restoration. Naiiiiimo B.C.. Canada Abstmcls. September 2001 not being actively farmed and may be available for establishing reef sanctuaries under agreement with leaseholders. Oyster shell is considered the best reef material, but supplies of shell are dwindling. The scale of reef restoration now anticipated will require development of alternative materials. Marine lime- stone rock (marl) has been shown to be a good setting substrate and may prove to be a valuable substitute for shell in reef con- struction. A second valuable function that marl may provide is that of a physical obstruction to poaching with traditional harvest gear. In a pilot project undertaken in the Nanticoke River, a tributary of Chesapeake Bay in Maryland, marl rocks were set remotely with hatchery-produced larvae and distributed on a one-acre area of leased ground to create a field of mini-reefs. In the process a new and promising approach to reef sanctuary creation was devel- oped. RESTORATION OF THE EASTERN OYSTER iCRASSOS- TREA VIRGINICA) THROUGH A VOLUNTEER PRO- GRAM IN MOBILE BA^ . ALABAMA. Kimberly A. Hamil- ton, D. LaDon Swann. Richard K. Wallace. Yolanda J. Brady. David B, Rouse, Scott Rikard. and Holly Hall. Department of Fisheries and Allied Aquacultures, Auburn Uni\ersity. Aubuin. AL 36849. USA. E-mail: hamilka@acesag.aubum.edu Restoration programs for the eastern oyster. Crassostrea vir- ginicu, in Mobile Bay, Alabama are used to increase the number of oysters on remaining beds and to renew ecological functions such as providing habitat and water filtration. To educate the general public of the ecological importance of oysters and oyster restora- tion efforts, an oyster gardening program was created for restora- tion of natural and constructed reefs. The volunteer program was implemented in Mobile and Baldwin counties along Mobile Bay and monitored for 7 months beginning in May 2001. A total of 30 volunteers representing sites located in unre- stricted oyster harvesting waters were provided a Taylor float and 1.000 oyster spat per volunteer. The 36.2 mm spat were provided to the first 15 volunteers and 12.5 mm spat were provided to a second group of 13 volunteers. Spat were stocked into the Taylor float (surface area of 0.74 m") and tied to a pier, piling, or weight. Growth and condition of oysters in Taylor floats were compared and correlated with water quality parameters (temperature, dis- solved oxygen, salinity, and water clarity) between each site. REPRODUCTIVE SANCTUARY FOR THE NORTHERN QUAHAUG. MERCENARIA MERCENARIA. IN PRINCE EDWARD ISLAND. Matthew Hardy,' T. Landry.- and A. Boghen,' Universite de Moncton. Moncton. N.-B.. El A 3E9; "De- partment of Fisheries and Oceans Canada. Science Branch, Mari- time Region. Moncton. N.-B.. EIC 9B6. Canada. West River was historically one of the most productive estu- aries for quahaugs in PEL However, due to the construction and subsequent removal of a causeway, in addition to extensive fish- ing, stocks are now considered to be too low to sustain the present level of commercial harvesting. Density estimates in West River ranged from 1.37 to 2.52 quahaugs • m"'. which are low in relation to other quahaug producing areas. Several years of research lead to the establishment of a repro- ductive sanctuary for large quahaug in the subtidal zone of West Ri\er. This was based on results showing that reproductive success could be optimized through low-level and inexpensive interven- tions. Large quahaugs are advantageous as broodstock because they have high fecundities and they are not significantly affected by high stocking densities. In vitro fertilization experiments showed that reproductive success increased significantly with higher gamete concentration and lower gamete ages. These related directly to the distance between a spawning pair of adults. The subtidal zone also provides ecological and socio-economic ben- efits. This type of enhancement offers certain advantages over hatch- ery produced seeding programs. Preliminary work tends to indicate that the sanctuary concept could be an effective restoration or enhancement technique for the quahaug. Plankton tows showed a greater presence of quahaug larvae in West River, following the establishment of the sanctuary, compared to previous years and other estuaries. Ultimately, it might be possible to enhance overall productivity to sustainable levels through properly managed broodstock areas. STATUS OF SHELLFISH ENHANCEMENT IN ALASKA. J. Hetrick, Chugach Regional Resource Commission. 4201 Tudor Center Drive. Anchorage. AK 99308. USA. The Alaskan shellfish aquatic farm industry has been growing steadily with an increase in the number of fanns and an instate shellfish hatchery for production of indigenous species. Recent attempts by native villages and local communities to enhance beaches for subsistence and recreational groups have been mired in bureaucratic gridlock. At issue is a directed use of a public re- source, management of enhanced stocks and their interaction with standing stocks and the lack of enabling legislation for shellfish enhancement. An outline and review of projects completed and underway will be presented. INNOVATIVE COMMUNITY PROCESSES PRODUCE IN- TEGRATIVE MANAGEMENT TOOLS. Barbara Joughin' and William Heath." 'Comox Valley Project Watershed Society. Box 3007. Courtenay, BC, V9N 5N3, Canada, "BC Ministry of Agriculture. Food and Fisheries. 2500 Cliffe Avenue, Courtenay, BC, V9N 5M6, Canada. Baynes Sound, located in western Canada on the east coast of Vancouver Island, is a highly productive area for shellfish culture, salmon, herring and waterbirds. Over the past decade. Baynes Sound has experienced increasingly degraded water quality from 312 Ahsiracts, September 2001 Shellfish, Restoration, Nanaimo B.C., Canada non-point source pollution, as well as loss and degradation of sensitive w ild habitat. The Baynes Sound Round Table was formed in 1994 to improve water quality in Baynes Sound, and has guided a series of collaborative community stewardship projects that ad- dress threats to environmental and economic hcallh m Baynes Sound. These projects create opportunities for citi/ens. govern- ments and shellfish growers to work together to identify and re- mediate non-point source pollution impacts in Baynes Sound. Hundreds of citizen volunteers have participated in recent stew- ardship programs. Community involvement and stakeholder col- laboration have established a strong foundation for proactive and integrated remedial planning and programming. An important tool has evolved from the Baynes Sound process - the development of the 'State of the Sound' Program. The State of the Sound Program is a long term monitoring, reporting and planning process that measures and reports the health of Baynes Sound. The program uses a geographic information system (GIS) to gather and analyse data for water quality and other indicators. Results are used to plan actions, and are communicated to the community to help increase public awareness and involvement. Information becomes acces- sible, 'crisis control" is replaced with comprehensive management, and an effective tool is available to assist with planning proces.ses such as local liquid waste management programs. ADVANCES IN SHELLFISH SAFETY ASSURANCE AND PARTICIPATION IN SANITARY GROWING WATER MONITORING PROGRAMS BY THE BC SHELLFISH AQUACULTURE INDUSTRY. Brian Kingzett. Kingzett Pro- fessional Services Ltd. .^21 St. Julian, St. Duncan B.C. V9L 3S5, Canada. The BC Shellfish Growers Association (BCSGA) represents the majority of shellfish production in British Columbia. Promot- ing shellfish safety and involvement in the growing water man- agement have become significant aspects of the work of the asso- ciation and its members. Decreases in regulatory monitoring bud- gets and increased demands for service by shellfish aquacullurists have led to proactive industry partnerships and programs. These involve industry participation in monitoring for Vibrio paia- bacinolxiiciis. an industry driven farm based Vp control program, involvement in sanitary growing water monitoring and remedia- tion, and maintenance of marine biotoxin sentinel sites. Currently the BCSGA is participating in a national program developing stan- dard tnethodologies and cuiriculum for industry growing water sampling and farm based HACCP for shellfish culturists. DEVELOPMENT OF INTENSIVE DEEPWATER OYSTER CULTURE SYSTEMS IN BRITISH COLUMBIA. Brian Kingzett, Kingzett Professional Services Ltd, .^21 St. Julian. St. Duncan B.C. V9L 3S5. Canada. Socio-political reasons have restricted the growth of the shell- fish culture industry in British Columbia during the last decade. The industry is relatively small and recent growth has primarily come about on small deep-water sites. Shellfish growers in British Columbia have developed a series of approaches to developing intensive oyster production in deep-water (off bottom) culture sys- tems. This has invohed adapting international techniques and de- veloping unique technologies, which allow significant production from small areas. USING INNOVATIVE STORMWATER CONTROLS FOR WATER QUALITY IMPROVEMENTS AND OYSTER HABITAT RESTORATION IN THE BARATARIA- TERREBONNE NATIONAL ESTUARY. K. E. Landruni,' K. M. St. Pe.' B. Ache.- and F. Kopfler,-' 'Barataria-Terrebonne National Estuary Program. P.O. Box 2663. Nicholls State Univer- sity. Thibodaux. LA 70310. USA; 'Battelle. 191 East Broad Street. Suite 315. Athens, GA 30601. USA; 'EPA/Gulf of Mexico Pro- gram. Stennis Space Center, Building 1 103, Room 202. MS 39329. USA. The loss of nearly 22 square miles of emergent wetlands per year in the Barataria-Tenebonne National Estuary represents the imminent loss of a nationally significant wetland resource and threatens the area's unique culture and local infrastructure. Oppor- tunities exist for rerouting some of the estuary's 260 stormwater pump station outfalls within the estuary to improve the water qual- ity associated with these discharges prior to entering historically productive oyster grounds. Runoff from rural and agricultural ar- eas are collected in borrow canals within the existing levee sys- tems and are generally pinnped into large man-made canals to ensure rapid evacuation of stormwater. The Barataria-Terrebonne National Estuary Program and EPA's Gulf of Mexico Program are spearheading efforts to moni- tor and assess changes in estuarine \'itality near rerouted pump station outfalls to demonstrate the benefits of this unique process. Qualitative evidence of the positive effects of the redistribution of stormwater into adjacent wetland areas is visually evident through- out both basins, although only limited quantitati\e assessment has occurred. Redirecting discharges so that freshwater is retained in adjacent wetlands rather than moved through them has been dem- onstrated to maintain lower salinities, promote vigorous plant growth through nutrient uptake, and lead to pathogen degradation. Numerous studies provide evidence of wetland uptake of pollut- ants and nutrients in constructed wetlands and riparian fringes, and the enhancement of marshes adjacent to hurricane protection levees would provide additional storm surge protection for prop- erties and local infrastructure. Shellfish, Restoration, Nanaimo B.C., Canada Abstracts. September 2001 313 WATER QUALITY IMPROVEMENT: A FOCUS ON NEW TECHNOLOGIES. Kevin L. LeBlanc, Fisheiies and Oceans Canada. Gulf Fisheries Centre, 343 University Street, Moncton. New Brunswick, EIC 9B6, Canada. Water quality improvement requires effective monitoring, fi- nancial support, partnerships, remediation and bio/socio-economic planning. However, tools that clearly define sources of contanu- nation remain the backbone for effective water quality improve- ment. Sewage is typically measured through the use of an indicator species such as faecal coliform bacteria, but effective remediation requires a clear link to the source of pollution. The South-western New Brunswick Clam Resource Committee (CRC), New Bruns- wick, Canada, has improved water quality and monitoring for the reclassification of shellfish growing areas since 1995 using the above mentioned strategies. By 2001, the CRC has increased the overall shellfish growing area by 277r. However, an additional 38% of shellfish growing areas, equivalent to 434 hectares ( 1 ,074 acres), could be available for harvest if the identification of the actual source of fecal coliform was possible. Tools such as genetic markers and gene sequencing of bacteria are examples of new approaches in differentiating between different sources of fecal coliform. These innovations will be critical in establishing feasible and effective courses of remediation in shellfish growing areas. Furthermore, such advances could lead to the development of mo- lecular-based kits for monitoring activities, similar to those used for biotoxin monitoring. The continued development of science in these fields is necessary for the continued success of water quality improvement initiatives. COMMERCIAL-SCALE OYSTER PRODUCTION FOR REEF RESTORATION AND STOCK ENHANCEMENT. A. T. Leggett. A. Blow, W . Goldsborough. R. D. Brumbaugh, Chesapeake Bay Foundation, 142 W. York Street, Suite 318, Nor- folk, VA 23510, USA. Oyster restoration in Virginia has focused on the construction of three-dimensional reefs made from oyster shells since the early 1990s. Since 1997, the Chesapeake Bay Foundation (CBF) has assisted with his effort by organizing and training citizens and students to grow oysters (Crassostrea virginica) using small-scale aquaculture techniques. To date, approximately I . I million oysters have been grown using these small-scale techniques, and stocked on Virginia Sanctuary reefs. In 2000, CBF initiated the Virginia Oyster Aquaculture Pro- gram to complement these volunteer-based efforts by producing an additional one million oysters per year for Virginia reefs. A com- mercial-scale oyster farm was designed and put into operation growing oysters in ADPl mesh bags contained in commercial oys- ter trays stacked on PCV racks. The oysters were placed directly in the trays when they reached an appropriate size (>25 mm) with approximately 1,500 oysters per tray. After eleven months, over 930,000 oysters (mean size = 52 mm) were harvested using vol- unteer labor and transplanted onto eight sanctuary reefs. Survival to transplanting for the three stocks used in the program was ap- proximately 85%, and first year capital expenses were approxi- mately $62,400. The oysters produced through this program were used as in-kind match for newly appropriated federal funds dedi- cated to Virginia oyster restoration. Future considerations of the program include genetic aspects of the oyster stocks used and the application of new techniques including the use of a floating up- weller (FLUPSY) for the nursery phase of the grow-out. PROTECTION AND RESTORATION: WASHINGTON STATE SHELLFISH PROGRAM. Donald Lennartson, Office of Food Safety and Shellfish Programs, Washington State Depart- ment of Health, 7171 Cleanwater Lane, Building 4, Olympia, WA 98504, USA. The Washington State Department of Health oversees 126 shellfish growing areas with over 1.400 marine water sampling stations. The state shellfish industry produces over 80% of the shellfish harvested on the west coast, not including geoducks. This rich shellfish resource faces a range of threats, from rural land use activities to rapid urban growth. Prevention of classification down- grades and the restoration of water quality require careful plan- ning, strong technical skills, tough regulatory tools, and creative partnerships among local, tribal, state, and federal agencies. The stimulus for shellfish restoration activities was the inno- vative Puget Sound Water Quality Management Plan, whose pur- pose is "to restore and protect the biological health and diversity of Puget Sound". A major component of this ongoing Plan is the Shellfish Protection section, which outlines seven action elements, the first two of which address shellfish protection policy and the restoration of commercial shellfish beds. In keeping with the spirit of the Plan, the Department of Health initiated an Early Warning System, a proactive measure which alerts county governments to the threat of classification down- grades. If preventative measures fail and a downgrade does occur, state law requires the county to establish a Shellfish Protection District, and a Closure Response group is convened to formulate actions in the watershed to identify and correct the pollution sources. ABALONE RESTORATION THROUGH COLLABORA- TIVE PARTNERSHIPS IN BRITISH COLUMBIA. B. G. Lu- cas, and A. Campbell, Stock Assessment Division. Science Branch. Fisheries and Oceans Canada. Pacific Biological Statio. Nanaimo. BC V9R 5K6, Canada. Diverse stakeholder groups are collaborating to test potential methods to restore threatened abalone populations in British Co- lumbia. Fisheries and Oceans Canada is working with six groups throughout coastal BC in an attempt to halt further decline of abalone stocks and restore populations to self-sustaining levels. 314 Abstracts. September 2001 Shellfish. Restoration. Nanainio B.C.. Canada Five aquacuiture project.s are underway to develop the culture technology required to provide northem abalone seed for pilot restocking experiments. In addition, four projects are currently promoting local community stewardship of abalone resources through public education, increased awareness and, in some cases, experimental manipulations of wild abalone populations. These projects are providing training and employment in local commu- nities which have been impacted by declining opportunities in the fishing industry. SITE ASSESSMENT. SELECTION AND MONITORING METHODS FOR ABALONE RESTORATION NF:AR KIT- KATLA. BRITISH COLUMBIA. B. G. Lucas, A. Campbell. and D. Brouwer. Stock Assessment Division, Science Branch, Fisheries & Oceans Canada, Pacific Biological Station, Nanaimo, BC V9R 5K6. Canada. Several pilot projects are currently underway to attempt to re- store abalone populations in British Columbia (BC). Near Kitkatia, on BC"s north coast, an extensive process of assessing, selecting and surveying sites for pilot abalone rebuilding experiments was recently completed. General areas for potential experiments were selected in conjunction with local First Nation advisors and Fish- eries & Oceans Canada staff. In September 2000, five divers spent 7 days conducting pre- liminary surveys to locate specific areas of suitable habitat that met predetermined site selection criteria. After analysis of the survey results, 16 potential sites with similar characteristics were chosen. In April 2001, three dive teams spent 6 days permanently marking 15 sites and collecting detailed baseline information at those sites. The proposed experiments will test the sur\ i\ al and recapture rates of seeded juvenile abalone while examining the effects of sea urchin sizes and densities in the experimental plots. Long teitn monitoring of the sites is expected to continue for at least five to ten years after the experiments begin. EFFECTS OF REEF ARCHITECTURE AND SCALE ON FISH UTILIZATION OF OYSTER REEFS IN VIRGINIA. Mark W. Luckenbach. ,|. Nestlerode, P. G. Ross and A. J. Birch, College of William and Mary, Virginia Institute of Marine Science, Eastern Shore Laboratory, Wachapreague, VA 23480. USA. Current efforts to restore oyster reefs in the Chesapeake Bay are directed toward establishing self-sustaining reef sanctuaries that provide valuable ecological functions, such as benthic-pelagic coupling and support of increased diversity and production of mac- robenthos and finfish. Recent evidence has revealed the impor- tance of two components of reef architecture — vertical relief and interstitial space — on the development of oyster populations on restored reefs. We will present data which show that finfish utili- zation varies between "reefs" with and w ithout viable oyster popu- lations. A third component of reef architecture — aerial extent or .scale — is expected to affect both reef development and utilization by fish. In conservation biology this topic has often been charac- terized as the SLOSS (Single Large or Several Small) debate, but is more generally about optimizing the scale of a bio-reserve or sanctuary to support the desired species. To investigate this issue for oyster reefs we have initiated a large-scale restoration experi- ment in the Chesapeake Bay. In a replicated block design, we have constructed high relief reef bases ranging in size from 400 sq ni to 8000 sq m and are characterizing the development of resident and transient assemblages of organisms on the reefs. We will present data from the first year of the study on finfish utilization of these reefs. THE ROLE OF AQUACULTURE IN THE RESTORATION EFFORT TO SAVE WHITE ABALONE [HAUOTIS SO- RENSENI) IN CALIFORNIA. Thomas B. McCormick. Chan nel Islands Marine Resource Institute. 323 E. Matilija Street. Ojai. CA 93023, USA. Stocks of white abalone {Haliolis sdicnscni) in southern Cali- fornia declined precipitously from an a\'erage of 2,000 to 10.000 abalone per hectare in the to 1.6 per hectare in the 1990s. A short-lived fishery in the 1970s landed 270 metric 1970s tons from depths of 20 to 50 m. The fishery was closed in 1997 and in 2001 the white abalone was listed as an endangered species, the first marine invertebrate to be .so classified in the USA. The White Abalone Working Group was formed by federal and state agencies, scientists, universities, non-profit organizations, and mariculturists as a proactive step towards preventing the ex- tinction of white abalone. The group developed the following four- step plan for recovery of the species: ( 1 ) survey historic fishing grounds to locate survivors: (2) collect and hold adults as breeding stock: (3) produce a new generation of young adult abalone in the hatchery: and (4) introduce hatchery grown adult animals into refugia to reestablish self-sustaining wild populations. Success has been achieved in maintaining and spawning white abalone in captivity. Abalone cultivation systems incorporate the use of the red macrophyte. Pacific dulse {Pciliiniria mollis) as both a nutritious feed and biofilter. Large numbers of ju\eniles are being raised using commercial techniques. This program differs from other enhancement efforts in that the abalone will be raised to adult size (10 cm shell length) prior to release. Larger animals such as this should be better able to resist predation and are ca- pable of spawning immediately. Shellfish, Restoration. Nanaimo B.C.. Canada Abstracts. September 2001 315 BOAT WASTE AND SHELLFISH CLOSURES: A COM- PARISON OF CANADIAN AND UNITED STATES POLI- CIES. Jim Mclsaac, Coastal Enterprise and Resource Coopera- tive Association. 202-1931 Mount Newton X Rd.. Sidney, BC V8M 29A. Canada. Diseases such as hepatitis, typhoid and cholera have been trans- mitted by untreated boat sewage. Popular beaches and shellfish areas attract boaters; the resulting contamination from boat sewage creates health concerns for millions of shellfish lovers. Swimmers that frequent contaminated waters have been known to develop skin rashes, amoeba dysentery, and parastic worm diseases. Boat sewage has closed beaches and shellfish harvesting areas around the world. In 1991. the Canadian government passed the Pleasure Craft Sewage Prevention Regulations intending to protect the marine environment from the negative impacts of sewage disposal. Since passing the Regulations, nine west coast marine waterbodies have been designated no-discharge zones (NDZl. A further 54 marine sites have been recommended for NDZ and are under review. However, no reduction in breach or shellfish closures has been noted. In contrast, the United States Congress passed the Clean Vessel Act in 1992. Today, all vessels inside US waters are required to have a marine sanitation device. Seven states have all their surface waters designated as NDZ. An additional eleven other states have segments of their waterbodies designated as NDZ. Washington and Massachusetts have already identified a reduction in beach and shellfish closures. With intentions from both federal governments so evident, why are the results so different? In the United States, the Clean Water Act and the Clean Vessel Act clearly lay out areas of jurisdictional responsibility and financing arrangements. Implementation plans are in State control where boat waste disposal plans were devel- oped. In Canada, boat waste policies are caught in multi jurisdic- tional bureaucracies with overlapping acts and conflicting regula- tions, resulting in no boat waste disposal being developed. SUCCESSION IN MUSSEL COMMUNITIES: THE IMPOR- TANCE OF WHAT IS MEASURED. C. W. McKindsey, En- vironmental Sciences Division. Department of Fisheries and Oceans, Maurice Lamontagne Institute, 850 Route de la Mer. PC Box 1000; Mont-Joli, QCG5H 3Z4. Canada. This study examines the community of organisms in crevices in the St. Lawrence estuary, Canada, through succession and shows how extrapolations from the study of a limited number of organ- isms to the entire coinmunity of macro-invertebrates may not be valid. I first examined how varying the lower size limit of the organ- isms considered in the analyses (organisms >0.5. I, 2, 4. and 8 mm) alters the observed trends in community structure (richness and diversity) through succession. Diversity was maximal in mid- succession for the >2 and >4 mm size groupings but continued to increase through succession for the >1 and >0.5 mm groupings whereas richness always increased through succession for all groupings. Examining one of four distinct zones in crevices gave the same result as examining the total community, improving sam- pling efficiency. However, the other three zones did not show this predictive power. I then examined whether variation in richness and diversity through succession was a function of the structural heterogeneity (SH) provided by the dominant taxa or of the age of the commu- nities per se. This was done by comparing control cre\ice com- munities at four successional stages to ones that had been modified such that only the dominant species remained following a brief period to allow for colonization (mimic crevices). Within three months, control and mimic communities resembled each other in terms of both diversity and richness and the trends min'ored those observed four months early, thus supporting SH. Nonparametric multivariate analyses supported these conclusions. AN ARCHAEOLOGICAL PERSPECTIVE ON THE CUL- TURAL SIGNIFICANCE OF SHELLFISH RESOURCES TO FIRST NATIONS IN THE GEORGIA BASIN. Eric McLay, Archaeologist, Hurqumi'num Treaty Group. Archaeology offers an invaluable perspective toward under- standing the fundamental importance of shellfish resources to coastal First Nations in British Columbia. This study examines the relationship between archaeological site locations and shellfish resources on Valdes Island, a large southern Gulf Island in the Georgia Basin. British Columbia. A close association is observed to exist between the location, size and content of archaeological shell deposits and the distribution of local shellfish resources. It is argued that Central Coast Salish settlement patterns on Valdes Island demonstrate a precontact economic orientation to- ward exploiting productive coastal resource zones, particularly sandy intertidal habits, where populations aggregated to collect localized, predictable and abundant bivalve shellfish and other sandy foreshore resources. This archaeological study has important implications for modelling strategies of precontact settlement and subsistence in the Georgia Basin, and for considering the cultural significance of shellfish resources to modem First Nations. RESTORING OYSTERS TO THE CHESAPEAKE BAY: A COORDINATED EFFORT INVOLVING GOVERNMENT. UNIVERSITIES. COMMUNITY GROUPS, AND INDUS- TRY. Donald W. Meritt," S. Tobash,' C. S. Frentz,' W. Goldsborough,' and S. Reynolds,^ 'University of Maryland. Cen- ter for Environmental Science, Box 775. Cambridge. MD 21613, USA; "Maryland Oyster Recovery Partnership, Box 6775, An- napolis, MD 21401, USA; ' Chesapeake Bay Foundation, 6 Hern- don Avenue, Annapolis, MD 21403, USA. Once the world leader Chesapeake Bay oyster harvests are near all-time lows today. Overexploitation, disease and habitat loss 316 Ah.slnuts. September 2001 Shellfish. Restoration. Nanaimo B.C.. Canada have all contributed to this decline. Traditional techniques of spreading shell to collect spat are no longer reliable to produce consistent numbers of high quality oyster seed because, oyster diseases are highest in areas where natural spatfall is most abun- dant. Utilizing hatcheries and a strategy designed to minimize the risk of infection, oyster seed have been produced with little or no Dermo. Hatchery spat have been used to accomplish a wide range of objectives including supplying citizens growing oysters for res- toration, re-seeding harvest grounds in areas sensitive to disease, involving commercial oystermen in restoration, and establishing ecological sanctuaries. This program has grown dramatically over the past decade and now is responsible for planting tens of millions of oyster spat each year. The Maryland Oyster Recovery Partnership along with the Uni- versity of Maryland Center for Environmental Science, the Chesa- peake Bay Foundation. Maryland Watermen's Association and local community groups have been successful in producing the seed oysters, preparing the bottom, and planting the seed in a coordinated effort. OYSTER RESTORATION IN CHESAPEAKE BAY: I CRI- TERIA FOR SITE SELECTION. Donald VV. Meritt,' S. To- bash,' K. T. Paynter," and T. Koles." 'University of Maryland. Center for Environment Science, Box 775, Cambridge, MD. 21613. USA; "Department of Biology. University of Maryland. College Park. MD 20742, USA. Interest in restoring historical oyster reefs or in creating new oyster reefs in Chesapeake Bay has heightened in recent years. The state of Maryland has a long history of activities aimed at oyster repletion for the public fishery. Additionally, private oyster culture was once a major producer of market oysters in the Bay region. Disease, overharvest, and habitat loss have all contributed to the decline in oyster populations and the resultant harvests are al near all time lows. The economic importance of a healthy oyster fishery to water- front communities is well documented. Of more recent interest is the concern for the ecological role of healthy oyster communities to the overall health of the Chesapeake Bay. Oyster restoration for non-commercial uses is becoming more commonplace while the more traditional repletion program activities are being modified in an attempt to become more efficient. Many community groups tmd it desirable to have an oyster reef in their local area. Not every site is suitable for oyster reef con- struction. Many exhibit some but not all of the characteristics needed for successful oyster growth and survival. Success of any given project depends upon accurate as.sessment of these charac- teristics and the use of proper construction techniques. GENETIC MONITORING OF OYSTER STOCK EN- HANCEMENT IN THE CHOPTANK RIVER. CHESA- PEAKE BAY, MARYLAND. USA. C. A. Milbury. and P.M. Gaffney, Graduate College of Marine Studies, University of Dela- ware, 700 Pilottown Road, Lewes, DE 19958, USA. The increased spread of parasitic diseases (primarily MSX and Dermo), in conjunction with overharvesting, has led to the rapid decline of many Eastern oyster (Ciassostrea virginica) popula- tions, especially in Chesapeake Bay. Regional variation in disease resistance to these parasites may be useful in restoration efforts. In collaboration with the University of Maryland Horn Point Lab. we have proposed to assess the success of recent enhancement efforts within the Chesapeake Bay using molecular genetic markers. In 1997. oysters propagated from Louisiana broodstock were planted at ten sites within the Choptank River. Maryland. C. vir- ,i;iiiica exhibits regionally diagnostic DNA profiles in the form of North Atlantic, South Atlantic, and Gulf Coast 16s mitochondrial haplotypes. The presence of the Gulf Coast haplotype in newly settled spat confirms the survival and propagation of the Louisiana broodstock. DNA sequencing techniques developed by Pyrose- quencing Inc. were used to determine the mitochondrial haplotypes of a large number of oyster spat collected at several bars through- out the Choptank River estuary. This rapid, mass screening method revealed that 94% of spat collected were of the North Atlantic haplotype and approximately 59c were South Atlantic. Of 2,466 spat screened, four possessed the Gulf Coast haplotype (0.2%). Haplotype identifications were confirmed using restriction frag- ment length polymorphisms in other regions of the mitochondrial genome. The use of these genetic markers has enabled us to as.sess the survival, propagation, and dispersal of the Louisiana oyster stock within the Choptank River. Chesapeake Bay. OPEN SAANICH INLET SHELLFISH BEDS: A COLLABO- RATIVE APPROACH. Rob Miller. RLN Cameron. Environ- mental Programs. Capital Regional District. 524 Yates Street. Vic- toria British Columbia, V8W 2S6, Canada. The Saanich Peninsula and Inlet are located on the southern tip of Vancouver Island near Victoria. British Columbia. The Saanich Inlet shellfishery is an imponant food source to three Saanich Peninsula First Nation Bands and the community. Many of the shellfish beds on the east coast of Saanich Inlet are closed for harvesting due to elevated levels of fecal coliform bacteria. The primary method of fecal coliform transmission from the land to the marine environment is stormwater. The Capital Regional District (CRD) Stomiwater Quality pro- gram works to limit the impacts of stormwater runoff to the envi- ronment and public health and protect freshwater and nearshore marine ecosystems. In 1999, Environment Canada and the CRD established a project titled Open Saaiiicli Inlet Shellfish Beds iOSISB). This collaborative project will run until 2003 and in- Shellfish. Restoration, Nanaimo B.C.. Canada Abstracls. September 2001 317 volves all levels of government and the community working to- wards opening shellfish beds. Under this project, the sources of fecal coliform contamination responsible for shellfish closures are identified. The jurisdiction(s) involved then work toward their reduction/elimination. Limited nearshore marine sampling is also undertaken to de- termine fecal coliform levels near shellfish beds and their associa- tion with stormwater. The data are particularly relevant to moni- toring changes over time and measuring the success of efforts to open shellfish beds. Since the project began there has been a significant decrease in the number of stormwater discharges with high fecal coliform le\els along this 27 kilometer coastline. Limited marine nearshore sampling has also shown a significant reduction in fecal coliform levels. HYDROACOUSTIC SE.ABED CLASSIFICATION TECH- NOLOGY APPLIED IN SHELLFISH PRODUCTIVITY RE- SEARCH. Marc Ouellette and T. Landry, Fisheries and Oceans Canada. Gulf Fisheries Centre. Science Branch. Moncton. NB. EIC 9B6. Canada. A good understanding of the relation between shellfish popu- lation dynamics and their habitat is essential in order to develop successful enhancement and/or restoration methods for our natural populations through shellfish or habitat management. The physi- cal, chemical and biological characteristics of the seabed are key elements in shellfish productivity. In the past, this element has proven to be difficult and expensive to evaluate in part due to our inability to efficiently classify seabed on a large scale. With the recent development of new data acquisition and analysis tools (Global Positioning System. Geographic Information System, geo- statistics and hydroacoustics), however, it is now possible to map seabed areas within a reasonable time and financial framework. Most importantly, these new technologies are capable of providing accurate and repeatable measurements. This will provide us with the ability to measure spatial and temporal variation of the benthic assemblage in relationship to mollusc productivity. A new shallow water seabed classification system, QTC View (Series V), is being used for the first time to survey sites in the Gulf of St. Lawrence and Fundy Bay were research is presently being carried out on.oyster bed restoration, quahaug population management and mussel farming. THE LIVING VENEER: CHARACTERIZING HABITAT STRUCTURE CREATED BY OYSTERS. Paynter, Kennedy. and Elizabeth Flynn, Department of Biology. University of Mary- land, College Park. MD 20742, USA. Oyster reefs, like coral reefs, are biogenic structures. The settle- ment and growth of oysters create them. Therefore, the density of settlement, growth rate, and the mortality rate of oysters on a given reef will greatly affect the structure of the reef and the habitat created. Natural densities of oysters up to 1 .000 indi\ iduais/m" have been reported while the mean density of exploited reefs in Maryland is less than 3 oysters/m". Although the large-scale, three-dimensional relief aspects of oyster reefs has receixed much attention, the structure created by oysters themselves — the spaces among and between living oysters — is less well studied. We have attempted to characterize the habitat created by dif- ferent densities of oysters both in the lab and in the field. A variety of approaches are available for this kind of measurement including estimates of the "space" created by oyster shells in various assem- blages, a chain index, and fractal dimension. While interstitial volume was not significantly different between clumps and loose shell, chain indices and fractal dimension estimates were quite different. Seabed roughness may also be a useful measure as it takes into account shell height and nearest-neighbor distances. Such characterization is important to understand the relationship between physical structure and habitat value. We hope to associate the structural differences between reefs with benthic fauna asso- ciated with them as an as.sessment of habitat value. OYSTER RESTORATION IN CHESAPEAKE BAY: II. IM- PACTS OF WATER QUALITY AND DISEASE. Paynter,' Kennedy,' T. Koles.' D. Meritt," and S. Tobash.' 'Department of Biology, University of Maryland, College Park, MD 20742, -Horn Point Laboratory, Center for Environmental Sciences, Uni- versity of Maryland, Cambridge, MD 21613, USA. The siting of oyster restoration projects is typically guided by several considerations. Physical habitat present (ie.. "hardness of the bottom") is typically the first consideration. Local disease prevalence is sometimes assessed and water quality is usually as- sumed. Dissolved oxygen (DO) levels are typically not measured prior to restoration. Oxygen dynamics in shallow zones of estuaries are not well understood. While general trends typical of eutrophic systems are apparent including surface overproductivity, stratification, and deep anoxia, relatively little attention has been paid to water qual- ity in shallow (<6m) nearshore. sub-littoral zones. Furthermore, the effects of hypoxia (<4mg Oj/l) on benthic communities and organisms are not well understood. We have observed chronic hypoxia on many oyster reefs in Chesapeake Bay. One reef was exposed to <3mg 0,/l for nearly 72 h with only 2 or 3 brief (<30 min) episodes of higher DO. DO lesels measured by continuous monitoring at this reef were <5 mg/l 90% of the time throughout August and September, 2000. and May and June. 2001. DO levels measured in 2001 at approximately 30 other reefs throughout the Maryland portion of the Bay was typically <5 mg/l and often <3 mg/l. These levels of hypoxia could have important detrimental affects on restoration projects. 318 Alxsiracts. September 2001 Slielltish. Restoration. Nanuimo B.C.. Canada EARLY COMMUNITY DEVELOPMENT OF OYSTER REEFS IN VIRGINIA: EFFECTS OF REEF SCALE. P. G. Ross, M. W. Luckenbach and A. J. Birch, College of William and Mary. Virginia Institute of Marine Science. Eastern Shore Laboratory. Wachapreague. VA 23480, USA. Traditionally, oyster reef restoration in the Chesapeake Bay region has primarily focused on oyster production. Increasingly, however, efforts have included restoration of associated commu- nities and the overall ecological function of self-sustaining reefs as management goals. The communities that develop in association with oyster reefs in Chesapeake Bay. VA have been shown to be diverse and ecologically important. Additionally, reef architecture, such as vertical relief and interstitial space, has been shown to be important to developing and maintaining community diversity. Another, previously unaddressed architectural component, aerial extent or scale, is also expected to affect community development. In a replicated block design, we have constructed high relief reef bases ranging in size from 400 sq m to 8000 sq m and are char- acterizing the development of the associated resident and transient assemblages of organisms. We will present data evaluating early community development on these different scale subtidal reefs. We compare reefs of differing scales (i.e.. small versus medium versus large size reefs), but also make comparisons within a reef (e.g.. inner portions of reef base versus reef edge). Substrate for constructing reefs is often limiting and expensive and resource managers face tough decisions on how to allocate scarce resources (e.g.. construct several large or many small reefs). When maxi- mizing diversity and ecological function of reefs is a management objective, knowing the impact of reef scale can be a valuable asset for makin" such decisions. GENETIC AND PATHOGENIC ASPECTS IN SHELLFISH RESTORATION OF SCALLOP POPULATIONS. Rejean Tremblay, and T. Landry, UQAR-MAPAQ. Centre Aquacole Marin, Grande-Riviere, Que.. GOC IVO; Gulf Fisheries Center. DFO. Moncton. N.B.. EIC 986. Canada. With recent declines in the scallop fishery in Atlantic Canada, several projects on stock enhancement are being conducted. The success of shellfish population restoration is directly related to the fitness of the scallop seed. Results obtained from restoration ac- tivities of Giant scallops (Placopecten magellanicus) populations will be used to discuss of the impacts of genetic and pathogenic aspects on the fitness of scallops and the success of these activities. Factors that will contribute to reduce the overall genetic variability may compromise the capacity of a species to adapt to environ- mental changes and to resist to pathogens. Thus, the long-term survival of that species may be compromise. Indeed, if genetic variation within individual populations is reduced, there will be less of a basis for future adaptation within a given population. Genetic changes often occur during the hatchery process. In Que- bec, scallop enhancement activities are carried out using juveniles produced in hatchery, or collected over scallop beds and that are either re-released in the same area or transfer to other areas. The impacts of all these practices on genetic variability of populations are not known. The genetic of shellfish produced in hatchery are frequently altered through inbreeding, selective breeding or do- mestication with an overall reduction in genetic variability. This can also be true for seed collected on artificial collectors. These enhancement activities could result in hybridization, with the pos- sibility of reducing the genetic variation and hence, the fitness of the enhanced population. WATER QUALITY AND OYSTER HEALTH {CltASSOS- TREA VIRGINICA): AN INTEGRATED APPROACH TO DETERMINING HABITAT RESTORATION POTENTIAL. Aswan! K. Volety.' S. Gregory Tolley,' and James T. Win- stead," 'Florida Gulf Coast University. Division of Ecological Studies. 10501 FGCU Blvd. S.. Fort Myers. FL 33965. USA: "U.S. Environmental Protection Agency, Gulf Ecology Division. 1 Sab- ine Island Drive. Gulf Breeze. FL 32561. USA. The influence of water quality and season on disease preva- lence and intensity, gonadal condition, recruitment potential, and growth of oysters was examined monthly at five locations along the Caloosahatchee River estuary. Florida. Habitat suitability of oyster reefs for fishes and decapod crustaceans was examined at three of these sites. Higher temperatures and salinities favored the parasite Perkinsiis mahuiis. and histological analyses revealed the presence of several additional parasitic or comniensalistic organ- isms. Comparison of mortality among sites indicated that juvenile oysters tolerated salinities of 15-38 ppt. Spat recruitment was higher at sudtidal (1-5 spat/shell) than at intertidal locations, where sparse oyster distribution and swift currents appeared to limit settlement success. The late peak in gameiogenesis (August to September) observed at all sites may have resulted from reduced salinities during May to Jul) or may imply that oysters spawn twice per season. Oyster-reef habitat supported a fish and decapod assemblage averaging 77 individuals m~" or 14 individuals 1 oyster cluster. Reef-resident fishes included Gobieso.x stnimosits, Chasmodes sahurrae. Gobiosoma rohiisnim. and Opsamis beta: xanthid and porcellanid crabs represented the dominant crustaceans. Species diversity (H') did not vary among sites: however, significant dif- ferences in density (individuals 1"' oyster cluster) were found in the fishes C. strumosiis and C sabuirae and in the crabs Paiwpeus herhstii and Petrolisthes arimitus. Overall, results suggest that pe- riodic freshwater releases may benefit oysters by lowering the salinity and thus the intensity of parasite infection. PerkinsKS imiri- mis. It should be cautioned that the long-term effects of low sa- linity on oysters have not been investigated. Shellfish, Restoration. Nanaimo B.C.. Canada Abstracts. .September 2001 319 THE NUTRIENT VALUE OF SHELLFISH AND OTHER TRADITIONAL FOODS. THEIR PAST AND CURRENT CONTRIBUTION TO THE DIET OF FIRST NATIONS PEOPLE. Pamela Winquist. First Nations and Inuit Health Branch. Pacific Region. Health Canada. #540-757 West Hastings Street. Vancouver, EC V6C 3E6, Canada. The nutrient value of shellfish and other foods traditionally used by First Nations people in British Columbia. Canada will be reviewed. Information on past and current consumption, and fac- tors affecting use. will be discussed. Shellfish and other traditional foods offer superior nutrient value compared to commercial alternatives. These foods have con- tributed significantly to the overall nutrient intake of First Nations people, in the past. Seafood and other traditional foods continue to contribute to the overall nutrient quality of the diet of First Nations pet)ple. Traditional foods provide nutrients that are often low in the commercial food diets of First Nations people, such as vitamin A, calcium, iron, polyunsaturated oils and omega-3 fatty acids and folic acid. When these foods are replaced by commercial alterna- tives, fat. sugar and salt intakes increase. Diets high in saturated fat. sugar and salt are one of the risk factors in the development of chronic diseases such as obesity, diabetes and coronary heart dis- ease. Higher rates of these illnesses are seen in First Nations people, compared to the general population. Factors infiuencing present use of traditional foods include ac- cess to a commercial food source, knowledge and skills of family members to fish, hunt and/or trap, amount of traditional food a\ ail- able, fish and wildlife regulations, contamination of traditional foods, and household income. In conclusion, shellfish and other traditional foods are of high nutrient \ alue. These foods should continue to be consumed as part of a nutritious diet, when they are available for safe harvest and preparation. PREDATION OF JUVENILE SEA SCALLOPS (PLA- COPECTEN MAGELLANICUS) DURING SEEDING TRI- ALS IN THE NORTHUMBERLAND STRAIT. Melisa C. Wong,' M. A. Barbiau," L. ,\. Grecian,' L-.\. Davidson," M. Niles," and Donna Murray,"' 'Department of Biology, University of New Brunswick. Fredericton. NB. E3B 6E1, Canada; "Depart- ment of Fisheries and Oceans, Gulf Fisheries Centre, Moncton, NB, EIC 9B6. Canada; 'Botsford Professional Fishermen's Asso- ciation Inc.. 1696 Route 955. Little Shemogue. NB, E4M 3M6, Canada. The Botsford Professional Fishermen's Association Inc. and the Department of Fisheries and Oceans (Moncton. NB) have con- ducted a scallop enhancement project in the Northumberland Strait since 1998. Predation of juvenile sea scallops by sea stars [Asterias vulgaris) and rock crabs {Cancer irroratiis) is a concern in the Northumberland Strait. Our goal was to quantify predator-induced mortalitv of seeded scallops and to determine how quickly mor- tality occurs after seeding. We deployed assays (consisting of scal- lops tethered to lead-weighted lines) to monitor scallop mortality at seeded and non-seeded sites, after seedings in October 1999 and October 2000. In both years, shell remains on the tethered lines indicated the presence of both sea stars (empty intact shells) and rock crabs (shell fragments) at both sites. In October 1999. scallop mortality did not differ between the seeded site and the non-seeded site or show a significant change over time. In October 2000, scallop inortality did not differ between sites, while scallop mor- tality was significantly higher 10 days after seeding than 2 days after. Joiinitil of Shellfish Research, Vol. 21, No. 1. 321-328. 2002. ABSTRACTS OF TECHNICAL PAPERS Presented at the 55 Annual Meeting NATIONAL SHELLFISHERIES ASSOCIATION & PACIFIC COAST OYSTER GROWERS ASSOCIATION (PACIFIC COAST SECTION) Silverdale, Washington September 20-22. 2001 321 Pacific Coast Section, Silverdale, Washington Abstracts. September 2001 323 CONTENTS Jeremy M. Breach and Jeimfer L. Ruesink Population and behavioral characteristics of introduced oyster drills. Cenitnstoma innnwtiini. in Willapa Bay 325 Atnilee Caffey and Are Strom Juvenile growth study on the geoduck clam, Panopea ahnipta 325 Hakan Calik, Michael T. Morrissey, Paul W. Reno, and Haejiing An Effect of high-pressure processing on Vibrio panihat'iiuilyiiciis strains in pure culture and Pacific oysters 325 Elyse K. Cronin and Brent Vadopalas Age determination in geoduck clams {Panopea abnipta) utilizing patterns in the shell annuli 325 Robyn M. Estes, Russell P. Herwig. and Ralph A. Elston Characterization of pathogenic and nonpathogenic bacteria associated with bivalve larvae and shellfish hatcheries 326 Stephen P. Ferraro and F. A. Cole Oyster grounds: A superior habitat for small, sediment-dwelling invertebrates 326 Graham E. Gillespie Status of the Olympia oyster. Ostrea concliapinla. in Canada 326 Geoff Hosak, David Armstrong, Bryce Semens, Brett Dutnbauld, and Steven Rumrill Oyster aquaculture as fish habitat in Pacific northwest coastal Estuaries 327 Chris Langdon, Ford Evans, John Brake, and Sean Matson Survival and growth effects on yields of Pacific oysters. Crassostrea gigas 327 Cynthia S. Marshall and Russell P. Herwig Research to examine use of high hydrostatic pressure to inacti\ate human enteric viruses in oysters 327 Sean E. Matson, Chris Langdon, Ford Evans, John Brake, and Dave Jacobson Predictability of grow-out performance from nursery performance of Pacific oyster, Crassostrea gigas 327 Daphne M. Munroe and Leah Bendell-Young The effects of clam aquaculture on intertidal ecosystem structure and function 328 Paul G. Olin and Jim Hobbs Habitat \ alue of commercial oyster culture gear 328 Pacific Coast Section, Silverdale, Washington Abstracts. September 2001 325 POPULATION AND BEHAVIORAL CHARACTERISTICS OF INTRODUCED OYSTER DRILLS, CERATOSTOMA IN- ORNATUM, IN WILLAPA BAY. Jeremy M. Breach and Jen- nifer L. Ruesink, Department of Zoology. University of Wash- ington. Seattle. WA 98195-1800. Japanese oyster drills in Willapa Bay were studied to discern population parameters including density, size-frequency distribu- tion, food preference, and movement rates. Drills were collected during the summer of 2001 from 0.0625 and 1 m" quadrats near the Washington Department of Fish and Wildlife lab at Nahcotta, Washington. Drill density averaged 31 m"". and the size distribu- tion included a peak of small individuals suggestive of this year's recruitment. Drills showed a preference for feeding on smaller oysters, and oysters appeared to escape drill predation at shell lengths exceeding 2 cm. Drills were labeled and released into three different habitats to .study drill movement. Significant differences were found between movement rates on eelgrass. oyster bed. and mudtlat. with drills traveling up to 1 m/day on mudtlat and eel- grass until reaching small patches of oysters. Risks of drill impacts are highest for seed oysters placed on the bottom in infested areas, particularly if few oysters are currently present. JUVENILE GROWTH STUDY ON THE GEODUCK CLAM, PANOPEA ABRUPTA. Amilee Caffey, Washington Department of Fish & Wildlife, Point Whitney Laboratory. Brinnon. WA 98320: Are Strom, University of Washington, WA 98062. The geoduck clam has proven to be a challenging species to culture in the hatchery setting. Water temperature, salinity, sun- light, food concentrations, and handling all affect levels of growth. The goal of this study was to determine what effect temperature and varying food concentration levels in the sunounding water had on the juvenile geoduck clam. The statistical analysis method was a two-way ANOVA in which juvenile clams were introduced to four varying environ- ments where temperature and food concentrations were aberrant. Clams were placed in 1-in. square, plastic trays, layered with per- meable fabric and sand. The stocking density was 100 juveniles per tray. Smaller juveniles were chosen, ranging between 14 and 19 mm in shell length in order to determine rapid growth rates. Three identical trays were subjected to each of the four treatments, with a total of 12 trays. The first and second treatments were subjected to a higher temperature, between 1 5 and 1 6°C. The third and fourth treatments were at a lower temperature, 10-1 1°C. The first and fourth treatment were held at a higher concentration of food. 45 k/mL. The second and third treatments were held at a lower food concentration of 15 k/mL. Flow rate was 6 L/min. Shell length did not vary significantly between treatments, however. Juveniles exposed to higher temperature (I5-16°C). at higher food concentration (45 k/mL algal cells), demonstrated the most growth. EFFECT OF HIGH-PRESSURE PROCESSING ON VIBRIO PARAHAEMOLYTICUS STRAINS IN PURE CULTURE AND PACIFIC OYSTERS. Hakan Calik, Michael T. Morris- sey, Paul VV. Reno, and Haejung An, Department of Food Sci- ence & Technology. Oregon State University Seafood Laboratory. 2001 Marine Drive. Room 253, Astoria. OR 97103. Several cases of Vibrio parahaemolyticiis (Vp)-induced gastro- enteritis occurred in the Pacific Northwest due to consumption of raw oysters. High-pressure process (HPP) technology has shown good potential in reducing pathogens. Environmental and clinical strains of Vp in broth cultures and Vp-inoculated live Pacific oys- ters (Crassostrea gigas) were subjected to HPP at different treat- ment settings ( 1-10 min at 241 MPa; 1-5 min at 276 MPa; 30-120 s at 310 MPa; 10-90 s at 345 MPa). Results showed that Vp numbers were reduced by HPP in both pure culture and whole oysters. Vp inactivation was dependant on treatment time and pressure. Optimum conditions for reducing Vp in pure culture and oysters from 109 to 101 CFU/mL were achieved at 345 MPa for 30 and 90 s. respectively. Resistance variations were detected be- tween Vp in pure culture and in oysters. Further high-pressure tests with clinical 03:K6 Vp strain isolated from an outbreak in Texas ( 1998) showed that 5 min at 310 MPa was necessary for complete elimination, making the isolate the most baro-resistant of all strains used in the study. HPP proved to be an efficient means of reducing Vp in oysters. AGE DETERMINATION IN GEODUCK CLAMS {PANO- PEA ABRUPTA) UTILIZING PATTERNS IN SHELL AN- NUEL Elyse K. Cronin and Brent Vadopalas, School of Aquatic and Fishery Sciences. University of Washington, Seattle, WA 98105. Geoducks (Ponupcti iihnipia) are deep-burrowing bivalves dis- tributed from Southeast Alaska to Baja, California. This extremely long-lived species supports a lucrative fishery in Washington state, yet little is known of geoduck population dynamics. Age data are being collected from various sites in Puget Sound to investigate both population dynamics and temporal genetic structure in geo- ducks. via collaborations between the Washington Department of Fish and Wildlife (WDFW), Washington Sea Grant, and the Uni- versity of Washington School of Aquatic and Fishery Sciences. To age geoducks, the hinge plate of the right valve was thin- sectioned using a diamond saw. polished, and finally etched with 19f HCl. Annual growth rings (annuli) were visualized via light microscopy, and two independent observer counts were made on three thin-sections for each clam. Annuli revealed year-specific width variation consistent across samples. These patterns may cor- relate with localized environmental factors or regional oceano- graphic conditions, and are used to establish year reference points in series of annuli. 326 Ahsinicls. September 2(1111 Paeitic Coast Section, Silverdale. Wasliuigtoii Ages determined from 200 geoducks collected from a Hood Canal site sampled in 2000 {N = 1010) ranged from 3 to 136 y. When complete, this age data set will be used in analyses of genetic population structure and population dynamics of the spe- cies in Puget Sound. CHARACTERIZATION OF PATHOGENIC AND NON- PATHOGENIC BACTERIA ASSOCIATED WITH BI- VALVE LARVAE AND SHELLFISH HATCHERIES. Robyn M. Estes, Russell P. Herwig, School of Aquatic and Fishery Sciences, Box 355020, University of Washington. Seattle. WA 98195-5020: Ralph A. Elston, AquaTechniques/Pacific Shellfish Institute, P.O. Box 687, Carlsborg. WA 98324. Bacterial diseases are a major cause of larval mortality in shell- fish hatcheries. Even with proper sanitation measures, bacterial pathogens cannot be eliminated. The proper identification of pathogens and the application of probiotics may help control dis- ease outbreaks. More than 100 bacterial isolates were collected from larval Pacific oysters, larval geoducks. and locations within hatcheries in the Pacific Northwest and Hawaii. Isolates were characterized by whole cell analysis and restriction fragment length polymoiphism (RFLP) of 16S rDNA using three restriction enzymes. Both of these methods show similar relationships between the isolates. Pathogenicity tests of isolates collected from Pacific oyster larvae from Washington and Oregon showed that 3 out of 33 isolates were highly pathogenic. These as.says examined larval mortality and the ability of larvae to remain suspended in a water column. Pathogenic and nonpathogenic bacteria strains appear closely re- lated to each other. These results could provide information for the development of probiotics in shellfish aquaculture. Future research includes sequencing the 16S rDNA of patho- genic bacteria, declaring new species if they cannot be identified as known species, and developing polymerase chain reaction (PCR) primers for rapid detection of pathogenic bacteria. This will help in early detection of pathogenic bacteria and in determining the source and ecology of the pathogenic organisms in hatcheries. OYSTER GROUNDS: A SUPERIOR HABITAT FOR SMALL, SEDIMENT-DWELLING INVERTEBRATES. Steven P. Ferraro and F. A. Cole, U.S. Environmental Protection Agency. 2111 S.E. Marine Science Drive. Newport. OR 97365- 5260. As part of a programmatic effort to determine estuarine habitat values for ecological risk assessments, quantitative tleld studies ol small, sediment-dwelling invertebrates were conducted in Willapa Bay, Washington, in July 1998 and Tillamook Bay, Oregon, in July 1999. The six habitats included in both studies were (1) "grow-out" (2-3 y old) oyster ground culture, (2) eelgrass, Zosleni marina. (3) mudshrimp, Upogebia piit-ctlciisis. (4) ghost shrimp. Neotnpaea califonuciisis. (5) bare mud. and (6) subtidal. un- dredged. About fifteen 0.01 nv x 5 cm deep. 0.5 mm mesh samples were collected randomly in each habitat throughout bulh estuaries. Multix ariate analyses of the data revealed that the invertebrate fauna on oyster grounds was much more similar to that in eelgrass and mud shrimp habitat than that in ghost shrimp, bare mud. and subtidal habitat. Among the six habitats studied, oyster grounds consistently tanked either first or second in terms of the number of species, abundance, and total biomass of invertebrates. Oyster grounds, which have high economic value in terms of oyster pro- duction, are also ecologically valuable because they provide a superior habitat for small invertebrates upon which many larger animals (e.a.. fish, crabs, waterfowl) feed. STATUS OF THE OLVMPIA OYSTER. OSTREA CON- CHAPHIIJi, IN CANADA. Graham E. Gillespie. Fisheries and Oceans Canada. Pacific Biological Station. Nanaimo. British Co- lumbia. V9R 5K6. The Olympia oyster. Ostrea amcluiphila. is the only oyster native to the Canadian Pacific coast. Olynipias supported commer- cial fisheries in British Columbia from the late 1800s to about 1930. when focus of the oyster industry shifted to Pacific oysters. Cnissostrea gigcis. Olympia oyster distribution in British Columbia is limited by specialized habitat requirements, and relatively low fecundity and dispersal. Olympias are vulnerable to temperature extremes, and are not resistant to haivests on a commercial scale. Habitats that once supported large aggregations in Georgia Strait no longer do. in part because of historic overharvests and en\ ironmental stresses, and because development of large oyster reefs tnay require centu- ries without disturbance. Small relict populations survive at low tide levels and under floating structures. Olympias are locally com- mon at sites on the west coast of Vancouver Island, and little information exists on populations in Johnstone Strait or in the Central Coast. They do not occur in the Queen Charlotte Islands. Olympia oysters are not likely facing imminent danger of ex- tinction or extirpation in Canada. Limiting factors have led to significant reductions to population levels in the past. From the limited data available, the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) assigned a status of Special Con- cern in November 2000. Proposed federal legislation, the Species At Risk Act (S.ARA). will require development of an Olympia oyster management plan within 3 y. Pacific Coast Section, Silverdale. Washincton Abstracts. September 2001 327 OYSTER AQUACULTURE AS FISH HABITAT IN PA- CIFIC NORTHWEST COASTAL ESTUARIES. Geoff Ho- sack. David Armstrong, Bryce Semens, School of Aquatic and Fishery Sciences. Box 355020, University of Washington. Seattle, WA 98195: Brett Dumbauld, Washington State Department of Fish and Wildlife. Willapa Bay Field Station. P.O. Box 190. Ocean Park. WA 98640; Steven Rumrill, South Slough Estuarine Re- search Reserve. P.O. Box 5417, Charleston. OR 97420. Increased pressure on traditionally managed stocks of marine and anadromous fish, calls for protection of essential fish habitat under the Magnuson-Stevens Act, and recent listings of several salmonid stocks under ESA, have brought aquaculture activities that take place in coastal estuaries under increased public scrutiny. We initiated a study designed to examine the ecological role that oyster aquaculture plays as habitat in coastal estuaries of the Pacific Northwest. The goal of the project is to identify and quan- tify beneficial and adverse impacts of shellfish farming on eel- grass, juvenile salmonids. and other selected estuarine fauna and flora, and to develop farming practices and recommend inanage- ment protocols that protect or enhance those resources. We present results of initial fish and invertebrate sampling in selected habitats from Willapa Bay during spring and summer 2001. and an experi- ment designed to examine the effects of oyster harvesting on eel- grass habitat. We make a plea to managers to consider oyster aquaculture areas as fish habitat on a broader estuarine scale. SURVIVAL AND GROWTH EFFECTS ON YIELDS OF PA- CIFIC OYSTERS, CRASSOSTREA GIGAS. Chris Langdon, Ford Evans, John Brake, and Sean Matson, Coastal Oregon Marine Experiment Station and Department of Fisheries and Wild- life. Oregon State University. Newport. OR 97365. In Spring 1999. 29 full-sib families derived from crossing non- selected "wild" oysters were planted at a subtidal site in Yaquina Bay. Oregon. In addition, in Fall 1999. 32 full-sib. families from crosses within two groups of six selected families were planted at a an intertidal site in Tomales Bay. California. After about 1 y of growth, each cohort was harvested and average yields, survival. and individual growth rates determined for each family. These parameters were adjusted for the effects of differences in average initial weights of planted spat per family if a significant {P < 0.05) effect was detected. Adjusted survival explained 517c and 68%. and growth ex- plained 25% and 21% of the variation in adjusted family yields at the Yaquina and Tomales sites, respectively. Furthermore, up to 79%' of the variation in family yields at the Tomales site was explained by differences in survival among families, if survival was not adjusted for a significant (regression, r = 0.38; P < 0.0001 ) positive effect of initial spat weight. These results indicate that both growth and. perhaps to a greater degree, survival should be considered when designing breeding programs to increase oys- ter yields. RESEARCH TO EXAMINE USE OF HIGH HYDROSTATIC PRESSURE TO INACTIVATE HUMAN ENTERIC VI- RUSES IN OYSTERS. Cynthia S. Marshall and Russell P. Herwig, School of Aquatic and Fishery Sciences, Box 355020, University of Washington. Seattle. WA 98195-5020. The desire for safe shellfish products in the retail market and food service establishments is paramount to the success and growth of the shellfish industry. Unfortunately, bacterial and viral pathogenic organisms may be associated with fresh and processed shellfish. These organisms can lead to seafood-borne illness that may result in severe economic impacts on the shellfish industry, causing shellfish bed closures, product recalls, and lost consumer confidence. Human enteric viruses are the causative agents asso- ciated with a large, but poorly understood, number of seafood- borne illnesses per year. The principle viruses associated with seafood include hepatitis A. Norwalk virus, Norwalk-like viruses, and astrovirus. Viruses are typically not monitored in seafood products and in shellfish growing waters because of the difficulty and expense of quantifying active virus particles. There is a large deinand for raw oysters in the market. Depu- ration or relaying may be used to reduce the number of pathogenic organisms present in live shellfish before they are harvested. In recent years, high hydrostatic pressure (HHP) treatment has been shown to reduce or eliminate bacterial pathogens from shellfish while retaining sensory properties of the raw product. We are beginning a new research project that will examine the effects of HHP on human enteric viruses. Poliovirus will be used as a model virus in our experiments. This virus was chosen because it can be safely used in the laboratory and can be cultured using standard protocols. In laboratory experiments, we will vary hydrostatic pressure, time of exposure. pH. and temperature on preparations containing poliovirus. PREDICTABILITY OF GROW-OUT PERFORMANCE FROM NURSERY PERFORMANCE OF PACIFIC OYS- TER, CRASSOSTREA GIGAS. Sean E. Matson, Chris Lang- don, Ford Evans, John Brake, and Dave Jacobson, Hatfield Marine Science Center. Oregon State University. 2030 S. Marine Science Dr. Newport. OR 97365-5296. The predictability of Pacific oyster (Crassostrea gigas) grow- out performance from nursery performance was investigated by measuring yield, survival, and growth of pedigreed families of Pacific oysters, in the nursery and at a grow-out site in Totten Inlet. Washington. Early prediction of grow-out performance could sig- nificantly reduce labor, materials, and space required to select pedigreed families on the basis of performance in an oyster breed- ing program. This research was conducted as part of the Molluscan 328 Abstracts. September 2001 Pacific Coast Section. Silverdale, Washington Broodstock Program, a breeding program for the sustainable in- crease in Pacific oyster yields through genetic selection. Yield was measured as oyster bag weight in grams and is a function of both survival and growth. Unadjusted yield, yield adjusted for initial size, and unadjusted individual growth of Pacific oyster seed in the nursery were used as predictors of unadjusted yield, yield adjusted for initial weight, and unadjusted mdividual growth in the field. Unadjusted nursery yield was found to be a significant predictor of unadjusted grow- out yield IP = 0.0009. r = 0.250). Initial planting weight, how- ever, was also a significant predictor of unadjusted grow-out yield (P < 0.0001. r = 0.283). Similarly, initial nursery weight was a highly significant predictor of nursery yield (P < 0.0001. r = 0.703). Thus, adjustments were employed to both nursery and grow-out yield, to remove the effect of initial weight. Adjusted nursery yield was not a significant predictor of adjusted grow-out yield (P = 0.9975, /" = 2.475 x 10" ). These data indicate that heavy spat grew into heavy oysters because they were planted as heavy spat. They also indicate that Pacific oyster families with high yields at grow-out (adjusted for initial weight) are not easily identified in the nursery using the methods described here. THE EFFECTS OF CLAM AQUACULTURE ON INTER- TIDAL ECOSYSTEM STRUCTURE AND FUNCTION. Daphne M. Munroe and Leah Bendell- Young. Department of Environmental Science. Simon Fraser University. 8888 University Drive. Burnaby. BC V5A IS6. Quadrat and core sampling was carried out to gather baseline data regarding ecosystem structure and function from three beaches on Denman Island. British Columbia. Ecosystem structure was examined through biodiversity measures (species richness, evenness, and heterogeneity I. community composition, and spe- cies distribution. Ecosystem function was evaluated using percent silt and percent organic matter from core samples. The three study sites experienced different levels of commercial aquaculture. One site was a recreational harvest beach and the other two were leased for commercial aquaculture: one was 159c covered with nets, the other was only 20% covered. We observed differences in ecosystem structure and function among the three sites. First, species richness was higher on the beach where no commercial practices occurred. Second, there were higher numbers of organisms per quadrat in the upper areas of the recreational beach and considerably more surface species on that beach. Third, we saw that the distribution of the most abun- dant clam species was limited to the upper half of the beach where no commercial clam culture was conducted; however, on the other two beaches, the distribution of this clam species extended the length of the study area. This research indicates a high probability that commercial clam culture causes changes in intenidal ecosy.stem structural and func- tional components. Further research is imperative to provide man- agers with the scientific information needed to develop sustainable and environmentally sound management protocols. HABITAT VALUE OF COMMERCIAL OYSTER CUL- TURE GE.AR. Paul G. Olin. University of California Sea Grant. Davis. CA 95616; Jim Hobbs. University of California, Davis, CA 95616. Oyster growers in Tomales Bay, California, produce deeply cupped single oysters for the half-shell market using plastic mesh socking attached to stakes, and plastic mesh bags on the bottom, on racks or floating attached to longlines. This oyster culture gear and the oysters that are grown form a complex three-dimensional habi- tat that interacts in a variety of ways with the biological and physical components of the estuarine ecosystem. This habitat is utilized by a myriad of fish and invertebrates, which are often prey for larger commercially important species such as halibut or dungeness crab. To document the habitat value of cultured oysters and gear, fish and invertebrates from 36 culture bags were collected and all mac- roorganisms were enumerated and identified to species. Ten phyla and 11 taxonomic classes were represented by the 51 different species identified. One oyster culture grow-out bag held more than 5.000 organisms, although more typically between 600 and 1.000 individuals were found in each bag. In Tomales Bay approximately 8% of the intertidal and subtidal bottom lands are leased for shellfish culture by the state. Of this S^f, around 1% is actively farmed. Although this represents a small portion of the Bay, it is highly productive and provides complex intertidal habitat that has been lost in many areas due to erosion and resultinc sedimentation. Journal of Shellfish Research. Vol. 2!. No. I. 329-356. 2002. ABSTRACTS OF TECHNICAL PAPERS Presented at the 22'"' Annual MILFORD AQUACULTURE SEMINAR Milford, Connecticut Fehruarv 25-27. 2002 329 Milford Aquaculture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 331 CONTENTS Walter Blogoslawski Overview, 22'"' Milford Aquaculture Seminar 335 Peter Adamik, Roxanna Andersen, Murray Croiis, Graham Mains, Vicke Starczak, Isabelle Williams, Diane Murphy, and Dale Leavitt Influence of qualiog grow-out nets on benthic diversity in Wellfleet, MA 335 John Aldred, hike Siinila, and Christopher Martin Possible lar\ al mycosis as a cause of bivalve seed mortality in a production hatchery 335 Standish K. Allen, Jr. New developments with nonnative shellfish species in the Chesapeake Bay 336 Brian J. Ball, Stephanie T. Rutkowski, Emily T. Griffiths, and Kim W. Tetraiilt The effects of varying cold water temperatures, size and population density on the growth and mortality rates of bay scallops. Argopecteii imidiaiis imidiiiiis. in an upweller 336 Amber L. Beitler and John J. Roy Catch statistics of Htmuinis aiiiericaiuis. the American lobster, from a 3-wk study conducted by students from the Sound School Regional Aquaculture Center aboard a commercial lobster vessel in the waters off Fishers Island. New York 337 David A. Bengtson, Stephen Willey, Erin McCaffrey, and David Alves Effects of water velocity on conditioning of summer flounder ParuUclitliys dciitatus for net pens 337 Luther Blount Reminiscences of early pioneers in oyster culture 338 Diane J. Brousseau and Ronald Goldberg "Crab sightings'" in Long Island Sound during 2001 338 Walter J. Canzonier Some classical personalities in the shellfish research arena: Eclectic observations of an erstwhile observer 339 Diane Carle Massachusetts Ocean Resource Information System (MORIS) 339 Joseph Choromanski, Sheila Stiles, Mark Dixon, and Christopher Cooper Habitat suitability ascertained by growth and survival of bay scallops in tiered cages 340 Carmela Cuomo, Paul R. Bartholomew, Leslie Angelini, Brian King, and Jeffrey Byczko Horseshoe crab aquaculture: Preliminary results from hatching and rearing studies 340 John J. Curtis, Sherry W. Lonergan, Paul J. Tnipp, Peimin He. Raqiiel Carmona, Charles Yarish, George P. Kraemer, Christopher D. Neefns, Thierry Chopin, and George i\'ardi A cooperative study on the aquaculture of Porphyra leiicosticta (Rhodophyta) for an integrated finfish/seaweed recirculating aquaculture system in an urban application 341 Gef Flinilin Update on shellfish restoration and review of local hatcheries in New Jersey 341 Michael J. Goedken and Sylvain De Guise Flow cytometry as a tool to quantify the oyster phagocytosis, respiratory burst, and apoptosis 342 Josh Goldman Cobia culture 342 Eric Goodman, Colleen Cook, and Michael Weiss Creating opportunities for student aquaculture projects 342 Helene Hegaret, Gary H. Wikfors, Philippe Soudant, Maryse Delaporte, Jeanne Moal, and Jean-Franfois Sainain An experimental investigation of dietary fatty acids and sterols and the immunology of the American oyster, Crassostreci virginica: A well-fed oyster is a healthy oyster, n'est-ge pas? 342 Andrea Hsu, Erin Summers, James Estrada, and Roxanna Smolowitz The role of three bacteria in shell disease of the American lobster [Hoiminis ainfiicanus) 343 Richard C. Karney, Amandine Siirier, David W. Grunden, and Thomas E. Berry Dermo investigations, razor clam nursery trials, and preliminary bay scallop adhesion culture efforts 343 Robin Katersky, Barry Smith, Dean Perry, and David Nelson Some culture strategies for growing rotifers (Brachioiiiis plii(itili\) as feed for aquaculture applications 344 Brian Kilpatrick, Joseph DeAlteris, and Robert Rheault Assessing habitat value of modified rack and bag aquaculture gear in comparison with submerged aquatic vegetation. in particular, an eelgrass (Zosteiu iiuiriini) bed 344 332 Ahslracls. 2002 Annual Meeting. February 25-27. 2002 Milford Aquaculture Seminar. Milford. Connecticut .Suspended aquaculture development in Connecticut "^^ It takes a coinnuinity to yrow a scallop '^ Gordon King The culture of black pearl oysters on subsurface longlines in Savu Savu. Fiji 345 Gordon King Urban commuiiit\ meets aquaculture: A case study in the Northwest 345 Dale Leavitt, William Bnrt, Diane Murphy, and Rebecca Hanson Progress with culturing the razor clam (£);.s(.s cliirctiis) 345 Kathryn R. Markey and John J. Roy A comparison of sur\ ival m luvenile Argopccieii irnidicms iinulians using various culture techniques at the Sound School Regional Aquaculture Center ■*" Paul D. Mangle Suspendei Mary F. Morgan, Kathleen K. Becker, and Kim TetraiiU It takes a coinnuinity to grow a scallop Diane Murphy, Dale Leavilt, Bill Burt, and Bill Clark Bay scallop (Argopectcn irnidicms irnidicms ) restoration on Cape Cod 347 Jennifer Mugg-Pietros and Michael A. Rice Effects of CnissDsircii viri^inica populations on sedimentation, phytoplankton species composUion. and ammonia 348 cycling in experimental mesocosms Steven Pitchford and Richard Rohohm A review of diseases in the bay scallop {Argopectcn irnidiims irnidicms) and some observations on mortalities at the 348 Milford Laboratory Perry Raso and Michael A. Rice Shellfish aquacullure's effect on total organic carbon (TOC) in the benthos 349 Edwin Rhodes Industrial-scale scallop culture in Chile— the C.M.I, experience 349 Karen Rivara, Amber L. Beitler, and John J. Roy The development of an instructional shellfish hatchery: A collaborative effort between Aeros Cultured Oyster Co. and the Sound School Regional Aquaculture Center "^^ Karen Rivara The East Coast Shellfish Growers Association: A work in progress 350 Rene Sanz, Sherry Lonergan, Jennifer Sutorius. and Dania Lieberthal Heavy metal survey of Fkciis spiralis collected from southv\esterii Long Island Sound 350 Martin P. Schreibman, Chester Zarnoch, John T. Tanacredi, Lucia Magliiilo-Cepriano, Jacob Raz, and Stefano Diomede Aquaculture activ ilies in Brooklyn. New York? Sandra E. Shumway Shellfish aquaculture: Good for the economy, good for the em ironment. good for you! 351 Roxanna Smolowitz, Susan Find, and Lisa Ragone-Calvo Health management guidelines for shellfish culture in the northeastern United States 351 Roxanna Smolowitz, hike Sunila, Nancy Stokes, and Lisa Ragone-Calvo Prevalence and mortality associated with SSO and SSO-like infections of Crcissaslrca virginica in the Northeast 352 Karin A. Tanimi, Najih Lazar. Arthur Ganz, James G. Turek, and John G. Catena Rhode Island's Shellfish Restoration Program in response to the North Cape oil spill 352 A7;// TetrauU, R. Michael Patricio, and Maty Morgan SPAT (Special Proizrams in Aquaculture Tramins) update. 2002. Establishing a model for community-bused shellfish : " 353 culture and restoration John Wadsworth, Tessa Simlick, and Nancy Balcom 353 A new clam for Connecticut Bethany A. Walton Life in a trailer— development of a new shellfish hatchery at the Massachusetts Maritime Academy 354 Donald Webster The new oyster wars: Policy perspectives on the introduction of Crassostrcci ciriakciisis in the Chesapeake Bay 354 Milford Aquacultiire Seminar, Milford. Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 333 Scott Weston. Mark Fregeau. and Joe Biittner Developments in softsheli clam hatchery and nursery production on Massachusetts" North Shore 3.'i4 James C. Widman, Jr. and David J. Veilleux Rapid grow th of ha\ scallops. Argopecteii irradiims irnulinns. in Long Island Sound 355 Gary H. Wikfors Livestock domestication in the third mnienniiun: All wet? 355 iMwrence Williams and Tessa Simlick Blue mussel aquaculturc m Long Island Sound 356 Stephen Willey, David A. Bengtson, and Moti Harel Arachidonic acid requirements in lar\ al summer flounder. Paraliclitlns dematiis 356 Milford Aquaciilture Seminar. Milford. Connecticut Absn-acts. 2002 Annual Meeting. February 2.S-27. 2002 .^.^.'^ OVERVIEW, 22"" MILFORD AQUACULTURE SEMINAR. Walter J. Blogoslawski. U.S. Departinenl of Commerce. National Oceanic & Atmospheric Administration. National Marine Fisher- ies Service. Northeast Fisheries Science Center. Milford Labora- tory. 212 Rogers Ave.. Milford. CT 06460. With more than 19.^ registrants, the 22'"' Annual Milford Aqua- culture Seminar was our largest gathering of industry, research, and academic interests. By blending both the theoretical and practical aspects of aqua- culture, the meeting permitted attendees an exchange of technol- ogy in aquaculture methods outside their own expertise and pro\ ided a forum where the latest innovations were introduced and discussed. Fifty formal papers and posters were presented by attendees from 10 U.S. coastal states and France. Meeting attendees repre- sented ? vocational aquaculture high schools. 16 universities. 6 marine labs, and several state and federal institutions involved in shellfish and finfish aquaculture. A highlight of the meeting was a set of papers reviewing the northeastern aquaculture pioneers, making us aware of the difficulties and successes of those who laid the foundation for our aquaculture ventures and studies. Other topics included information on new hatcheries, education, disease, nutrition, and culture techniques. The seminar has developed a tradition of offering the latest information available in the field in an informal atmosphere. This has successfully promoted a free exchange among all with an interest in the success and future of aquaculture. This seminar continued that approach which allowed all attendees to enjoy and learn from the formal presentations, and afforded informal oppor- tunities to discuss the latest developments pertinent to this impor- tant expanding field. At this year's seminar. 45 separate aquaculture companies met in an evening session to discuss the feasibility of forrning an in- dustry group tentatively titled "The East Coast Shellfish Growers Association." The proposed Association's goals are to promote and protect association members' needs in state and regional con- texts and involve all stakeholders in the task of enhancing the shellfish aquaculture industry. The main reason for joining to- gether is to promote industry unity and to counteract recent anti- aquaculture litigation and publicity. The meeting was sponsored by the National Marine Fisheries Service, Northeast Fisheries Science Center, Milford Laboratory. Milford. Connecticut. INFLUENCE OF QUAHOG GROW-OUT NETS ON BENTHIC DIVERSITY IN WELLFLEET. MA. Peter Ad- amik. Roxanna Andersen. Murray Crous, Graham Mains, Vicke Starczak, Isabelle Williams, Diane Murphy, and Dale Leavitt, SouthEastern Massachusetts Aquaculture Center. Massa- chusetts Maritime Academy. 101 Academy Dr.. Buzzards Bay. MA 02532. The question of environmental impacts tiom shellfish aquacul- ture has been posed recently both locally and at an international level. An often-used indicator of environmental health is the di- versity of species within the ecosystem in question. The prevailing assumption is that a decrease in biodiversity suggests a decrease in the quality of the environment. To begin addressing the issue of en\ ironmental impacts of shellfish aquaculture on local intertidal Hats, we initiated a study investigating benthic diversity associated with netted raceways used for quahog (Menenaria inerceiicuia) grow-out as compared to uncovered, unimpacted intertidal flats. The specific question asked was. "Does the presence of aquacul- ture netting structures and high quahog planting densities in the intertidal marine habitat alter the diversity of macrobenthic species in proximity to the nets?" The study plan was to visit two sites having a history of quahog aquaculture and collect replicated core samples within and outside of the netted raceway. The samples were preserved, stained, and returned to the laboratory. In the laboratory, the samples were sieved to 500 p.ni and all living tissue, as stained with rose bengal, was sorted and identified to the lowest taxonomic level possible. A total of ."^9 macrobenthic species were identified in the core samples from sample site 1 (Town Landing. Welltleet. MA) and 27 from site 2 (Old Wharf. Wellfleet, MA). The dominant species in all samples at Town Landing was the glassy tube worm. Spio- chaetoptents ociilatiis: at Old Wharf, the dominant species were the capitellid worm. Heieromastus fdifonmis and the mud snail. Uxanassa ohsolela. The impact of shellfish aquaculture on envi- ronmental quality was analyzed in terms of the species richness, species diversity, and distribution of dominant species between sites and treatments. In general, there were minor or no differences between netted and nonnetted treatments at the two sites investigated. Many other factors can influence diversity, species richness, and evenness of the intertidal macrofauna. including hydrography and fluid inter- action under the netted area, human activities related to shellfish farming, and the historical background of the netted site (long-term culture, mid-term culture, or short-term culture). It was concluded that further study was warranted to include a seasonal and long- term component to the study. POSSIBLE LARVAL MYCOSIS AS A CAUSE OF BIVALVE SEED MORTALITY IN A PRODUCTION HATCHERY. John Aldred, Town of East Hampton. Shellfish Hatchery. 159 Panligo Road, East Hampton. NY 11937; Inke Sunila, State of Connecticut, Department of Agriculture. Bureau of Aquaculture, P.O. Box 97. Milford. CT 06460; Christopher Martin, USDOC. NOAA. National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford. CT 06460. Since 1996. the shellfish hatchery at East Hampton has expe- rienced sporadic unexplained mortalities in cultures of hard clams {Mcrccnaria mcrcciiaiio) and bay scallops (Argopecten irradians iiradiaiis). Typically, a population of larvae progressed normally through metamorphosis and then, eariy in the juvenile stage (post- set) stopped feeding. In such cultures, examination revealed many 336 Ahstmct.s. 2002 Anniiiil Meeting. February 25-27. 2002 Milford Aquaculture Seminiir. Milt'ord. Connecticut dead or dying individuals. Within 2-4 days of the first indication of the problem most animals were dead. Affected juveniles ranged in shell length from 0.2 to \.5 mm. Eastern oysters {Cnissosrreci virginiiu) reared at the same hatchery have not been affected. Preventative measures have been unsuccessful. These included switching algal diets, carelul monitoring of pH. changing water more frequently, and giving special attention to cleaning and dis- infecting the system. On occasion, it has been possible to rescue some juNcniles from affected cultures by mo\ uiy them to a tlow ing water system, with clams surviving better than scallops. During the 2001 growing season, similar mortalities were ob- served. Samples of ju\enile clams (approximately 10 mm in shell length) from two cohorts were preserved in Davidson's fixative. Scallop juveniles also from two cohorts (ranging from approxi- mately 0.2 to O.-S mm in shell length) were similarly fixed. Fol- lowing decalcification, the specimens were stabilized in agarose, embedded in paraffin, sectioned to 6 (xm. and stained with hema- toxylin-eosin according to standard histopathological procedures. Microscopic examination revealed invasion of most tissues and the shell by fungal mycelia. Mycelia were detected in the gill filaments where they often completely obstructed the heniolymph canals. Fungal hyphae were observed within the mantle cavity, with invasion of all epithelia (mantle, gill, and foot). The hyphae appeared to have some affinity to mucus. Cilia were entrapped. All layers of shell were invaded, with penetration to the exterior. The hyphae stained dark blue with hematoxylin, measured approxi- mately 2-3 (jLin in diameter, and were rarely branched and septate. Evidence of starvation was apparent. In most instances, food particles were absent from the digestive system and adductor muscles were atrophied. In adsanced stages of invasion, tissues were disintegrated, with complete loss of vesicular connective tis- sue in some specimens. We concluded that larval mycosis was the cause of the observed mortalities. The fungus appears to be an opportunistic pathogen causing significant morbidity and mortality when conditions are optimal for its pathogenic development. Bi- valve cultures in hatcheries often collapse and new cultures are initiated. The cause or causes of mortality are rarely determined. We suggest that larval mycosis may be a common, under- recognized cause of such events. NEW DEVELOPMENTS WITH NONNATIVE SHELLFISH SPECIES IN THE CHESAPEAKE BAY. Standish K. Allen. Jr., Aquacultuie Genetics and Breeding Technology Center. Vir- ginia Institute of Marine Science. Gloucester Point. VA 23062. VIMS has been systematically examining the potential of sev- eral nonnative species to alleviate the serious decline of oyster stocks in the Virginia portion of the Chesapeake Bay. One that has emerged as quite promising is the Suminoe oyster, Crassostiea ariakt'iisis. Field trials with sterile triploids have shown a general resistance to disease, rapid growth rate, and high smvival. Formal and informal test marketing of the product has been similarly successful. As with any nonnative species, serious concerns exist about the long-term implications of introduction. Some of these issues are associated with simultaneous introduction of pests or pathogens, and some are associated with the ecological effects of sustained population growth in the Bay. By culturing this species ill the hatchery, pest and pathogen issues are largely, but not wholly, addressed, Aquaculture of triploid-only individuals miti- gates, but does not eliminate, most of the ecological concerns. Aquaculture of hatchery-raised sterile triploid seed represents an intermediate solution to assisting the industry between abandoning research on nonnative species and wholesale introduction of dip- loids. The industry potential is enormous, but there are lingering questions about how precisely this "revolution" will or won't pro- ceed. THE EFFECTS OF VARYING COLD WATER TEMPERA- TURES, SIZE AND POPULATION DENSITY ON THE GROWTH AND MORTALITY RATES OF BAY SCAL- LOPS. ARGOPECTEN IRRADIANS IRR.ADIANS. IN AN UP- WELLER. Brian J. Ball, Stephanie T. RutkowskI, Emily T. Griffiths, Mattituck High School. LSI 2.5 Main Road. Mattituck, NY 1 1952; Kim W. Tetrault, Cornell Cooperative Extension — Suffolk County Marine Program, 3690 Cedar Beach Road, Southold, NY 11971. The growth of scallops has been known to slow in cold tem- peratures. The objective of this experiment was to observe the growth and mortality rates of larger and smaller sized bay scallops sorted into silos of varying densities in an upweller with decreas- ing coldwater temperatures. To set up the experiment, five silos were filled with smaller scallops (10-20 mm) and five silos were filled with larger scallops (21-30 mm). Each silo of larger and smaller scallops varied in population density. The five silos of smaller scallops had 300. 700, SOO, 900. and 1 ,200 scallops per silo, respectively. The five silos of larger scallops had 150. 1 80. 250, 330, and 350 scalk)ps per silo, respectively. Once a week, data were collected from a sample size of 30 scallops from each silo. Using a caliper, the size of the scallops was recorded to observe any growth. Any mortality within the sample population was also noted. Eight weeks into the experi- ment, January 10, 2002, all of the scallops were counted in each silo and all of the deaths were recorded. Using the data, mortality ratios of the entire population for each silo were calculated. A total count was done to verify the accuracy of our sample populations. The scallops were then returned to the silos for further study. The results of this experiment show that tor the varying popu- lation densities that we measured, the larger scallops continued to grow and had a low mortality rate in coldwater temperatures rang- ing from 13.21 to 0.27°C. The population density of each silo for the larger scallops had little effect on the growth and mortality rates. The growth and mortality rates in the smaller scallops were Milford Aquaculture Seminar. Milford, Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 .\^7 greatly affected by the population densities in our experiment. The silos with the fewest scallops (296 and 659) showed the most growth, whereas the silos with the highest density of scallops (1,162 and 844) showed less growth. For the smaller scallops, the silos with the highest density had the greatest mortality rate. The silo containing 1,200 scallops had a mortality rate of 10.4%, whereas the mortality rates for the remaining smaller groups of scallops had a mortality rate of approximately 5'yf . Experimental testing will continue through April in order to reach valid conclu- sions from this study. CATCH STATISTICS OF HOMARUS AMERICANUS. THE AMERICAN LOBSTER, FROM A 3-WK STUDY CON- DUCTED BY STUDENTS FROM THE SOUND SCHOOL REGIONAL AQUACULTURE CENTER ABOARD A COM- MERCIAL LOBSTER VESSEL IN THE WATERS OFF FISHERS ISLAND. NEW YORK. Amber L. Beitler and John J. Roy, The Sound School. 60 South Water St.. New Haven. CT 06519 During the summer of 2001. from July 9 to August 1. six students from the Sound School Regional Aquaculture Center kept catch statistics on Hoiminis aniericaiuis. the American lobster, while interning as deck hands aboard the UP FOR GRABS, a commercial lobster vessel berthed in Noank. Connecticut. The student time aboard the vessel was credited toward their Super- vised Occupational Experience (SOE) compliance requirements. The UP FOR GRABS fishes out of Noank and possesses both a Connecticut and a New York State Commercial Lobstering Li- cense. Eighty-five percent of the gear was fished on grounds that lie between an east-west line that can be drawn through Ram Island and Sea Flower Reef on the northern (Connecticut) side and a line running east to west 0.5 miles north of Fishers Island on the southern (New York) side of Fishers Island Sound. The remaining 15% of the gear was fished in an area just north of Ram Island. No differentiation was made between the areas being fished. Trawls were at times moved within this zone from areas of low produc- tivity to areas where the catch was found to be higher, during a given week. The time of the students" internship corresponded with the peak season for lobster capture in that area. Traditionally termed "the run" in the commercial lobster community, the month of July is considered to be the peak month for lobstering in the entire year. Forty-seven pot trawls were fished each week. All of the pots that were fished were wire. An average of 10 trawls, or 70 traps, were hauled each day. 4 days each week. Each trawl was hauled once each week. The students recorded the catch statistics from the 280 traps as each trawl was landed. The data that were taken included: the number of legal lobsters that were caught in each trawl; the number of sub-legal lobsters (or shorts) that were returned to the water from each trawl; the number of male and female legal lob- sters found in each trawl; the number of dead lobsters found in each trawl; and the number of lobsters that exhibited shell-rot disease in each trawl. All categories were totaled daily. The daily totals were then compiled by week for 4 wk. A total of 2.018 lobsters were caught during the study. Forty-five percent of the lobsters (191) landed in the first week were of legal size. The number of legal lobsters (341) that was landed in the second week increased by 56%. The third week of the study had a 4%' drop in the number of legal lobsters (322) taken. There was a 38% drop in the number of legal lobsters (201) landed in the final week of the study. The percentage of sub-legal lobsters varied between 44% and 54% of the total number of lobsters taken each week during the study. More legal male lobsters were caught in the first and second week, whereas legal female lobsters ap- peared in greater numbers during the final 2 wk of the program. The number of dead lob.sters found in the trawls ranged from 0.65% to 1.44%. All deaths were attributed to cannibalism, pre- dation. or physical impact of the trap or trap components. The number of lobsters exhibiting shell-rot disease was less than 1% throughout the entire study. However, because of the proximity to the molt, this percentage may become greater later in the season. The students at the Sound School had first hand experience with the dramatic declines in the lobster populations in western Long Island Sound during the late 1990's. We believe that it is becoming increasingly important to monitor accurately the exist- ing stocks of Homants ameiicanus at all levels of the fishery, as well as in the scientific community. A concerted effort will be required to ensure the survival of this important natural resource. EFFECTS OF WATER VELOCITY ON CONDITIONING OF SUMMER FLOUNDER PARALICHTHYS DENTATUS FOR NET PENS. David A. Bengtson, Stephen Willey, and Erin McCaffrey, Department of Fisheries. Animal and Veterinary Sci- ence. University of Rhode Island, Kingston. RI 02881; David Alves, Coastal Resources Management Council, Stedman Govern- ment Center, Wakefield. RI 02879. Attempts in the late 199()s to rear summer flounder in net pens suffered from high mortality during the month after fish were transferred from the nursery facility to pens. As part of the Uni- versity of New Hampshire's Open Ocean Aquaculture Demonstra- tion Project (GOADP). we investigated whether exposing suinmer flounder to increased (and constant) current velocity in the nursery stage would condition them for better performance in cages placed in Narragansett Bay. Rhode Island (not the OOADP site). Three experiments, two of 60-day duration and one of 30-day duration, were conducted with different water velocities in tanks, using fish of 1 24 ± 4 g (exposed to 0. 1 5. or 30 cm/sec for 60 days). 257 ± 12 g (exposed to 0. 20. or 40 cm/sec for 60 days), and 387 ± 13 g (exposed to 0. 15. or 30 cm/sec for 30 days), in a raceway system with adjustable paddlewheels. For all of the size groups of fish, survival was significantly reduced at the highest current ve- locity. For the 124-g fish, survival in high velocity (26 ± 1%) was 338 Abstracts. 2002 Annual Meeting. Febriiury 25-27. 2002 Milford AquacLilture Seminar. Millord. Connecticut significantly lower than that in medium xelocity (."^7 ± 7% ) and in low velocity (67 ± 69f )• For the 2.^7-g fish, survival in high ve- locity (35 ± 19%) was significantly lower than that in medium and low velocities ( 1 00% in both cases). For the 387-g fish, survival in high velocity (50 ± 11%) was significantly lower than that m medium velocity (98 + 2%); all 387-g fish in the low-velocity treatment were lost due to a system malfunction. For both 1 24- and 257-g fish, growth in the medium-velocity treatment was signifi- cantly better than that in the low-velocity treatment, which in turn was better than that in the high velocity treatment. For 1 24-g fish, growth was 76 ± 1 2 g. 49 ± 8 g, and 39 ± 0 g in the medium, low. and high velocities, respectively. For 257-g fish, growth was 47 ± 10 g, 25 ± 4 g, and -7 ± 17 g (weight loss) in the medium, low. and high velocities, respectively. For 387-g fish, growth at medium velocity (26 ± 6 g) was significantly greater than that at high velocity (-22 ± 8 g). Food consumption data from the 257-g fish showed that the fish in medium velocity grew most because they consumed significantly more food during the experiment (1.622 + 128 g per tank) than did fish in low velocity (915 ± 65 g). which in turn consumed significantly more than fish in high velocity (640 ± 90 g). Nevertheless, there was no significant difference in food conversion ratio (FCR) between fish at low velocity ( 1.54 ± 0.37) and those at medium velocity ( 1.37 ± 0.23). At the end of the experiment with 1 24-g fish, fish from the low-velocity and medium-velocity treatments were moved to cages in Narragansett Bay. where currents of about I knot (approx. 55 cm/sec) are routine. After 3 wk in the cages, no significant differences in survival were observed (low velocity = 83% ± 12%; medium velocity = 81% ± 2%). Subsequent damage to some of the cages and escapement of the fish precluded funher statistical analysis of survival, as well as any growth measure- ments. We conclude that cuirent velocities of 15-20 cm/sec in the nursery improve growth of juvenile summer flounder, that current velocities of 30— lO cm/sec are excessive, but that increased current velocity in the nursery does not improve fish survival upon transfer to cages. REMINISCENCES OF EARLY PIONEERS IN OYSTER CULTURE. Luther Blount, Blount Shipyards, Warren, Rl 02885. In the late 1920s and early 1930s, there were about six oyster companies in Warren, Rhode Island, which not only worked closely with the Connecticut oyster people but also had connec- tions in the nearby Taunton River and Assonet Bay areas in Mas- sachusetts, where there was always a good oyster set. In those days, oyster fishermen in Rhode Island were really into summer clam bakes, where all the oystermen gathered. My uncle. Byron Blount, of E.B. Blount and Sons, often invited Dr. Paul Galtsoff there. In fact. Dr. Galtsoff would drive over from Woods Hole to talk oysters with my uncle. So 1 knew him as a white- haired scientist discussing good and bad oyster sets and this always was his primary subject. Dr. Victor Loosanoff of the Milford Laboratory, Milford. CT followed Dr. Galtsoffs visits to my uncle and 1 happened to be there the day he showed us a paper egg crate coaled not only with cement, but also literally loaded with thou- sands of oyster set. For the past half century 1 found myself work- ing with these shellfish pioneers and learning from them as I built Prudence Island Farms. Because we had previously bought spat on scallop shells from Dr. George Matthiessen on Fishers Island and knew how he got them, we looked for a marine biologist to help us get a set from Green Hill Pond on the Rhode Island south shore. We hired a young man who just graduated from the University of Rhode Is- land, called him "John Oyster,"" and began to buy scallop shells and get rafts made, which are used today in my salt water oyster pond on Prudence Island. "CRAB SIGHTINGS" IN LONG ISLAND SOUND DURING 2001. Diane J. Brousseau, Biology Department, Fairfield Univer- sity, Fairfield. CT 06430: Ronald (Joldberg. USDOC. NCAA. National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford. CT 06460. During summer and fall of 2001. there were a number of in- quiries to the Milford Laboratory about observations of crusta- ceans in Long Island Sound. In July, swarms of niegalopae of the Asian shore crab. Hemii>rapsiis sanguineus, were seen both in the water column and rafting on floating rockweed near Charles Island in Milford. Connecticut. These larvae were likely the result of an early summer spawning. Recent increases in population size of Hcuiigrapsus may account for greater abundance of the larvae than in the past. In early fall, reports were again made of dense aggre- gations of small crabs swimming close to the surface. Initially mistaken for Asian shore crabs by many observers, they were later identified by Nizinski (pers. comni.) as sub-adult pinnotherids, Pinni.ui cluietoptenuui. This small crab is often found living as a commensal within the tube of the polychaete worm Chaetopterus. Pinnixa sightings were widespread, occurring in the Thimble Is- lands, Milford Harbor, and Housatonic River in Connecticut, and near Orient Shoals off Long Island. Fall swarming of Pinnixa prior to settlement has been observed previously by Mroczka (pers. comm.). but the early life history of this species has not been described fully. In October, an adult male shame-faced crab. Cat- (ippa tlaiuiuea. was collected in shallow water by a scuba diver in Stoningtoii. Connecticut. Calappa is predominantly tropical to subtropical, but larvae can drift as far north as southem New England, accounting for occasional occurrence of adults in this region. This molluscivorous crab spends much of its time buried in the sand, making brief excursions to search for food. The obser- vations described above are not unique, but nia\ ha\e been more apparent during 2001 because of interannual variability in climate or the population dynamics of the species involved. Milf'oid Aquaculture Seininur, Milford, Ctniiiccticut Abstracts. 2002 Anmiiii Meeting. Fehruaiy 25-27, 2002 339 SOME CLASSICAL PERSONALITIES IN THE SHELL- FISH RESEARCH ARENA: ECLECTIC OBSERVATIONS OF AN ERSTWHILE OBSERVER. Walter J. Canzonier, M.R.O.C.F.. P. O. Box 662. Port Norris. NJ 08349. The personalities of many of the scientists associated in the past with the shellfish research and development community might be of general interest to those currently engaged in this field. The personalities of these indi\iduals. other than makuig them colorful and intriguing characters, probably also had a considerable influ- ence on the approaches they used in addressing problems requiring scientific or technological intervention. Ergo, to better appreciate the contributions of some of the members of the community in- volved with the recent, and not-so-recent evokition of shellfish research, it has been suggested that it might be both entertaining and educational to have a glimpse at their personae. Using a cap- sule review of some of the key players in the shellfish research and development saga. I will attempt to share with the more recent entrants in the game a few anecdotal sketches of some of those individuals with whom I have had contact in my 45 years of involvement with the academic and the industry elements of mol- luscadom on the East Coast. This informal documentary, as dis- torted as it might be as a result of the undoubtedly biased perspec- tive of the reporter, is offered to the curious in hope that it will help them gain a better insight into the personalities of thiise whom they have encountered merely as names in the nondigitized literature. Some of those mentioned were colleagues, others mentors, still others known to me primarily through accounts by relatives or close associates. Interest in documentation of personalities was first stimulated by the efforts of Sewell Hopkins, who prepared a set of three unpublished but extremely interesting and informative memoirs, two of which (University of Illinois and Project Nine) present enlightening and amusing sketches of his teachers and colleagues. In this rambling account of the members of the "Old Guard." 1 will attempt to reconstruct the images of some of the well- recognized, as well as the lesser-known individuals. Certainly the names of Julius Nelson (known to me through the stories from his son); Galtsoff and Loosanoff, who worked in this immediate area; T. C. Nelson; J. G. Mackin and Sammy Ray of dermo fame; J. D. Andrews; and H. H. Haskin will probably be familiar to many currently working in this field. Other, less commonly recognized names include W. F. Wells, known to me only from exchanges of vitriolic correspondence with T. C. Nelson; L. A. Stauber. a poly- valent researcher more noted in the field of parasites of vertebrates, but always available to his students and colleagues as a valid anchor in the basic principles of shellfish pathology and physiol- ogy; his students S. Y. Feng and M. R. Tripp, among others; Joe Glancy. who doggedly pursued development of commercial shell- fish hatchery technology despite strident attempts to rectify his errant behavior by his neighbor across the Sound; Mike Castagna who pursued a similar course in more southern waters; and M. R. Carriker. Carl Shuster. Herb Hidu. Tom Cheng, and others who at some point in their careers had close ties with the Depart- ment of Oyster Culture of the New Jersey Agricultural Experiment Station. If asked what might be a common trait of these individuals, my observations would lead me to conclude that it was a capacity to mobilize their imagination to devise a plan of action that effica- ciously utilized the often very limited resources available to them — whether this he in the laboratory or in the field. This in- trinsic ability to adapt their efforts to the resources at hand enabled them to make significant contributions in the realm of academia. and to cost-effectively assist the shellfish industry in its quest for more productive culture practices and strategies. For this vanish- ing breed, a lack of sophisticated facilities and equipment was never considered to be an insurmounlablc limitation, but merely a challenging impediment to be circumvented by innovative tactics. MASSACHUSETTS OCEAN RESOURCE INFORMATION SYSTEM (MORIS). Diane Carle. Office of Coastal Zone Man- agement, Executive Office of Environmental Affairs, 251 Cause- way Street. Suite 900. Boston. MA 021 14. Massachusetts Coastal Zone Management (CZM) has em- barked on a long-term project to develop the Massachusetts Ocean Resource Information System (MORIS). MORIS will be a com- prehensive database and ArcView GIS extension providing access to the broad range of information necessary to carry out the CZM mission. The first phases of the MORIS project were completed in 2001 and focus on information and tools useful for screening for potential aquaculture sites. CZM contracted EVS Environmental Consultants to conduct the initial data mining and GIS application development. CZM also teamed with NOAA's Coastal Services Center (CSC). Mas- sachusetts Division of Marine Fisheries (DMF), and Massachusetts Department of Environmental Protection (DEP) to add a "georegu- lation" tool that allows users to query an area for aquaculture- related regulations. Users are presented with a list of relevant regulations and can view summaries as well as the actual legisla- tion. Users can also view the web sites of the relevant regulatory agencies. The CSC is also creating new data layers for the project, including benthic maps of selected areas of the Massachusetts coast and maps of areas suitable for sustaining shelltlsh growth. CZM is completing maps of existing aquaculture leases in Mas- sachusetts. Future phases of the project will develop new water quality data layers and enhance the applications' water quality mapping capabilities. CZM is also pursuing funding to create an Internet mapping site for the project. The MORIS CD containing the ArcView extension and data- base is available at no cost from CZM. 340 Ahstmcls. 2002 Annual Meeting. February 25-27, 2002 Milt'ord Aquaculture Seminar, Milford. Connecticut HABITAT SUITABILITY ASCERTAINED BY GROWTH AND SURVIVAL OF BAY SCALLOPS IN TIERED CAGES. Joseph Choromanski, Sheila Stiles, Mark Dixon, and David Veilleux USDOC, NCAA, National Marine Fisheries Service, Northeast Fisheries Science Center, Milford Laboratory, Milford, CT 06460; Christopher Cooper, Ocean Technology Foundation, UCONN-Avery Point Campus, Groton, CT 06340. A laboratory-spawned line of bay scallops (Argopecten iinuli- ans irnidiuns) was used to evaluate the effectiveness of commer- cial three-tier, rigid-mesh cages for growing scallops in two proxi- mate hut dynamically different sites in eastern Long Island Sound. With permission of the Gi-oton Shellfish Commission and the State of Connecticut Aquaculture Division, we established comparative field sites on the western side of Ram Island (low dynamic, gentle tides), and the southern end of the island (high dynamic, tidal current of up to 2 knots). The scallops were spawned in March and held m temperature- controlled tanks in the Milford Laboratory at 22"C until May when ambient seawater temperature reached about IS'C. The scallops were then acclimated to the lower temperature and distributed in outdoor raceway tanks using densities determined as optimal — 5 L biomass per tank. In late July, scallops with a mean size of 25 mm were deployed at each site, with a starting biomass of 2 L (ap- proximately 400 scallops) in the top tiers of a set of two cages at each site. The cages were made of plastic-coated wire with a 7.5-cm mesh. Each cage measured 56 x 56 x 94 cm and was divided horizontally into three sections or tiers. Two ballast areas below the bottom tier provided an offset from the sea floor of approximately 15 cm. Cage inserts of smaller mesh (10 and 17.5 mm) measuring 41 x 10 x 81 cm were used to hold the scallops. After I month, the scallops were divided into three densities of 50. 100, and greater than 1 50 scallops per tier in each of the cages. The cages were checked monthly to the end of the experiment to de- termine survi\al and growth, with the added attention to shell indentations that might indicate density problems, and to check and remove fouling organisms. Results from the field experiment indicated that satisfactory growth of scallops occurred at both sites. The initial growth rate was rapid, but then leveled off as the water temperature decreased. Growth of scallops was slightly better at the western side of the island (mean size of 51 mm) than at the southern end (mean size of 48 mm). There was no difference in the mean size of scallops in the top and middle tiers, with 50 and 100 scallops, respectively. Bottom tiers of all cages, stocked with greater than 150 scallops, did show slightly slower growth and higher mortality. Scallops in the cages from the southern, high-dynamic area exhibited more shell indentations: this could be attributable to the strong currents pushing the scallops together for periods of time, thereby causing a temporary decrease in available cage area that may mimic effects of higher shelf densities. Further studies are waiTanted in similar habitats to corroborate these results. HORSESHOE CRAB AQUACULTURE: PRELIMINARY RESULTS FROM HATCHING AND REARING STUDIES. Carmela Cuomo, Yale University. New Haven, CT 06520 and Uni\ersity of New Haven, West Haven, CT 06516; Paul R. Bar- tholomew, SUPERB Technical & Environmental, Hamden, CT 06517; Leslie Angelini, Brian King, and Jeffrey Bytzko, The Sound School, 60 South Water St., New Haven, CT 06519. Previous studies undertaken by the two senior authors during the summer of 2000 at the National .Marine Fisheries Laboratory in Milford, CT resulted in the successful spawning of captive adult horseshoe crabs, Liiiutlii.s polxplwiuns. The fertilized eggs were allowed to develop under conditions approximating field condi- tions, although predators were kept to a minimum. The eggs had a hatching rate of 60% over an initial 2-mo period, followed by a 98'/r hatching rate over the course of 10 mo. Evaluation of the long-term survival (greater than 1 y ) of Llnuilm was not completed successfully because of a malfunction m the rearing tanks. This study was undertaken in an effort to evaluate the role of different food sources on the growth and survival of post-hatch horseshoe crabs. Approximately 4.500 eggs were kept in small, experimental bowls filled with Long Island Sound (LIS) seawater and aerated. Initial egg densities varied per bowl, with a maximum density of 1,500 and a minimum density of 50. All bowls were checked daily for hatchlings. Upon hatching, all hatchlings from an individual bowl were removed to a separate bowl, with no more than 50 per bowl. Hatchlings were kept in aerated LIS seawater and a series of behavioral observations were made on them. Hatchlings in bowls were checked daily for tiiolting. Molted individuals were removed frotn hatchling bowls and placed in individual bowls of aerated seawater. The number of post-hatch molted individuals per bowl ranged between 10 and 30. Horseshoe crabs at this stage were separated into three groups for preliminary experiments examining the role of diet on growth and survival. They were fed one of three food items: newly hatched brine shrimp, rotifers, or concentrated dried food flakes. The ju- veniles were checked every day and any secondary molts or deaths were noted. When an organism underwent a secondary molt, that organism was transferred to a new bowl and fed one of the three food choices. This same procedure was repeated for every new molt stage reached. At the conclusion of the experiment, all sur- viving post-hatch molts (second and upward) were removed and placed in aerated, 10-gal aquaria containing artificial seawater and a bottom covered with 3 cm of fine sand. The results suggest that diet may play a supporting role in the growth and sur\ ival of post-hatch Liniiilu.s juveniles, although this factor needs further investigation. In general, all juveniles reached their first post-hatch molt between 5 and 12 days. The time to second post-hatch molt varied with diet. There was a slight dif- ference in molt timing among the three test groups. Juveniles (first post-hatch molt) rea)ed on a diet of rotifers underwent a second post-hatch molt within 7-12 days, and those reared on a diet of brine shrimp generally molted between 10 and 14 days. Juveniles Milford Aquaculture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meetins. February 25-27. 2002 .Ul reared on flaked food generally molted between 9 and 12 days. Of these juveniles, those fed on a diet of brine shrimp experienced a mortality rate of up to 5%, whereas those fed on rotifers or flaked food experienced a mortality rate of 85%) than those fed either rotifers or brine shrimp. The results from these initial studies suggest that food quality and type has only a slight effect on the two earliest post-hatch molt stages of Limulus polyphenuis juveniles, but has a strong effect upon the third post-hatch molt. It appears likely that this effect is related not only to the food type, but also to the life habit of the horseshoe crab, and that any rearing plan for this species must include at least a two-phase food supply ciinsisting initially of zooplankton. followed by food that is mixed in with the sediments. Research on the effects of food quality on the growth of Limulus juveniles continues at this time. A COOPERATIVE STUDY ON THE AQUACULTURE OF PORPHYRA LEUCOSTICTA (RHODOPHYTA) FOR AN IN- TEGRATED FINFISH/SEAWEED RECIRCULATING AQUACULTURE SYSTEM IN AN URBAN APPLICATION. John J. Curtis. Sherry W. Lonergan, and Paul J. Trupp. Bridgeport Regional Vocational Aquaculture .School. Bridgeport. CT 06605; Peimin He, Shanghai Fisheries University. Shanghai, People's Republic of China; Raquel Carniona and Charles Yarish. University of Connecticut, Stamford. CT 06901; George P. Kraemer, State University of New York. Purchase. NY 10577; Christopher D. Neefus, University of New Hampshire, Durham, NH 0,^824; Thierry Chopin, University of New Brunswick, Saint, John, NB E2L 4L5; George Nardi, GreatBay Aquaculture LLC, Portsmouth. NH 03801, Aquaculture represents an excellent opportunity to help reju- venate blighted coastal urban areas on the Northeast coast. On land, aquaculture requires a relatively small amount of space; building space can often be acquired at reduced cost in ungentri- fied city areas and aquaculture can represent an attractive, envi- ronmentally benign form of commerce. However, finfish and shellfish aquaculture operations are a source of an effluent with high concentrations of dissolved inorganic nutrients (N, P). To prevent eutrophication, the EPA is developing stringent guidelines for the release of N and P into coastal waters. An integrated re- circulating aquaculture .system, coupling the growth of seaweed and fish, can solve these problems for urban aquaculture facilities. Not only is the effluent remediated (the seaweed requires for growth the waste products produced by the fish), but an additional multiproduct, high-value crop can be generated. Marine aquacul- ture in the Northeast consists primarily of coastal pen-based salmon production and shallow benthic production of shellfish (mussels, clams, and oysters). There are very few tank-based (on land) marine aquaculture operations in the Northeast. One opera- tion is GreatBay Aquaculture LLC (Portsmouth. NH). a land-based hatchery and grow-out facility for summer flounder and cod. high- value fish that are being sold to the U.S. and Japanese sushi and Sashimi markets and to "white tablecloth" restaurants. Our work is part of a multi-university effort (University of Connecticut. State University of New York at Purchase. University of New Hampshire. University of New Brunswick, and Shanghai Fisheries University! with GreatBay Aquaculture LLC to develop an integrated finfish/seaweed recirculating aquaculture system (RAS) suitable for urban aquaculture. The unique aspect of our RAS system is that it will be integrating the culture of finfish (i.e.. summer flounder) and native species of seaweed (i.e.. Porphyra). The red alga. "Pdiphyra". is the most valuable maricultured sea- weed in the world, with an annual value of more than $1.8 billion. Porphyra (nori) is primarily used for food as the wrapping around sushi rolls; it is a major dietary source of taurine (controls blood cholesterol levels) and is rich in proteins, vitamins, trace minerals, and dietary fiber. On a dry weight basis. Porphyra can be worth more as a source of biochemicals than as food. It is the preferred source of the pigment, r-phycoerythrin, utilized as a fluorescent tag for biotechnological applications. The United States is dependent primarily upon foreign sources (i.e.. China. Japan, and Korea) for nori via coastal net culture. BRVAS students are working along- side undergraduate and graduate students in the construction and operation of these systems. The life history of Porphyra includes a microscopic filamen- tous stage (the conchocelis stage) and the more conspicuous mac- roscopic blade stage that grows attached to intertidal and shallow subtidal substrate. There are at least seven recognized species of Porphyra in the Northeast. We have begun evaluating one of these native species {P. leucosticta) as a candidate for the integrated RAS, because it may be one of the best sources for the sushi food industry and for r-phycoerythrin. We are developing the mass culture techniques (in both free culture or attached to nets) for this native species of Porphyra. We will report on the mass seeding technologies that we have developed and the specific growth rates of P. leucosticta at the BRVAS culture facilities. UPDATE ON SHELLFISH RESTORATION AND REVIEW OF LOCAL HATCHERIES IN NEW JERSEY. Gef FHnilin. Rutgers Cooperative Extension. Toms River. NJ 08755. Cooperation among several sectors that work with water and shellfishery issues has led to two new accomplishments that com- bine an interest in improving both the ecology of the waterways and the potential economy of the shellfish industry in New Jersey. In the summer of 2001, 10,000 bushels of washed oyster shell were deposited to construct a new reef in a near shore area in Keyport Harbor in western Raritan Bay. The project was spear- headed by the New York/New Jersey Baykeeper, with assistance from the National Marine Fisheries Service, Rutgers Cooperative Extension, local shelUishermen, and the NJ Department of Envi- ronmental Protection's Bureau of Shellfisheries. In a community- 342 Abstracts. 2002 Annual Meeting. February 25-27, 2002 Miltord Aquaeulture Semuiar. Millord, Connecticut supported effort. 10.000 oysters, which had been raised in Taylor Floats by volunteers from the area, were deposited on the reef to establish a community. This effort of growing oysters by the vol- unteers continues. Later that year in the fall, with the support from the South Jersey Economic Development District, the last oyster boat on the New Jersey Atlantic Coast moved 2.000 bushels of oyster seed to an old bed at the mouth of the Mullica Riser. The Fitney Bit bed will have been closed by the Bureau of Shellfisheries for a year when it is opened for public harvest. Industi7 representatives and Rutgers Cooperative Extension, who acquired the funds to do this work, are presently looking for more funds to expand this work. Shellfish hatcheries in New Jersey have been reevaluating their production in light of challenges from Brown Tide blooms and unexplained hatchery mortalities. FLOW CYTOMETRY AS A TOOL TO QUANTIFY THE OYSTER PHAGOCYTOSIS. RESPIRATORY BURST. AND APOPTOSIS. Michael J. Goedken and Sylvain l)e Guise, De- partment of Pathobiology. University of Connecticut. 61 N Ea- gleville Road. U-89, Storrs. CT 06269. Infectious diseases are a significant problem in oyster aquacul- ture and cause immense production losses. The protozoan parasites Perkinsus mariniis and Haplosporidium nclsam have generated losses estimated in the hundreds of millions of dollars over the last 35^5 y in the Middle Atlantic states. The relationship between parasites and oyster defense mecha- nisms is unclear. A better understanding of the immunopathologic association may reduce these economic losses. Defense mecha- nisms of the eastern oyster {Crassostrea virt>iiuca) were quantified at the single-cell level utilizing flow cytometry. Phagocytosis was measured using fluorescent beads. Respiratory burst activity was quantified as the increase in dichlorofluorescein-associated fluo- rescence upon stimulation. Apoptosis was evaluated with TUNEL assay. Three subpopulations of hemocytes (granulocytes, hyali- nocytes, and intermediate cells) were identified with unique func- tional characteristics. Granulocytes were most active at phagocy- tosis and peroxide production, whereas hyalinocytes were rela- tively inactive. TUNEL assay application allowed quantification of hemocyte programmed cell death with temperature-dependant changes. Flow cytometry can rapidly, accurately, and directly quantify the morphology and function of a large number of indi- vidual cells, and will lead to a better understanding of the bivalve immune system. COBIA CULTURE. Josh Goldman. Fins Technology. Inc.. l.'S Industrial Rd., Turners Fall. MA 01376. Cobia {Rcichycentron canadiim). is considered a prime candi- date for culture, given its extremely fast growth rate and excelleni flesh quality. Interest in cobia culture has grown substantialh in the last 5 y. fueled by reported successes in extensive, net-pen operations in Taiwan, and as U.S. researchers have gained some familiarity with captive fish. Fins Technologs. with support from NOAA"s National Sea Grant, has investigated a variety of aspects of intensive cobia culture in an effort to develop protocols for commercial production. We have evaluated growth and feed con- version efficiency at 10. 20. and 30 ppl salinity in artificial sea- water, and have instituted routine bacterial and histological moni- toring of our populations to assess the risks and develop appropri- ate health management strategies needed to support commercial production. During the last 2 y. we and our collaborators hope to demonstrate commercial feasibility of this promising species. CREATING OPPORTUNITIES FOR STUDENT AQUACUL- TURE PROJECTS. Eric Goodman, Colleen Cook, and Michael Weiss. Friends Academy. Duck Pond Rd.. Locust Valley, NY 1 1560. The goal of our ongoing project is to increase awareness and participation in shellfish biology, habitat preservation, and aqua- culture techniques among our peers. This group currently includes seven students in Friends Academy, our high school, as well as five students from Locust Valley and Oyster Bay, two nearby public schools on the North Shore of Long Island. The project began w ith a program designed as a basic introduction to shellfish aquaculture for local educators to motivate them to start similar projects in their schools. We held a series of seminars for students who showed an interest in our work and who wished to design an aquaculture project of their own. The topics of these seminars included working in the lab with algae and constructing grow-out equipment. Our next initiative was to construct a facility that was open to the public to increase the number of educational and re- search opportunities in the community. This facility, which is un- der development, is a hands-on center that includes a classroom as well as a fully functioning shellfish hatchery. We will be growing the Atlantic bay scallop (Argopecten iinulwiis inadians) as well as the eastern oyster iCrassostrea virgiiiica). Lessons in hatchery operation and maintenance, shellfish life cycles and anatomy, and the use of a FLUPSY are also taught in the hatchery. We hope that through our work in the center, students will use the hatchery to conduct their research relating to shellfish life cycles during the school year. AN EXPERIMENTAL INVESTIGATION OF DIETARY FATTY ACIDS AND STEROLS AND THE IMMUNOLOGY OF THE AMERICAN OYSTER, CRASSOSTREA VIR- GINICA: A WELL-FED OYSTER IS A HEALTHY OYSTER. N'EST-CE PAS? Helene Hegaret. Ecole Nationale Superieure Agronomique de Rennes. France; Gary H. Wlkfors, NCAA Fish- eries. NEFSC. Milford. CT 06460; Philippe Soudant, Universite de Brest. France; Maryse Delaporte. Jeanne Moal, and Jean- Fran(;ois Samain, Laboratoire de Physiologic des Invertebres. IIREMER. Brest. France. The lipid composition of the algae fed to oysters is very im- portant because fatty acids and sterols in the membranes of oyster cells to some extent have a dietary origin. The fatty acid and sterol Milford AquacultLire Seminar. Miltord, Connecticut Abstracts. 2002 Annual Meeting. February 25-27. 2002 343 composition of hemocyte membranes is thought to affect immune function, thereby Hnking nutritional status with abiUty to respond to environmental and health stresses. To investigate this linkage, we designed an experiment in which two replicates of seven feed- ing treatments were applied to the oyster. Crassostrea virginica, and hemocyte function was evaluated before and after a subse- quent high-temperature stress. Twelve oysters (ca. 50 mm) were put into each of 14 computer-controlled feeding chambers. Algal diets fed at 10% and 509f^ daily rations (dry wt/dry wt) were LB 1077/lB (Skeletonemu aistatuin). PLY429 {Tetmselinis chiii). and a 50/50 (dry wt) mix; we also included two unfed controls. These two algae have different lipids: LB 1077/1 B contains the fatty acids C20:5(n-3) and C22:6(n-3) and cholesterol; whereas. PLY429 con- tains C20:5(n-3). but no C22:6(n-3). and 24-methyl and 24- niethylene cholesterol. After 5 wk of feeding at 20"C. we sampled half of the oysters for hemocyte analyses and gill-membrane lipid composition, subjected the remaining oysters to I wk of high- temperature (28"C) stress, and sampled as previously. Gill mem- brane lipids will be analyzed by GLC (work in progress). Hemocyte function was evaluated by five methods employing the FACSCAN flow cytometer: identification of hemocytes by SYBR green fluorescence, viability by propidium iodide fluorescence, adherence and aggregation by forward- and side-scatter, phagocy- tosis using fluorescent plastic beads, and respiratory burst by a newly modified method using Zymosan A as the activator. These response variables were tested statistically, first using a nested ANOVA model, with feeding ration and regime as independent variables, and in second way using discriminant analysis. Individual hemocyte functions were not affected significantly by feeding ration or regime; however, a consistent trend relating the higher ration with enhanced hemocyte function was apparent. By contrast, differences between unfed and fed oysters were, in many cases, highly significant and appreciable, with unfed oysters showing decreased function in most subpopulations of hemocytes (granulocytes, hyalinocytes. and intennediate cells). Discriminant analysis, using data from multiple hemocyte measurements, was able to differentiate between high- and low-quantity feeding treat- ments, as well as between the different algal diets. These findings indicate that nutrition does affect immune function in oysters, thereby mediating their response to stress. THE ROLE OF THREE BACTERIA IN SHELL DISEASE OF THE AMERICAN LOBSTER {HOMARUS AMERICA- NVS), Andrea Hsu. Erin Summers, and James Estrada, Boston University Marine Program. Marine Biological Laboratory. 7 MBL St., Woods Hole. MA. 02543; Roxanna Smolowitz, Marine Biological Laboratory, 7 MBL St., Woods Hole, MA 02543. Although typically infecting impounded lobsters, lobster shell disease is becoming increasingly more prevalent in wild popula- tions throughout the New England region. This study utilizes scan- ning electron microscopy (SEM) and histological analyses to de- scribe the morphology and prevalence of bacterial cells present on carapace samples taken from two wild-caught lobsters with shell disease and one lacking any noticeable infection. SEM analysis revealed and statistical tests verified three separate morphological types of bacteria present on both carapace samples and cultures taken from shell lesions. Results from bacterial identification sug- gest two bacteria to be of the genus Vibrio, whereas the third is a Pasteiirclla-Wke organism. Bacteria on infected carapace were seen to concentrate on intact epicuticle. the edge of lesions, mi- croscopic cracks and holes, and setal pores. Halo-like holes sur- rounded all bacterial types, closely matching the shape of the bacteria, suggesting that each bacterium is boring into the epicu- ticle. Healthy carapace showed substantial bacterial concentrations present only around carapace setae. Several of these bacteria were seen with small-bore holes surrounding them, but active boring and degradation of the epicuticle was minimal in contrast to the infected carapace. Histological observations show that there is a difference between the breakdown of wild and impoundment le- sions. Shell lesions of wild lobsters show lattice-like cuticular remnants attached to underlying less degraded cuticle in several eroded foci. This study documents not only the presence of three bacterial species in shell disease of wild-caught New England lobsters, but also illustrates their role in the degradation of the carapace. This is the first evidence of the mechanism that is used to break down the epicuticle of the lobster shell, and suggests that different organisms are involved in causing wild and impoundment shell disease. DERMO INVESTIGATIONS, RAZOR CLAM NURSERY TRIALS, AND PRELIMINARY BAY SCALLOP ADHE- SION CULTURE EFFORTS. Richard C. Karney and Aman- dine Surier, Martha's Vineyard Shellfish Group. Inc.. Box 1552. Oak Bluffs. MA 02557; David W. Grunden, Town of Oak Bluffs, Box 1327, Oak Bluffs, MA 02557; Tliomas E. Berry. Martha's Vineyard Shellfish, Box 1660, Edgartown, MA 02539. To compare the infection patterns of dermo disease (Perkiiisiis tnarimts) in Edgartown Great Pond and Tisbury Great Pond, cages of wild and disease-free cultured oysters were deployed in both ponds on June 20 and 21 and sampled monthly for mortality and dermo infection progression. Man-made breaches of the salt ponds' barrier beaches allowed for some manipulation of water chemistry within the ponds. Edgartown Great Pond was managed to maximize its exchange with the sea to reduce eutrophic condi- tions believed to be stressing its oyster population. Management in Tisbury Great Pond sought to limit exchange with the ocean in hopes of achieving salinities unfavorable to the dermo parasite. By October, infection rates in both ponds for all of the wild and two of the three groups of cultured oysters were 100%. The infection rate of one group of the cultured oysters in Tisbury Great Pond did not rise above 76%. By November, cumulative mortality of the wild oysters was 19.8% in Tisbury Great Pond and 29.5% in 344 Ahslnicts. 2002 Aniuuil Meeting. February 25-27, 2002 Miltord Aquaculture Seminar. Milford. Connecticut Edgartown Great Pcind. Mortality of the cultured oysters was 4.4% in Edgartown. The mortalities of cultured oysters at two sites in Tisbury Great Pond were 5.5% and 3.4%. The variation in mor- tality of the cultured oysters appeared to correlate with the densi- ties of natural oyster beds at the sites. In late August, seed razor clams {Eiisis direcliis) with an av- erage length of 20. 1 mm were planted at two densities (54.5/ft" and 163.5/tr) in four different nursery systems — a tidal upweller. mesh-covered bottom boxes, and mesh-covered and open floating sand boxes. Final growth measurements taken in early November were poorest for the high-density tidal upweller (28.8 mm) and best for the low-density open floating sandbox (54.1 mm). In No- vember, clam survival in the sandbox nurseries was as follows: low density/no mesh 56%; high density /no mesh 50%; high den- sity/mesh 48%: low density/mesh 39%. .Several adhesives have been tested in efforts to develop a cul- ture technology based on gluing bay scallops to plastic nets. A suitable adhesive has yet to be located. SOME CULTURE STRATEGIES FOR GROWING ROTI- FERS (BRACHIONVS PUCATILIS) AS FEED FOR AQUA- CULTURE APPLICATIONS. Robin Katersky. Barry Smith, Dean Perry, and David Nelson, USDOC, NCAA, National Ma- rine Fisheries Service, Northeast Fisheries Science Center. Milfnrd Laboratory. Milford, CT ()64(i(). Rotifers are provided as first feed to larval finfish when they are absorbing their yolk sacs and their digestive systems have developed sufficiently to consume food. Marine finfish aquacul- ture, therefore, requires them in large numbers at these times. Rotifers can be fed marine microalgae for growth and reproduction as well as for enrichment just before they are fed to the fish. Rotifers typically are grown in large tanks with algal culture added to the volume to replace the algal biomass previously con- sumed by the rotifers. Culture water is usually changed by draining the tank (and remaining algae), catching the rotifers on a screen, and resuspending them in clean water. Rotifers can consume large quantities of algae. Finfish studies at the Milford Laboratory re- quire production of millions of rotifers. The finfish are fed daily at a density of 10 rotifers/mL in 893-L tanks. With an algal supply of 1 X 10" cells/niL. how does one get enough algal biomass for maximal rotifer growth in an economical space and time? We investigated two culture methods to achieve our requirements. The first culture method investigated was to concentrate the rotifer tank using a hollow-fiber filter to remove 40 L/day of water only. The rotifer tank then was refilled with algal culture. This procedure was repeated daily, providing an average feeding of 200,000 algal cells/mL in 200- to 240-L rotifer tanks. Rotifer tanks were started with 46 rotifers/mL and reached 233 rotifers/mL in 4.8 days. The second culture method used a different strategy. Rotifers were introduced into a full tank volume of algal culture (150 L) at a density of -300.000 cells/mL. The rotifers then were left to grow for 4 days with only experimental samples removed. Rotifer den- sities consistently went from 59/mL to 232/mL in 3 days. In all cases algae was consumed to below countable levels. The second culture strategy was much less labor intensive than both the traditional and the first method. The second, or all-algae method, reduced labor by over 50% and required three culture vessels to meet demand. For the remainder of the larval rearing period (sum- mer 2001), the all-algae system was used for rotifer production because of its reliability and ease of use. This system produced an average of 30 x 10" rotifers/day. These experiments, together with past work, indicate that there is much more potential for optimizing the culture of rotifers. ASSESSING HABITAT VALUE OF MODIFIED RACK AND BAG AQUACULTURE GEAR IN COMPARISON WITH SUBMERGED AQUATIC VEGETATION, IN PARTICU- LAR, AN EELGRASS [ZOSTERA MARINA) BED. Brian Kil- patrick, Joseph DeAlteris. and Robert Rheault, Department of Fisheries. Animal, and Veterinary Science, Uni\ersity of Rhode Island, Kingston, RI 02882. Submerged aquatic vegetation (SAV) has attracted consider- able attention in recent years because of its role as an essential fish habitat necessary for sustainable fish production. Recent regula- tions that protect SAV have been critical in conserving and restor- ing this resource. Current policies that serve to protect SAV have affected applications for construction, docks, dredging, and aqua- culture. Opponents of shellfish aquaculture operations argue that the gear used for the grow-out phase of the shellfish reduces the potential for SAV restoration, and may negatively impact estuarine ecosystems. This study was conducted to compare and contrast habitat value of modified rack and bag aquaculture gear (MRBAG), submerged aquatic vegetation (SAV), and a nonvegetated shallow seabed (NVSB). Habitat value is defined herein by descriptive species diversity statistics and each habitat's ability to support an abun- dance of organisms throughout the year. The study took place over the course of 1 y, in which each of the three habitats was studied at the end of each season (three replicate samples per habitat). Sampling was performed in Pt. Judith Pond, an estuarine pond in Rhode Island that discharges directly into Block Island Sound. Specially constructed lift nets were used to sample the aquaculture gear, whereas the remaining two habitats were sampled using a drop-net of identical size and a venturi-driven suction sampler. Organisms (>5 mm) in each sample were identified, enumerated, and measured to the nearest millimeter. Environmental data were collected at the time of each sampling period to discern similarities and/or differences among the three habitats. Biofouling organisms were measured in terms of percent cover, and an average surface area (square centimeters per square meter of seabed) ot each bio- fouling phyla and/or class was estimated for each habitat and sea- Milford Aquaculture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meeting. February 23-27. 2002 345 son. Structural components, in particular, emergent surface area, were measured to determine their role in providing habitat. En\ ironmental parameters (temperature, salinity, and dissolved o.xygen) were not significant between habitats but were signifi- cantly different between seasons. Sediment type was primarily sand and was not found to be significantly different (P > 0.05) between habitats when the gravel size component (shell hash due to the aquaculture operation) was removed from the MRBAG habitat. The MRBAG habitat supports a significantly higher abun- dance of organisms per unit area (m") of seabed throughout the year. Total numbers of specimens collected are as follows: MR- BAG. 6.777 individuals: SAV. 577 individuals; NVSB, 211 indi- viduals. Species richness is consistently higher throughout the sampling periods in the MRBAG habitat as compared to the SAV and NVSB habitats. A two-way ANOVA using the richness esti- mates generated by the jackknife procedure using quadrat samples (Heltshe and Forrester 1983) indicated significant differences be- tween all three habitats (P < 0.001 ). A Tukey HSD test indicated that MRBAG > SAV > NVSB in terms of species richness values. The species diversity of each habitat was computed using the Shannon-Weiner Index and showed a significant difference (P < 0.001 ) using a two-way ANOVA between habitats. A Tukey HSD test indicated that there were no differences between MRBAG and SAV habitats, but there were differences between MRBAG and NVSB habitats. The Smith and Wilson measure of evenness was used to describe how each habitat was organized in terms of spe- cies abundance proportions. MRBAG habitat had a consistently lower evenness value because the MRBAG habitat was dominated by a few species {Neopanopeiis sayi. Tautogolabnis adspersiis, Mytitus edulisj. Habitat structural complexity was determined us- ing emergent surface area of the structures found within each habitat. A two-way ANOVA indicated a significant difference {P < 0.001) between the MRBAG and SAV habitats in terms of surface area of structure (MRBAG or SAV) per .square meter of seabed. These findings suggest that shellfish aquaculture gear pro- vides habitat to many organisms throughout the year and may be ecologically beneficial to many native species of recreationally and commercially important fish and invertebrates, especially in the early life stages of these resources. This research is the first step in determining habitat value for shellfish aquaculture gear. THE CULTURE OF BLACK PEARL OYSTERS ON SUB- SURFACE LONGLINES IN SAVU SAVU. FIJI. Gordon King, Taylor Resources. Inc.. 130 SE Lynch Rd.. Shelton. WA 98584. Continuing an informal company policy to diversify into new species and geographical areas. Taylor United. Inc.. used family knowledge and contacts to develop a Black Pearl Oyster Farm in Savu Savu Bay on the Island of Vanua Levu in the island nation of Fiji. The subsurface longline system as promoted by Dr. John Bonardelli was adapted for grow-out of the black pearl oyster. Pinctada inarguritifera. The installation and development of the farm was complicated by political instability and an armed coup in Fiji. Despite these difficulties, the farm now has more than 30 longlines and 30.000 oysters, with several hundred pearls already harvested and several thousand due for harvest; many more are being implanted in 2002. Taylor Resources is employing a three- pronged approach for production of oysters for implantation: spat collection, buying mature oysters, and a joint-venture hatchery. It is believed that the farm will be a solid source of income for the company and is already providing important employment and rev- enue for the local Fijian population. URBAN COMMUNITY MEETS AQUACULTURE: A CASE STUDY IN THE NORTHWEST. Gordon King. Taylor Re- sources. Inc.. 130 SE Lynch Rd.. Shelton. WA 98584. Taylor United, Inc.. an established shellfish farming company in Washington State, sought to expand their suspension mussel farming operation beginning in 1995. This led to a series of events including the formation of a local association to stop further aqua- culture development; the production of a sophisticated anti- aquaculture propaganda video; many hundreds of protest letters to the local politicians, the press, and agencies; the requirement by Thurston County for Taylor United to complete an EIS; and a citizen law suit under the Clean Water Act claiming that the com- pany should be required to get an NPDES permit. Seven years and many tens of thousands of dollars later, the expansion is still on hold. The presentation documents this series of events and explores some of the various parties' attitudes, and asks how much of the friction is due to bona fide environmental concerns and how much is due to the gulf in knowledge and understanding between urban Americans and the industries that priiduce their food? PROGRESS WITH CULTURING THE RAZOR CLAM (£,V- SIS DIRECTUS). Dale Leavitt, William Burt, Diane Murphy, and Rebecca Hanson, SouthEastem Massachusetts Aquaculture Center. Massachusetts Maritime Academy, Buzzards Bay, MA 02532. As reported in 200 1 . SEMAC has embarked on a study to assess the feasibility of farming the razor clam {Ensis directus) in the northeast. Operating on research funds from the Northeast Regional Aquaculture Center (USDA-CSREES), we have con- tracted with a commercial shellfish hatchery (.Aquaculture Re- search Corporation. Dennis. MA) to produce 1-cm seed razor clams for distribution to commercial shellfish growers throughout the northeast. The growers' role is to test existing or new technol- ogy for grow-out of a product for market. This presentation will provide an update of the results of this study after year 1. Approximately 4 million razor clam larvae were spawned in the hatchery and reared through the nursery stage using conventional 346 Ahstmcr.s. 2002 Annual Meeting. February 25-27. 2002 Milford Aquaculture Seminar. Milfurd. Connecticut hatchery technology similar to that used lor the surf clam (Spisula solidissiina). Roughly 150.000 I -cm juveniles were delivered to SEMAC in July lor distribution to the growers. Although the hatchery process needs refinement to increase the larval and early juvenile survival rate, we demonstrated that the hatchery stage of razor clam production is feasible and can potentially produce enough juveniles to sustain a commercial effort in razor clam farming. Adjustments to the hatchery procedures are currently be- ing made as we enter our second year of hatchery production under this project. The 2-cm juvenile razor clams were distributed to 10 commer- cial shellfish growers, ranging froin New Jersey to Massachusetts, who had been selected through a competitive proposal process. Each grower received an allotment of seed w ith funds to construct their proposed culture system. Technology being tested ranges from conventional quahog culture techniques using netted race- ways to floating culture trays and containment systems deployed on the bottom. The seed were distributed late in the growing sea- son this year, but it allowed the growers to field test their proposed grow-out system for modification and improvement in anticipation of next year's larger scale seed distribution. The two parameters of interest during this preliminary stage of the study were recovery of planted individuals and their growth as measured by a change in length. Recovery of juvenile razor clams using a variety of field grow-out technologies varied from very poor, where intensive digging within the grow-out area uncovered few individuals using a conventional quahog netted raceway, to very high, where two 4-in. cores produced more than 50 living individuals in a boarded and netted raceway. Growth rate also varied from very little increase in seed size during the I l-wk test period to significant growth where the 2-cm seed had grown to more than 5.4 cm in length. Given these preliminary results, the prospect of farming razor clams seems to be achievable and the imprinement of hatchery and rearing technology will continue thrt)Ui;h the next growina season. A COMPARISON OF SURVIVAL IN JUVENILE AR- GOPECTEN IRRADIANS IRRADIANS USING VARIOUS CULTURE TECHNIQUES AT THE SOUND SCHOOL RE- GIONAL AQUACULTURE CENTER. Kathryn R. Markey and John J. Roy, Ttie Sound School. 60 South Water St.. New Haven. CT 06519. Shellfish aquaculture is expanding in the northeastern Linited States. With the increased interest in commercial aquaculture has come the advancement of the techniques employed by the growers. As such. The Sound School Regional Aquaculture Center is rap- idly expanding the portion of the school's curriculum that deals with shellfish aquaculture. We have successfully sought assistance from both commercial growers and scientists dedicated to the ad- vancement of aquaculture. They have supported us in our endeav- ors to provide our students with a state-of-the-art education when- ever possible. We have been involved in the culture of both oysters and hard clams for several years and are currently engaged in our second year of working with Argnpectcn irnidimis irnuUuns. the bay scallop, at the school. This year, in August, the National Marine Fisheries Service in Milford. Connecticut, donated approximately 3,000 Argopecten inaditins irradians to the school. The Groton Shellfish Commis- sion made arrangements with our students to over-winter 6.000 >2() mm and 26,000 10- to l5-mni bay scallops at the school. In addition, the Noank Aquaculture Cooperative made 20.000 4- to 6-mm bay scallops available to our program for a nominal charge. In our previous attempts to work with bay scallops, we concerned ourselves with monitoring growth as well as survival. We have learned, as have many in the industry, that when mortality in a scallop crop reaches UJO^i. most concerns with growth are un- founded. This year we have refocused our experimental regimes to deal with the issues of survival of the scallops over winter. A variety of culture techniques ha\e been employed to over- winter the juvenile bay scallops. Cages and ADPI bags, holding scallops at +25 mm in low densities, have been successful to date, with a 91% survival rate. Three upwellers have been designed. One is located on a fixed pier outside the school. The other two were built inside a wet lab in the school. All three upwellers have unit|ue design modifications. The outside upweller holds approxi- mately 1.200 +25-mm scallops and mortalities have been <3% of the total. More than 40.000 bay scallops of various sizes have been successfully held in the inside upwellers. where we attempt to maintain water temperature between 13 and 18"C and provide the juvenile bay scallops with supplemental feedings of microalgae. To date, mortalities in the.se systems have been 50 mm, they are tumbled and transfened to suspended oyster rearing trays for rear- ing to market size. To accommodate sail boating and other recreational activities, these tray units hang at regular intervals from a submerged long- lute 10 ft below the surface in waters that range from 18 to 28 ft at MLT. Sustained rearing operation will enhance and sustain recre- ational shellfish activities because each spawn will bring new shellfish seed into the environment. These operations will increase essential fish nursery habitat, remove bacterial and nitrogen from coastal waters, mitigate coastal eutrophication, and serve as an environmental sentinel. IT TAKES A COMMUNITY TO GROW A SCALLOP. Mary F. Morgan, Kathleen K. Becker, and Kim Tetrault, Cornell Cooperative Extension of Suffolk County. Marine Environmental Learning Center, Southold, NY 11791. Cornell Cooperative Extension of Suffolk County. New York, is in the second year of an expansion of its Marine Program to include a community-based shellfish restoration model to foster stewardship of the marine environment. The project, called SPAT (Special Projects in Aquaculture Training) is based on the under- standing that enhancement of shellfish beds contributes greatly to the health of estuarine ecosystems, and that local communities can play a significant role in stewardship and restoration. Bay scallops, Argopecten irradians irradians, hard clams. Mercenaria merce- naria notata. and eastern oysters, Crassostrea virginica. are com- mercially, recreationally, ecologically, and historically important species to the Peconic Estuary. However, cuirently the estuary supports only 1% of its historic oyster stocks and less than 1% of its historic scallop stocks, due in large part to the occurrence of brown tide algal blooms and its effects on habitat and shellfish health. The SPAT project incorporates a community-based shellfish enhancement effort with intensive aquaculture training and infor- mation gathering. The active membership is currently 191 families from 41 different communities throughout Suffolk County. The families are currently in training to become Master Shellfish Gar- deners, learning the process of growing local shellfish species in containment and collecting data on growth rates and survival. Each participant has been licensed by the NYS Department of Environ- mental Conservation and local townships to possess shellfish un- der a scientific collector's permit. In addition, more than 2001) volunteer hours have been donated to date to assist in culturing and planting millions of seed clams, oysters, and scallops in local waterways and select test plots. Dozens of participants assisted in a 250.000 bay scallop relay effort in March at Goose Creek. Southold. A planting of millions of seed clams took place with more than 30 community members at Cedar Beach Creek, in Southold. in August. Thousand of eastern oysters are being grown in containment to adult size in 48 local creeks and embayments. From June to December, two dozen participants have lent their time, effort, and expertise to build a community-operated shellfish hatchery on location, which will concentrate on the culture of bay scallops. The hands-on transfer of techniques that enable commu- nity participants, be they high school students, local business own- ers, or retired professionals, to restore locally important marine resources goes a long way toward raising awareness and develop- ing a stewardship ethic within communities. BAY SCALLOP {ARGOPECTEN IRRADIANS IRRADIANS) RESTORATION ON CAPE COD. Diane Murphy, Dale Lea- vitt. Bill Burt, and Bill Clark, Cape Cod Cooperative Extension, P.O. Box 367, Deeds & Probate Building, Barnstable, MA 02630. The bay scallop {Argnpecieii irradians irradians) fishery has long held a historic supplemental niche for Cape Cod and south- eastern Massachusetts fishermen. However, within the last 15-20 y. bay scallop populations have experienced a dramatic decline, with some localized extirpations. Some suggested causes for this de- cline include habitat degradation in the form of nutrient enrich- ment and loss of eelgrass beds as well as increased predation from introduced species such as green crabs (Carcinus maenus). In 1999, in response to the diminished stocks of wild scallops on Cape Cod, a collaborative restoration effort was initiated be- tween Cape Cod Cooperative Extension, SouthEastern Massachu- setts Aquaculture Center (SEMAC). and selected Barnstable County municipalities. Why bay scallops? Rapid growth rate, high market value, unstable supply, coupled with the evidence of de- clining and/or absent natural populations from historically produc- tive areas suggest that bay scallops would be viable candidates for enhancement purposes. Pursuant to an exhaustive review of cur- rent information available on the bay scallop, a regional workshop 348 Abstracts. 2002 Annual Meeting. February 25-27. 2002 Milford Aquaculture Seminar. Milf'ord. Connecticut was held to design an effective restoration program. It was con- cluded that spawning sanctuaries would provide the best means of creating self-sustaining scallop stocks for commercial and recre- ational fishing purposes. Now in its third year, the Barnstable County restoration pro- gram consists of purchasing small scallop seed derived from local broodstock in late summer to over-winter in off-bottom cages. These cages make optimal use of the water column for filter- feeding, as well as reducing predation and slltation. The following spring, scallop survival is assessed and scallops are redeployed into floating spawning sanctuaries sited in historically productive areas. Embayments are chosen with the greatest potential of larval entrainment — avoiding sites with extreme tidal fluxes to ensure localized settlement of seed. Optimal stocking densities also in- crease the likelihood iif successful spawning. Spat bags are de- ployed in close proximity to the spawning sanctuaries in order to monitor recruitment. In addition, genetic fingerprinting (using RAPD-PCR) is being used to evaluate the success of the bay scallop restoration program. EFFECTS OF CRASSOSTREA VIRGINICA POPILATIONS ON SEDIMENTATION. PHVTOPLANKTON SPECIES COMPOSITION, AND AMMONIA CYCLING IN EXPERI- MENTAL MESOCOSMS. Jennifer Mugg-Pietros and Michael A. Rice, Department of Fisheries, Animal and Veterinary Science. University of Rhode Island. Kingston, RI 02881. To determine the effects of oyster populations on water quality, a mesocosm study was performed from June to October 2000. Mesocosms with a volume of 1.^.000 L were used, in which there were triplicate control tanks without oysters and triplicate experi- mental tanks each with 200 oysters (=35 mm in valve height; nominally filtering about 55 L day"' ind'' ). Experiments were run sequentially in time for 3-wk periods, with water exchange rates ranguig from 0% to 100% per day (0-13.000 L day''). Several parameters were measured and compared between the control and experimental tanks, including chlorophyll-a, particulate organic and inorganic matter, sedimentation rates, nitrate, ammonia, phy- toplankton species and numbers, and oyster growth rates. There were no significant (P < 0.05) differences between tanks for most parameters, with the exceptions of rates of sedimentation and spe- cies composition of phytoplankton in the water column. Diatoms of the genus Nitzsclun were predominant in mesocosms with oys- ters; in control tanks. Skeletoiwma were dominant. Rates of atn- monia excretion by oysters of various sizes were determined by the sensitive salicylate-hypochlorite method, allowing for rapid deter- mination of excretion rates to minimize biases introduced by vola- tilization or transformation of the ammonia over time. The excre- tion of ammonia by oysters can be described by the allometric equation E = 50.65\r"'^'''''-, when E is the excretion rate in |jLg NH3-N hr"' and w is the dry soft tissue weight in g. On the basis of these data, it would be expected in the 3-wk experimental period that an additional 470 p.g L"' ammonia above the average control concentrations of about 40 |j.g L" would be present in each me- socosm with oysters, but no significant difference was noted be- tween the experimental and control tanks. This suggests that there is rapid cycling of ammonia, perhaps by uptake by the rapidly regenerating phytoplankton populations. This is publication number 3910 of the College of the Envi- ronment and Life Sciences at the University of Rhode Island, with support from the RIAES under project number H-886. A REVIEW OF DISEASES IN THE BAY SCALLOP [AR- GOPECTEN IRRADIANS IRRADIANS) AND SOME OBSER- VATIONS ON MORTALITIES AT THE MILFORD LABO- RATORY. Steven Pitchford and Richard Robohm, USDOC. NOAA. National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford. CT 06460. Various diseases and other causes of mortality in the bay scal- lop, Argopcctc'ii imulians irnuUans. will be reviewed, especially those that have been encountered historically and reported during hatchery and grow-out in the northeast Atlantic region. Unlike some of the other cultured bivalve species such as the eastern oyster. Cnissostrea virginica. which is affected by MSX and dermo. the adult bay scallop does not appear to be susceptible to specific pathogenic agents that can cause widespread epizootics. Examples of infections in bay scallops to be discussed briefly include those caused by prokaryotes (rickettsia. chlamydia. Vibrio ssp.). protozoans, algae, and fungi. As part of larger studies relating to immune system function and disease resistance in bay scallops, a program to screen for potential pathogenic targets was started in 1994. More than 70 bacterial isolates, the majority of which were Vibrio sp., were recovered from dead and moribund larval and adult bay scallops. High-concentration (approximately 10'' bacteria/larvae) screening assays were conducted with 46 of the strains. Seven of these caused >80Vf mortality. Next. 1.000 2-day-old larvae/L were ex- posed in 48 h. using serial dilution challenges with the seven bacteria. Only two isolates were considered to be pathogenic for larvae, with median lethal concentrations (LC^d) of 8.65 x 10^ and 1 .98 X 10' colony forming units (cfu)/niL after 48 h. Follow ing the crash of a bay scallop larval culture in 1998 at the Milford labo- ratory, monitoring of the cultures, the ambient seawater, and algal food sources for potential bacterial targets also was initiated. Highlights of the monitoring program include consistent high counts of Vibrio sp. for 1 wk following periodic, hot, freshwater flushing of the seawater lines. These high bacterial loads may have resulted from the large amount of dissolved and undissolved or- ganic matter that remained in the piping system. In addition, newly established mass algal cultures also would invariably show very high numbers of Vibrio sp. OlO"^ cfu/mL) soon after being started; however, the counts from these cultures would decrease to near 0 cfu/mL. usualh after 4-S w k. Milford Aquaculture Seminar. Milford, Connecticut Absrmcts. 2002 Annual Meeting. February 25-27. 2002 349 Other occurrences of high mortality at the Milford laboratory also will be discussed; these include events with a possible infec- tious disease etiology and those where increased mortality resulted from biotic and abiotic causes, such as overcrowding and fouling. Several episodes of gas bubble trauma have resulted in mass mor- talities, killing VS^-lOO^c of adult scallops in very short periods. Over-wintering mortalities routinely reach 409^ or even higher in cold winters, when water temperatures approach 0"C. SHELLFISH AQUACULTURES EFFECT ON TOTAL OR- GANIC CARBON (TOO IN THE BENTHOS. Perry Raso and Michael A. Rice, Department of Fisheries. Animal & Veterinary Science. University of Rhode Island, Kingston. RI 02881. Rhode Island waters provide a suitable habitat for shellfish aquaculture. Rhode Island waters are also highly valued by both local residents and tourists. If shellfish aquaculture is going to expand as a successful industry in Rhode Island, it must progress in a manner that does not negatively affect the estuary or bay in which it is conducted. To examine the effects of shellfish aqua- culture on the benthos, sediment samples were tested for total organic carbon (TOC). Fifteen 0.5-L sediment sainples were taken at each of 14 different shellfish aquaculture leases in Rhode Island waters. Five samples were taken within the lease; five within 10 m of the lease, and five no less than 100 m from the lease in a similar water body (depth, flow, distance from shore). Results showed normal variance between TOC levels of samples taken within, around, and away from aquaculture leases. Mean TOC of all samples away from the lease was 1 .48%, 1 .49% near the lease, and 1.21% inside the lease, with confidence intervals overlapping. This study provides data showing that shellfish aquaculture. at the scale that it is currently practiced in Rhode Island, does not significantly affect TOC levels in the benthos. This study is partially funded by RIAES project number H-886 and is publication number .^918 of CELS-URI. INDUSTRIAL-SCALE SCALLOP CULTURE IN CHILE— THE C.M.I. EXPERIENCE. Edwin Rhodes, Aquatecnics, LLC, Milford. CT 06460. Cultivos Marinos Internacionales, S.A. (CMl) is the largest scallop aquaculture company in the world. CMI grows Argopecten piirpiiniliis. native to Chile and Peru. Seed for the operation are primarily hatchery-produced, but seed collection is also employed. The original concept of producing 5-mm seed from a land-based hatchery and nursery did not work reliably, but settling hatchery- produced larvae on plastic mesh and putting them directly into the sea suspended from longlines proved to be successful. The scallops are subsequently grown in two pearl net stages, and three lantern net stages, all on longlines. CMl leases more than 1 100 hectares of growing area, split about evenly between two bays about .300 miles apart, and uses a total of about 1 .500 longlines. each 200 m long. Net changes to control fouling and to adjust scallop density are done on land in one bay, and from barges in the other. Scallops reach the market size of 80 mm shell height in 18 mo. and have an adductor muscle plus roe weight yield of 20 g each. CMFs pro- duction in the mid 1990s exceeded 5 million scallops/mo. Final product form is usually individually quick frozen (IQF), and vir- tually all of the scallops are e.xported from Chile to France. THE DEVELOPMENT OF AN INSTRUCTIONAL SHELL- FISH HATCHERY: A COLLABORATIVE EFFORT BE- TWEEN AEROS CULTURED OYSTER CO. AND THE SOUND SCHOOL REGIONAL AQUACULTURE CENTER. Karen Rivera, Aeros Cultured Oyster Company. 100 Main St.. Noank, CT 06340; Amber L. Beitler and John J. Roy, The Sound School. 60 South Water St.. New Haven, CT 06519. The Sound School Regional Aquaculture Center, in coopera- tion with Aeros Cultured Oyster Company, designed the Interdis- trict Marine Educational Program's Instructional Shellfish Hatch- ery (IMEP/ISH). The Instructional Shellfish Hatchery program is an Interdistrict Cooperative Grant and is funded by the Office of Urban and Priority School Districts of the Connecticut State De- partment of Education. The IMEP/ISH project was formed to pro- mote outcome-based learning between science classes from sev- eral coastal Connecticut high schools. The IMEP/ISH incorporates the vocational agriculture attitude of "learning through doing" as the method of education that best enables young researchers to conceptualize abstract points in their investigations as well as pro- viding them with the techniques necessary to achieve the definable outcomes being sought. The IMEP/ISH was constructed in the Purity Processed Sea- food building at the Noank Shipyard in Noank, Connecticut. The program began on May 7 and finished on May 31, 2001. During that time period, Karen Rivara of Aeros, in conjunction with staff from The Sound School and instructors from the participating schools successfully implemented 10 laboratory lessons that had been designed for the IMEP/ISH cuiriculum. The laboratory les- sons each considered an aspect of shellfish husbandry. The lessons included the following topics: water quality, shellfish biology, bi- valve reproduction and spawning techniques, microalgae culture, hatchery technology, nursery systems, grow-out technology, shell- fish health, harvest and handling methods, and depuration tech- niques. The Sound School acted as host school for the project. Ella T. Gras.so/Southeastern Vocational Technical School, Groton, Con- necticut, functioned as a partner school in the program, providing the necessary benchtop space (in the school's BET lab facilities) for the shellfish dissection and the shellfish health laboratories. Students from Ledyard High School's aquaculture classes (VoAg Department) participated in the program as well. When the pro- gram was completed. 55 different students had participated in 199 student days of laboratory activities involving the major aspects of shellfish culture as practiced by professional aquaculturalisis in Long Island Sound. 350 Ahstracts. 2002 Annual Meeting. February 25-27. 2002 Miltbrd Aqiiacullure Seminar. Milford. Connecticut THE EAST COAST SHELLFISH GROWERS ASSOCLA- TION: A WORK IN PROGRESS. Karen Rivara, East End Marine Farmers Association. Soiitlioid. NY I 1971. During the last 25 y. shellfish aquaculture has grown on the East Coast of the United States despite many obstacles. Newcom- ers in the industry come from many sectors of the economy, from retired schoolteachers and recent college graduates, to members of the traditional fisheries who look to aquaculture to maintain a sustainable living on the water. Established shellfish companies continue to use and improve various culture methods to meet the challenges of consistent production presented by losses due to disea.se and the loss of good cultivation areas. Other stakeholders in the marine environment have challenged the growth of this industry. This impediment to the growth and survival of shellfish aquaculture stems predominantly from a lack of understanding regarding the actual impacts of the shellfish aquaculture industry. Those who are opposed to the growth of this industry overlook many of the benefits of shellfish aquaculture. In other cases, shellfish aquaculture is viewed as an impediment to the development of other industries in the marine environment. Unfortunately, those who are against the growth of the industry have in many cases taken their grievances to legislators and the press, further fostering a lack of understanding and animosity to- ward the industry. Too often aquaculturalists are put in the position of having to take time from growing their businesses to defend them against unreasonable public opposition. The shellfish aquaculture industry clearly needs to become or- ganized. We need to establish an East Coast Shellfish Growers Association (ECSGA) that will identify socio-political obstacles and work to remove them, will work in a proactive way with other stakeholders, and will develop a public outreach/public awareness protocol that will prevent the industry from constantly having to defend itself. The ECSGA would also take the lead to implement best management practices to guide individuals involved in shell- fish cultivation as well as to inform legislators and other policy makers. It is important that this association be industry-driven, but in- volve participation from related areas of the industry such as pub- lic shellfish aquaculture facilities, state extension programs, aca- demia. government, and regulatory agencies. This year presents a unique opportunity to organize an East Coast Shellfish Growers Association. A roundtable discussion at the 22"*^ Milford Aqua- culture Seminar will be devoted to the formulation of this asso- ciation. We will discuss organizational considerations such as geo- graphic scope, types of membership, mission statement, and the concerns of the industry that this association will address. In April, the National Shellfisheries .'Association annual meeting will be held m Mystic. Connecticut. This affords the shellfish aquaculttire industry another opportunity to continue the process of forming an association. The goal of the ECSGA meeting at NSA in Mystic would be to continue discussion regarding the formation of the association and establish a committee of interested individuals that would also serve as the first Board of Directors. These individuals would complete the legal process of forming an association, es- tablishing an office, setting meeting dates, and establishing mem- bership. There are many reasons why the East Coast shellfish aquacul- ture industry should begin to organize. We need to take advantage of the opportunity to do so this year. HEAVY METAL SURVEY OF FUCVS SPIRALIS COL- LECTED FROM SOUTHWESTERN LONG ISLAND SOIIND. Rene Sanz. Sherry Lonergan. Jennifer Sutorius. and Dania Lieberthal. Bridgeport Regional Vocational Aquaculture School, 60 St. Stephens Road. Bridgeport, CT 06605. Finns spinilis. commonly know n as rockweed. is a brown alga found in the upper intertidal and niidtidal /ones of the North At- lantic. Similar to red algae. Fiiciis spinilis contains phycocolloids. which are valuable emulsifiers used in the manufacture of food, pharmaceutical, and healthcare products. In general, inacroalgae take in heavy metals, some of which are beneficial for nutrition and others that are possibly harmful to algae and humans in high levels. These heavy metals accumulate in algae, which makes them useful in biomonitoring. marine pollution assessment, and phytore- mediation. Because algae are becoming more popular for use in food, industry, and environmental applications, determining heavy metal concentrations is important to a wide range of audiences. Samples from five sites known as. sites A. B. C. D. and E, located in southwestern Long Island Sound, were surveyed to de- termine the concentration of heavy metal intake by Fiicus spiralis. The heavy metals being tested include: cadmium (Cd), chromium (Ca), copper (Cu), lead (Pb), and zinc (Zn). Samples were col- lected within 1 h of low tide. After samples were collected, the alga was washed, oven-dried, ground, and digested using a micro- wave lab station. Samples were then analyzed using atomic ab- sorption spectroscopy. Some samples were analyzed in replicate to ensure quality control. Collection for test samples began on Oc- tober 26, 2001. with the most current being December 19. 2001. Results of the sampling were as follows: • Cadmium (Cd) at site A ranged from below detectable limits (n/d) to 0.04 |jig/g: site B value was 0.04 jxg/g: values at sites C and D were all n/d: and site E value ranged from n/d to 0.04 M-g/g. • Copper (Cu) at site A was 0.18 p.g/g; site B value was 0.2.^ jjig/g; site C values ranged from 0.036 to 0.0582 (Ag/g: site D values ranged from 0.048 to 0.1104 (xg/g; and site E values ranged from 0.048 to 0.26 |J.g/g. • Chromium (Ca) at site A was 0.02 |xg/g: site B value was 0.03 pig/g; site E values ranged from n/d to 0.026 (j.g/g; site D values ranged from n/d to 0.082 [xg/g; and site D values ranged from n/d to 0.083 (jig/g. • Lead (Pb) at site A was 0.48 jig/g: site B value was 0.5 |j.g/g: site C values ranged from n/d to 0.134 |j.g/g: site D values ranged from 0.022 to 0.14 |jLg/g; and site E values ranged from 0.02 to 0.51 |ig/g. Milford Aquaciilture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meetina. February 25-27. 2002 351 • Zinc (Zn) at site A was 0.64 jjis/g: site B value was 0.7 (Jig/g; site C values ranged from 0.1882 to 0.2332 |ji.g/g: site D ranged from 0.06 to 0.214 [jLg/g; and site E ranged from 0.136 to 0.86 M-g/g. Analysis of these metals will continue at additional sites in Long Island Sound. Baseline data will be used in future compara- tive research of this and other algal species. in our recirculating aquaculture systems in order to study the in- cidence of liver tumors and mortality in Atlantic tomcods. Educational outreach programs: AREAC is developing cur- ricula for pre-K-1 2'^ grade students, teachers, and their families to enhance their knowledge and teaching ability on issues relating to aquaculture and the environment. Our programs have been funded by NPS, USAGE, DEC. Con Edison, and CUNY. AQUACULTURE ACTIVITIES IN BROOKLYN. NEW YORK? Martin P. Schreibnian. Chester Zarnoch. John T. Tanacredi, Lucia Magliulo-Cepriano, Jacob Raz, and Stefano Diomede, Aquatic Research and Environmental Assessment Cen- ter ( AREAC 1. Brooklyn College. 2900 Bedford Avenue, Brook- lyn. NY 11210. In Brooklyn College's Aquatic Research and Environmental Assessment Center (AREAC), state-of-the-art recirculating aqua- culture systems (RAS) have been used to conduct a number of diverse aquaculture and enviriinmental assessment/restoration pro- grams. These projects include the following topics. Finfish aquaculture; Our program consists of induction of spawning and/or grow-out of commercially important species. Our achievements include production of market-si/e walleye and tila- pia, as well as the culture of winter and summer flounder. Bivalve aquaculture: Recirculating systems for downwelling and broodstock conditioning were utilized in the culture of hard clams (Merceiiaria moxenaria). Seawater used in the static algal and larval cultures was biologically and chemically treated and then reused. In its first season of operation, the hatchery produced 2 million hard clams; when 2.0 nmi in shell length, they were moved out to a field site in Jamaica Bay, New York. An intensive study is underway to measure the survival and physiological con- dition of juvenile hard clams during the winter period. In addition, oyster seed were cultured in Taylor float systems at two sites in Jamaica Bay to monitor growth and water quality. Ornamental and research aquaculture: AREAC has more than 600 aquaria dedicated to the culture of freshwater species for fish hobbyists and scientific research. Soft coral is also being propa- gated. Captive breeding of horseshoe crabs: Adult horseshoe crabs were spawned in AREAC in July 2001. Developing animals were cultured to determine optimum temperature, medium, and nutri- tional parameters in recirculating systems. Currently, we have 2,500 horseshoe crabs with carapace widths ranging between 2 and 35 mm. Environmental assessment and restoration: The impact of en- docrine-disrupting chemical pollutants on freshwater and saltwater fishes has been studied for u number of years in AREAC. A program of field and laboratory experiments has begun to examine the effects of nonylphenols on winter flounder reproduction (in collaboration with Dr. Anne McElroy, SUNYSB). In another proj- ect, we have simulated the Hudson River by creating microcosms SHELLFISH AQUACULTURE: GOOD EOR THE ECONOMY, GOOD FOR THE ENVIRONMENT, GOOD FOR YOU! Sandra E. Shuniway, Department of Marine Sci- ences, University of Connecticut, 1080 Shennecossett Road, Gro- ton, CT 06340. The United States ranks 3"' worldwide in the consumption of seafood, yet it ranks 1 I "' in aquaculture production, with just 1.1% of global production by weight. World production of bivalve mol- lusks has increased steadily over the past decade, and 80'/f of world bivalve production is cultured product. Oysters comprise 35% of that total; clams and arkshells combined constitute about 33%. with China producing approximately 64% of the world total. The United States ranks i"' among bi\alve-producing nations and only 19"' in production via aquaculture. Possible reasons for these discrepancies and the benefits of shellfish aquaculture will be de- lineated and discussed. HEALTH MANAGEMENT GUIDELINES FOR SHELL- FISH CULTURE IN THE NORTHEASTERN UNITED STATES. Roxanna Smolowitz, Marine Biological Laboratory, Woods Hole, MA 02543; Susan Ford, Rutgers University, Port Norris, NJ 08349; Lisa Ragone-Calvo, Virginia Institute of Ma- rine Science, Gloucester Point, VA 23062. Bivalve diseases can cause significant losses of both cultured and wild stocks. To prevent the spread of disease among popula- tions, most states generally do not allow the importation of bivalve shellfish unless the animals are considered to be "disease free."" As a result, examinations for disease have become a necessary pre- requisite for determining health status. However, shellfish transfer regulations vary by state, often appear capricious, and complicate the sale and transport of commercially important seed. Further- more, both regulators and producers have few guidelines that would help them respond to and manage disease outbreaks, when they occur. Lines of communication need to be developed between diverse but knowledgeable groups to produce a set of standardized monitoring/management guidelines for use by state regulators. We describe a project intended to provide a set of uniform, scientifi- cally based recommendations for the health management of com- mercially important bivalve species in the northeastern L'nited States. The guidelines will be produced as a result of a series of work- shops and meetings proposed for the next 2 y that will include 352 Abstracts, 2002 Annual Meeting, February 25-27, 2002 Milford Aquaculture Seminar. Milford. Connecticut scientists, industry, regulators, and extension agents in the north- eastern United .States. This interchange will first identity the strengths and weaknesses of, as well as alternatives to. current approaches to bivalve health management in the region, and in- vestigate those in use or proposed by other states, by the federal government, and by other countries. The guidelines that follow from these discussions will be published in booklet form and will be posted on the Web. In addition to the guidelines, the document will also contain sections pertaining to ( I ) concepts in disease spread, control, and diagnosis; (2) descriptions of standard diagnostic methods; (3) an examination of the potential application of novel molecular-based diagnostics, including an assessment of their accuracy and sensi- tivity, and hov\ their outcome should be interpreted; (4) a fact sheet for each disease or disease agent; and (5) a glossary of general terms. The fact sheet will include the common and scientific names of the disease agent and its host or hosts, known geographic dis- tribution, known environmental limitations, life cycle, method of transmission, recommended diagnostic procedures, and treatment or amelioration methods. Each disease agent will be classified according \.o its potential danger to bivalve stocks. Although the guidelines will have no force of law, we anticipate that the inclu- sion of all interested parties in their development should lead to their use by all northeastern states because the recommendations are reasonable, scientifically based, and because they will benefit the shellfish uidustries in the recion. PREVALENCE AND MORTALITY ASSOCIATED WITH SSO AND SSO-LIKE INFECTIONS OF CRASSOSTREA VIRGINICA IN THE NORTHEAST. Roxanna Smolowitz, Ma rine Biological Laboratory, 7 MBL St.. Woods Hole, MA 02543; Inke Sunila. State of Connecticut. Dept. of Agriculture. Milford. CT ()(i4f)(); Nancy Stokes and Lisa Ragone-Calvo, Virginia In- stitute of Marine Science. College of William and Mary. Glouc- ester Point, VA 23062. Haplosporidium costale (seaside organism. SSO) was identi- fied as a cause of mortality in the eastern oyster (Ciassostiea virainwa) on the Atlantic coast of Maryland and Virginia in 1962 and is now endemic there. Early ( 1962) investigations in Virginia showed mortality associated with SSO could reach 60% in some years. SSO plasmodia are first identified in tissue sections in early spring. Mortality resulting from synchronous sporulation in the connective tissues occurs in May-June each year. Until recently, post-sporulation SSO plasmodia have not been positively identi- fied in oyster tissues until the following spring. SSO-infected oysters have also been found along coastlines of the more northern states, but historically. SSO morbidity and mor- tality has not been considered significant. Northeast regulatory agencies do not restrict shipments of oysters on the basis of posi- tive SSO findings. In late spring 1998, oyster culturists in Katama Bay, Martha's Vineyard. Massachusetts, observed IWc-lWr mor- tality of cultured stocks. The cause was identified as SSO. thus indicating that SSO can cause significant mortalities in some years in the northeast. Notably, in I99S in both Connecticut and Mas- sachusetts. SSO-like sporulating plasmodia were noted in sections of oysters in the late fall of the year. Although no other cases have since been identified in Massachusetts. SSO-like. fall sporulating organisms continue to be identified in Connecticut. Haplosporidiiiin nclsoni (MSX) also produces plasmodia in oyster tissues and is a cause of significant mortality in mid- Atlantic and northeast oyster populations. It is difficult to differ- entiate H. nt'lsoni and //. costule plasmodia using traditional diag- nostic methods, and it is possible that SSO and SSO-like plasinodia have been misidentitied as MSX over the last several years, resulting in the confusion between mortality associated with MSX and SSO. In this study, species-specific DNA-based diagnostic PCR methods and in situ hybridization (ISH) were employed in con- junction with traditional histological examination to differentiate H. nelsoni and H. castalc infections in cultured oyster populations in Massachusetts and Connecticut, with the main objective of de- termining to what extent mortality is attributable to SSO and/or MSX. Additional objectives were to identify the SSO-like organ- ism and to determine if fall and winter infection characteristics (i.e.. prevalence, tissue forms, and locations) of SSO are of value in predicting SSO-related disease severity in the following spring. Results indicate that pathologists cannot reliably differentiate MSX. SSO. and SSO-like plasmodia in traditionally stained tissue sections. Using species-specific DNA primers and probes, PCR and ISH methods can differentiate between these infections. In addition. ISH can help quantity the relative proportion of SSO and MSX Plasmodia within tissue sections. Some plasmodia identified in oyster tissues collected in the fall, which might have been iden- tified as MSX, are positive for SSO and SSO-like organisms. The relationship of SSO-like organisms to SSO and MSX plasmodia continues to be studied. Combining the history of oyster mortality in a specific location with results from various diagnostic tests is the best way to predict morbidity and mortality associated with these oyster diseases m any population. This work is sponsored by a NOAA/Sea Grant Oyster Disease Research Award. RHODE ISLAND'S SHELLFISH RESTORATION PRO- GRAM IN RESPONSE TO THE NORTH CAPE OIL SPILL. Karin A. Tanimi. Najih Lazar. and .Vrthur Ganz. Rhode Island Department of Environmental Management. Coastal Fisheries Laboratory. 1231 Succotash Road, Wakefield. RI 02879; James (;. Turek, National Oceanic and Atmospheric Administration Restoration Center, 28 Tarzwell Drive. Narragansett, RI 02882; and John G. Catena, National Oceanic and Atmospheric Admin- istration Restoration Center, One Blackburn Drive. Gloucester, MA 01930. On the evening of January 1 9. 1 996. the tank barge North Cape struck ground off Point Judith. Rhode Island, and began leaking oil in the vicinity of two National Wildlife Refuges, several salt Milt'ord Aquaculture Seminar. Milford. Connecticut Abstracts. 2002 Annual Meetinc, Februarv 23-27, 2002 353 ponds, and public and private beaclies. Wmd and wave action dispersed the oil into the atmosphere, throughout the water col- umn, and into the benthic sediment. Approximately 828,000 gal- lons of heating oil were released into the surrounding offshore and inshore environment, affecting large numbers of crustaceans, mol- lusks. birds, amphipods, and fish. It was determined that the spill was responsible for the loss of about 150 million surfclams, Spisuki solidissiiiKi. with a total bioniass of 379,000 kg. for a value of $1.5 million. The spill resulted in the formation of a natural resource trustee group, composed of Rhode Island Department of Environmental Management, the National Oceanic and Atmo- spheric Administration, and the United States Fish and Wildlife Service, to evaluate the injury to the natural resources and to plan the resulting restoration activities. Because the surfclam popula- tion should recover to natural baseline levels within 3-5 y. a com- pensatory shellfish restoration program will be launched in Nar- ragansett Bay and in the coastal salt ponds. Beginning in 2002. the trustees will initiate a multifaceted and multispecies approach to shellfish restoration with programs for the eastern oyster. Cnis- sostrea virginica: Northern quahog. Mercciiaiici iiifireiuiriii. and the bay scallop, Argopecten inaJiaiis iinnUans. The shellfish res- toration strategy will utilize many techniques, which include a remote setting program for C. virgincia and spawning sanctuaries and spat collection for bay scallops. A. irradians irnidiuns. The shellfish restoration initiatives in response to the North Cape oil spill disaster offer tremendous opportunities for Rhode Island's shellfish resources. operated hatchery on location is expected to be operational for the 2002 growing season, which will concentrate on the culture of bay scallops {Argopecten irradians irradians). Various projects are planned for the 2002 season. These include restoration work on two community spawner sanctuaries funded by NOAA (oysters) and 5-Star (clams/scallop) grants. Two addi- tional spawner sanctuaries, one for oysters and another for scal- lops, have been designated by the town of Southold. .All sanctu- aries will be stocked and monitored by SPAT trainees. The focus will be on early field planting and predatiir control techniques for select species of clams, oyster bed preparation and reef building, and intensification of scallop deployment at multiple stages within healthy eelgrass beds. The community hatchery is expected to significantly increase the production of bay scallops with a target goal of 5-10 million post-set for the 2002 season. A grant to examine the sociological components of the SPAT initiative has been awarded by an anonymous foundation. Two pilot educational programs, one at the third-grade level and one at the teenage level, are actively in progress. New internships in community-based shellfish restoration activities are available. It is the intention of the Cornell Cooperative Extension SPAT initiative to establish a model for community-supported shellfish restoration efforts thiough intensive training and active participa- tion. The founding members of SPAT are excited and prepared to meet the challenges in the upcoming year. SPAT (SPECIAL PROGRAMS IN AQUACULTURE TRAIN- ING) UPDATE. 2002. ESTABLISHING A MODEL FOR COMMUNITY-BASED SHELLFISH CULTURE AND RES- TORATION. Kim Tetrault, R. Michael Patricio, and Mary Morgan. Cornell Cooperative Extension of Suffolk County, Ma- rine Environmental Learning Center, Southold, NY 1 1971. Cornell Cooperative Extension of Suffolk County, New York, has expanded a component of their marine program division to include a series of educational and training initiatives collectively referred to as SPAT (Special Programs in Aquaculture Training). The SPAT campaign is now 1 y old and has made tremendous progress. The active membership of SPAT Master Shellfish Gar- deners is currently 191 families strong, with attendance at the monthly workshop series (II, 2-h lectures) consistently exceeding KJO participants. More than 2,000 h have been logged into the community hands-on training sessions held weekly throughout the year. SPAT members assisted in culturing and planting millions of seed clams, oysters, and scallops in local waterways and selected test plots, as well as maintaining personal "garden" stocks. A new web site (www.cce.cornell.edu/Suffolk/MARprograms/ Aquacultruemain.htm) now allows members to record data that have been collected on a monthly basis for growth and survival of cultured oysters {Crassostreu virginica). A community-built and A NEW CLAM FOR CONNECTICUT. John Wadsworth, Niantic Bay Shellfish. LLC. 15 First Street. Waterford, CT 06385; Tessa Simlicli and Nancy Balcom. Connecticut Sea Grant, Uni- versity of Connecticut. 1084 Shennecossett Road. Groton, CT 06340. The razor clam Ensis directus. is one of the few untapped resources in shellfish aquaculture in the northeastern United States. The market supply of razor clams is low and inconsistent, particu- larly because of the difficulty in harvesting these fast-digging shellfish. If razor clam production were to become economically feasible and less labor intensive, there would be great opportunity to expand this underutilized species to niche inarkets. The North- eastern Regional Aquaculture Center (NRAC) has provided fund- ing for the aquaculture industry to test various clain grow-out methods in four stales: Connecticut, Massachusetts, New York, and Rhode Island. In Connecticut, a project was initiated to inves- tigate razor clam grow-out in cages. Seed clams were obtained from the Aquaculture Research Corporation (ARC) in Dennis, Massachusetts, in September 2001. The clams were held initially in upwellers and then transferred to cages for field experiments. The project consisted of two experiments, the first of which was designed to compare razor clam growth rates in cages lined with felt set at two different heights. Replicate cages were lined with felt to the top of the cages or. the high position (HI). The remain- ing cages were lined up to the level of the sediment, or low posi- 354 Abstracts. 2002 Annual Meeting. February 25-27. 2002 Miltord Aquacullure .Seminar. Milford. Conneclieut tion (H2). The second experiment was designed to compare razor clam growth rates between two different growing areas, the Nian- tic River (NR) and the Niantic Bay (NB) in Waterford. Connecti- cut. The clams were divided among replicate cages and stocked at a density of one clam/2.5 cm- (one clam/in.-). Monthly inventories to determine clam density and growth rate (length and width to ±0.01 mm) were performed beginning in September 2001. As of early February, preliminary results showed that there was no sig- nificant difference in growth rates between felt heights (HI vs. H2) or growing area (NR vs. NB ) ( ANOVA. P < 0.05 1. The clams grew in length from 18.84 ± 2.22 mm to 34.71 ± 4.46 mm. High den- sities were observed in the NR cages (>907f of original stocking densities), however, the clams were present in very low densities in the NB cages. LIFE IN A TRAILER— DEVELOP.MENT OE A NEW SHELLFISH HATCHERY AT THE MASSACHUSETTS MARITIME ACADEMY. Bethany A. Walton, Aquaculture Laboratory. Massachusetts Maritime Academy. 101 Academy Drive. Buzzards Bay. MA 02532. The Aquaculture Lab at the Massachusetts Maritime Academy (MMA) in Buzzards Bay. Massachusetts, officially opened its doors in April 2001. Located at the southern entrance to the Cape Cod Canal on Taylor's Point, the hatchery is a cooperative venture between MMA and Barnstable County; the SouthEastern Massa- chusetts Aquaculture Center (SEMAC) also provides technical support. This facility is not a production-scale shellfish hatchery, but rather, a "teaching hatchery."" This teaching hatchery ser\es as an integral component of an introductory aquaculture course taught at MMA to provide "hands-on"" training for students in shellfish biology, hatchery techniques, and intermediate culture. The building (a refurbished trailer that measures appro.ximately 1100 ft") consists of two primary areas, one devoted to algae production and the other to larval production. The greenhouse area holds twelve 250-L Kalwall tubes and has space for several 3-L carboys and 1-L jugs. Microalgae production consists primarily of batch culture of more than 10 species. The larval production area has a capacity to produce approximately 30-50 million 1-mm lar- vae and holds four 937.5-L larval tanks; there is also a small area devoted to setting animals and broodstock holding tanks. A limited amount of upweller space is also on site. Two innovative features of the hatchery are its seawater system and protected interior sur- faces. The seawater system is a multiple pump system that feeds into a common manifold consisting of four titanium submersible pumps with a capacity of 50 gal/min. In addition, the interior surfaces of the facility are protected by a commercial polyurethane formulation (typically used for spray-on truck bed liners) applied to the floor and walls to seal them from conosion and water damage. Not only will we continue to use the hatchery as a teaching tool. we will also be coordinating our 2002 spawning activities with the Eastham Aquaculture Technology and Training Center to maxi- mize shellfish propagation and enhancement efforts in the Cape Cod area. We plan to produce razor clams tEiisis directiis). bay scallops iArgopecteii irradians irradians). and eastern oyster iCrassostrea viri;iiuca) larvae for remote sets in various Cape Cod towns. Currently, we are culturing a small number of razor clam larvae. THE NEW OYSTER WARS: POLICY PERSPECTIVES ON THE INTRODUCTION OF CRASSOSTREA ARIAKENSIS IN THE CHESAPEAKE BAY. Donald Webster. University of Mar\land. Wye Research & Education Center. P.O. Box 169. Queenstown. MD 21658. Oyster harvests in the Chesapeake Bay have declined more than 907f in the past 50 y. largely due to the inlluence of the oyster diseases Haptosporidiim uelsoni and Perkinsus mariniis. Recent studies regarding the potential of the species Cnissostrea ariak- ensis have show n that this animal may pros ide beneficial attributes desirable both for reconstruction of the oyster fishery as well as for en\ ironmental management of the Bay. However, because it is a nonindigenous species, there are factors to be considered prior to an\ large-scale introduction. Variations in the current status of the Cnissostrea virfiinica resource, as well as historical differences of management techniques inherent in the two states, have led to challenges regarding open-water introductions, as well as plans to increase stocks of the nonnative oyster in the future. This paper focuses on the policy differences between Maryland and Virginia and the attitudes of \arious user groups that are interested parties in the potential introduction of C. ariakcnsis in the Chesapeake Bay. DEVELOPMENTS IN SOFTSHELL CLAM HATCHERY AND NURSERY PRODUCTION ON MASSACHUSETTS' NORTH SHORE. Scott Weston, Mark Fregeau, and Joe Butt- ner. Northeastern Massachusetts Aquaculture Center and Depart- ment of Biology. Salem State College. Salem. MA 01970. A major goal of the Northeastern Massachusetts Aquaculture Center (NEMAC) focuses on nurturing a sustainable aquaculture industry on Massachusetts North Shore amenable to existing so- cial, economic, and en\ ironmental conditions. Several North Shore communities are exploring aquaculture as a means to diversify and supplement their capture fisheries while restoring and enhancing endemic populations. Historically, the softshell clam {Mya ari'iuiria) supported significant commercial and recreational har- vests. Efforts initiated in the 1990s to augment natural populations now in\ol\e a half dozen towns. Culture protocols adapted to local conditions have demonstrated the efficacy of stocking 10- to 15- mm spat in spring. Availability of suitable seed is limited and has impeded stocking efforts. With the encouragement and participa- tion of local communities. NEMAC has targeted reliable and ul- timately large-scale production of spat as a priority objective. NEMAC"s second year of operation (2001) started eariy as Milford Aquaculture Seminar, Milford, Connecticut Abslracts. 2002 Annual Meeting, February 25-27. 2002 355 200.000 juvenile clams were collected by local shellfishers from the Rowley River in November 200(1 and successfully over- wintered in the Cat Cove Marine Laboratory. Clam survival ap- proached lOO'^'K though growth at 15-17°C averaged -0.5 inm/ino between 3 January and 7 May. Clams were returned to the Rowley community and released in early May 2001 at approxed sites. Starting in early July 2001 and continuing to the present. NEMAC personnel and North Shore shellfishers have monitored sites in the Rowley River to ascertain water quality, clam abundance, and optimal release sites. NEMAC personnel induced four spawns of local clams be- tween early May and mid July. Inadequate setups for settling lar- \ae diminished survival of initial spawns and polyspermy ad- versely impacted larval development during the fourth spawn. NEMAC produced 75.000 post-set spat, supplemented by 500.000 clams imported from the Beals Island Regional Shellfish Hatchery (BIRSH) in Maine. All clams were cultured in the laboratory on Tetiaselmis clniii and T-ISO. As clams reached -3.5 mm in length, they were transferred to screen-bottom trays at 10.000 clams/tray (-3.600 cm"). Appro.ximately 30 periwinkles (Littorina sp.) were added to each tray to crop macroalgae and other fouling organisms. The first tray was stocked in mid July and stocking continued throughout the summer. All trays were covered with white plastic, floated in Smith Pool, and exposed to tidal flush. Initial growth averaged 2.5-5 mm/mo. At 8-9 mm. growth of clams diminished, which is indicative of overcrowding. Approximately 70% of the clams stocked in upwellers survived. Clams are being over- wintered. 250.000 (8-15 mm) in spat bags suspended in Smith Pool and 150.000 (3-8 mm) are distributed in trays stacked in a trough with a continuous flow of filtered seawater. Laboratory- housed clams are fed 1-2 times daily with a Telrasetmis chuii and T-ISO mix. The objective is to release 200.000 spat of suitable size onto approved tidal fiats in spring 2002. It is hoped and anticipated that on-going, collaborative restoration/enhancement efforts will evolve into commercial enterprises conceived and pursued by local shellfishers. RAPID GROWTH OF BAY SCALLOPS. ARGOPECTEN IR- RADIASS IRR^iDIAyS. IN LONG ISLAND SOUND. James C. Widman. Jr. and David J. Veilleux, USDOC, NOAA, Na- tional Marine Fisheries Service, Northeast Fisheries Science Cen- ter, Milford Laboratory. Milford. CT 06460. Bay scallops. Argopecten irradians irradiuns. were held in pearl nets to evaluate the suitability of western Long Island Sound as a culture site for scallops. Scallops were deployed at six loca- tions off the coast of Darien. Connecticut. Pearl nets were an- chored with a cement block and vertical lift was provided by an attached subsurface buoy. Nets were deployed singularly or in groups of three. Thirty scallops were measured to the nearest 0.1 mm. placed in each of nine pearl nets, and transported to the site in coolers filled with ambient seawater. Initial deployment was on July 25. 2001. and the experiment ended on February 6. 2002. Survival was high, although there was some initial mortality. Survival for the entire experiment averaged 71% and ranged from 53% to 83%. After the initial mortality, survival after September 1 1 averaged 93% and ranged from 79% to 100%. The increase in survival after the September 11 sampling period indicates that there may have been some handling/transport problems. Scallops with initial mean shell heights ranging from 6.8 to 7.7 mm on July 24. 2001. grew to mean shell heights ranging from 45.4 to 50.6 mm by February 6. 2001. Growth rates were high from July 24 until October 16. when water teinperatures began to decline below 15"C. Less than 1 mm of growth occurred after December 4. when water temperatures were low. Rapid growth rates ranging from 0.36 to 0.49 mm/day were observed during the period from September 1 1 to October 16. 2001. a period of de- clining water temperatures. For the experimental regime. Western Long Island Sound ap- pears to be a suitable site for culturing bay scallops. LIVESTOCK DOMESTICATION IN THE THIRD MILLEN- NIUM: ALL WET? Gary H. Wikfors. USDOC, NOAA, Na- tional Marine Fisheries Service, Northeast Fisheries Science Cen- ter, Milford Laboratory, Milford. CT 06460. The dictionary defines "domestic" animals as those "kept by and for the use of man." The first domestication of land animals (sheep in what is now Turkey) is thought to have occurred between 13,000 and 9.000 y ago during the Neolithic-to-Paleolithic transi- tion in human history. Since that time, the trajectory of human food acquisition has been away from the hunter-gatherer mode to increasingly sophisticated husbandry of both plants and animals. The one exception to this historical paradigm has been seafood. It appears, however, that mankind now is entering a transition period from harvest of wild populations in the sea to domestication for the same reasons that drove land-animal domestication — rising de- mand and limited supply of wildlife for harvest. We have the benefit of about 10.000 y of human experience to guide decisions about marine plant and animal domestication — both successes and failures. There has, however, been limited effort to transfer the broad principles of animal husbandry downstream; therefore, the first objective of this presentation will be to advance this transfer. At this early stage in the transition to aquaculture. the first few global, industrial-scale aquaculture products — salmon and shrimp — have reached a point of development to impact world seafood markets, as well as generate controversy about the future of aquaculture expansion on several fronts, chietly economic and environmental. Unquestionably, mistakes have been made with shrimp and salmon, as they have with agricultural livestock pro- duction methods. What lessons can be learned from modem agri- culture and our limited experience with industrial aquaculture and how can these be related to broad principles identified from the history of animal husbandry? These questions will be discussed comparatively. 356 Ahsrmcls. 2002 Aniuuil Meeting. February 25-27. 2002 Mllford Aquaculture Seminar. Millord. Conneetieut BLUE MUSSEL AQUACULTURE IN LONG ISLAND SOUND. Lawrence Williams, Jessie D.. Inc.. 68 Anchorage Drive. Milford, CT 06460: and Tessa Simlick. Connecticut Sea Grant, University of Connecticut. 1084 Shennecossett Road. Gro- ton, CT 06340. A pilot-scale investigation into the longline culture of blue mussels Mytilus ediilis in Long Island Sound has been initiated. The project, in progress since the spring of 2001. includes collect- ing and grading seed for grow-out on dynamic longlines at three sites in Long Island Sound and comparing production rates. A horizontal head rope (100 ft in length) was submerged 6 ft below the surface (at MLW) and anchored with concrete blocks (150 lb total weight). Vertical seed collecting lines (11 ft in length) were attached to the head rope at 3-ft intervals. Seed collectors were placed over submerged mussels beds in Branford. Milford, Fair- field, and Stonington. Connecticut, in late April 2001. A large seed-set appeared in Stonington in early June 2001 . however, mus- sels were not visible on the collectors in western LIS until mid- July 2001. In late September 2001. a second set of seed attached to the seed collectors. ARACHIDONIC ACID REQUIREMENTS IN LARVAL SUMMER FLOUNDER, PARAUCHTHYS DENTATUS. Stephen Willey and David A. Bengtson, Department of Fisheries. Animal and Veterinary Science, University of Rhode Island, Kingston. RI 02881; Moti Harel, University of Maryland Center of Marine Biotechnology, 701 East Pratt Street, Baltimore. MD 21202. Highly unsaturated fatty acids (HUFAs) have been identified as essential fatty acids in marine fish and the dietaiy requirements for the n-3 (omega-3) HUFAs eicosapentaenoic acid (2():5n-3. EPA) and docosahexaenoic acid (22:6n-3. DHA) have been well docu- mented. Only recently has attention been given to HUFAs of the n-6 series, in paiticular arachidonic acid (20:4n-6. AA). The im- petus for much of this attention is the fact that AA is highly conserved during periods of starvation and also serves as a pre- cursor in the biosynthesis of eicosanoids. physiologically active compounds that aid in stress response, among other things. The AA requirements of larval summer flounder were deter- mined for the rotifer- and Artemia-feeding stages. Experimental emulsions contained adequate n-3 HUFA ratios and emulsion lev- els of AA were set at 0%, 3%. 6%. 9%. and 12% (AAO. AA3. AA6. A9. and AA12). Examination of fatty acid levels in live feeds and larval tissues confirmed the physiological incorporation of fatty acids relative to dietary levels. In the first experiment. survival, growth, and salinity tolerance (2 h in 70%c) were mea- sured at 18 days after hatch (dah) after feeding the larvae the various levels of AA. Larvae fed AA6-enriched rotifers were better able to survive the salinity tolerance test. AA enrichment up to 12Vf had no effect on growth and survival. In the second experi- ment, larvae fed AAO- or AA6-enriched rotifers until 23 dah. followed by unenriched 24- and 48-h Artcnua nauplii until 32 dah. These larvae were then subdivided and fed AA-enriched Anemia from 33 to 45 dah. At the end of this experiment, larvae fed AA6-enriched rotifers had higher survival, increased growth, and survived better in the salinity tolerance test (2 h in 809ff ) than did those fed AAO (unenriched) rotifers. The AA enrichment oi Ar- temia did not have any significant effect on these variables. Thus, the provision of AA6-enriched rotifers early in larval development may serve to enhance larval stress tolerance at the rotifer stage, while also increasing larval survival, growth, and stress tolerance later in the Arlcniiii stage. Joimuil of SlwUfish Research. Vol. 21. No. 21, 357^4U. 2UU2 ABSTRACTS OF TECHNICAL PAPERS Presented at The 94th Annual Meeting NATIONAL SHELLFISHERIES ASSOCIATION Mystic, Connecticut April 14-18, 2002 357 National Shellfisheries Association, Mystic, Connecticut Abstracts, April 2002 359 CONTENTS I'ERKINSUS George R. Abhe, Brian W. Albright. Carol B. McColloitgh. Christopher F. Diingan and Stephen J. Jordan Environmental effects on Perkiusiis nuiriinis infection rates, growth and survival among Dermo-disease-free juvenile oysters planted at three salinity regimes in the Patuxent River, Maryland 37 1 Bassem Allam and Susan E. Ford Transport of particles across epithelia from oyster mantle cavity: a model for Perkinsiis inarimis invasion 371 Louis E. Burnett and Christopher S. Milardo Effects of o\ ster emersion on the grow th and the metaboiisin of Perkinsiis iinirimis 371 David Bushek, John Scarpa and Susan E. Laramore Susceptibility of the Caribbean oyster Crassoslrea rliizopliorac to Perkinsiis iiuinniis 37 1 Fu-Lin E. Chu, Eric Lund and Ellen Harvey Does the unique fatty acid synthetic capability of Perkinsiis iiianniis have implication for virulence? 372 Lewis E. Deaton and Percy J. Jordan Effect of Perkinsiis inanniis on phenoloxidase activity in bi\alve hemolymph 372 Christopher F. Dungan, Rosalee M. Hamilton, Carol B. McCollough, Kimberly S. Reece and Karen L. Hudson Epizootic diseases in Chesapeake Bay clams 372 Vincent G. Encomio, Shawn Stickler and Fu-Lin Chu E\ aluation of physiological condition in Dermo resistant oysters 373 Tarquin Dorrington, Maria Gomez-Chiarri and Lenore Martin Evaluation of antimicrobial peptides for recombinant feed-based delivery in shellfish aquaculture 373 Julie D. Gauthier, Jerome F. La Peyre and Jill A, Jenkins Flow cytometric analysis of lectin binding to in vitm cultured Perkinsiis nuiriniis surface carbohydrates 373 Eileen E. Hofmann, John M. Klinck, Eric N. Powell, Susan E. Ford, Stephen Jordan and Eugene Burreson Climate variability and Dermo disease in Chesapeake Bay 374 Stephen L. Kaattari, E. Alanna Maclntyre and Christopher G. Earnhart Modulation of Perkinsiis nuiriniis functions by host-derived products 374 Jerome F. La Peyre, Amy D. Nickens, Sandra M. Casas and Antonio Villalba Viability and growth of Perkinsus marinus and Perkinsiis ailanticiis at three temperatures 374 Eric D. Lund, Fu-Lin E. Chu and Philippe Soudant Is the temperature and salinity-dependent virulence of Perkinsiis nuiriniis associated with increased lipid metabolism? 374 Carol B. McCollough, Christopher F. Dungan, Stephen J. Jordan, George R. Abbe and Brian W. Albright Perkinsiis marinus infection rates in specific-pathogen-free juvenile oysters planted at three salinity regimes in the Patuxent River. Maryland 375 Kennedy T. Paynter, Tim Koles and Don Meritt Perkinsiis prevalence in oysters produced in the hatchery and planted in the field 375 Sammy M. Ray, Thomas M. Soniat, Enrique V. Kortright and Lance Robinson Recent trends in levels of infection of Perkinsiis marinus in oysters from Galveston Bay. Texas: results of the Dermowatch Monitoring Program 375 Kimberly S. Reece Utilization of molecular genetic data for detecting, identifying and describing Perkinsiis species 376 Pilar Mui'ioz Ruiz and Maria Gomez-Chiarri Protease activity in the eastern oyster Crassoslrea virginica after experimental infection with the protozoan parasite Perkinsus marinus 376 S. M. Stickler, V. G. Encomio, S. K. Allen, Jr., F-L. E. Chu and J. F. Im Peyre "Natural Dermo resistance"" in eastern oyster stocks: Chesapeake studies and defense-related activities 376 THE BIOLOGY AND CONSERVATION OF FRESHWATER MUSSELS Alan D. Christian. David J. Berg and B. Crump Spatial and temporal analysis of ecosystem processing of freshwater mussels in two headwater streams 377 Susan Rogers, Richard Biggins and Steven Ahlstedt Conservation acti\ities tor freshwater mussels by the United States Fish and Wildlife Service 377 William F. Henley and Richard J. Neves Diet evaluation for the freshwater mussel Elliptio comptanala ( Bivalvia: Unionidae) 377 360 Ahslimts April 2002 National Shelltisheries Association. Mystic, Connecticut Variability in condition index and tissue biochemistry of Elliplio loniplanalti held in the field and laboratory 378 Curt L. Elderkin. David J. Berg. Janice L. Metcalfe-Smith. Caryii C. Vaughn. Alan D. Christian and Sheldon I. Guttman Hierarchical analysis of MtDNA variation in Amhlema plicata. a widespread mussel species 378 Jeffrey T. Garner and Thomas M. Haggerty Gametoizenesis. spawning, brooding and glochidial discharge in Mci^aUmuuis iicrvd.sa (Bivlavia: Unionidae) from the ^ ^ ^ ^ 378 Tennessee River in Alabama '° Catherine M. Gatenhy. Daniel A. Kreeger. Rohyn Reinmiller and Richard J. Neves Clearance rate and filtration efficiencv of EUiptic compUmaw (Bivahia: Unionidae) exposed to different laboratory ,.,. ' 378 conditions Daniel A. Kreeger, Catherine M. Gatenby and Deborah Raksany lity Richard J. Neves, Jess W. Jones and William F. Henley Propagation of endangered freshwater mussels in recirculating culture systems 379 Daniel E. Spooner and Caryn C. Vaughn Small-scale ecological function of freshwater mussels (Family: Unionidae) in the Kiamichi River. Oklahoma 379 Caryn C. Vaughn, Daniel E. Spooner, Melissa Moore and Keith R. Gido Comparative and experimental evidence for the functional role of freshwater mussels in streams 379 W.-H. Wong, Jeffrey S. Levinton and Benjamin Twining Assimilation of micro/.ooplankton by zebra mussels: the benthic zooplankton loop 380 GENETICS AND MOLECULAR BIOLOGY Charles L. Brockhouse and Gillian Richard Unscrambling the lobster genome: complex heterozygosity in the American lobster? 380 Bruno Ernande, Joel Haurc, Lionel Degremont, Edouard Bedier and Pierre Boudry Genetical basis of the plasticity of the resource allocation in the Pacific oyster Crassostrea gigas 380 Ximing Guo, Jian Wang. Brenda J. Landau. Li Li. Gregory A. DeBrosse and Krista D. Buono The successful production of tetraploid eastern oyster. Crassostrea virginica Gmelin 380 Matthew P. Hare and Stephen R. Palumbi Genetic distinctiveness of inshore and offshore Spisula clams Dennis Hedgecock, Sophie Hubert, Gang Li and Katharine Bucklin A genetic linkage map of 100 microsatellite markers for the Pacific oyster Crassostrea gigas 381 Gregg G. Hoffman. Ami E. Wilbur, Martin H. Posey and Troy D. Alphin A sin>'le-step multiplex PCR identification assav to distinguish megalopae of Calliiiecles sapuliis trom Callinectes siinilis in plankton samples Brenda J. luindau, Arnaud Tanguy and Ximing Guo Searching for differentially expressed genes in diploid and Iriploid eastern oyster. Crassostrea virginica Gmelin 382 Ziniu Yu and Ximing Guo A basic AFLP linkage map for the eastern oyster. Crassostrea virginica Gmelin 382 Mingjdng Zhou and Standish K. Allen, Jr. Reversion revisited in Crassostrea ariakciisis: chromosome set instability in field trials across salinity regimes 382 EAST COAST BIVALVE INDUSTRY SESSION Standish K. Allen, Jr. An overview of the potential for Crassostrea ariakensis in the Chesapeake Bay Gregory A. DeBrosse, David R. Jones and Eric N. Powell Surf clam [Snisula solidissima) culture in southern New Jersey: hatchery culture proKK'ols and preliminary field grow , ' 383 out results M. Richard DeVoe and G. Ross Nelson Coastal states permitting procedures for deploying a tidal powered upwelling nursery system 383 George E. Flimlin, Jr. Relevant issues for the east coast shellfish aquaculture industry: finding commonality to foster unity 383 Nation;il Shellfisheries Association. Mystic. Connecticut Ahsrracts. April 2002 361 Aiiiaii Liithra and William Walton Stakeholders" perceptions and challenges to participatory management: the case of the soft-shell clam. Mya arenaria L.. industry in Maine 384 Sandra Macfarlane Embracing aquaculture BMP's; a concept whose time has come as a good neighbor or just one more thing to deal with? 384 Dana L. Morse and John Riley Optimization of tidal upweller design: project wrap-up 384 Robert B. Rheault and Jeffrey T. Gardner Growth and economics of shellfish nursery culture in a commercially available floating upweller 385 Leslie N. Stunner, John E. Supan and Charles M. Adams Enhancing seed availability for the hard clam aquaculture industry through application of remote setting techniques ... 385 William C. Walton Using shellfish seed as a public enhancement tool: a review of its use and success in the northeast US 385 Christopher G. Warfel, P.E. Growth and economic advantages of distributed powered upwellers: creating a new aquaculture niche 385 Donald Webster The new oyster wars: policy perspectives in the introduction of Crassostrea ariakensis in the Chesapeake Bay 386 BIVALVE DISEASE STATUS AND TRENDS Bruce J. Barber and hatherine J. Boettcher Recent expansion of juvenile oyster disease ( JOD) in Maine 386 David Bushek, Dwayne Porter, Loren D. Coen, M. Yvonne Bobo and Donnia L. Richardson Status and trends of Dermo and MSX in South Carolina 386 Lisa M. Ragone Calvo and Eugene M. Burreson A decade of disease: a report of oyster disease monitoring efforts in the lower Chesapeake Bay 1990-2000 387 Ryan B. Carnegie, Gary R. Meyer, Janice Blackbourn, Susan M. Bower and Nathalie Cochennec-Laureau Development and application of a PCR for Mikrocytos macktni. the causative agent of Denmaii Island disease in oysters 387 Susan E. Ford Development of high disease resistance in a wild oyster population 387 Stephen J. Jordan and Jessica Vanisko The long-term role of parasitic diseases in oyster population dynamics 387 Dale Leavitt, D. Murphy, W. Burt, W. Clark, M. Hickey, J. Moles and R. Smolowitz Cape Cod bivalve diseases monitoring program. Year I 388 Karen L. Mareiro, Marta Gomez-Chiarri, Katherine Kerr, Emily Carrington and Arthur Ganz Prevalence and intensity of parasitic diseases in bivalves from Rhode Island waters 388 Spencer Russell, Salvatore Frasca, Jr., Richard A. French and Inke Sunila Modification and field trials of a multiplex PCR for the detection of three protozoan pathogens of the eastern oyster. Crassostrea \iri;inica. Gmelin 1 87 1 388 Thomas M. Soniat, Enrique V. Kortright and Sammy M. Ray Dermowatch: a web-based approach for monitoring the oyster parasite Perkinsus marinus (Dennocystidiuin inarinuin} 389 Mary F. Stephenson, Sharon E. McGladdery and Nancy A. Stokes Detection of a previously undescribed Haplosporidian-like infection of a blue mussel (Myfihis ediilis) in Atlantic Canada 389 hike Sunila Monitoring bivalve health in Long Island Sound 389 DISEASES OF CRUSTACEA Donald C. Behringer, Jr., Mark J. Butler, IV and Jeffrey D. Shields Ecological ramifications of disease in the Caribbean spiny lobster. Paniilirus argus 390 362 Abstracts. April 2002 Nation;il Shclirisheries Association. Mystic. Connecticut Kathleen Castro and Thomas Angell Field observations on the development and progress of a shell disease episode tor American lobster in Rhode Island: 1 993-200 1 ^^^ Terry Glover, Lauren Bergey and Judith S. Weis Effects of paiasites on beha\ lor oi grass shrimp. Pcilaciiioiuics />iigio ?90 Barbara Homey, Andrea Battison and Allan Mackenzie Cytocentrifu>'e preparations: an alternate method to examine the hemocytes of the American lobster " . ^91 Himiartis amencainis Gretchen A. Messick and Tom F. Nalepa Parasites in Dtpi'icia spp. amphipods from Lakes Michigan and Huron ^^ 1 Jeffrey D. Shields Pathological alterations in the eyes of the American lob.ster, Homanis cimcriinnii.s. infected with Panimocba sp 391 Jeffrey D. Shields, Donald C. Behringer, Jr. and Mark J. Butler, IV A pathogenic herpes-like virus from the spiny lobster, Piundirus urgus 391 BLUE MUSSEL BIOLOGY AND CULTURE John Brake, Jeffrey Davidson and Jonathan Davis Observations on growth, gametogenesis, and sex ratio of Iriploid and diploid Mytilns eihilis 392 Jeff Davidson. Frank Boolhroyd, Neil McNair and Thomas Landry The distribution and biology of an invasive tunicate in Prince Edward Island. Canada 392 Ellen L. R. henchington, Kenneth R. Freeman, Benedikte M. Vercaemer and Barry W. MacDonald A lield study of settlement depths of A/\7(7i/,s cdidis and M. trossidus in Nova Scotia, Canada, 1998-2000 392 Angeline R. LeBlanc, Gilles Miron and Thomas Landry Nutrient uptake and release from fouling organisms associated with cultured mussels in Tracadie Bay, PEI 393 Afton McGowen. Matthew Gordon and Paul D. Rawson Species-specific settlement patterns of blue mussels in Cobscook Bay. Maine 393 Marcelo Miranda, David Innes and Raymond Thompson Genetic aspects of the blue mussel (Mxriliis cdidis and M\tihis trossidus) hybrid zone in Atlantic Canada 393 Valerie Moreau, Edwin Bourget and Rejean Tremhiay Small scale distribution of A/vr//;/,v edulis and M. irossiilu.s m the bale des Chalcurs and the Gaspe Peninsula 393 Randy W. Penney, M. J. Hart and N. Templeman spei Paul D. Rawson Cladistic analysis of genetic differentiation between populations of the blue mussel, Mxtdus trossidus 394 Judith Senechal and Jon Grant Behavior and growth of juvenile mussels (Myldiis spp.) in suspended culture socks 394 Benoit Thomas, Valerie Moreau and Rejean Tremblay Update on the distribution of two mussels species {Mxtiliis cdidis and Mytiliis trossulus) in the Quebec maritime 394 regions Rejean Tremblav, Valerie Moreau. Thomas Landry, Bruno Myrand and Cyr Couturier Performance of Mxiiliis cdiilis and AA//7((,s irossidits in the Gulf of St. Lawrence: a laboratory experiment 395 J. Evan Ward, Sandra E. Shumway and Jeffrey S. Levinton Blue mussels as model systems to investigate pallial cavity function in bivalves 395 HARMFUL ALGAL BLOOMS Marie-Claude Archambauh, Jon Grant, Monica Bricelj and Don Anderson Effects of clay, used to control harmful algal blooms, on juvenile hard clams, Mcrccnaria mercemuia 395 Rita A. Horner History, some recent HAB events, and their impacts on shellfish and finfish m Washington State 396 Jay R. Leverone and Norman J. Blake Effects of the toxic dinotlaaellate, Karciiiu hrcvis. on lar\al mortality and juvenile feeding behavior in the bay „ ^ ■ r" 396 scallop, Argopeclcn irnuhans Mussel culture in a mixed species (A/, cdidis and M. trossidus) /.one — some commercial implications 394 National Shellfisheries Association. Mystic, Connecticut Abstracts. April 2002 363 Gaiy H. Wikfors, Jennifer H. Alix, Roxanna M. Smolowitz, iMcey Wallace and Helene Hegaret Detrimental effects of a recent Pryiiim-siiiiii isolate from Boothbay Harbor. Maine (USA) upon juvenile bay scallops Argopecten irradians 397 SHELLFISH BIOLOGY Richard R. Alexander and Robert M. Baron BuiTowing-induced internal fractures and external abrasion in shells of the hard clam Mercenaria mercenaria from Raritan Bay, New Jersey 397 Eleanor A. Bochenek, Eric N. Powell, John M. Klinck and Eileen E. Hofmann Influence of environment and food supply on survival of Crassostrea gigas larvae: a modeling study 397 Andrew T. Cogswell and Sainia Sarkis Growth characteristics of Argopecten gihhiis juveniles reared in tw o suspended culture systems 398 W. R. Congleton, Jr., B. R. Pearce and M. Parker Growth and dispersal studies of Mya arenaria using a numerical flow model 398 Zaiil Garcia-Esquivel, Marco A. Gonzalez-Gomez and Francisco Ley-Lou Estimation of ingestion and biodeposition rates of the Pacific oyster. Crassostrea gigas. in a coastal lagoon of NW Mexico 398 iMiira E. Gomez, Ma. Teresa Viana, Zaiil Garcia-Esquivel, Armando Shimada and Louis R. D'Abramo Effect of dietary protein/energy ratio on growth and metabolism of juvenile green abalone (Haliotis fulgens) 399 John Kraeuter, Eric N. Powell, Eileen E. Hofmann, John M. Klinck, Ray Grizzle, Monica Bricelj and Stuart Btickner Modeling the growth of the hard clam. Mercenaria inerceiiana 399 Eric N. Powell, Eleanor A. Bochenek. John M. Klinck and Eileen Hofmann Influence of short term variations in food supply and critical periods on survival of Crassostrea gigas larvae 399 Melissa J. Southworth, Juliana M. Harding and Roger Mann Population structure of the hard clam. Mercenaria mercenaria. in Hampton Roads. Virginia 400 Helen Woods, Ken Moore and Carl Hershner An examination of potential conflict between hard clam aquaculture and SAV in the lower Chesapeake Bay 400 SCALLOP BIOLOGY AND CULTURE V. Monica Bricelj, Anne Veniot, Celine Bane and Peter Beninger Postlarval development of the gills and implications for feeding in the sea scallop. Placopecten magellaniciis 400 Maureen Davidson The effects of stocking density in pearl nets on survival, growth, and reproductive potential of the bay scallop. Argopecten irradians irradians 40 1 Scott Feindel and Daniel Schick Developing a coastal Maine sea scallop enhancement program 401 Brad Harris and Kevin Slokesbury Growth and movement of sea scallops in the southern part of the Great South Channel on Georges Bank: a tagging study 40 1 Daniel L. Jackson, Barry W. MacDonald, Shaka James, Benedikte Vercaemer, Ellen L. Kenchington and Andre Mallet Investigations w ith triploid Atlantic sea scallops. Placopecten luagellanicus 401 Lisa M. Milke, V. Monica Bricelj and Christopher C. Parrish A comparison of microalgal diets for enhanced production of Placopecten magellaniciis postlarvae 402 Shawn M. C. Robinson, Jim D. Martin and Eddy J. Kennedy Impacts and control of the spionid polychaete. Polydora wchsteri on the sea scallop. Placopecten magellaniciis in midwater culture 402 Gregg E. Rosenkranz and Douglas Pengilly Development of a stock assessment program for weathervane scallops in Alaska 402 Fabrice Pernet. Rejean Tremblay and Edwin Bourget Biochemical indicator of giant scallop PUuopecten magellaniciis quality: larval growth, competency and settlement ... 403 Kevin D. E. Stokesbury and Brad Harris Examination of sea scallop. Placopecten magellaniciis. aggregations using a video survey in closed areas of Georges Bank 403 364 Ahstracts. April 2002 National Shellfislieries Association. Mystic. Connecticut PARASITE AND HOST DEFENSES Steven M. Allen and Louis Burnett The effect of pH on the kilhng activity of heniocytes m the Pacific oyster, Crassostrea gificis 403 Robert S. Anderson, Brenda S. Kraiis. Sharon McGladdery and Roxanna Smolowitz Mucoid secretions protect QPX Ironi antimicrobial agents 404 Gwenaelle Choqnet, Philippe Soudant, Christophe Lambert, Christine Paillard and Jean-Louis Nicolas Measurement of \'ihiinamics of the Atlantic sea scallop fisheiy 416 Farhad Rajahipour Population structure of the Indian squid. Lolii^o iliivuKielii Orbigny. 1848. in Iranian waters of the Oman Sea 417 Anne Richards, Michael Fogarty. David Mountain and Mirta Teichberg Recruitment dynamics of northern shrmip {PainUilii.s horealis) in the Gulf of Maine 417 James R. Weinberg and Charles Keith Deep sea red crabs off southern New England: has there been a fishery impact on the population? 417 DISEASES OF SHELLFISH: CONTRIBUTED SESSION B. K. Diggles, P. M. Hine, V. L. Webb, E. W. Maas, J. Nichol, S. Wakefield, R. Roberts. C. S. Friedman, N. Cochennec-Laureau, K. S. Reece and N. A. Stokes Mortalities of cultured abalone. Hiiliciis iris, infected by a novel haplosporidian 418 Ralph Elston, Dan Cheney, Brian MacDonald and Andrew Suhrbier Low salinity tolerance in Manila clams Vciicnipis pliilippiiwntm 418 Sean E. Matson and Christopher Langdon A specific pathogen free culture system for C. gigas larvae and spat 418 Gary R. Meyer and Susan M. Bower Intramitochondrial crystals within the haemocytes of mussels [Mytihis cdiilis) experiencing unexplained mortalities.... 419 James D. Moore and Carolyn S. Friedman Environmental detection of the rickettsiales-like prokaryote causing withering syndrome in abalone 419 J.-L. Nicolas, M. Gamier, M. Gay and F. Lerou.x Vibrio associated with juvenile oyster during summer mortality in France 419 Wolf T. Pecher, Jose A. F. Robledo. Calhleen A. Coss and Gerardo R. Vasta Further molecular characterization of Perkiiisiis aiulrewsi and related isolates 420 Jose A. F. Robledo, Gerardo R. Vasta. Patricia A. Nunes and M. Leonor Cancela III villi) culture of Peikinsiis alhiiiucus trom clams Tapes deciissatiis: characterization of the rRNA gene 420 Jose A. F. Robledo, Eric. J. Schott. Adam G. Marsh and Gerardo R. Vasta Gene discovery in Pcrkiiisiis iiniriiiiis using expression sequence tags (F.ST) 4-0 Jose A. F. Robledo and Gerardo R. Vasta The Nramp gene and competition for available iron between Crassostrea virgiiiica and Perkiiisiis imirinus 420 Arnaud Tanguy, Susan E. Ford and Ximing Giio Characterisation of gene expression in response to Perkiiisiis imiriniis and HuplosporiJiiim iiclsoiii infections in the eastern and Pacific oysters BIVALVE HABITAT SUITABILITY AND THE ROLE OF BIVALVES IN ECOSYSTEMS Loren D. Coen, Richard K. Wallace and Nancy Hadley How community-based oyster restoration can enhance research efforts: examples from South Carolina and Alabama William S. Fisher Oyster habitat suitability as a component of resource management 4-1 Dana M. Frank and J. Evan Ward Continuous monitoring of pumping pressure and valve gape in the oyster Ciassosiicci virginica in response to changes m environmental parameters ^ — Niitidiial Shellfisheries Association, Mystic. Connecticut Abstracts. April 2002 367 Raymond Grizzle, Jennifer Greene and Mark Luckenbach A simplitied sestt>n uptake model for bivalves: preliminary field tests 422 Mark Luckenbach, Janet Nestlerode, Paige Ross, Jr. and Alan Birch The influence of reef architecture and scale on success of oyster reef restoration 422 Jennifer Miigg Pielros and Michael A. Rice Effects of Crassostrea rirglntca populations on sedimentation, phytoplankton species composition and ammonia cycling in experimental mesocosms 423 Thomas M. Soniat A modified habitat suitability index for the eastern oyster. Crassastrea virginicu 423 S. Gregory Tolley, Aswani K. Volety, Emily C. Lindland and James T. Winstead Use of oyster habitat by reef-resident fishes and decapod crustaceans in the Caloosahatchee Estuary, Florida 423 Aswani K. Volety, S. Gregory Tolley and James T. Winstead Effects of freshwater releases and season on oysters {Crassostrea virginica) in Caloosahatchee Estuary. Florida 424 James T. Winstead. Aswani K. Volety and S. Greg Tolley Parasitic and symbiotic fauna inhabiting oysters (Crassostrea virginicci] and mud crabs {Panopeus hcrhstii) sampled from Caloosahatchee Estuary. Florida 424 POSTERS Patrick Baker and Amy Benson Habitat and ecology of green mussels, Perna viridis. in Florida 424 Shirley Baker, Ed Phlips, David Heuberger, Clay Montague and Leslie Stunner Introducing the CLAMMRS project: clam lease assessment, management and modeling using remote sensing 425 Andrea Battison, Barbara Horney. Richard Cawthorn and Allan Mackenzie Hemocytes of Homarus americanus stained with a modified Wright-Giemsa stain: description and comparison to current classification schemes 425 Karine Bouilly, Alexandra Leitao, Helen McCombie and Sylvie Lapegue Impact of atrazine on aneuploidy in the Pacific oyster, Crassostrea gigas 425 Francois Bourque, Bruno Myrand and Marcel Roussy Status of the Mytiliis edtdis stock which supplies high-quality spat to mussel growers in lles-de-Ia-Madeleine (Gulf of St. Lawrence ) 425 Jason P. Breves and .Andrew E. Tate Predator induced phenotypic response by the common periwinkle (Littoriiia littorea) 426 Sandra M. Casas, Antonio Villalba, Jerome F. La Peyre, Kimberly S. Reece and Carlos Azevedo Continuous in viiro culture of Pcrkiiisiis atlanticus, parasite of the carpet shell clam Tapes decitssatiis 426 Lise Chevarie, Bruno Myrand, Francois Bourque, Michel Giguere, Lizon Provencher, Philippe Archambault and Rejean Tremblay A R&D program to develop Mya arenaria culture in Iles-de-la-Madeleine (Gulf of St. Lawrence) 426 Vania R. Coelho and Jeffrey D. Shields Loss of eye pigmentation in two gammaridean amphipods from the biosphere 427 Tricia L. Cranmer, Daniela Zima and Richard R. Alexander Shell repair rates in surgically damaged valves of the blue mussel {Mytiliis edulis) and the ribbed mussel (Geiikensia demissa) from New Jersey 427 Alan J. Erskine and Standish K. Allen, Jr. Over-w intering of Crassostrea ariakensis in land-based systems in Virginia 427 Chwan-Hong Foo and Jerome F. La Peyre IiTadiation of oyster primary cell cultures with ultraviolet-light to eliminate bacterial and protozoal contaminants 428 B. Gagnaire, H. Thomas-Guyon and T. Renauh In vitro effects of heavy metals and atrazine on Pacific oyster, Crassostrea gigas, haemocytes 428 Holly A. Gefroh, Matthew J. Jenny, Ryan B. Carnegie, Kevin L. Schey and Robert W. Chapman Inducible antibacterial activity in oyster {Crasso.strea virginica) hemolymph 428 368 Ahsnvcls. April 2002 National Shellfisheries Association. Mystic. Connecticut Jennifer Greene. Raymond Grizzle, David Burdick, Larry Ward and Ann Reid Constructing shellfish reefs in a polluted, urban estuary: scientists join with the community to work for a common goal -+29 Rashel V. Grindberg. Erin C. Rasnake, Michael Savarese and Aswani K. Volety The eastei-n oyster as an indicator species to establish restoration targets in southwest Florida estuaries 429 Raymond Grizzle, Jamie Adams and Linda Wallers Long-term changes in intertidal oyster reefs in a Florida lagoon potentially caused by boating activities: an analysis of aenal photographs from 1 943-2(100 430 Raymond Grizzle, Jennifer Greene, Stephen Jones, Mark Luckenbach and Roger Mann An oyster (Crassosirea \ir)>iiuca) reef restoration experiment m Nev\ Hampshire inxolving CROSBreed stock and native transplants 430 William J. Hargis, Jr.. Helen E. Woods, Rebecca Arenson, Sharon Dewing, Arman Kaltayev, Elizabeth Mountz, Marcia R. Berman and Dexter S. Haven What certain 19"' and early 20"' century navigational and species purpose survey charts reveal about changes in the oyster reef morphology of the lower Chesapeake Bay 430 William J. Hargis, Jr., Helen E. Woods, Rebecca Arenson, Elizabeth Mountz, Marcia R. Berman and Sharon Dewing Morphology of a Chesapeake Bay oyster reef system in 1871-1S73 431 Cindi A. Hoover and Patrick M. Gaffney Geographic variation in nuclear genes of the eastern oyster Cmssostrea viri^inicu 431 Nicola J. Kernaghan, Eileen Monck, Carlo Wieser and Timothy S. Gross Characterization and manipulation of sex steroids and \ itellogenin in freshwater mussels 43 1 Edouard Kraffe. Philippe Soudant. Yanic Marty. Nelly Kervarec and Pierre Guenot A specific association of docosahexaenoic fatty acid with cardiolipins of some marine bivalves 431 Brenda S. Kraus. Amy E. Beaven and Robert S. Anderson Hemocyte-mediated defense responses of the lobster Hoiminis amciicaniis 432 Megan K. G. La Peyre. Amy D. Nickens and Jerome F. Im Peyre Potential elimination of the protozoan pathogen Perkiusus luaiinus from eastern oysters by freshet events 432 Jean Lavallee and Donald J. Rainnie A field investigation of the effects of V-notching on the health and susceptibility to infection of ovigerous female American lobsters 432 Li Li and Ximing Gito A preliminary linkage map for the Pacific oyster Cmssostrea aiU'^i^' consti-ucted with RAPD and AFLP markers 433 Karin B. Lohrmann. Elisabeth von Brand and Cristian Gallardo Gonadal maturation of triploid \cMo\-\s Aiiiopccteii jniipitniuis Lamarck. 1819 433 Bortolini R. Jose Luis. Torres G. M. Pilar and Montoya R. Leobardo Detection of white spot syndrome virus (WSSV) in carcinologic faun associated to shrimp culture of Sinaloa. Mexico. using Polymerase Chain Reaction (PCR) and in situ hybridization 433 Gregory MacCallum, Jeffrey Davidson. Garth Arsenault. Sharon McGladdery, Michelle Maillet and Neil MacNair Factors, risks and significance of emergent neoplasia diseases in cultured and wild soft-shell clams {Mya areiuiha) in Atlantic Canada 434 Clyde L. MacKenzie. Jr. Identity of United States mollusk production declines in the 1900s 434 Aaron P. Malay, Katherine J. Boettcher and Bruce J. Barber Development of a PCR-based assay for detection of the JOD-associated Roseohacter 434 Nassrin Mashaii Preliminary study about feeding ecology of the rock lobster. Paindinis hoinanis. Linnaeus. 1785, at Iranian seashores of Oman Sea 434 Sean McDermott, David Burdick, Raymond Grizzle and Jennifer Greene Evaluation of the structure and function of a created blue mussel (Mytihis ediilis ) reef 435 Coren A. Milbury and Patrick M. Gaffney Genetic monitoring of oyster stock enhancement in the Choptank River. Chesapeake Bay 435 National Shellfisheries Association, Mystic. Connecticut Abstracts. April 2002 369 Daniel P. Molloy, Denise A. Mayer, Michael J. Gaylo, Kathleen T. Presti, Alexander Y. Karatayev and Lyiibov E. Burlakova Progress in the hiological control of zebra mussels with microbial toxin 435 Thomas E. Mullen, Jr. and Salvatore Frasca, Jr. Characterization of a parasitic amoeba in the American lobster by molecular systematics 436 Kim-Lien T. Nguyen, Jerome F. La Peyre and Terrence R. Tiersch Seasonal changes in cell proliferation of oyster tissues 436 Amy D. Nickens, Jerome F. La Peyre and Sandra M. Casas Development of a medium to induce hypnospore formation and zoosporulation of Pcrkiiisiis maritius 436 Rodney Peters and David Raftos The effects of Maitcilia sydneyi on the host defense responses of the Sydney rock oyster. Saccostrea gloinerata 437 Alan J. Power, Mary Sweeney-Reeves, Todd C. Recicar, Dodie M. Thompson and Randal L. Walker Population biology of melongenid whelks in the intertidal /one in Wassaw Sound, Georgia 437 Deanna L. Prince, Robert Bayer. Christina Congleton, Shannon Colby, Danielle La Vine, Danielle Volmulh, Katrina Brooks, Margaret Berry, William Congleton and John Vetelino A rapid method for assessing stress in the American lobster using a hand held glucometer 437 Michael Sheppard, Florian Rambow, Marc E. Frischer and Richard F. Lee Routes of Heiinitihliiuiiin sp. transmission into blue crabs 438 Shelly M. L. Tallack The relationship between black spot disease and limb-loss in Cancer pugunis from the Shetland Islands. Scotland 438 Anne Thessen, Q. Dortch, T. M. Soniat and G. J. Doucette Oyster grazing on toxic and non-toxic PscnJo-nilzschia and Thiilassisinra wcisfhiggii. and Diryhiiii lirighlwclUi 438 Jessica M. Vanisko, Mary Christman, Kennedy T. Paynter and Stephen J. Jordan Historic eastern oyster (Crassosirea virginica) dredge and patent long surveys in the Maryland portion of the Chesapeake Bay 439 Anna Walker. Michael Sheppard. Richard F. Lee and Marc Frischer Heinatodiniuin infection in blue crabs, spider crabs and stone crabs 439 Linda Walters, Kevin Johnson, Lisa M. Wall, Neysa Martinez and Ray Grizzle Shell movement and juvenile survival of the oyster Crassnstrea virginica on intertidal reefs adjacent to waters with intense boating activity in the Indian River Lagoon. Florida 439 Win Watson, Steve Jury, Jennifer Wishinski, Dan O'Grady, Walter Golet, Darren Scopel, Heidi Pye and Chris Rillahan Development and testing of a simple field system for monitoring mussel shell gape size 439 National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18, 2002 371 PERKINSUS ENVIRONMENTAL EFFECTS ON PERKINSUS MARINUS INFECTION RATES. GROWTH AND SURVIVAL AMONG DERMO-DISEASE-FREE JUVENILE OYSTERS PLANTED AT THREE SALINITY REGIMES IN THE PATUXENT RIVER, MARYLAND. George R. Abbe* and Brian W. Al- bright, Academy of Natural Sciences Estuarine Research Center. St. Leonard. MD 206S.'S; Carol B. McCollough. Christopher F. Dungan. and Stephen J. Jordan. Sarbanes Cooperative Oxford Laboratory, Oxford, MD 21654. Specific pathogen-free {SPFl oysters were set on oyster shell and transplanted to three sites in the Patuxent River, Maryland along a salinity gradient to investigate environmental effects of Perkinsus mariiuis on infection rates, growth and survival. Oysters were held in trays on PVC structures 0. 1 m off bottom on natural oyster bars. Several thousand spat were deployed at each site, and 100 oysters in a separate tray were followed for growth and mor- tality. From September 2000 to September 2001 salinities at Hol- land Point (HP, upper river). Gatton (GAT, mid) and Town Creek (TC, lower) averaged 11.1, 1.3.0 and 14.4. respectively. Oysters were examined monthly for growth and mortality and 30 were collected from each site for assay of P. iiuiriniis infections by v\hole body burden technique. This allowed determination of time to initial infection and subsequent progression of disease. An ad- ditional 30 from the natural population at each site were also examined monthly by rectal tissue assay. Oysters (initially 25 mm) at HP, GAT and TC grew 23, 34 and 27 mm, respectively, and survival was 95, 98 and 949f during the first 12 months. Mean intensity of dermo disease among feral populations (on a scale of 0-7) at HP, GAT and TC ranged from 1 . 1 to 4.2. 0.7 to 4.6 and 0.7 to 4.7, respectively, and averaged 2.51, 2.72 and 2.79. It appears that salinity had little effect on growth, survival and infection intensity during the first year; however, it is generally later that damage occurs. Preliminary data suggest tlial a mid-river site might be the best area to locate oysters, but additional data during subsequent years may dictate otherwise. TRANSPORT OF PARTICLES ACROSS EPITHELIA FROM OYSTER MANTLE CAVITY: A MODEL FOR PER- KINSUS MARINUS INVASION. Bassem Allam and Susan E. Ford.* Haskin Shellfish Research Laboratory. Rutgers University. Port Norris, NJ 08349. The digestive tract has long been considered the major portal of entry for the oyster parasite, Perkinsus marimis. but recent studies suggest that the mantle and gill may also be import invasion sites. We used 5-|a.ni latex beads, placed in the mantle cavity, to deter- mine whether P. marinua could be actively transported across mantle and gill epithelia. At intervals after incubation, oysters were processed for histological and cytological analysis. After 2 h, beads were observed in hemocytes at the epithelial surface and within the epithelium of the gill and mantle. After 6 to 16 h. beads were found within hemocytes in the underlying tissues. Sixteen hours following contact, only about 1% of hemocytes withdrawn from the adductor muscle contained beads, although most of those that did contained multiple beads. Over the next 96 h, the fraction of hemocytes with beads gradually declined. Meanwhile, tissue sections showed that beads were progressively transferred toward the digestive tract. These results emphasize the role of mantle and gill epithelia as portals of entry for P. iminiuis and other micro- organisms, and suggest a role for hemocytes in their transport. EFFECTS OF OYSTER EMERSION ON THE GROWTH AND THE METABOLISM OF PERKINSUS MARINUS. Louis E. Burnett* and Christopher S. Milardo. Grice Marine Laboratory, University of Charleston, SC, 205 Fort Johnson, Charleston, SC 29412 We examined the effects of pH, oxygen, carbon dioxide, and temperature on the metabolism of the oyster parasite Perkinsus nuiriniis simulating conditions that occur within the tissues of the Eastern oyster Crassoslrea virginica during air exposure in the summer. P. inarimis was cultured in a modified JL-ODRP medium in ambient air at high humidity. The culture medium was gassed with appropriate CO,. N, and O, mixtures, and pH adjusted in order to simulate the conditions present in oyster hemolymph. Oxygen uptake of the parasite decreased proportionately with de- creasing ambient oxygen, and decreasing pH increased P. muriiuis respiration under low CO, conditions (p = 0.0006); under 15 torr CO,, there was no relationship between pH and oxygen uptake. Increased CO, levels (simulating conditions during oyster air ex- posure) significantly increased parasite O, uptake (p < 0.0001 ). as did increased temperature (p < 0.0001). Cycling temperature (at pH 6.3, 7.1 and 7.6) in 6-hour intervals from 25°C-35°C did not affect culture growth; however, when cycled from 25°C— 10°C, cells showed a significant decrease in growth (p < O.OOOl ). Simi- larly, cells grown at 35''C and 15 torr ambient CO, showed de- creased growth. These results suggest that summer environmental conditions in the intertidal zone and especially in the tissues of oysters play a role in keeping P. muriniis infections of C. virginica at sub-lethal levels (SC Sea Grant R/ER-14). SUSCEPTIBILITY OF THE CARIBBEAN OYSTER CRAS- SOSTREA RHIZOPHORAE TO PERKINSUS MARINUS. David Bushek.* Baruch Marine Field Laboratory. PO Box 1630. University of South Carolina, Georgetown. SC 29442; John Scarpa and Susan E. Laramore. Harbor Branch Oceanographic Institution. Ft. Pierce, FL 34946. The oyster pathogen Perkinsus marinus is a formidable prob- lem for the American oyster, Crassostrea virginica. The Caribbean oyster C. rliizopliorae is a closely related species of C. virginica. 372 Ahsmicfs. 2002 Annual Meeting. April 1 4- IS. 2002 Nutidnal .Shellfisheries Association. Mvstic. Connecticut but little is known of its resistance to P. marinus (Dermo). There- fore. Dermo resistance was compared between the Caribbean and American oyster. Two Dernio-free families were produced and reared separately, under quarantine, for each species. Oysters from each family were challenged once via shell-cavity inoculations with either saline (control) or one of two genetically distinct isolates of P. nniriinis: ATCC 50762 from Fort Pierce. FL or ATCC 30783 from Cotuit Bay. MA. Parasite dosages were adjusted to I x \(y P. imiriniis per gram of wet tissue weight for each oyster. Oysters were fed phy- toplankton twice a day and water was exchanged weekly for 16 wk. A natural infection study was also performed by placing oys- ters from each family in concrete tanks that received 7-10 L/min unfiltered seawater from the Indian River Lagoon and examining the oysters for infection over the course of 214 days. Infections in each experiment were determined using the whole-body burden method. Fifteen America oysters and one Caribbean oyster died during the challenge experiment. Log 10 transformed P. mariiiiis burdens in surviving oysters were significantly heavier for Caribbean oys- ters (1.25 ± 0.08) compared to American oysters (0.72 ± 0.06). Control oysters did not develop infections (0.09 ± 0.01). In the natural infection study, parasites were detected in all groups after 103 days, but intensities were negligible and there was no differ- ence in burdens between the two species. At the end of the study (day 214), most oysters had died (survival: 0 to 277f ). Peikinsiis prevalence was 100% in all surviving C. \iii 200 ij,M), whereas three algal strains (Skeletonema costatiim. Chlamydomoiuis rein- hardtii and C. Pulsatilla] were sensitive (MIC,,, < 50 (jlM). While proteases from P. marinus significantly reduced the antimicrobial activity of pleurocidin. they did not affect the activity of tac- hyplesin. These results are consistent with the finding that the substrate specificity of Perkiiisus proteases is similar to that of elastase. Oyster clearance rates of C. reinhardtii. C. Pulsatilla, and P. pastoris were similar to those of Tetraselmis hicia. a commer- cially available alga used in aquaculture. FLOW CYTOMETRIC ANALYSIS OF LECTIN BINDING TO IN VITRO CULTURED PERKINSUS MARINUS SUR- FACE CARBOHYDRATES. Julie D. Gauthier,* Biology De partment. Nicholls State University, Thibodaux, LA 70310; Jer- ome F. La Peyre, Cooperative Aquatic Animal Health Research Program, Department of Veterinary Science. Louisiana State Uni- versity Agricultural Center, Baton Rouge, LA 70803; Jill A. Jen- liins. National Wetlands Research Center, U.S. Geological Survey, Lafayette, LA 70506. Parasite surface glycoconjugates are frequently involved in cel- lular recognition and colonization of host. The present study iden- tifies surface carbohydrates by flow cytometric analysis of fluo- rescein isothiocyanate-conjugated lectin binding. Lectin binding specificity was confirmed by sugar inhibition and Kolmogorov- Smirnov statistics. Clear, measurable separation between fluores- cence peaks and no parasite autotluorescence were observed. Para- sites (GTLA-5 & P-l strains) harvested at log phase growth from a protein-free medium reacted strongly with wheat germ agglutinin (WGA) and concanavalin A (Con A), reflecting presence of N- acetyl-D-glucosamine and glucose/mannose moieties, respec- tively. Both strains also bound, although with lower intensity. Madura pomifera agglutinin (MPA) and Baiiliinia purpurea ag- glutinin (BPA) (N-acetyl-D-galactosamine specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffhnia sim- plivifolia II (GSII) (N-acetyl-D-glucosamine specific). Back- ground fluorescence levels were detected for Ulex europaeus ag- glutinin I (L-fucose specific) and Limulus polyplwmus agglutinin (sialic acid specific). The order of lectin binding intensity differed between strains: GTLA-5 reacted with MPA>GSII>PNA>BPA, whereas P-l reacted with PNA>MPA>BPA>GSII. which may re- sult from differential expression of glycoconjugates throughout log phase growth. Fluorescence microscopy revealed that PNA bound with high intensity primarily to schizonts (dividing cells), whereas the other lectins bound with approximately equal intensity among parasite growth stages. Future efforts will determine if P. marinus rapid recognition and internalization involves a specific lectin- carbohydrate interaction. 374 Ahslnicl.s. 2002 Aiiiuuil Meeting. April 14-1 S. 2(102 National Shellfisheries Association. Mystic. Connecticut CLIMATE VARIABILITY AND DERMO DISEASE IN CHESAPEAKE BAY. Eiletn E. Hofniann* and John M. Klinck. Center for Coastal Physical Oceanography. Old Dominion Llniversity. Norfolk. VA 23529; Eric N. Powell and Susan E. P'ord. Haskin Shellfish Research Laboratory. Rutgers University. Port Norris. NJ 08349: Stephen Jordan, Sarbanes Cooperative Oxford Laboratory, 904 South Morris Street. Oxford. MD 21654; Eugene Burreson. Virginia Institute of Marine Science. P.O. Box 1346. College of William and Mary. Gloucester Point. VA 23062. A mathematical model that includes biological and environ- mental processes that contribute to the observed annual cycles of intensity and prevalence of the disease. Dermo. has been used to determine the effect of predicted climate variations on Eastern oyster (Crassosin-ii virginica) populations in Chesapeake Bay. Simulations that use the salinity change predicted for Chesapeake Bay by the Hadley Center Climate model for conditions of doubled CO, suggest that the present pattern of Dermo disease in the Bay will be altered to the benefit of the oysters. The predicted salinity change in Chesapeake Bay for conditions of doubled CO, pro- duces an overall decrease in Bay salinity, which results in a de- crease in the prevalence and intensity of Dermo disease. However, for reduced salinity conditions, there is a trade-off between re- duced disease levels and reduced reproductive capacity of the oys- ters. Also, conditions of increased food can offset the effects of either increased or decreased salinity on Dermo disease levels. The simulated total oyster biomass changes suggest that for conditions of doubled CO,, northern Chesapeake Bay oyster populations may not be viable over the long term without external inputs of juve- niles. However, oyster populations in the Rappahannock and York Rivers increase or have stable biomass over time for most climate change conditions. One implication of these results is that oyster populations in southern Chesapeake Bay may sustain the Bay-wide oyster fishery. These simulations provide a basis for suggesting management plans for diseased oyster populations under condi- tions of climate change. MODULATION OF PERKINSUS MARINUS FUNCTIONS BY HOST-DERIVED PRODUCTS. Stephen L. Kaattari,* E. Alanna Maclntyre, and Christopher G. Earnhart, Department of Environmental Sciences. Virginia Institute of Marine Science. College of William and Mary. Gloucester Point, VA 23062. In an attempt to induce physiological changes in vitro that would be comparable to those elicited by natural infection, we have co-incubated P. mariiuts cells with extracts of Crassostrea spp tissues and plasma. Co-incubation with these host-derived ma- terials gave rise to a variety of complex effects including altered differentiation, protease expression, growth rates, infectivity. and in vitro parasite mortality. Preliminary evidence is suggestive that a suite of such functions could be employed as a prognostic tool for the selection of resistant oyster stocks. However, variations in the relative activity of these materials suggest that preparative procedures and/or conditions of storage could confound such analyses. Thus, while indicative of prognostic value, the precise mechanisms of these biological effects must be resolved. VIABILITY AND GROWTH OF PERKINSUS MARINUS AND PERKINSUS ATI^NTICUS AT THREE TEMPERA- TURES. Jerome F. La Peyre* and Amy D. Nickcns, Coopera- tive Aquatic Animal Health Research Program. Department of Veterinary Science. Louisiana State University Agricultural Cen- ter. Baton Rouge. LA 70803; Sandra M. Casas and Antctnio Villalba, Centro de Investigacions Marinas. Xunta de Galicia. aptdo. 13. E-36620 Vilanova de Arousa. Spain. The ability to culture several protozoan parasites of the genus Perkinsus allows comparison of their growth and tolerance given different environmental conditions. This information is important to understand the distribution ofthe.se molluscan parasites and can be used to predict zones for high risk of mortalities of host popu- lations. The effects of temperature on P. marinus and P. atlanticus were determined because temperature is a major factor controlling the prevalence and intensity of infection in their respective host, the eastern oyster. Crassostrea virginica, and the carpet shell clam. Tapes decussatus. Three isolates of each species were added to 96-well plates at a seeding density of 10'^ cells/ml and incubated at 4°C, 15°C and 28°C for 15 days. The parasite density, viability. size and metabolic activity were measured every other day starting on day 2. Parasite density was measured with a hemacytometer. Parasite viability was determined by neutral red uptake. Parasite metabolic activity was assessed by measuring the cellular biore- duction of the fluorescent dye Alamar Blue. The mo.st significant result was that all three isolates of P. atlanticus multiplied at I5°C whereas no increase in parasite number of any P. marinus isolates was detected at LS'^C. The growth rate of P. marinus and P. at- lanticus was comparable at 28"C and neither species grew at 4''C. While perkinsosis is generally considered a warm water disease. this study suggests there will be differences in the temperature at which different Perkinsus species kill their respective hosts. IS THE TEMPERATURE AND SALINITY-DEPENDENT VIRULENCE OF PERKINSUS I^ARINUS ASSOCIATED WITH INCREASED LIPID METABOLISM? Eric D. Lund* and Fu-Lin E. Chu. Virginia Institute of Marine Science. College of William and Mary. Gloucester Point. VA 23062; Philippe Soudant, Universite de Bretagne Occidentals Place Nicolas Co- pernic. 29280 Plouzane. France. Teinperature and salinity significantly affect the rate of prolif- eration and development of Perkinsus marinus and the progression of the disease in its host, the eastern oyster. To better understand how these environmental parameters affect the nutritional metabo- lism of this parasite, the effects of temperature and salinity on the lipid metabolism of P. marinus were investigated. Using axenic Nation:il Shellt'isheries Association. Mystic, Connecticut Abstracts. 2002 Annual Meeting, April 1-1-1 8. 2002 375 cultures grown at 3 temperatures and 2 salinities, the uptake, in- corporation and nielabolisni of two fluorescent lipid analogues in meronts and the lipolytic activities of nieronts and extracellular proteins (ECPs) were determined. Uptake and bioconversion of the lipid analogues were positively coirelated to temperature. Salinity did not affect the uptake, but bioconversion of these two compo- nents was lower at low salinity. Triacylglycerol lipase activity of cell homogenates and ECPs were positively correlated to tempera- ture. Phospholipase activity was not detectable in ECP. This re- search was supported by NSF (MCB9728284). PERKINSUS MARINUS INFECTION RATES IN SPECIFIC- PATHOGEN-FREE JUVENILE OYSTERS PLANTED AT THREE SALINITY REGIMES IN THE PATUXENT RIVER, MARYLAND. Carol B. McCollough.* Christopher F. Dungan, and Stephen J. Jordan, Sarbanes Cooperatix e Oxford Laboratory, Oxford, MD 21654; George R. Abbe and Brian W. Albright. Academy of Natural Sciences Estuarine Research Center. St. Leonard, MD 20685. Specific-pathogen-free (SPF) seed oysters were set and reared in filtered and sterilized Patuxent River water. They were trans- ferred to three natural oyster bars in the Patuxent River along a salinity gradient. Deployment sites were located at Town Creek (TC, down river), Gatton (GA, mid-river), and Holland Point (HP, upriver). Samples of 30 oysters were assayed at 2, 4. and 8 weeks post-deployment for infection by Perkinsus mariniis. using an en- hanced RFTM whole body burden technique. Assays continued at 4-week intervals until three consecutive samples from each area tested positive for presence of P. mariuiis. Three discrete spat sets and deployments were made: September 2000, June 2001, and August 2001. Animals at sites TC and GA September 2000 de- ployments acquired P. marinus infections within 2 weeks, with prevalences of 10% and 3% respectively. Positive results at low prevalences and intensities continued for 8 weeks. Sites TC and GA June 2001 deployment acquiied infections within 2 weeks, with 10% and 13% prevalences respectively, and positive results continued for 8 weeks with increasing prevalences. Site HP ac- quired one infection (3%) between 2 and 4 weeks. Positive results continued through two additional 4-week sampling intervals, with increasing prevalences. All August 2001 deployments acquired infections within 2 weeks, with prevalences of 7% at TC, 87% at GA, and 3% at HP, and positive results continued for 8 weeks. Infection intensities among these samples ranged from 1-23 hyp- nospores per host animal. The GA deployment was 100% infected by 8 weeks post-deployment. These results show that juvenile oysters acquire P. iiuiriiiiis infections as early as 2 weeks after placeinent in dermo disease endemic areas, and that these infec- tions persist in the planted populations over time. PERKINSUS PREVALENCE IN OYSTERS PRODUCED IN THE HATCHERY AND PLANTED IN THE FIELD. Kennedy T. Paynter* and Tim Koles, Department of Biology. University of Maryland. College Park. MD 20742; Don Meritt, Horn Point Laboratory, University of Maryland Center of Envi- ronmental Science, Cambridge, MD 21613. Since 1995, millions of oyster spat have been produced by the Horn Point oyster hatchery. Most of those oysters have been planted on oyster bars in various locations in the Maryland portion of Chesapeake Bay in association with oyster restoration effoils. Oysters planted in most of these areas were tested for P. marinus prior to being planted and then regularly tested, in some cases for as many as five years, after planting. Salinity, temperature and dissolved oxygen data were also collected at most sites. Data were analyzed from all monitored sites planted with Pcrkinsiis-ixec spat over the past 5 years. Analysis showed that very few oysters were infected with P. marinus during their first year. In fact, very few oysters were infected during their second and even third years. Of the nearly 2,000 oysters collected in 2001 that were planted on restoration sites in 1998, 1999 or 2000, only 44 were infected. In addition, infections appear to be more related to local infection foci than salinity. Infection rates in hatchery-produced oysters are lower than expected compared to the naturally set oysters trans- planted by the State as part of the repletion program. RECENT TRENDS IN LEVELS OF INFECTION OF PER- KINSUS MARINUS IN OYSTERS FROM GALVESTON BAY, TEXAS: RESULTS OF THE DERMOWATCH MONI- TORING PROGRAM. Sammy M. Ray,* Department of Marine Biology. Texas A&M University, Galveston. TX 77553; Thomas M. Soniat, Department of Biology, Nicholls State University. Thi- bodaux. LA 70310; Enrique V. Kortright. Kortright Corporation, 102 Allendale Dr.. Thibodaux, LA 70301; Lance Robinson, Texas Parks & Wildlife Department, 1018 Todville Rd.. Seabrook. TX 77586. Since December 1998 oysters in Galveston Bay have been monitored for levels of the parasite Perkinsus marinus as part of the Dermo Watch Program. Monthly sampling of six reefs (Red- fish, Hannah's, Frenchy's. Fisher's, Confederate and April Fool) and three leases provides good spatial coverage and adequate tem- poral resolution for management purposes. Each month oysters are collected, their length (L) measured, and checked for weighted incidence ( WI) of infection; water temperature and salinity are also determined. Initial WI, T, S, and L are used by a model embedded in the DermoWatch web site (www.blueblee.com/dermo) to cal- culate a time to critical level of disease (t-crit). The t-crit is the number of days that it would take to reach a critical WI of 1.5 (assuming no change in T and S). Values of t-crit are low when T and S are high. The effects of drought conditions during 1999 and 2000 were reflected in higher WI and lower t-crit values through- out the Bay. Oyster growers and managers can use estimates of 376 Abstracts. 2002 Annual Meeting. April 14-18. 2002 National Shellfisheries Association. Mystic. Connecticut t-crit to manage disease. For example, heavily infected populations of oysters with t-crit values equal to or approaching zero can be moved to lower salinity sites or harvested before they die of dis- ease. UTILIZATION OF MOLECULAR GENETIC DATA FOR DETECTING. IDENTIFYING AND DESCRIBING PERKIN- SUS SPECIES. Kimberly S. Reece,* Virginia Institute of Marine Science. The College of William and Mary. Gloucester Point. VA 23062. Morphological characters are unreliable for distinguishing among Perkinsiis species. In addition, traditional detection meth- ods for Perkinsiis species such as histological analyses or the Ray's fluid thioglycollate assay do not differentiate among the species. Therefore, Perkinsiis species designations are based largely on differences in hosts and/or geographic ranges. In recent years several species-specific molecular detection assays have been developed targeting DNA sequence differences among de- scribed species. DNA sequence data is also being used to support and validate descriptions of new Perkinsiis species. Results from several molecular genetic studies indicate that caution must be employed, however, to ensure that adequate molecular data is available to appropriately develop molecular diagno,stics and to discriminate among species. Intra-, as well as inter-specific se- quence variation should be examined to confidently target a par- ticular nucleic acid sequence in molecular diagnostic assays or to use DNA sequence data from a locus in phylogenetic analyses or for taxonomic discrimination. Data will be presented suggesting that genetic variation that was previously thought to represent sequence differences between two Perkinsiis species is observed within the genome of a single Perkinsiis species isolated from two different hosts. In another study, a comprehensive examination of sequence variation among Perkinsiis species within the internal transcribed spacer region of the ribosomal RNA gene complex supports designation of a new Perkinsiis species that is clo.sely related to P. atlanticus and P. olseni. Currently, P. iitlanticiis and P. olseni cannot be distinguished based on available molecular sequence data suggesting that synonymization of these two species may be warranted pending results of more extensive and complete analyses. PROTEASE ACTIVITY IN THE EASTERN OYSTER CRAS- SOSTREA VIRGINICA AFTER EXPERIMENTAL INFEC- TION WITH THE PROTOZOAN PARASITE PERKINSIIS MARINUS. Pilar Muiioz Ruiz and Marta Gomez-Chiarri,* De- partment of Fisheries, Animal and Veterinary .Sciences, University of Rhode Island, Kingston, RI 02881. Perkinsns mariniis. an endoparasitic protozoan parasite, has long been recognized as a serious oy.ster pathogen that is often blamed for widespread mortality of the Eastern oyster Crassotrca virginica. In order to investigate the molecular interactions be- tween P. mariniis and oyster hemocytes during early infection. Perkinsiis-free Eastern oysters (Taylor Shellfish. Washington. USA), were inoculated either in the mantle cavity or the aductor muscle with cultured cells of P. mariniis. Protease and antimicro- bial activity were measured in filtered supernatants of hemolymph collected 4 hours. 24 hours. 4 days and 2 months after experimen- tal infection. No antimicrobial activity was detected in hemolymph supernatants of parasitized or control oysters. Protease activity in hemolymph supernatants collected 4 h after experimental infection was significantly higher in parasitized oysters than in control oys- ters. There were no differences in secreted protease activity be- tween control and parasitized oysters sampled 24 hours, 4 days and 2 months after experimental infection. Gelatin-gel electrophoresis (zymography) analysis showed the presence of 5 high inolecular weight bands (60-220 KDa) with protease activity in both control and parasitized oysters. Large variations in both the amount of protease activity and the relative abundance of each protease were observed between individual oysters. The presence of low molecu- lar weight proteases (35-50 KDa). corresponding to P. mariniis serine proteases, was observed in hemolymph supernatants of a small proportion of experimentally infected oysters. "NATURAL DERMO RESISTANCE" IN EASTERN OYS- TER STOCKS: CHESAPEAKE STUDIES AND DEFENSE- RELATF:D ACTIVITIES. S. M. Sticliler.* V. G. Encomio. S. K. Allen, Jr. and F-L. E. Chu, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062; J. F. La Peyre, Louisiana State University. Baton Rouge, LA 70803. The restoration of Eastern Oyster populations can be acceler- ated, in part, by using stocks of oysters resistant to the parasitic disease Dermo, caused by Perkinsiis mariniis. Similarly, identify- ing correlated defense activities will expedite the breeding process for disease resistance. We used putatively Demio-resistant wild stocks from both the Gulf of Mexico and the Chesapeake Bay and a disease-resistant hatchery strain, and compared the resistance of their progeny to Dermo disease in a two-year common garden experiment at two Dermo-enzootic Chesapeake Bay sites. Using a modified body burden assay, we determined Perkinsiis infection levels and Dermo-related mortality were considerably higher in Chesapeake stocks than in Louisiana stocks. Chesapeake stocks experienced wide variation in both disease infection levels and mortality. These results indicate a genetic basis for disease resis- tance, even within a single region. Monthly hernocyte counts and hemocyte and serum potential for killing P. mariniis. and plasma protein, lysozyme, and protease inhibition levels were obtained (or correlation with Perkinsiis infection levels. This project was sup- ported by ODRP. NCAA (Virginia Sea Grant # VA-OD-99-3). National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-1 «. 2002 377 THE BIOLOGY AND CONSERVATION OF FRESHWATER MUSSELS SPATIAL AND TEMPORAL ANALYSIS OF ECOSYSTEM PROCESSING OF FRESHWATER MUSSELS IN TWO HEADWATER STREAMS. Alan D. Christian, Department of Zoology. Miami University, Oxford, OH 45056; David J. Berg, Department of Zoology. Miami University. 1601 Peck Blvd.. Hamilton. OH 4501 1 ; B. Crump, Caddo Ranger District. Ouachita National Forest, 912 Smokey Bear Lane, Glenwood. AR 71943. The objectives were to determine food resources, food resource nutrient composition, tissue nutrient composition and N and P excretion rates of two species of mussels from two Little Darby Creek. OH and two Ouachita River, AR headwater sites. Seston stable isotope analysis revealed no differences in 8'^N values be- tween sites within a streams or seasons, however, summer b"C values were significantly different than spring and fall S'^'C and 8''^N values. No differences in mussel 8'''N values were observed between sites within a stream, but were significantly different seasonally and different regionally. Seston nutrient concentrations were similar between sites within a stream and among seasons. No significant differences in nutrient tissue concentrations were ob- served between species within a stream, sites, or seasons. Excre- tion rates of N and P were lowest in spring and highest in fall for all species. Average N:P ratios were below 16:1 for some species and above 16:1 for other species. Nutrient limitation experiments may have indicated N and P limitation in the fall. Isotope values were regionally different, locally similar, temporally variable and suggest mussels utilize a subset of the seston, possibly bacteria. Mussels may be providing an important ecosystem role by pro- viding limiting nutrients to primary producers. CONSERVATION ACTIVITIES FOR FRESHWATER MUSSELS BY THE UNITED STATES FISH AND WILD- LIFE SERVICE. Susan Rogers,* US Fish and Wildlife Service. Conway. AK 72032; Richard Biggins, US Fish and Wildlife Ser- vice. Asheville. NC 28801; Steven Ahlstedt. US Geological Sur- vey, Knoxville. TN 37828. The Fish and Wildlife Service (FWS) is active in freshwater mussel conservation through several approaches: habitat restora- tion, propagation and reintroduction of juveniles, collaboration with partners, and conservation research. The FWS is involved in conserving both federally listed and unlisted species. Before any recovery program can be successful, however, habitat must be restored and protected. A program such as that enacted in partner- ship with The Nature Conservancy on the Clinch River can be particularly successful in reaching this goal. Partnerships are inte- gral to the FWS's freshwater mussel recovery programs. For ex- ample, the Tennessee Valley Authority and the Army Corps of Engineers also have worked with the FWS on mussel conservation in relation to their water resource projects. In addition to habitat restoration, propagation efforts have been ongoing for approxi- mately 10-15 y. and endangered mussel populations throughout the country are being augmented with propagated juveniles. Fi- nally research is being conducted to determine the causes of the bottleneck in survival that juvenile mussels experience around 60 d of age. Further information needs that would aid the FWS's conservation efforts inckide quantification of the impacts of sedi- mentation from dredging and navigation projects on mussel popu- lations, and the impacts of pesticides on glochidial transformation success. DIET EVALUATION FOR THE FRESHWATER MUSSEL ELLIPTIO COMPLANATA (BIVALVIA: UNIONIDAE). Wil- liam F. Henley* and Richard J. Neves, Virginia Cooperative Fish and Wildlife Research Unit. Department of Fisheries and Wildlife Sciences. Virginia Tech. Blacksburg. VA 24061. To determine the effects of holding conditions and different algal diets on freshwater mussels, the tissues of adult eastern el- liptio freshwater mussels. EUiptio coinpkmnta (56-106 mm) were tested for physiological and gametogenic condition over 8 sam- pling events. Treatments included a reference group from the source population, the Nottoway River. Virginia (NR), and 3 cap- tive treatments. Captive treatments were mussels fed Scencdesmus quMtricaiuta (S). Neocliloris nleouhiindiins (N). and a no feed treatment (NF). Patterns in levels of soluble protein, glycogen, and percentages of moisture differed over the course of the experiment (p < 0.01 ). Production of ripe and developing gametes did not differ in the treatments (p = 0.22). but the NF mussels ceased gametogenesis in spring. Carbohydrate levels of the N and S ani- mals did not differ from those of the NF mussels (p = 0.99). Soluble protein levels of the N and S mussels remained relatively high throughout the experiment, those of the NF animals declined, and those of the NR varied. Percent moisture in tissues of the treatments differed over time (p < 0.001 ). Levels of total lipids in the mantle tissues of the treatment groups, as well as muscle fiber diameters in the foot, provided useful complimentary data for as- sessing body condition. 378 Ah.stmct.s. 2002 Annual Meeting. April 14-18. 2002 National Shcllfisheries Association. Mystic. Connecticut HIERARCHICAL ANALYSIS OF MTDNA VARIATION IN AMBLEMA PLICATA, A WIDESPREAD MUSSEL SPECIES. Curt L. Elderkin,* Dept. of Zoology, Miami University, Oxford, OH 4.'S036: David J. Berg, Dept. of Zoology, Miami University, Hamilton. OH 4.S01 1; Janice L. Metcalfe-Smith. National Water Research Institute, Burlington, ON, Canada L7R 4A(i; Caryn C. Vaughn, Dept. of Zoology and Oklahoma Biological Survey. Uni- versity of Oklahoma. Norman, OK 73019; Alan D. Christian, Dept. of Zoology. Miami University, O.xford, OH 4-^056; Sheldon I. Guttman, Dept. of Zoology, Miami University, Oxford. OH 45056. Knowledge of genetic structure of target species is essential for the development of effective conservation plans. Amhiema plicata is a common, widespread freshwater mussel species. Six or seven individuals from six populations of Aiuhlcma plicata within the Lake Erie and Ohio River drainages were analyzed by sequencing restriction fragments from a 652-base portion of the mitochondrial cytochrome oxidase I (COI) gene and a 530-base portion of the nuclear internally transcribed spacer (ITS- 1 1 gene. Percent se- quence divergence was determined and the results were analyzed by calculating Tamura-Nei distances between individuals and then constructing neighbor-joining trees from the distance matrix. Two haplotypes were identified for the COI gene and all populations shared these haplotypes. Greater diversity was found for the ITS- 1 gene, with a total of six haplotypes, although each population had only one or two haplotypes. Cluster analysis revealed no real geo- graphic structuring for either gene. These preliminary results are in striking contrast to those we have found using allozymes. The latter group of loci showed significant differentiation between the Ohio River and Lake Erie basins. Further investigation is required in order to explain this apparent contradiction. GAMETOGENESIS, SPAWNING, BROODING AND GLOCHIDIAL DISCHARGE IN MEGALONAIAS NERVOSA (BIVALVIA: UNIONIDAE) FROM THE TENNESSEE RIVER IN ALABAMA. Jeffrey T. Garner,* Alabama Division of Wildlife and Freshwater Fisheries, 350 Co. Rd. 275. Florence, AL 35633: Thomas M. Haggerty, Department of Biology, Uni- versity of North Alabama, Florence, AL 35632, Rebekah L. Rog- ers. Bradshaw High School. Florence, AL 35630. The annual gametogenic cycle of Megalonuias iwrvasa was monitored over a two-year period by observing temporal changes in histological preparations of gonadal tissues. In male specimens, temporal changes in cell numbers and relative abundance were evaluated by counting different germ cell types along transects through gonadal acini. Temporal changes in female specimens were quantified by measuring diameters of oocytes and counting the numbers of oocytes per acinus. The cycles observed in both male and female M. nervosa differed from those observed in other members of the Ambleminae. Little activity was observed through most of the year, culminating in a Hurry of activity in late simimer and early autiniin. In male specimens, acini filled with spermato- zoa in July and August and all were released in September. In female specimens, oocytes developed and grew quickly during the period in which spermatogenesis occurred in males and all oocytes were released to the marsupia over a short period, .showing a high degree of spawning synchronicity with the males. Glochidia quickly matured and were present in both inner and outer gill demibranchs until December. CLEARANCE RATE AND FILTRATION EFFICIENCY OF ELLIPTIO COMPIANATA (BIVALVIA: UNIONIDAE) EX- POSED TO DIFFERENT LABORATORY CONDITIONS. Catherine M. Gatenby.* Daniel .A. Kreeger and Robyn Rein- miller, Patrick Center for Environmental Research. Academy of Natural Sciences, Philadelphia, PA 19103; Richard J. Neves, Vir- ginal Cooperative Fish And Wildlife Research Unit, Department of Fisheries and Wildlife Sciences, Virginia Tech, Blacksburg, VA 24061-0321. As part of an ongoing research program improve laboratory culture protocols for native freshwater mussels, we quantified and compared feeding rates and particle size preferences of mussels. Elliptio complaiuihi. that were exposed to different holding con- ditions in the laboratory. Two culture parameters were examined: the importance of a burrowing substrate (sand) and the effect of food quality (natural seston vs. lab-cultured niicroalgae). A Multi- Sizer was used to measure and compare clearance rates for the whole diet (2-63 (xm diameter), specific size fractions of the diet (e.g.. 2-3 p-m), and for various sized carboxylate microspheres. By comparing clearance rates for different sized particles, we calcu- lated their relative filtration efficiency, which tended to be greatest for moderately large sized particles averaging 7-10 (xm in diam- eter. Both clearance rate and filtration efficiency varied consider- ably between sediment/no sediment conditions, and among dietary treatments of different quality. These parameters were also moni- tored for periods of up to 60 d to determine whether £. complanata adjust either feeding rates or particle size preferences as an adap- tive response to differences in physical (e.g. sediment) or nutri- tional conditions. VARIABILITY IN CONDITION INDEX AND TISSUE BIO- CHEMISTRY OF ELLIPTIO COMPlJiNATA HELD IN THE FIELD AND LABORATORY. Daniel A. Kreeger,* Catherine M. Gatenby and Deborah Raksany, Patrick Center for Environ- mental Research. Academy o\ Natural Sciences. Philadelphia, PA 19103. Like then marine counterparts, native freshwater mussels can be sufficiently abundant to affect key ecological functions (e.g., energy, biogeochemical cycles) and perform important ecosystem services (e.g., removal of suspended particulate matter, use as bioindicators). The effectiveness of freshwater mussels depends in National Shelltisheries Association. Mystic, Connecticut Ahsrnuts. 2002 Annual Meeting, April 14-18, 2002 379 part on their physiological rate functions, which may vary with their physiological condition. Our goal w as to determine the level of variability in physiological condition of Elliptio conipUinata, a common unionid in the Atlantic drainage. Adults were subjected to a variety of laboratory and field conditions. In the lab mussels were held in continuous flow chambers with or without sediment and fed isocaloric rations of either natural seston or lab-cultured mi- croalgae. Field populations were caged and transplanted to waters of varying quality. Condition index and pro,\imate tissue biochem- istry (protein, lipid, carbohydrate, ash) were monitored for 6 weeks in the lab and 1 year in the field. Both condition and tissue bio- chemistry of wild mussels varied seasonally, as expected for adults undergoing reproductive cycling. However, irrespective of these normal seasonal patterns, condition and tissue biochemistry also varied among all experimental comparisons; food quality (seston > algal diets), sediment conditions (sediment > no sediment) and environmental conditions (field site differences). Although E. cnmplanata is considered to be one of the more hardy unionids. these results indicate that its physiological condition is sensitive to nutritional and environmental cues. Therefore, physiological fit- ness measures for E. complanata should prove useful as bioindi- cators, and studies of ecological processing by these animals should quantify physiological rate functions under a variety of conditions. SMALL-SCALE ECOLOGICAL FUNCTION OF FRESH- WATER MUSSELS (FAMILY: UNIONIDAE) IN THE KIAMICHI RIVER, OKLAHOMA. Daniel E. Spooner* and Caryn C. Vaughn, Oklahoma Biological Survey and Department of Zoology. University of Oklahoma. Norman, OK 73019. Freshwater mussel (Family Unionidael biomass and species diversity is declining as a result of recent and historic anthropo- genic impacts. Despite this marked trend, very little is known concerning the ecology of unionids with respect to stream ecosys- tem function and local macroinvertebrate assemblages. We per- formed a 12-month field enclosure experiment to examine the role of unionids in streams and document their associated sediment and epilithic communities. Treatments were two mussel species, Acti- nonaias ligameiuina and Ainhlenia plicaiii. shell treatment, and a non-mussel sediment treatment. We observed colonization of al- gae, ash-free dry mass and invertebrates in the sediment and on the shells over three time periods: 1 month. 3 months, and 12 months. Chlorophyll a, AFDM and invertebrate abundance were higher in treatments containing live mussels. This trend was significant for months where stream flows were low, but non-significant during periods of high flow. However, there were no significant differ- ences between species despite observed trends. This study indi- cates that freshwater mussels have the capacity to impact local algae, invertebrate and organic matter dynamics. However this relationship appears to be context dependent and may be overrid- den by advective forces such as flow. PROPAGATION OF ENDANGERED FRESHWATER MUS- SELS IN RECIRCULATING CULTURE SYSTEMS. Richard J. Neves,* Jess W. Jones and William F. Henley, Virginia Co- operative Fish and Wildlife Research Unit. Department of Fisher- ies and Wildlife Sciences. Virginia Tech. Blacksburg. VA 24061. A propagation facility for the production, culture, and release of endangered ju\enile mussels has been established at Virginia Tech, to address the conservation needs of 70 federally listed mus.sel species in the United States. After years of research on species' life histories, substratum and food requirements, and ef- ficacy of culture systems, the facility has been successful in cul- turing juvenile mussels for release to natal rivers. Nine species have been cultured thus far. and over 250,000 juveniles have been released to 3 rivers in Tennessee and 2 streams in Virginia. These culture systems are seemingly suitable for holding and condition- ing adult mussels for spawning, and graduate student experiments are ongoing to test the range of applications to mussel conserva- tion. A recent grant from the National Fish and Wildlife Founda- tion has allowed construction of a new facility to test the recircu- lating culture systems on a larger production scale, to begin in 2002. The new facility, coupled with an adjacent pond, will pro- vide additional research opportunities and conditions for long-term rearing of juvenile and adult mussels. COMPARATIVE AND EXPERIMENTAL p:VIDENCE FOR THE FUNCTIONAL ROLE OF FRESHWATER MUSSELS IN STREAMS. Caryn C. Vaughn,* Daniel E. Spooner, Melissa Moore and Keith B. Gido. Oklahoma Biological Survey and De- partment of Zoology, University of Oklahoma, Norman, OK 73019. Both the overall abundance and species richness of freshwater mussels are declining at an alarming rate. If mussels play critical roles in the functioning of river ecosystems, this significant loss of benthic biomass may result in alterations of ecosystem processes and functions, and also may impact other stream organisms that have co-evolved with mussel assemblages. We used a comparative field study and laboratory experiments to investigate the functional role of unionids in streams. From 1999-2001, we sampled benthic macroinvertebrates, meiofauna and fishes living in and around mussel beds in nine rivers of the Quachita Uplands of Oklahoma and Arkansas. Data analyzed to date indicate that densities of some benthic groups, notably oligochaetes and filtering caddisflies, are significantly related to unionid density. Laboratory experiments compared community respiration rates, water column nutrient con- centrations, algal clearance rates, excretion rates, and biodeposi- tion rates of Aclin(n)aius Ugaincuthia and Aiiihlcimi plicahi. Both species exhibited a strong, linear relationship between biomass and most ecosystem processes at small spatial scales. However, there 380 Abstracts. 2002 Annual Meeting. April 14-18, 2002 National Shellfisheries Association. Mystic. Connecticut were only subtle differences between species in their effects. Our results indicate that unionids can have strong effects on ecosystem processes when bioniass is high, but that these effects are context dependent and can be oveiridden by physical forces. ASSIMILATION OF MICROZOOPLANKTON BY ZEBRA MUSSELS: THE BENTHIC ZOOPLANKTON LOOP. W.-H. Wong* and Jeffrey S. Levinton, Department of Ecology and Evolution. State University of New York. Stony Brook. NY 1 1794-5245; Benjamin Twining, Marine Sciences Research Cen- ter. State Uni\ersity of New York. Stony Brook. NY 1 1794-5000. Bivahes living at high densities exert strong effects on the water column. Following their invasion of North American rivers and lakes, zebra mussels (Dreisseini polyiiiorpha) caused major declines in both phytoplanklon and microzooplankton. We tested the hypothesis that the microzooplankton are a potential food source for zebra mussels, and by extension, other bivalves. We labeled phytoplankton with '""C and fed them to two species of rotifers found abundantly in the Hudson River, which had declined following the zebra mussel invasion. The labeled rotifers were fed to 7.ebra mussels and we estimated clearance rates and assimilation of carbon. For both species of rotifers the assimilation efficiency was 40-50%. Before zebra mussels dominated the Hudson River in 1992. the assimilation From rotifers was about 2 to 3 times higher than its riiutine metabolic rate and also contributed about 0.349 (J/h) to 0.662 (J/h) to mussels growth and reproduction, conferring a positive scope for growth. Since the zebra mussels became abundant, the assimilation is still sufficient to explain about 16.4% to 23.1% of the mussels routine metabolic rate. Therefore, rotifers play a conceivably large role in the zebra mus- sels energy budget whether at high rotifer concentrations (before the invasion) or at low rotifer concentrations {after the invasion). GENETICS AND MOLECULAR BIOLOGY UNSCRAMBLING THE LOBSTER GENOME: COMPLEX HETEROZYGOSITY IN THE AMERICAN LOBSTER? Charles L. Brockhouse* and Gillian Richard, Marine Aquatic Genetics. Biological Sciences, U. South Alabama. Mobile. AL 36688. The American Lobster. Hoiiiarus amcricaniis Edwards, is both a highly important economic species and a model for invertebrate physiology. In spite of this, remarkably little is known about any aspect of the genetics of this species. In view of the long breeding cycle, genomics approaches are clearly preferable to classical breed- ing genetics. As a prelude to a genomics program, we have un- dertaken genome size and meiotic studies. Using Feulgen staining densitometry, we determined the H. aiiu'ruaiiiis genome size to be approximately 2.7 x 10'' base pairs. Confocal microscopy was used to study meiosis in male lobsters. The chromosome number was found to be n = 108, in approximate agreement with previous studies. Large numbers of univalents with chiasma to multiple other univalents were observed at metaphase I. Most or all of the chromosomes were observed in these conformations. This is strongly indicative of heterozygous interchanges among the chro- mosomes, a condition which has the genetic effect of linking many chromosomes into a single linkage group. GENETICAL BASIS OF THE PLASTICITY OF RE- SOURCE ALLOCATION IN THE PACIFIC OYSTER CRASSOSTREA GIGAS. Bruno Ernande, CNRS-CREMA. 17137 L'Houmeau. France; Joel Haure, IFREMER-LCPL 85230 Bouin. France; Lionel Degremont, Edouard Bedier and Pierre Boudry* IFREMER-LGP. 1 7390 La Tremblade. France. Phenotypic and genetic correlations between fitness-related life-history traits — such as survival, growth and reproductive al- location— have to be considered to better understand selective pro- cesses, both in the wild and in breeding programs. Quantitative genetic experiments, based on nested half-sib mating designs, were performed in the Pacific oyster C. f;igas ( 1 ) to estimate genetic variance and the response to selection and (2) to document phe- notypic and genetic trade-offs between life history traits. Physi- ological trade-offs. i.e. plastcity of resource allocation, were shown to vary among genotypes. Genetic polymorphism was pri- marily observed for the plasticity of reproductive effort. In addi- tion, the degree of plasticity in reproductive effort appeared to be genetically cortelated with mean survival. The signs of some sig- nificant genetic correlations were found to reverse accross envi- ronments. These results could explain the maintenance of genetic polymorphism for the studied traits. These may help us to better understand the causes of summer mortalities in C. gificis juveniles, on which a national program recently started in France. THE SUCCESSFUL PRODUCTION OF TETRAPLOID EASTERN OYSTER, CRASSOSTREA VIRGINICA GMELIN. Ximing Guo,* Jian Wang, Brenda J. Landau, Li Li, Gregory A. DeBrosse and Krista D. Buono, Haskin Shellfish Research Laboratory, Rutgers University, 6959 Mdler Avenue, Port NoiTis, NJ 08349, Triploid eastern oyster grow 30—40% faster than normal dip- loids. Fast growth reduces not only culture duration and cost, but also losses from diseases inflicted mortality, which represents a serious problem in the eastern oyster. The ideal way to produce triploids is through diploid x tetraploid mating. Triploid production from tetraploid is as simple as producing normal diploids, 100% effective and free from genetic defects caused by polar body in- hibition. Tetraploid Pacific oysters have been successfully pro- duced anil commercialized. A small number of tetraploid eastern oysters were previously produced hut lost. Here we reported the N;iti(iii;il Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 381 production of a large number of tetraploid eastern oysters. The tetraploids were produced using the Guo-Allen method — inhibiting polar body I in eggs from triploids fertilized with normal sperm. Triploid females were individually confirmed by flow cy- tometry before use. Fertilized eggs were treated with 0.5 mg/L cytochalasin B from about 10 to 30 min post-fertilization (PF). Thirteen replicates were made, and eight produced significant numbers of spats OlOO). Ploidy analysis at two months PF found tetraploid spat in all replicates, ranging from 10 to 100%. Over 4000 tetraploid spats were produced from the eight surviving rep- licates. Tetraploids were larger than triploids and diploids within groups. At five months PF. about 10% of the tetraploids changed to primarily triploid/Ietraploid mosaics. The tetraploids were made from a Rutgers disease-resistant stock, which demonstrated strong resistance to MSX. a parasitic disease. The combination of supe- rior growth and disease-resistance may make triploid eastern oys- ters extremely valuable for aquaculture production. published microsatellite DNA markers, we obtained 1 14 microsat- ellite DNA markers for testing on three outbred families, using 1 1 -day-old larvae, to reduce segregation distortion caused by re- cessive deleterious mutations (Launey & Hedgecock 2001 Genet- ics 139:255). Of the 102 markers that are informative in at least one family. 98 are placed on a consensus map comprising 10 linkage groups and 880 centimorgans (cM; cf. karyotype of 10 chromosome pairs with an estimated length of 600-1000 cM based on chiasma frequency). Map distances and, occasionally, marker order differ between parents and among families. Of the 79 new microsatellites tested on four other Cnissostrca species. 76 may be amplified from C. angidatii. 65. from C. sikuinea. 30. from C. ariakensis, and 8. from C. virginica. Decline in ability to amplify these markers across congeneric species, together with a high fre- quency of null alleles within C. gigas (40% of the loci are segre- gating for a null allele in at least one family), suggests that primer- target DNA sequences are rapidly evolving in oysters. GENETIC DISTINCTIVENESS OF INSHORE AND OFF- SHORE SPISULA CLAMS. Matthew P. Hare.* Biology De- partment. University of Maryland. College Park. MD 20742: Stephen R. Palumbi, Center for Conservation and Evolutionary Genetics. 16 Divinity Ave.. Cambridge. MA 02138 SpisiiUi solidissiiiiii is restricted to the western North Atlantic where it is commercially harvested by dredge from shelf waters between Georges Bank and Cape Hatteras. A closely related taxon. S. solidissima similis. inhabits the Gulf of Mexico and shallow coastal waters along the Eastern Seaboard. Although studies of the pi)pulation biology and demography of Spisitia clams have been productive because of the ability to age cohorts from annular growth rings, few data have been published on range distributions and patterns of genetic variation. The mitochondrial and nuclear DNA sequences presented here represent a preliminary effort to describe the phylogeny and biogeography of Spisida clams and relatives in the North Atlantic. Mitochondrial cytochrome oxidase I and nuclear calmodulin intron sequence data show the southern/ inshore subspecies is genetically very distinct from Spisiila solUlis- siimi populations from the Gulf of St. Lawrence (Canada). Georges Bank and offshore from Ocean City. Maryland. Despite the re- stricted inshore habitat of S.i. similis. genetic variation within this taxon is exceptionally high, suggesting a large effective population size or population substructure among coastal populations. A GENETIC LINKAGE MAP OF 100 MICROSATELLITE MARKERS FOR THE PACIFIC OYSTER CRASSOSTREA GIGAS. Dennis Hedgecock,* Sophie Hubert, Gang Li, and Katharine Bucl1 lobster/m~. significantly higher than before reef placement. Settlement of young-of-the-year lobsters significantly increased. We used microwire tags to mark hatchery reared lobsters and released them as 5"'-6'^ stage lobsters onto three of the reefs at a density of 4.5/nr for three years. However, subsequent density of young-of-the-year lobsters on the enhanced treatment sites was not significantly different from the non- enhanced sites. Despite intensive sampling, only three of these tagged lobsters were recovered after placement. Placement meth- ods may contribute to survival possibilities. Field and lab obser- vations confirm behavioral differences between 4"' stage and 5''" stage hatchery-reared lobsters as compared to field-caught lob- sters. Predation rates in the lab were significantly higher for the 4"' stage hatchery-reared lobsters raised in the URI facility compared to the RI field-caught lobsters and hatchery-reared lobsters ob- tained from Maine. were collected in the Eastern Long Island Sound. NY (ELIS). Buzzards Bay and Vineyard Sound, MA. Preliminary denaturing gradient gel electrophoresis (DGGE) data indicate that up to five independent phylotypes of bacteria are present in lobster lesions. At least two of them were found in all lobsters from ELIS. This is consistent with the five to six morphotypes of bacteria which were cultured on a marine agar. All isolated bacteria were either aerobic or facultatively anaerobic. No strictly anaerobic or microaerophilic bacteria were isolated from lesions. Only two different types of fastidious chitinolytic bacteria were isolated on a medium contain- ing chitin. One of the two is a motile bacterium forming bright yellow colonies, which was present in all analyzed lobsters (from Buzzards Bay, Vineyard Sound and ELIS). The second is a bac- terium forming white colonies. It was isolated from only two Buz- zards Bay lobsters. No correlation has been found between the presence or severity of shell disease and the presence of bacteria in hemolymph. CHANGES IN LOBSTER POPULATIONS IN NARRAGAN- SETT BAY. RHODE ISLAND. 1959-2(»00. J. S. Cobb and M. Clancy, Department of Biological Sciences, Unisersity of Rhode Island, Kingston, RI 02881. The past 35 years have seen an extraordinary increase in the catch of lobster (Homanis americanus) along the northeast coast of North America. Rhode Island landings ha\e tripled since the late 1960s. We explored a fishery-independent data set of lobster abundance developed from weekly research otter trawls made at two locations in Narragansett Bay. RI by the University of Rhode Island on a continuing basis since 1959. The patterns of abundance over time differed substantially at the two stations; one of the stations was highly correlated with the NMFS catch data set for the same time period. Water temperature, which has increased steadily since 1960, was significantly correlated with commercial landings and with abundance at one of the stations. Lobster abundance at both stations and the commercial landings were significantly cor- related with the NAG winter index. At one of the stations the timing of greatest abundance in the trawl samples shifted to earlier in the summer. It seems likely that there have been effects of climate on lobster abundance, however the effect appears to have acted differentially at the two stations in Narragansett Bay. CHARACTERIZATION OF MICROBIAL ASSEMBLA- GIES INVOLVED IN THE DEVELOPMENT OF SHELL DISEASE IN THE AMERICAN LOBSTER. HOMARUS AMERICANUS. Andrei V. Chistoserdov* and Fellza Mirasol. Marine Sciences Research Center, SUNY at Stony Brook, Stony Brook, NY 1 1720; Roxanna Smolnwitz. Marine Biological Labo- ratory, Woods Hole. MA 02543. A combination of culture-based and molecular techniques was used to study the consortia of bacteria isolated from shell lesions and hemolymph of lobsters. Diseased lobsters used in this study THE ROLE OF VIBRIO FLUVIALIS AND OTHER BACTE- RIAL SPECIES IN LOBSTER MORTALITIES IN MAINE. Ceni Giray* and Deborah .\. Bouchard. Micro Technologies, Inc.. 41 Main Street, Richmond, ME 04357. Weak lobsters {Httiiuinis aiiwrUaiuis) and mortalities have been reported by pounds in Maine for several years with Vibrio fliiviiilis implicated as the etiological agent responsible. Bacterial screening of moribund lobsters was pertbrmed by Micro Tech- nologies, Inc. through a contract with the Maine Department of National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 41 1 Marine Resources. The majority of isolates collected were puta- tively identified as Hyplioiuicrobium iiuliciini through 16S rRNA sequencing while V. fhivia/is was not isolated from any of the lobsters. Further characterization demonstrated that H. iiidicuin could grow at a temperature range from 3'C to a higher limit between I6-20°C while V. fluvialis grew best between 16-30°C with minimal growth at 8°C. The virulence of H. iiulicum and V. fluvialis was tested by injection into lobster hemolymph at various dosages. Both isolates resulted in 75-100% mortality withm 24 hours when injected at 10** colony-forming units (CFU). but only H. incliciiiii resulted in mortalities (12.59!-) when injected at 10" CFU/lobster. V. fischeri was used as a negative control while Li- stonella ani^uillarum and Aerococcus viridans. both indicated as lobster pathogens, were utilized as positive controls. The identifi- cation of H. indiciiin as the primary isolate collected from mori- bund lobsters and the observation of lobster mortalities mainly during lowered water temperatures suggest that the isolate respon- sible could be H. indiciini hut not \'. fhiviidis. SHELL DISEASE PREVALENCE AND SEVERITY IN OFF- SHORE AMERICAN LOBSTER POPULATIONS. Diane Ka- pareiko, Richard A. Robohm. Joiin J. Ziskowski, George R. Sennefelder, and Anthony Calabrese, National Marine Fisheries Service. Milford Laboratory. 212 Rogers Avenue. Milford. CT 06460. During the period 1990-1992, 15.004 lobster from 146 com- mercial catches at nine offshore canyon sites surrounding the 106- Mile Sewage-Sludge Disposal Site were examined for signs of shell disease. Overall. 1.184 lobster (7.9%) had lesions. Females were more affected by this condition than males. Shell-lesion oc- currence was independent of carapace length (CL), but strongly related to location (proximity to the 106-Mile Dumpsite as well as to the 12-Mile Dumpsite). Data collection for the shell-disease study included not only evaluation of presence or absence of dis- ease, but also measurements of lesion size and carapace length. This was done in anticipation of developing a method that would determine the percentage of total surface area of each lobster af- fected by shell disease: this percentage is the basis of a Disease Severity Index (DSI). An estimate of lobster surface area could be derived mathematically from carapace length, for both male and female lobster in our database, using the formula y = 1.1034 + 1 .9677 * log(CL). The percentage of surface area covered by shell lesions, multiplied by 10\ provides a DSI that may allow better statistical correlations between mean disease severity and site of lobster collection. Regression Tree analysis of this multi-variate database indi- cated that, unlike prevalence, the most important variable affecting the DSI was carapace length. Overall. DSI's for smaller lobsters (CL < 95). were significantly higher (p < 0.02) than larger lobsters, regardless of sex or location. Small females had significantly higher DSI's (p < 0.04) than large females. Males showed no significant differences when similarly compared. A complete non- parametric regression analysis of our DSI in relation to proximity to the 106-Mile Site, may indicate whether sewage sludge dump- ing had any effect on the severity of shell disease lesions in off- shore American lobster populations. DEPTH-RELATED PREDATION PRESSURE ON LARVAL LOBSTERS {HOMARUS AMERICANVS) PRIOR TO SETTLEMENT. Wendy Norden and J. Stanley Cobb, Univer- sity of Rhode Island. Kingston. Rl 02880. Prior to settlement, lobster postlarvae swim near the surface of the water for several days. Settlement behavior begins between two and six days after metamorphosis from the third larval stage. During settlement, postlarvae dive from the surface to the benthos to seek out suitable habitat. This potentially exposes the postlarvae to a variety of predators not present at the surface. Understanding where in the water column predation pressure is the greatest will help in understanding behavioral strategies used by lobster post- larvae when seeking appropriate habitat. We tethered postlarvae at various depths to experimentally test the potential predation pres- sure throughout the water column. Tether lines were deployed over mud and cobble substrate types to look at the difference in preda- tion pressure as it relates to habitat w ith four depths (0.5m. 1 .5m. 2.5m. 4m) above substrate. Higher predation occurred over cobble substrate than over mud. Most of the predation occuired within 1.5m of the bottom and was probably primarily due to the cunner. Tautogolabrus adspersiis. Laboratory observations with epibenthic fish species confirmed their ability to consumer post- larvae. THE EFFECT OF TEMPERATURE ON SEXUAL MATU- RITY IN THE FEMALE LOBSTER, HOMARUS AMERICA- NVS. Susan A. Little* and Win Watson, University of New Hampshire, Zoology Department, Durham, NH 03824; Bonnie Splnazzola, Atlantic Offshore Lobstermen's Association, I 14 Ad- ams Road. Candia. NH 03034. The purpose of this study was to determine if female American lobsters {Homarus americaniis) inhabiting areas with dissimilar annual temperature profiles (measured in degree days >I0°C), reached sexual maturity at different sizes. We compared the size at maturity of lobsters captured in 3 different offshore locations (two southern and one northern). 1 inshore site (Isles of Shoals. NH) and one estuarine site (Great Bay Estuary. NH). Lobsters inhabit- ing the Great Bay Estuary experienced the warmest temperatures ( 1320 degree-days), followed by the southern offshore sites (876: 906). the northern offshore site (669), and the Isles of Shoals (416). Lobsters that migrate could gain a considerable thermal advantage. For example, southern offshore lobsters would increase their de- gree-days from 216 to 906. For this reason temperature exposure for the offshore lobsters was calculated assuming a seasonal mi- 412 Abstracts. 2002 Annual Meeting. April 14-18. 2002 National Shelltisheries Association. Mvstic. Connecticut gration. The maturity of female lobsters was determined using a combination of abdominal width/carapace length ratios, cement gland stages, and egg stages. As expected, lobsters that experi- enced the most degree-days above 10°C reached sexual maturity at smaller sizes. The size at which 50% of the lobsters were mature was 84 mm CL for the Great Bay estuary (n = 7 1 ). <83 mm CL for the two southern offshore sites (n = 80 lobsters), 91 mm CL for the northern offshore site (n=40), and 89 mm CL for the Isle of Shoals (n = 33). This difference in size at maturity was also mani- fested in the average size, and range of sizes of berried females captured in each location. These data suggest that regional differ- ences in size at maturity should be taken into account when man- aging the fishery. However, it is important to fully understand all the behavioral, developmental and oceanographic processes that influence size at maturity before implementing any new regula- tions. This project was funded by the Northeast Consortium. ENVIRONMENTAL MONITORS ON LOBSTER TRAPS. James Manning, Northeast Fisheries Science Center, Woods Hole. MA. Bonnie Spinazzola, Atlantic Offshore Lobster Asso- ciation; Patrice Farrey. Maine Lobstermen Association; David Casoni, Mass Lobstermen Association; Clare GrindaU Downcast Lobstermen Association. Beginning in early 2001, electronic temperature probes were distributed to over 70 New England lobstermen by the four largest associations. These units are recording hourly temperatures at fixed locations throughout the Gulf of Maine including many off- shore canyons sites. The objective is to occupy these same loca- tions year-after-year for documenting interannual variability of the deeper bottom water. Phase II of the project, just getting underway in 2002, will deploy several salinity sensors around the region. The primary objective is to characterize the water mass and to as.sess the influence of remote source water. Participating lobsteren are asked to record catch wherever probes are attached. Understanding the biological significance of the physical variability is a second- ary but a potentially worthwhile bi-product of the study. Details are posted on the project website http://www.nefsc.nmfs.gov/ -jmanning/emolthtml. LESIONS ASSOCIATED WITH RECENT EPIZOOTIC SHELL DISEASE IN HOMARLS AMERICANLS ON THE NORTHEAST COAST. Roxanna Smolowitz,* Andrea Hsu. and Erin Summers, MBL. 7 MBL St.. Woods Hole. MA 02543; Andrei Christoserdov, Marine Sciences Research Center, SUNY, Stony Brook. NY 11794. Shell disea.se is a commonly recognized problem in lobsters held in impoundments during winter months. However, recently, the economically devastating disease has reached high proportions in free-living populations along the coast from Eastern Long Island Sound, New York, to the Vineyard Sound, Massachusetts. The disease is characterized by multifocal to confluent, shallow to deep erosions, primarily of the dorsal carapace, often resulting in thin, easily compressed shells and, rarely, ulcerations. Grossly, lesions usually show marked melanization. but no obvious pathogen. Histopathologically, carapace erosions are of variable depth; but deeply extensive erosions are common. Carapace matrix in the erosions is usually missing, but in some instances, unlike lesions noted in impoundment disease, pillars of carapace matrix still re- main attached in the eroded tissue. Intlammation is composed of increased numbers of hemocytes in the underlying connective tis- sues and accumulations of usually necrotic hemocytes between layers of carapace. A second type of intlammation consists of variable amounts of carapace proliferation produced by the intact hyperplastic epithelium underlying the eroded site. Various organ- isms are identified in the erosions, but the predominate organism found at the interface of necrotic and li\e shell are bacteria. Work is underway to identify the pathogenic bacteria at the lesion inter- face. WHAT CAN DATA FROM INDUSTRY CONDUCTED TAG-RECAPTURE PROGRAMS TELL US? A STUDY OF THE AMERICAN LOBSTER {HOMARUS AMERICANUS). Barbara A. Soniers,* and Kathleen M. Castro, University of Rhode Island. Fisheries Center. East Farm. Kingston. RI 02881; John Sorlien, Rhode Island Lobstermen's Association. Box 421, Wakefield, RI 02880; Tom Angell, Department of Environmental Management, Coastal Fisheries Lab. Wakefield. RI 02880. Tag-recapture data for the American lobster {Himuiriis ameri- cciiuis) compiled from a two year experimental tag-recapture pro- gram done by the Rhode Island Lobstennen's Association (R1L.\) in cooperation with Rhode Island Sea Grant and the Rhode Island DEM is being used to determine migration and life-history char- acteristics for the Southern New England lobster stocks. Twenty boats participated in the program; number of lobsters tagged and recaptured varied among fishing areas, taggers and recapture re- porters. A total of 1 1 .964 lobsters were tagged and released over a 16-month period. The majority of the lobsters were tagged in August and September of 2000 and the months with the highest recapture rates were Jul\, August and September of 2000 and 2001. 982 lobsters have been recaptured to date giving a recapture rate of 8.29(^. Recaptures were received by phone and by mail with a total of 91 fishermen returning tags. Biological data estimating growth, egg frequency, shell disease and movement will be re- ported as well as perceptions about cooperatise research from the project participants. National Shellfisheries Association. Mystic, Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 413 OYSTER REEFS AND RESTORATION A CHARACTERIZATION OF IDEAL HABITAT STRUC- TURE FOR THE STRIPED BLENNY CHASMODES BOS- QUIASUS. Elizabeth M. Flynn* and Kennedy T. Paynter, Jr.. Department of Biology. University of Maryland. College Park. MD 20742. Historically, oyster reefs dominated the trophic interactions of the Chesapeake Bay. Besides improving water quality and forming an integral link in the food web. oysters provided the physical structure that facilitated the development of a complex benthic community. In the past 150 years, disease, overfishing, and pol- lution have decimated the oyster population and destructive har- \ esting methods have reduced complex, high relief reef structures to flat "footprint" bars. The impact of this systemic loss of vital habitat on the small demersal reef fish has gone relatively unstud- ied. The objective of this study is to separate and identify the structural components of shell aggregations that constitute desir- able habitat for the striped blenny, Chasmodes hosqiiiainis. Disarticulated oyster shells were arranged in a 30-gallon glass aquariumin various vertical, horizontal and angled arrays to tested for spatial preferences. Individuals were released into the tank and remotely observed via overhead video for one hour. Mean perch duration, perch frequency, minimum and maximum perch dura- tions, and total duration of association with the shell arrays were analyzed. The results indicate that C bosqidaniis prefer 1-2 cm NN distances. C. bosquianus similarly preferred 60 degree arrays as compared to 90 degreeswith regard to total association, maxi- mum perch duration, and frequency. The short-term behavioral patterns analyzed here suggest that purely structural elements of oyster reefs are critical factors influencing demersal fish popula- tion distributions. A COMPARISON OF ACOUSTIC TECHNIQUES, VIDEOGRAPHY, AND QUADRAT SAMPLING FOR CHARACTERIZING SUBTIDAL OYSTER REEFS. Ray- mond Grizzle,* Larry Ward, and Jamie Adams, Jackson Estua- rine Laboratory, University of New Hampshire, Durham. NH 03824; Semme Dijkstra, Center for Coastal & Ocean Mapping. University of New Hampshire; John Nelson, New Hampshire Fish & Game Department. Marine Fisheries Division. Durham. NH 03824. Acoustic techniques, videography. and quadrat sampling were used to characterize several subtidal oyster reefs in the Great Bay Estuary in New Hampshire and to compare their effectiveness, with the long-term goal being a general protocol for reef mapping and monitoring. The acoustic techniques consisted of single beam, multibeam. and sidescan sonar. Preliminary analysis of the acous- tics data indicated that reef boundaries could readily be mapped. Videography was conducted by systematically imaging each of 40 sampling cells in a grid covering the approximate area of each reef. A single drop was made in each cell and a 5 to 10-s recording made of a 0.25 m" area. A still image was produced for each of the 40 cells and all were combined into a montage that revealed the approximate boundaries of the reef. Five to ten cells on each reef were randomly chosen and sampled by divers using a 0.25 m" quadrat; all live oysters were measured (shell height) to nearest millimeter using calipers. Oyster counts were also made directly from each video image and compared to the quadrat data from the same photo; preliminary analyses showed good correlations be- tween these counts. Overall, our results to date indicate that acous- tic techniques generally can delimit the boundaries of oyster reefs, as has been demonstrated in other studies. Their potential for in- ferring other reef characteristics (e.g., oyster densities), however, is being assessed. Thus far, the major finding is that videography may be a powerful and relatively inexpensive tool for detailed reef mapping, including inferring oyster densities and perhaps other characteristics. PREVALENCE OF ENTERIC MICROORGANISMS IN THE EASTERN OYSTER {CRASSOSTREA VIRGINICA) AND THEIR OVERLYING WATERS AT REPRESENTA- TIVE SITES OF AN OYSTER GARDENING PROGRAM IN MOBILE BAY. ALABAMA. Kimberly A. Hamilton,* D. LaDon Swann. and William Burkhardt. III. Department of Fish- eries and Allied Aquacultures, Auburn Uni\ersity. Auburn. AL 36849 and U.S. Food and Drug Administration. 1 Iber\ille Dr., Dauphin Island. AL 36528-0158. An oyster gardening program for the restoration of the eastern oyster. Crassostrea virgiiiica. in Mobile Bay. AL began in May 2001. Thirty study locations were established in Mobile and Bald- win counties along Mobile Bay. Of these sites three from Mobile county and two from Baldwin county for a total of five sites were purposefully selected in July 2001 to determine if a relationship exists between enteric microorganisms in oysters and their over- lying waters at different geographic locations along Mobile Bay. The oysters were suspended under piers inside Eastfields floats (surface area of 0.35 m") which positioned the oysters directly below the surface of the water. Oyster and water samples were taken from each site on a monthly basis and levels of fecal coliforms. Escherichia coli. and male-specific bacteriophage (an enteric viral simulant) were quantified using pre-established pro- tocols. The results have shown intermittent high levels of coliforms in the shellfish, but levels in their overlying waters were low. The bacteriophage levels have been consistently lower than the level of detection; thus indicating that human wastewater treatment source is not a likely cause of fecal coliforms. A high number of birds and their droppings have been observed at some of the study sites from which the Eastfields floats are suspended. These results may have 414 Absli-acts. 2002 Aiiiuial Meeting. April 14-IX. 2002 National Shelltisheries Assdciation, Mystic. Connecticut implications tor future site selection of the gardening program. Findings from these analyses will be used to inform local com- munities and regulatory agencies of any impacts water quality may have on shellfish quality. USE AND VALUE OF OYSTER REEFS AMONG RECRE- ATIONAL FISHERMEN IN LOUISIANA. J. C. Isaacs.* Loui siana Department of Wildlife and Fisheries, Baton Rouge. LA 70898; W. R. Keithly, Coastal Fisheries Institute, Louisiana State University. Baton Rouge. LA 7080,^; A. Diagne, University of Arkansas at Pine Bluff. Department of Business Administration, 1200 North University - Mail Slot 4976. Pine Bluff. AR 71601, Oyster reefs serve a wide variety of purposes. Until recently, the majority of research has focused on the relationship between the quantity and quality of reefs and commercial harvests derived therefrom. Recreational fishermen in Louisiana, however, also make extensive use of oyster reefs. This study serves two pur- poses. First, it provides an examination of catch rates and species diversity among inshore recreational fishermen fishing over oyster reefs in comparison to inshore fishermen not fishing over oyster reefs. Second, it provides an estimate of "willingness to pay" among individual recreational fishermen for the privilege of fish- ing over oyster reefs. This study employed a telephone survey of Louisiana anglers to generate an estimate of the \alue of oyster reefs in Louisiana to the recreational fishing sector. Overall, the value is found to be significant, gives a justification for maintain- ing oyster reefs in addition to commercial harvest. RELAYING AS A METHOD TO REMOVE HOOKED MUS- SELS FROM OYSTERS PRIOR TO REHARVEST FOR SALE. Earl J. Melancon, Jr.,* Biology Department. Nicholls State University, Thibodaux. LA 70.^10; Dale Diaz, Mississippi Department of Marine Resources, Biloxi, MS 39530; Badiollah Asrabadi, Math Department. Nicholls State University, The hooked mussel. Ischadiuin recumtm, is considered a na- tive species of the northern Gulf of Mexico, but introduced in the Chesapeake, It is a small bivalve that can be found in great num- bers attached to subtidal oysters located in low salinity environ- ments. In the northern Gulf of Mexico, oystermen have tradition- ally relayed mussel-laden oysters to higher salinity waters with the expected results of killing them, and thereby removing them, be- fore reharvest of the cleaned oysters for sale. Results of laboratory, small-scale field and two commercial- scale field experiments indicate that removal of mussels due to transplanting to higher salinity waters is less a function of physi- ological salinity stress and more a function of the transplanting process itself. The hooked mussels were crushed, dislodged or stressed to the point of permanent gaping during the commercial- scale relaying operations. This resulted in an immediate 32-34% mussel moilality, with a corresponding negligible oyster mortality. The dead and dying mussels attracted predators, such as the blue crab. CalliiH'ctes sapidus. and the southern oyster drill, Stranumita hcu'imi.\iiniHi. which resulted in continued mussel losses in the ensuing weeks. In the more coastal location, where salinity is relatively high, mussel-fouled oysters were cleaned by the third week after a sum- mer transplant. The dilemma is that oysters in the higher salinity waters may also become more vulnerable to predators and the oysteriiian must consider reharvest in a short time. In the more inland location, where salinity is more intermediate, and perhaps with less abundant oyster predators present, mussel removal was a longer process with 76% removed by the fifth week. Results of these experiments strongly suggest that salinity is a key element in the removal of mussels from oysters, but more in the role of a habitat response and less as a physiological response to salinity. MODELING THE INFLUENCE OF FILTRATION BY OYS- TER STOCKS ON TURBIDITY AND SEAGRASS GROWTH. Roger I. E. Newell,* Evamaria VV. Koch, Melissa K. Wood, Ray E. Grizzle, and Raleigh R. Hood, Horn Point Laboratory, University of Maryland Center for Environmental Sci- ence, PC Box 775, Cambridge, MD 21613-0775, Chesapeake Bay has undergone severe ecological changes. Oysters are almost completely absent due to overharvest and dis- eases and seagrass beds either are in decline or have disappeared due to high water turbidity reducing light availability. Oyster reefs and seagrass beds tended to impro\e water quality by actively filtering particles out of the water, reducing wave energy, and minimizing sediment resuspension. In part because of the loss of these ecosystem functions water quality is poor and seagrass res- toration not always successful. We hypothesized that the reestab- lishment of oyster stocks, either via restoration of reefs or aqua- culture, could benefit seagrass populations. Results from labora- tory experiments and field studies were used to paramterize a model predicting the extent to which oysters and seagrasses can affect water quality and enhance the chances of survival and ex- pansion of seagrasses. Oysters were capable o\ increasing light penetration due to high summer filtration rates. The ability of seagrass beds to minimize resuspension was a function ot water depth and their capacity to attenuate waves. Large reproductive seagrass plants that occupied the entire water column were more effective at minimizing resuspension than small vegetative plants. Our model results suggest that increasing oyster stocks can dra- matically enhance light penetration and thereby increase the area of Bay bottom where seagrasses can grow. National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 1-H8. 2002 415 THE EFFECT OF STOCKING DENSITY ON BENTHIC COMMUNITY DEVELOPMENT AND SHELL HEIGHT IN THE EASTERN OYSTER. CRASSOSTKEA VIRGINICA. Stewart Harris* and Kennedy T. Paynter, University of Mary- land. College Park. MD 20742. The eastern oyster. Crassostrea virginica. is a gregarious, reet- formins organism. Oyster populations that once dominated the Chesapeake have declined significantly and interest has recently arisen to restore the economic and ecological benefits of native oyster populations. Understanding the ecological importance of oysters and oyster reefs is critical to the restoration of the estuary's ecosystem as a whole. Oyster densities on most Maryland reefs are very low (about 3 oysters/m"). however, natural reefs formed in other areas are comprised of high densities of oysters (>500/m^). In order to maximize the effectiveness of oyster restoration, it is important to determine how oyster density may affect oyster growth, parasite prevalence and the formation of reef habitat uti- lized by the benthic community. In the fall of 1999, twelve 0.2-acre experimental plots were constructed in the Patuxent River by placing fossil oyster shell on a barren natural oyster bar. The plots were assigned one of four treatments, zero. 124, 247. 494 oysters/m". in a randomized de- sign. Oyster growth was 0.12 (± 0.004 SEM) mm/day for the 2000 season and slowed to 0.07 (± 0.006) mm/day in 2001. The data did not show any effect in shell height due to density of oysters. Colonization of the oyster reefs with fouling organisms was related to density of oysters. In summer and fall of 2000. barnacle density declined with increasing oyster density. EVALUATING THE CONTRIBUTION OF COMMERCIAL OYSTER AQUACULTURE TO RECRUITMENT. Rachel E. Sackett,* Russ Peterson, and Ami E. Wilbur, Department of Biological Sciences and the Center for Marine Science. .5600 Mar- vin K. Moss Lane. University of North Carolina at Wilmington. Wilmington, NC 28409; Jim Swartzenberg, J&B AquaFood. Holly Ridge. NC 28445. Commercial oyster {Crassostrea virginica) aquacullure opera- tions concentrate large numbers of potentially reproductive ani- mals. Such aggregations may function to produce large numbers of larvae that subsequently recruit to natural beds. Anecdotal obser- vations have suggested the enhancement of recruitment in the vi- cinity of commercial aquaculture operations; however, confirma- tion of such an effect has generally not been possible, as new recruits derived from the cultured stock generally cannot be dis- tinguished from those resulting from the spawning of wild oysters. Recent stocking of commercial leases in North Carolina with oys- ters produced in a hatchery in Louisiana has provided an oppor- tunity to evaluate the potential for the enhancement of local re- cruitment due to the spawning of cultured stock. Oysters from the Gulf coast and Atlantic coast have been previously shown to ex- hibit genetic differences in mitochondrial DNA (mtDNA) haplo- type. PCR (polymerase chain reaction) amplification and DNA sequencing of 409 base pairs of the 16s ribosomal gene are being used to screen recent recruits collected from 7 natural beds sur- rounding 1.5 acres of leased bottom (stocked with -70, 000 Gulf oysters), as well as a sample of the culture stock. Sequence data will be analyzed to determine what proportion of recent settlers exhibited mtDNA haplotypes consistent with the hypothesis that they were produced by the spawning activity of the culture stock. Preliminary analyses revealed some recruits exhibit mtDNA hap- lotypes identical to those exhibited by the culture stock, suggesting that there is some local recruitment resulting from reproduction of commercial aquaculture stock. RECRUITMENT OF THE OYSTER CRASSOSTREA VIR- GINCA ON INTERTIDAL REEFS IN AREAS WITH IN- TENSE BOATING ACTIVITY IN THE INDIAN RIVER LA- GOON, FLORIDA. Lisa WalL* Linda Walters, Kevin Johnson, and Neysa Martinez, Department of Biology. Univer- sity of Central Florida, 4000 Central Florida Blvd. Orlando, FL 32816; Ray Grizzle, Jackson Estuarine Laboratory. University of New Hampshire, Durham. NH 03824. Productivity, diversity and survival of estuaries are threatened by explosive coastal population growth and associated recreational activities. One major area of recreational growth has been the number of people motoring in small pleasure craft at high rates of speed. In counties bordering Mosquito Lagoon (northernmost sec- tion of the Indian River Lagoon systein. east coast of central Florida), there were 51.000 registered boaters in 1998. Numbers have increased 10% annually since 1986 and continue to grow. In areas of Mosquito Lagoon with intense boating activity, intertidal reefs of Crassostrea virginica with dead margins commonly occur. The dead margins consist of mounds of disarticulated shells. The cause(s) of the reef die-offs is unclear. However, the disarticulated shells may be reducing reef sustainability if these surfaces are unavailable for oyster recruitment. Recruitment trials were run on eight reefs (4 impacted. 4 healthy) in two eight-week trials in Summer 2001 and Winter 2001-2002. Sediment loads, tempera- ture and water motion at all sites were monitored. In the Summer 2001 trial, no significant differences were found between or within sites for settlement or recruitment. However, temperatures reached over 40°C on portions of the dead reef. Data of this type is needed to identify causes of reef declines, habitat-specific management protocols and appropriate restoration techniques. 416 Abstnicts, 2002 Annual Meeting. April 14-18, 2002 National Shelltisheries Association, Mystic, Connecticut OFFSHORE FISHERIES INTEGRATING VESSEL TRACKING, CATCH DATA. AND DEPLETION MODELS TO ESTIMATE COMMER- CIAL SCALLOP DREDGE EFFICIENCY. Todd Gedamke* and William DuPaul. Virginia Institute of Marine Science, School of Marine Science. College of William and Mary, Gloucester Point, VA 23062. In June of 1999, Georges Bank Closed Area II was opened to the United States sea scallop fleet after a five-year multi-species fishing closure. During the five-month opening, nearly six million pounds of scallop meats were harvested, catch-per-unit effort data were collected from o\er 1.000 commercial tows, and the tine- .scale distribution of fishing effort was recorded hourly by manda- tory vessel monitoring systems. A spatial analysis of both catch and effort data was perfoimed to locate areas consistent with the DeLury model assumptions. Gear efficiency was estimated to be 45'7c utilizing a combined maximum likelihood analysis of CPUE declines in all suitable regions. An additional mdependent estimate of efficiency was generated from survey stations that were sampled before and after the opening. A kriging analysis was used to determine mean catch rates and the index-removal method was applied to compare the change in catch rates to the total landings reported for the opening. Dredge efficiency was estimated to be 54Vc. The results of this study suggest that the 25% efficiency esti- mate used in calculations prior to the opening resulted in the over- estimation of absolute biomass and the setting of a quota that exceeded target exploitation levels. In addition, the results ot this study suggest that information from the vessel monitoring systems. now in use on many commercial fleets, can provide the fine scale spatial details necessary to successfully apply depletion models to open-ocean commercial fishing operations. (<80 mm) scallops ha\e stimulated interest in developing an area wide management strategy for sea scallops. During commercial openings of these areas during 2000-2001, the performance of a 4-inch (101 nnii) ring scallop dredge was evaluated against the standard 3.5-inch (88 mm) ring dredge. De- tailed catch data from 208 tows on eight commercial trips showed a reduction in scallop harvest of up to 42.5% for scallops <90 mm and increases in harvest efficiency of up to 12.9% for scallops >l 15 mm. These results are closely related to the size frequency distribution of the scallop resource. When the size frequency dis- tribution of the scallop population has a modal shell height of gi eater than I 1 0 mm, the use of a 4-inch ring dredge may play a significant role in realizing the benefits of area-based management. REBUILDING SEA SCALLOP (PLACOPECTEN MAGEL- LANICUS) STOCKS USING AREA CLOSURES AND RO- TATIONAL FISHING. Deborah Hart* and Paul Rago, NCAA Fisheries, Northeast Fisheries Science Center, 166 Water St.. Woods Hole, MA 02543. In the seven years since the closure of three large areas on Georges Bank to trawling and dredging in December, 1994, sea scallop biomass on Georges Bank has increased by more than a factor of twenty. Limited fishing in the closed areas in 1999 and 2000 yielded about 1 1 million lbs of scallop meats while still allowing for increases in biomass in these areas. Dramatic in- creases in biomass have also been seen in the Mid-Atlantic Bight after two areas there were closed to scallop fishing for three years in 1998. These experiences indicate that area closures can be an effective way to rebuild shellfish stocks and alleviate growth over- fishing. Strong recruitment observed since the closures suggests that the closed areas have also become important sources of scal- lop larva. Both theory and practice indicate that rotational man- agement, where areas are successively closed and then opened to fishing, can increase both yield and spawning-stock biomass in the sea scallop fishery. THE USE OF A 4-INCH (101 MM) SEA SCALLOP {PLA- COPECTEN MAGELLANICUS) RING DREDGES IN THE CONTEXT OF AN AREA MANAGEMENT STRATEGY. Kevin D. Goff, William I). DuPaul.* and David B. Rudders. Virginia Institute of Marine Science. College of William and Mary, Gloucester Point, VA 23062. Early attempts to manage the sea scallop (Placopecten magel- lanicus) fishery focused on establishing age-at-entry controls. Since 1994, the primary management strategy shifted to an effort control program and an increase in scallop dredge ring size to 3.5 inches (88 mm) from 3.0 inches (76 mm), with the intent of in- creasing yield per recruit. Dramatic increases in scallop biomass in three areas of Georges Bank closed to mobile fishing gear in 1994 and two areas in the mid-Atlantic closed in 1998 to protect concentrations of small FLEET DYNAMICS OF THE ATLANTIC SEA SCALLOP FISHERY. Paul J. Rago and Deborah R. Hart, NCAA Fisher- ies, Northeast Fisheries Science Center. 166 Water St.. Woods Hole, MA 02543. The sea scallop (Placopecten magellanicus) supports the sec- ond most valuable commercial fishery in the northeast USA. Clo- sure of large areas of Georges Bank to scallopers in 1994 not only resulted in rapid increases in scallop biomass but also altered the spatial distribution of fishing effort. Detailed electronic informa- tion on the hourly position of each vessel was used to study the behavior of the fishing fleet for the period 1998 to 2000. Addi- tional information from at-sea observers cortoborated the analyses National Shellfisheries Association. Mystic. Connecticut Ahstruit.s. 2002 Annual Meeting. April 14-18, 2002 417 of vessel monitoring data. Limited re-openings of the closure areas in 1999 and 2000 induced marked shifts in fishing effort. Fine- scale changes in fishing patterns can be related to the scallop density, size composition and economic value as well as the pres- ence of bycatch species. The scallop fishery is conducted over an area of about 1 2.000 nni', but more than 75% of fishing activity is concentrated within an area of about 3,000 nni". When scallop density is low, however, fishing vessels disperse more widely. Such variations in the concentration of fishing activity have im- portant implications for impacts on habitat and finfish bycatch and provides insights into management strategies for bivalve fisheries. POPULATION STRUCTURE OF THE INDIAN SQUID, LO- LIGO DUVAUCELII ORBIGNY, 1848. IN IRANIAN WA- TERS OF THE OMAN SEA. Farhad Rajabipour,* Offshore Fisheries Research Centre, Chabahar, Iran. The population biology of the Indian squid Loligo duvauceli Orbigny, 1848 was described from monthly sampling. December 1997 to January 1999. Samples were caught as by-catch of bottom fishing trawlers from continental shelf area of Oman Sea at south- east of Iran. 1281 samples including 694 males and 587 females with the mean length of dorsal mantle and weight of 109.8 ± 34.95 mm and 48.32 ± 36.23 gr for males, and 1 10.6 ± 22.4 mm and 54.34 ± 28.35 gr for females, were collected. Maximum length (ML) of dorsal mantle in males and females was observed in January and June. Length-weight relationship was acquired. It is reversed at 72 mm length of dorsal mantle in logarithmic model. ML50 of males and females is 15.4 cm and 8.7 cm. There is no significant difference between females and males frequencies at the beginning of simimcr and ending of winter. NGI and GSl indices detected that the pick of reproduction occurs at the begin- ning of summer and winter. Males have three length classes, two recruitment peaks at beginning of summer (17.4%) and winter (82.6%), but females have only one length class and two recruit- ment peaks at beginning of winter {58.979'f ) and summer (41.3%); (tO = 1.4/year). Age and monality indices were measured. Catch rate of L. diivcniceli in Iranian waters of Oman Sea was 0-100 kg/hour. RECRUITMENT DYNAMICS OF NORTHERN SHRIMP (PANDALUS BOREAUS) IN THE GULF OF MAINE. Anne Richards,* Michael Fogarty, and David Mountain, NCAA Northeast Fisheries Science Center. Woods Hole, MA 02543; Mirta Teichberg, Boston University Marine Program, Woods Hole, MA 02543. The western Gulf of Maine is the southern limit of distribution for northern shrimp Pandalus borealis. Previous studies showed that recruitment of northern shrimp in the Gulf of Maine is sig- nificantly affected by spring surface temperatures as well as spawning stock biomass. The temperature effect corresponds to the period of planktonic larval development. The purpose of this study was to extend these analyses by examining the influence of addi- tional environinental factors (freshwater runoff, wind patterns, and climate indicators) on recruitment and to consider mechanisms for the spring surface temperature effect. Runoff and wind patterns strongly affect circulation within the western Gulf of Maine and thus may affect recruitment through effects on retention and/or transport of larvae. Results to date indicate no significant effect of mean river discharge, timing of peak discharge, other temperature effects, or the NAO winter index; however, the spring surface temperature effect continues to be significant. The match- mismatch hypothesis is a possible explanation for this effect, and we are evaluating the hypothesis by developing estimates of timing of onset of the spring phytoplankton bloom along with estimates of timing of the shrimp hatch. DEEP SEA RED CRABS OFF SOUTHERN NEW EN- GLAND: HAS THERE BEEN A FISHERY IMPACT ON THE POPULATION? James R. Weinberg* and Charles Keith, NMFS. NEFSC. 166 Water St., Woods Hole, MA 02543. A directed trap fishery for the deep-sea red crab, Chaceon (formerly GeiyoiO i/iiiiit/Kcdeiis Smith, has taken place off the coast of southern New England since the mid-1970s. These slow- growing crabs occur from about 250-1000 m, and males grow larger than females. The National Marine Fisheries Service con- ducted a survey of population size-structure in 2001 and measured the relationship between carapace width and body weight. This inforination had not been updated since the late 1970s. Given that the fishery targeted large males, we examined whether there was a decline over tmie in the percentage of large males in the population and in the individual weights of large males. Overall, the percent- age of large males in 2001 was lower than in 1974, whereas the fraction of large females either stayed the same or increased slightly during the same time period. Data collected between 1973 and 2001 suggested that male body weight, for a given body size, has declined over time. There was no trend over time in female body weight. Thus, compared to the 1970s, the population appears to have a lower percentage of large males in 2001, and these males have lower biomass for their carapace size. The results might be due to harvesting of large, heavy males over time; however, the surveys are too widely spaced in time, and too few in number, to draw strong conclusions about causality. 418 Abstracts. 2002 Aniiiwl Meeting, April 14-18. 2002 National Slicllfisheries Association. Mystic. Connecticut DISEASES OF SHELLFISH: CONTRIBUTED SESSION MORTALITIES OF CULTURED ABALONE, HALIOTIS IRIS, INFECTED BY A NOVEL HAPLOSPORIDIAN. B. K. Diggles, P. M. Hine, V. L. Webb, and E. W. Maas, National Institute of Water and Atmospheric Research, Kilhirnie. Welling- ton. New Zealand; J. Nichol and S. Wakefield, School of Medi- cine. University of Otago. Wellington South. New Zealand: R. Roberts, Cawthron Institute. Nelson. New Zealand; C. S. Fried- man. School of Aquatic and Fishery Sciences. University of Washington. Seattle. WA; N. Cochennec-Laureau, Laboratoire de Genetique et Pathologic. IFREMER. La Tremblade. France; K. S. Reece and N. A. Stokes.* Virginia Institute of Marine Sci- ence. Gloucester Point. VA. Chronic mortalities of juvenile abalone, Haliotis iris, occurred in a commercial culture facility in New Zealand during the Austral summer and autumn of 2000 and 2001. Histopathology of mori- bund abalone showed heavy systemic infections of a uni- to multi- nucleate protozoan parasite associated with severe tissue damage. Heavily infected abalone exhibited lethargy, loss of righting reflex, and weak surface adherence. Mortality levels reached 90% in the affected raceways. The parasite was identified as a haplosporidian based on TEM and molecular analyses. Ultrastructural character- istics of the parasite included the presence of multinucleate Plas- modia, lipid droplets, an anastomosing endoplasmic reticulum and the production of haplosporosome-like bodies from nuclear mem- brane-bound golgi apparatus that matured into haplosporosomes. Molecular confirmation of the TEM identification was accom- plished by performing in situ hybridization (ISH) and by PCR- amplifying and sequencing the parasite's small subunit ribosomal RNA (SSU rRNA) gene. A DNA probe specific for several mem- bers of the haplosporidia exhibited hybridization to the Plasmodia in ISH of infected animals. The SSU rRNA gene sequence was novel, but phylogenetic analyses strongly supported grouping this parasite with the haplosporidians. The parasite was at the base of the phylum Haplosporidia. ancestral to Urosporidiiim crescens and the Hiiplosporidtiitn. Mincliiniu. and Bonamia species. LOW SALINTY TOLERANCE IN MANILA CLAMS VENERUPIS PHILIPPINARUM. Ralph Elston, AquaTechnics/ Pacific Shellfish Institute. PC Box 687. Carlsborg. WA 98324 USA; Dan Cheney. Brian MacDonald. and Andrew Suhrbier, Pacific Shellfish Institute, Olympia. WA. The manila clam. Venenipis philippiiuiniin. is the second most important product of molluscan aquaculture on the west coast of the United States and is grown on several other continents. We evaluated the tolerance and the low salinity response in this clam species using experimental exposures of 17 populations of manila clams. Ambient seawater (30 psu) and freshwater were first tem- perature conditioned, then mixed using aperture controlled flows into incremental salinity head tanks that supplied the test cham- bers. Water supplied to the test tanks was supplemented with algal feeds. Low salinity tolerance of clams was determined by perform- ing exposures of up to four weeks in duration at constant salinities, followed by a recovery period. Clams were tested in salinity in- crements of 2.5 psu ( =ppt). Tolerance to low salinity events in clams is both a function of survival response (duration of shell closure) and the physiological adaptability of the clams to exist in low salinity environments. All individual clams from all sources were tolerant to \5 psu but populations showed variable proportions of individuals with tol- erance to 12.5 psu and none were tolerant to long term exposure to 10 psu. Variable tolerance to 12.5 psu appears to be a function of the effectiveness and duration of the shell closure response. All clains could withstand 6 days at 5 psu. but complete group mor- tality occuned by 12 days at 5 psu. All clams could withstand 8 days exposure to 10 psu but complete group mortality occurred by 14 days at 10 psu. Little effect of temperature was found on the clams" tolerance to marginal low salinities. The low salinity ex- posures resulted in alterations in the digestive gland epithelium including loss of granulation, cellular swelling and cellular slough- ing. We concluded that candidates to .select for low salinity toler- ance are available in most populations but some populations are enriched with individuals with tolerance to the marginal salinity (12.5 psu). Research supported by Saltonstall-Kennedy project NA96FD0194. National Marine Fisheries Service. U.S. Depart- ment of Commerce to Pacific Shellfish Institute. Olympia. Wash- ini;ton. A SPECIFIC PATHOGEN FREE CULTURE SYSTEM FOR C. GIGAS LARVAE AND SPAT. Sean E. Matson* and Chris- topher Langdon. Hatfield Marine Science Center. Oregon State University, Newport, OR 97365. The Molluscan Broodstock Program (MBP). a selective breed- ing program for the Pacific oyster. Crasso.slrca .(•ificis. uses a Spe- cific Pathogen Free culture system for all production and mainte- nance of larvae, spat, broodstock and microalgae. This system is necessary to exclude infectious agents of Haplosporidian costale (Seaside Organism, SSO). which has been found in Pacific oysters grown in Yaquina Bay, Oregon, where the program draws its seawater. and for the safe outplanting of MBP spat in field test sites along the West coast (USA). All seawater entering MBP facilities is filtered through sand, diatomaceous earth, and a series of 20. 5. and 1 jjLm cartridge filters. Seawater to mass algal cultures and the nurserv is afso irradiated with UV-light at >30.000 micro- National Shellfisheries Association. Mystic. Connecticut Abslnicts. 2002 Annual Meeting. April 14-18. 2002 419 Watts-sec/cni" (MWS) as a back-up precaution. .Since the system's inception, no MBP spat have been identified as being contami- nated with SSO. or any other infectious agent. A series of labora- tory experiments was performed to assess the effects of UV v\'ater on larval growth and survival, spat growth and sur\i\al. and mi- croalgal culture density. Experiments with oyster larvae indicated that both the micro-filtration system and UV water treatment had a significant negative effect on larval growth (p = O.O(H)l). A significant reduction in growth was evident at UV intensities as low as 10.000 MWS (p < 0.05). Methods that have significantly improved larval growth, survival, speed to metamorphosis and spat growth within the SPF culture system include substituting a 0.2p.m filter and charcoal for a UV filter when rearing larvae, and the addition of calcium bentonite (2mg/ml/day) or calcium montmo- rillonite (3mg/ml/day) to larvae and spat cultures (p < 0.05). INTRAMITOCHONDRIAL CRYSTALS WITHIN THE HAEMOCYTES OF MUSSELS {MYTILUS EDULIS) EXPE- RIENCING UNEXPLAINED MORTALITIES. Gary R. Meyer,* and Susan M. Bovver, Department of Fisheries and Oceans, Pacific Biological Station. Nanaimo. British Columbia. V9R 5K6. Canada. Persistent mortality (having a cumulative total of about 75%) was observed between August 1999 and April 2000 amongst a stock of cultured blond mussels {Mytilus editUs selectively bred for their blond coloured shell trait) grown near Quadra Island. British Columbia, Canada. During this period, 7 samples of mussels (n = 30 per sample) were examined. Up to 27% had macroscopic le- sions (orange pustules) and up to 50% had histopathology consist- ing of multifocal and or diffuse haemocyte infiltration that con- tained abundant phagocytic cells and necrotic cellular debris usu- ally in the mantle/gonad and digestive gland. However in some instances, this pathology was also observed in the adductor muscle, gills, kidney or heart tissue. No etiological agent was evident. Intracellular mycobacteria-like organisms were observed within a few haemocytes in up to 20% of the mussels examined however their role in the disease remains unknown. During ultra- structural examinations, intramitochondrial crystalline arrays were commonly observed within necrotic or lysed haemocytes. These crystals were not observed "free" within the cytoplasm of affected cells and never observed in adjacent "healthy-looking" cells. The chemical composition and significance of these crystals is un- known. However, we speculate that they are likely composed of condensed protein. Similar crystals have been reported from the mitochondria of a wide variety of organisms and were usually affiliated with an abnormal situation such as disease, pathological process, or response to cell damage. ENVIRONMENTAL DETECTION OF THE RICKETTSI- ALES-LIKE PROKARYOTE CAUSING WITHERING SYN- DROME IN ABALONE. James D. Moore.* California Depart- ment of Fish and Game. UC Bodega Marine Laboratory. 2099 Westside Road. Bodega Bay. CA 94923: Carolyn S. Friedman, School of Aquatic and Fishery Sciences. Box 355020. University of Washington. Seattle. WA 98195. A gastrointestinal Rickettsiales-like prokaryote (WS-RLP) was recently identified as the causative agent of Withering Syndrome, a chronic, fatal disease of wild and cultured California abalone. Using a polymerase chain reaction (PCR) diagnostic method de- veloped to detect the WS-RLP in abalone gut tissue samples, we have amplified WS-RLP DNA from necrotic tissue held at room temperature for up to 7 days, from samples fixed and embedded in paraffin, and from seed abalone as small as 2mm. A non- destructive WS-RLP sampling method is desired since both wild and farmed abalone are highly valued. We developed a method involving tangential flow filtration that concentrates particles > 0.1 |j.m from large volumes of seawater. allowing purification of DNA for PCR from 15-(- liter samples. Although sample processing is laborious, using this method we have detected the pathogen DNA in effluent from abalone holding units in both experimental and culture facilities. The WS-RLP is present in feces from infected animals, and PCR using fecal material appears to be useful for efficiently detecting the WS-RLP in various stages of farm pro- duction. We are currently investigating an hypothesis that filter- feeding organisms concentrate the WS-RLP from endemic waters and can. thereby, be used to monitor WS-RLP presence in natural populations. Initial attempts to amplify WS-RLP DNA from gill or gut tissue of mussels (Mytilus spp.) being held with WS-RLP positive abalone have been unsuccessful. Yet it is clear that mo- lecular-based tools will significantly aid management of this dev- astating disease. Supported, in part, by California Sea Grant Col- lege and the Marine Region, California Department of Fish and Game. VIBRIO ASSOCIATED WITH JUVENILE OYSTER DUR- ING SUMMER MORTALITY IN FRANCE. J.-L. Nicolas and M. Garnier Laboratoire de Physiologie des Invertebres. Ifremer BP 70. 29 280 Plouzane. France; M. Gay and F. Leroux. Labo- ratoire de Genetique et de Pathologic. Ifremer. Ronces Les Bains, BPI33. 17390 La Tremblade. Ainong the hypotheses advanced to explain summer mortalities of oyster in France (Morest program), the bacterial disease ap- peared one of most relevant. Indeed, recent works of Lacoste (2fJOI ) and Waechter (thesis 2001 ) showed that of Vibrio spleiuU- dus (I and II) strains were able to kill the juvenile or adult oysters by bath or injection. To verify this hypothesis, oysters were sampled along the French coasts and analysed during mortality period. Only the half of moribund oysters were invaded by dom- inant bacteiia. Haemolyniph of other diseased oysters as well as 420 Ahstiacts. 2002 Annual Meeting. April 14-1 «, 2002 National Shellfisheries Associatum. Mystic, Connecticut healthy oysters was often colonised by more or less abundant and diversified bacterial flora. The isolation of a same dominant bac- terial strain on several oysters of the same batch, occurred only in hatchery and on only one oyster park. The taxonomic studies (by ARDRA of gyrase B. and sequences of 16S DNAr) made it pos- sible to classify in ? species these opportunist bacteria; V aestn- ariaims (3 types). I', splciulidiis (II) and V cifii;iiilUiriim. Except V. aestuarianus (type II). Phenotypical characters of these strains were fickle and could not be used to identify the bacteria. a P. alkmticiis and compare by alignnienl uilh those pailically characterize rRNA loci from other PcrkinsKs species. Actively growing cultures exhibited considerable si/e heterogeneity with a large proportion of clusters constituted by small cells. Behavior of P. iiilaiiticiis in culture resembles that of P. nuinnus. Optimization of the culture conditions for P. atlanticus is underway. Established clonal cultures will be useful for genetic characterization of this species, and to increase our understanding of its pathogenicity. [Supported by Grant NAO6RGOI0I-5 from ODRP. NOAA, through the MD Sea Grant College, to GRV, and the Portuguese Government PERKLAM Program to MLC). FURTHER MOLECULAR CHARACTERIZATION OF PERKINSUS ANDREWSI AND RELATED ISOLATES. Wolf T. Pecher, Jose A. F. Robledo, Cathleen A. Coss, and Gerardo R. Vasta, COMB. UMBI, University of Maryland, Baltimore. MD 21202. We previously described ultrastructure. behaviour in culture, and the rRNA locus of a Pfikiiisns species isolated from the baltic clam Macoma halthica. The morphological characterization did not reveal features remarkable enough to cleariy indicate that the isolate was a distinct Perkinxus species (Coss et al. 2001a). How- ever, the degree of difference of all rRNA genes and intergenic regions examined was comparable to or greater than differences between accepted Perkiii.siis species, which lead to its designation as P. aiulieusi n. sp. (Coss et al 2001a. b). Here we report further molecular characterization of P. aiulrewsi and the characterization of an additional isolate from the hard clam Mcneiuiria merce- luiriu. Based on the molecular characterization both isolates are closely related. Their relationship with other recognized Perkinsus species will be discussed. [Supported by Grant No. NA06RG010I-5 from ODRP. NOAA. through the Maryland Sea Grant Colleae. to GRV[. IN VITRO CULTURE OF PERKINSUS ATLANTICUS FROM CLAMS TAPES DECUSSATUS: CHARACTERIZATION OF THE rRNA gene. Jose A. F. Robledo* and Gerardo R. Vasta. COMB. UMBI. University of Maryland. Baltimore. MD 21202; Patricia A. Nunes and M. Leonor Cancela. Molecular Biology and Biotechnology. CMS-CCMar. University Algar\e-UCTRA. Campus Gambelas. XOOO-SIO Faro. Portugal. Perkinsus allaiiiicus cultures were derived from fresh hemolymph and gills, and from tissues incubated in fluid thiogly- collate medium from infected clams Tapes (Icciissanis from Al- garve (South of Portugal). P. uilaiuiciis cultures were established in the medium developed and optimized for P. mariniis. Prolifer- ating cells were cloned by limiting dilution, and the species iden- tity was established by applying two PCR-based P. uihmticiis- specific diagnostic assays and by sequencing the RNA locus. We provide for first time the complete sequence of the rRNA locus of GENE DISCOVERY IN PERKINSUS MARINUS USING EX- PRESSION SEQUENCE TAGS (EST). Jose A. F. Robledo,* Eric J. Schott. Adam G. Marsh, and Gerardo R. Vasta, COMB. UMBI. University of Maryland. Baltimore. MD 21202. Since the late 1940s the protistan parasite Perkinsus marinus has been associated with mass mortalities of the eastern oyster. Crassostrea virginica. Based on decades of research, several ap- proaches have been proposed to control ""Dernio" disease includ- ing eariy disease detection, selective breeding of disease-resistant oysters, and treatment with anti-parasitic agents. However, none of these approaches has proven effective. In recent years virtually all fields of biology have benefited from the information generated by genomic approaches. We are building a P. marinus EST database sequencing cDNAs from two P. marinus Lambda ZAP libraries constructed using P. marinus propagated in standard culture me- dium and in medium supplemented with C. virginica serum. From the first 300 ESTs two findings are evident; there are many ex- pressed genes whose products are known to be involved in \iru- lence of other parasites and there are notable differences in the classes of genes expressed in control versus serum-supplemented cultures. As the number of ESTs increases, so will prospects for identifying new targets for therapy and for understanding the bio- logical basis of parasite virulence, infectivity. and pathology. THE NRAMP GENE AND COMPETITION FOR AVAIL- ABLE IRON BETWEEN CRASSOSTREA VIRGINICA AND PERKINSUS MARINUS. Jose A. F. Robledo* and Gerardo R. Vasta. COMB. UMBI. University of Maryland. Baltimore. MD 21202. In the past decades Perkinsus marinus has produced extensive damage to oyster bars with catastrophic consequences for shell- fisheries and the health of coastal waters. Despite intense parasite pressure, no Dermo-resistant oysters have been identified so far in natural populations. The identification of genes that are directly linked to disease-resistance and assessment of their value as ge- netic markers, may facilitate the establishment of disease-resistant National Shellfisheries Association. Mystic, Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 421 oyster strains. Most parasites have strong iron requirements and have developed efficient mechanisms for iron acquisition from their hosts. Reciprocally, most hosts have developed mechanisms to prevent pathogens from acquiring iron while maintaining avail- ability for their own cells. Iron sequestration from the pathogen is also a non-specific host response to infection (nutritional immu- nity). The mammalian natural resistance-associated macrophage protein (Nramp) is a membrane transporter thought to be a deter- minant of resistance/susceptibility to intracellular pathogens. We have cloned Nramp from C. virgiuica and P. iiniri)iiis. used them to screen genomic libraries, and examine their gene organization. The characterization of these genes and their products in both host and parasite will provide insight into their competition for iron, and yield information on the mechanisms underlying disease sus- ceptibility [Supported by Grant No NA06RG0101-5 from ODRP. NOAA, through the Maryland Sea Grant College, to GRV]. CHARACTERISATION OF GENE EXPRESSION IN RE- SPONSE TO PERKINSUS MARINUS AND HAPLOSPO- RIDIUM NELSONl INFECTIONS IN THE EASTERN AND PACIFIC OYSTERS. Arnaud Tanguy.* Susan E. Ford, and Ximing Guo, Haskin Shellfish Research Laboratory, Institute of Coastal and Marine Sciences, Rutgers University, 6959 Miller Av- enue. Port Norris, NJ 08349. The eastern oyster Crassostrea virginica has two major dis- eases: Dermo caused by the parasite Perkinsiis mariniis and MSX by Haplosporidiwn nelsoni. Effects of infection in C. virginica range from reductions in condition index, hemolymph protein con- centrations and lysozyme activity to decline in reproductive out- put, retarded growth and death. MSX-resistant strains of C. vir- ginica have been developed at Rutgers University, and the Pacific oyster C, gigas appears to be resistant to both diseases. At present, however, we know little about molecular mechanisms of infection and host defense against the two parasites. We have begun a study searching for genes involved in host response to Dermo and MSX infections, using subtractive expression analyses. For Dermo, con- trolled artificial infections are conducted in both C. virginica and C. gigas. For MSX, infected wild oysters and uninfected resistant oysters, which were naturally exposed to the parasite, were used to construct the subtractive library. Nine subtractive libraries were made: six for Dermo and three for MSX exposures. About 70 clones were selected for sequencing from each library. Among the first batch of sequences analyzed, one-third matched with known genes in GenBank. and the other represent unknown genes. A detailed characterization of all sequences will be presented at the meeting. BIVALVE HABITAT SUITABILITY AND THE ROLE OF BIVALVES IN ECOSYSTEMS HOW COMMUNITY-BASED OYSTER RESTORATION CAN ENHANCE RESEARCH EFFORTS: EXAMPLES FROM SOUTH CAROLINA AND ALABAMA. Loren D. Coen,* Marine Resources Research Institute. SCDNR. 217 Fort Johnson Rd.. Charleston, SC 29412; Richard K. Wallace, Auburn University Marine Extension and Research Center. 4170 Com- manders Dr.. Mobile. AL 36615; Nancy Hadley, Marine Re- sources Research Institute, SCDNR. 217 Fort Johnson Rd.. Charleston. SC 29412. In both South Carolina and Alabama, we have been utilizing community-based programs to construct and/or enhance larger scale oyster restoration efforts at selected sites using criteria based on science and best management policies. We are trying to use these sites as research platforms to test various restoration meth- odologies such as type of shell, alternative substrates, reef con- figurations, time/method of planting, and stabilizing meshes. In South Carolina we are actually placing "habitat' (over 40 tons of shell in 2001 ). rather than oysters into tidal creeks across the state. Routine monitoring (chemical, physcial and biological parameters) is being conducted by both trained volunteers (students and adults) and researchers. In South Carolina, we have also meshed our reef monitoring efforts with HAB efforts through the NOS's South Carolina Phytoplankton Monitoring Network (SCPMN) and we are using the reefs also as 'living classrooms' for a variety of groups (e.g.. one built adjacent to the SC Aquarium). In Alabama, oysters produced by volunteers using gardening techniques are being planted at relic reef sites that were already enhanced for sportfishing (as artificial reef fish habitat). This strategy creates opportunities for restoration research which can be supported by multiple interest groups. By constructing oyster reefs, we are im- proving the suitability of sites for oyster reestablishment, thereby improx ing habitat quality for reef residents and transient species. OYSTER HABITAT SUITABILITY AS A COMPONENT OF RESOURCE MANAGEMENT. William S. Fisher,* US En vironmental Protection Agency, National Health and Ecological Effects Research Laboratory, Gulf Ecology Division. Gulf Breeze. FL 32561. Economic and ecological issues have led resource managers to examine depletion of eastern oyster reefs along the U. S. Gulf of Mexico and Atlantic coasts. Crassostrea virginica is a lucrative commercial species (over $60M in 2000) that also supports eco- system integrity by providing enhanced vigor, organization and 422 Abstracls. 2002 Annual Meetnig, April 1 4- IS. 2002 National Shellfisheries Association. Mystic. Connecticut resilience. These values are threatened by overfishing, habitat per- turbation and land use changes that are also economically-driven, but diminish the survival of both oysters and reefs. Relevant and defensible scientific information is required to assess costs and benefits of healthy and productive oyster reefs and to establish management goals for their conservation, mitigation and restora- tion. Characterization of environmental conditions that support oyster survival and propagation is a requisite for this assessment. In this regard, habitat suitability indices (HSI) have been devel- oped that can be used to identify potential sites for initigation and restoration. However, greater attention is needed on conditions for spat settlement, a bottleneck life-stage for oyster populations. Al- though spat may settle on many substrates, success is greatest on bivalve shells and limited by even thin layers of sediment depo- sition. Better understanding of eunents. turbidity, sediment depo- sition and other conditions that influence larv al settling are needed to improve HSI evaluations and the potential for successful oyster reefs. CONTINUOUS MONITORING OF PUMPING PRESSURE AND VALVE GAPE IN THE O'i STER CRASSOSTREA VIR- GINICA IN RESPONSE TO CHANGES IN ENVIRONMEN- TAL PARAMETERS. Dana M. Frank* and J. Evan Ward. University of Connecticut. Department of Marine Sciences, 1080 Shennecossett Road. Groton. CT (16340. We have developed an optical biomonitor capable of continu- ous short and long-term recording of pumping pressure and valve gape in bivalve molluscs. The pressure sensor is situated in the suprabranchial chamber of the oyster. The valve gape sensor is attached to the right valve during experimental trials. With this arrangement, we are able to examine the relationship between valve gape and pumping pressure, in response to measured changes in environmental parameters such as temperature, food availability, dissolved oxygen concentrations and current velocity. Laboratory trials are cuirently underway to establish responses to some of these variables with the goal of deploying the systetn. along with sensor arrays to measure these parameters, in the field. Using this method, we hope to expand the scope of our under- standing about the compensatory responses of bivalve molluscs to changes in environinental parameters //; situ. Additionally, it al- lows us to explore more thoroughly the mechanisms available to shellfish for controlling pumping rates. Results of laboratory trials have revealed multiple patterns of association between pumping and valve gape and have established that the system is able to record changes in these physiological paiameters in real lime. A SIMPLIFIED SESTON UPTAKE MODEL FOR BI- VALVES: PRELIMINARY FIELD TESTS. Raymond Grizzle* and Jennifer Greene. University of New Hampshire, Jackson Estuarine Laboratory. Durham. NH 03824: Mark Luck- enbach, Virginia Institute of Marine Science. Gloucester Point, VA 23062. A spreadsheet-based model was developed to predict the per- cent of the total water column cleared of seston by suspension feeding bivalve molluscs on a daily basis: 9c Water Clearance = (A x B x C)/(D x E) x 100 where A = mean bivalve density (# ind/nr). B = mean individual clearance rate (mVindividual/da). C = bottom area of reef (nr). D = cross-sectional area of water column (ni"). and E = mean water flow speed (m/da). It is designed for use in estimating the impacts of shellfish reef restoration projects on water quality. The tnajor simplifying assumption is a completely mixed water column, and mean clearance rates are based on literature values. We tested the model over a portion of one tidal cycle at two different sites, both blue mussel (Myrilus edulis) reefs, one natural and one con- structed. On both occasions, upstreain and downstream locations were sampled concurrently at 30-min intervals for 2 to 5 hr for in situ fluorometry and/or chlorophyll a from a fixed height -10 cm above the bottom, and water depth and mid-depth water flow speed were measured. Replicate 0.16 nr quadrats were sampled on each reef, and mean density and size (shell length) of the live mussels were determined. The model predicted average clearance/seston uptake for the ambient water flow conditions, mussels densities, and other conditions measured on the constructed reef to be <2% of the total water flow. The field trial data cortoborated the inodel predictions, showing no significant differences between mean up- stream and downstream samples of chlorophv II a. For data from the natural reef, the inodel predicted a mean of 1 1 .b'k total clear- ance. During the field trial, measured chlorophyll concentrations were significantly lower downstream with a mean difference (rep- resenting seston uptake) of l6.3'/f . In situ fluorometry indicated an overall uptake of 28%. In conclusion, both preliminary tests showed good agreement with model predictions. Further testing is underway to determine the model's general applicability. THE INFLUENCE OF REEF ARCHITECTURE AND SCALE ON SUCCESS OF OYSTER REEF RESTORATION. Mark Luckenbach.* Janet Nestlerode, Paige Ross. Jr. and Alan Birch, Virginia Institute of Marine Science. College of William and Mary. Wachapreague, VA 23480. Current efforts to restore oyster reefs in the Chesapeake Bay entail the placement of substrate on the shallow seabed to promote oyster settlement and reef development. Because oyster shell and other alternative cultch are in short supply and/or costly, it is important that we optimize our placement of this material to inaxi- mize restoration success. In several field experiments we have National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 423 been investigating the role of three components of reef architec- ture— vertical relief, interstitial space and spatial scale — on the development of oyster populations and associated fauna on re- stored reefs. Our results indicate that modest differences in vertical relief and interstitial space have dramatic effects on early post- settlement survival of oysters and the development of viable reefs. To investigate the issue of scale and the role of reef size we have initiated a large-scale restoration experiment in the Chesapeake Bay. In a replicated block design, we have consti-ucted high relief reef bases ranging in size from 400 ni" to 8000 nr and are char- acterizing the development of oyster populations and associated assemblages on the reefs. Oyster recruitment and survival patterns from the first year of this study reveal significant effects of scale. EFFECTS OF CRASSOSTREA VIRGINICA POPULATIONS ON SEDIMENTATION. PHYTOPLANKTON SPECIES COMPOSITION AND AMMONIA CYCLING IN EXPERI- MENTAL MESOCOSMS. Jennifer Mugg Pietros and Michael A. Rice. Department of Fisheries. Animal and Veterinary Science. University of Rhode Island, Kingston, RI 0288 1 . To determine the effects of oyster populations on water quality, a mesocosm study was performed from June to October 2000. Mesocosms with a volume of 13.000 L were used, in which there was triplicate control tanks without oysters and triplicate experi- mental tanks each with 200 oysters (=35 mm in valve height; nominally filtering about 55L day"' ind"'). Experiments were run sequentially in time for three week periods with water exchange rates ranging from 0% to 100% per day (0 to 13,000 L day"'). Several parameters were measured and compared between the con- trol and experimental tanks, including chlorophyll-a, particulate organic and inorganic matter, sedimentation rates, nitrate, ammo- nia, phytoplankton species and numbers, and oyster growth rates. There were no significant (P < 0.05) differences between tanks for most parameters, with the exceptions of rates of sedimentation and species composition of phytoplankton in the water column. Dia- toms of the genus Nitzschia were predominant in mesocosms with oysters, and in control tanks, Skeletonema were dominant. Rates of ammonia excretion by oysters of various sizes was determined by the sensitive salicylate-hypochlorite method, allowing for rapid determination of excretion rates to minimize biases introduced by volatilization or tranformation of the ammonia over time. The excretion of ammonia by oysters can be described by the allome- tric equation E = 50.65w"'''^"-. when E is the excretion rate in |j.g NH,-N hr"' and w is the dry soft tissue weight in g. Based upon these data, it would be expected in the three-week experimental period that an additional 470 |xg L"' ammonia above the average control concentrations of about 40 jxg L"' would be present in each mesocosm with oysters, but no significant difference was noted between the experimental and control tanks. This suggests that there is rapid cycling of ammonia, perhaps by uptake by the rapidly regenerating phytoplankton populations. This is publica- tion number 3910 of the College of the Environment an Life Sci- ences at the University of Rhode Island, with support from the RIALS under project number H-886. A MODIFIED HABITAT SUITABILITY INDEX FOR THE EASTERN OYSTER. CRASSOSTREA VIRGINICA. Thomas M. Soniat.* Department of Biology. Nicholls State University. Thibodaux. LA 703 10. A series of habitat suitability index (HSI) models have been developed for the U.S. Fish and Wildlife Service for use in envi- ronmental impact and planning studies. HSI models assess habitat quality for particular species; their output is a numerical index from 0 to 1. where 1 represents optimal habitat and 0 represents unsuitable habitat. An HSI for eastern oysters was developed by Cake (1983) and modified by Soniat and Brody ( 1988). The Cake model includes variables important to larval stages (presence of cultch. mean summer salinity and a gregarious settling factor, mea- surable as the mean abundance of living oysters) and post- settlement stages (historic mean salinity, frequency of killing floods, substrate firmness, oyster drill density and intensity of the parasite Perkiiisiis murimis). The modification of Soniat and Brody removes the gregarious settling factor from the model, simplifies model structure, and better accounts for the negative effects of high salinity, parasitism and drill predation on oysters. The modi- fied HSI should be tested against a new, independently-collected data set. USE OF OYSTER HABITAT BY REEF-RESIDENT FISHES AND DECAPOD CRUSTACEANS IN THE CALOOSA- HATCHEE ESTUARY, FLORIDA. S. Gregory Tolley.* Aswani K. Volety. and Emily C. Lindland. Florida Gulf Coast University. Fort Myers. PL 33965; James T. Winstead. United States Environmental Protection Agency, Gulf Breeze, FL 32561. Habitat suitability of oyster reefs for fishes and decapod crus- taceans was examined monthly at three sites in the lower Caloosa- hatchee Estuary. At each site, I-m" lift nets containing approxi- mately 5 liters (volume displacement) of oyster clumps were de- ployed for a period of two weeks. Fishes and decapods sampled averaged 94 individuals m"~ or 23 individuals 1"' oyster cluster. Nine species of decapods and 18 species of fishes were identified: reef-resident fishes included Gobiesox slntmosus. Chasmodes saburrae. Gobiosoma robustum. and Opsanus beta: the xanthid Eui-\panopeus depressus and the porcellanid Petrolisthes armatus represented the dominant decapod crustaceans. Organism abun- dance ranged from 30-197 individuals m"", density ranged from 5.2-63.3 individuals 1"' oysters, species diversity (H') ranged from 0.05-1.75, species richness ranged from 4—11 species per sample, and species dominance ranged from 24.8-97.7%. Species diversity, species dominance, and organism abundance all differed significantly among stations. Among-station differences in organ- 424 Ahstmcts. 2002 Annual Meeting. April 14-1 S. 2002 National Shellfisheries Association. Mystic. Connecticut ism density (individuals 1"' oyster cluster) were found for the fishes Chasmades sulntrrae, Gohiesox stnimosiis. and Opscmus beta, and foi' the crabs Eiirypaiiopciis ili'pressiis. Menippe mene- naria, Panopeiis sp.. and Perriil is/lies annaliis. Of these, only E. depressus exhibited significant seasonal variation in density (Kruskal-Wallis test; n = 79, p = 0.00007). being more abundant durina the warmer months. EFFECTS OF FRESHWATER RELEASES AND SEASON ON OYSTERS {CRASSOSTREA VIRGINICA) IN CALOOSA- HATCHEE ESTUARY, FLORIDA. Aswani K. Volety* and S. Gregory Tolley, Division of Ecological Studies, Florida Gulf Coast University, Fort Myers, PL 3396.^; James T. Winstead, U.S. Environmental Protection Agency. Gulf Ecology Division. Gulf Breeze. FL 32561. The influence of freshwater releases and season on disease prevalence and intensity of Perkiiisii.s luuriinis. condition index, gonadal condition, recruitment potential, and growth of oysters was examined monthly at five locations along the Caloosahatchee estuary. Florida. Temperatures and salinities at the study sites ranged from I6-33°C and 3-39 ppt respectively. Higher tempera- tures and salinities favored P. mahniis. While prevalence of P. mariniis ranged from 0 (after heavy rainfall and/or freshwater wa- ter releases) -70"7f (during dry winter months), overall intensity was light (0-1.3). Comparison of mortality among sites indicated that juvenile oysters tolerated salinities of 5-38 ppt. Condition index of oysters was influenced by reproductive cycle and spawn- ing events. Spat recruitment (1-5 spat/shell) and growth of juve- nile oysters was higher at sudtidal than at intertidal locations, where sparse oyster distribution and swift currents appeared to limit growth and .settlement success. Oysters were reproductively active between March and October, with peak reproduction occur- ring from June-October. Overall, results suggest that periodic short-term freshwater releases may benefit oysters by lowering the salinity and thus the intensity of Perkiiisus nniriiuis. Laboratory studies suggest that adult oysters can tolerate low salinities (3ppt) for 1-2 weeks. Given high salinities and infection intensities dur- ing winter months, it is recommended that freshwater releases take place during winter instead of current summer releases. PARASITIC AND SYMBIOTIC FAUNA INHABITING OYSTERS [CRASSOSTREA VIRGINICA) AND MUD CRABS [PANOPEVS HERBSTII) SAMPLED FROM THE CA- LOOSAHATCHEE ESTUARY, FLORIDA. James T. Win- stead,* United States Environmental Protection Agency. Gulf Breeze, FL 32563: Aswani K. Volety and S. Greg Tolley, Florida Gulf Coast University, Fort Myers, FL 33965. Oysters, Crassostrea virginica. inhabiting five sites in the Ca- loosahatchee River estuary were studied over a 13 month period to determine the suitability of oyster habitat in relation to their health and condition. Histological examination of 650 oysters ( 10 animals per station per month) revealed a varied parasitic and symbiotic fauna inhabiting these molluscs at all stations. Organisms found included protozoans (Nematopsis sp. and Perkinsus mariniiin). di- genetic trematodes (Bucephalus cucuhis and the first report from this area of what appears to be Echinosloma sp.), ceslode larvae [Tylocephaliim sp.) and a hydrozoan inquiline symbiont [Eutima sp.). In addition, a significant number of mud crabs, Panopeus iicrhstii. inhabiting oyster habitat at two sites were found to be parasitized by rhizocephalan barnacles, Loxothylacus sp. Preva- lence and pathological consequences of these organisms and the role oyster-parasite relationships may play as an indicator of eco- system complexity (biodiversity) is discussed. POSTERS HABITAT AND ECOLOGY OF GREEN MUSSELS, PERNA VIRIDIS, IN FLORIDA. Patrick Baker,* Fisheries and Aquatic Sciences. University of Florida. Gainesville. FL 3261 1 ; Amy Ben- son, Florida Caribbean Science Center. USGS. Gainesville. FL 32653. The green mussel. Pcrnii \iridi.\. was introduced to Tampa Bay, Florida, prior to 1999. It is abundant from Tampa Bay to Charlotte Harbor and has appeared elsewhere in Florida. As with invasive freshwater zebra mussels (Dreissena polymorpha). early green mussel sightings were in industrial water intake systems, and green mussels currently foul many industrial and municipal structures. Green mussels show other parallels to invasive zebra mussels, such as reaching densities of over 10.000 per m" in an ecosystem that lacks a close native analog. It was with zebra mussels in mind that we initiated a study to examine the habitat and ecological impacts of green mussels. Based on preliminary results, however, predic- tions of green mussel ecological impacts should not be modeled upon zebra mussel impacts, for several reasons. First, green mus- sels in Tampa Bay have been limited to artificial substrata, such as pilings and buoys. Contrary to expectations, they rarely appear on mangroves, nor do they overgrow benthic sediments. Second, green mussels have invaded a habitat that already has a high di- versity of potential competitors, such as barnacles and oysters, and known molluscivores, including flatworms, crabs, stingrays, and fish. Third, there is no early evidence of negative impacts on native taxa, with the possible exception of oysters on artificial substrata. Green mussels increase the structural diversity of the fouling com- munity on pilings, and both the shells and the interstitial space are occupied by numerous other taxa, both native and nonindigenous. At least five native bivalves commonly occur in green mussel aggregations, for example. Ecosystem impacts of this abundant invader are almost certain to develop, but may differ considerably from those of other mussel-like invaders. Green mussels have survived several winters in Florida, and appear to reproduce more than once annually. Their initial spread National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting, April 14-18. 2002 425 southward is consistent with dominant coastal currents, which could eventually can'y larvae around to Florida's east coast. Ad- ditionally, juveniles have become abundant in aquaculture systems in Charlotte Harbor, and culturists are concerned about both their impacts on seawater systems and their potential spread via human vectors. The northern (thermal) limit of green mussels has not yet been determined. INTRODUCING THE CLAMMRS PROJECT: CLAM LEASE ASSESSMENT. MANAGEMENT AND MODELING USING REMOTE SENSING. Shirley Baker,* Ed Phlips, and David Heuberger, Department of Fisheries and Aquatic Sciences, Institute of Food and Agricultural Sciences. University of Florida, Gainesville. FL 3265.^; Clay Montague. Department of Environ- mental Engineering Sciences, University of Florida, Gainesville, FL 32653; Leslie Sturnier. Cooperative Extension Service, Insti- tute of Food and Agricultural Sciences, University of Florida, Ce- dar Key, FL 32625. In cooperation with the Florida Department of Agriculture and Consumer Services, Division of Aquaculture, we are addressing the needs of the emerging Florida hard clam (Mercenaria inerce- riaria) aquaculture industry. The needs we are addressing include: 1) Risk of catastrophic crop loss; 2) Optimum farm management practices; and 3 ) Selection of new aquaculture areas. Adoption of remote sensing technologies in management practices will enhance the sustainable development of open-water clam farming by in- creasing production, farm efficiency, and profitability. Specific objectives are to; 1 ) Install remote water-quality and weather sta- tions in clam aquaculture areas to provide growers with timely information important to their management decisions; 2) Create a water-quality database to be used by the pilot Cultivated Clam Crop Insurance program to document events associated with crop loss; 3 1 Develop new techniques to monitor changes in natural food abundance and quality, such as the use of satellite imaging; 4) Develop a clam production model to examine optimum manage- ment practices to increase production and profitability; and 4) Use the production model and remote water-quality monitoring in se- lection of new highly productive aquaculture areas in Florida. HEMOCYTES OF HOMARVS AMERICANUS STAINED WITH A MODIFIED WRIGHT-GIEMSA STAIN: DE- SCRIPTION AND COMPARISON TO CURRENT CLASSI- FICATION SCHEMES. Andrea Battison* and Barbara Hor- ney, Department of Pathology and Microbiology; Richard Caw- thorn and Allan Mackenzie, The AVC Lobster Science Centre. Atlantic Veterinary College. Charlottetown. University of Prince Edward Island. Charlottetown. PE, Canada. CIA 4P3. Transmission electron microscopy and phase contrast light mi- croscopy are common methods used to examine crustacean hemocytes. Using these techniques, three types of hemocytes are currently recognised in Hoinanis americanns and many other de- capod crustaceans; Large Granule. Small Granule, and Hyaline hemocytes. These procedures are. however, either time consum- ing, costly or. do not provide a permanent record of the results. Hemocytes of H. uiiu-iicaiuis were classified, based on cytoplas- mic and nuclear morphology, after staining cytocentrifuged prepa- rations with a modified Wright-Giemsa stain. Up to 1 1 different hemocytes. Types 1 to 11, could be identified in some samples. Morphologic similarities to the Large Granule and Small Granule hemocytes were observed. Based on similarities in appearance and alterations in number during Aerococcus viriclmis infections, the Type 1 hemocyte is considered the morphologic and functional equivalent of hemocytes in the Small Granule hemocyte category with the Type 6 hemocyte being its immediate precursor. Types 2, 7, and 8 may be subsets of hemocytes included in the Large Gran- ule hemocyte category. A counterpart of the hyaline cell has yet to be conclusively identified. This technique should prove useful in a research setting when alterations in number and types of hemocytes are being evaluated for assessment of lobster health. IMPACT OF ATRAZINE ON ANEUPLOIDY IN THE PA- CIFIC OYSTER. CRASSOSTREA GIGAS. Karine Bouilly. Alexandra Leitao, Helen McConibie, and Sylvie Lapegue. IFREMER. Laboratoire de Genetique et Pathologic. B.P. 133. 17390 La Tremblade. France. Aneuploidy has previously been reported in the Pacific oyster. Crassostrea gigas, and has been shown to be negatively correlated with growth. This is especially important since high variability of growth rate is one of the major problems in the aquacultural pro- duction of the species. The present study investigated the effect of an environmental factor on the level of aneuploidy. Crassostrea gigas animals at adult and spat stages were subjected to different concentrations of atrazine representing peak values found in the Marennes-Oleron Bay (A; 0.01 mg/1) and a value ten times higher (B: 0.1 mg/1). Although atrazine did not show any effect on the oyster mortality, significant differences in aneuploidy level were observed between the different treatments (8% for control, 16% for treatment A and 20% for treatment B). Moreover, the same level of response was observed at adult and spat stages. Finally, the offspring of oysters previously exposed did not show any differ- ence in larval growth but the hatching rate differed (74% for con- trol, 62% for treatment A and 55% for treatment B). This is the first evidence for an environmental cause of aneuploidy in the Pacific oyster. Crassostrea gigas. STATUS OF THE MYTILUS EDULIS STOCK WHICH SUP- PLIES HIGH-QUALITY SPAT TO MUSSEL GROWERS IN ILES-DE-LA-MADELEINE (GULF OF ST. LAWRENCE). Francois Bourque and Bruno Myrand, Station technologique maricole des Iles-de-la-Madeleine. Cap-aux-Meules. Canada. GOB IBO; Marcel Roussy, Centre aquacole marin. Grande-Riviere, Canada. GOC 1 VO. Mussel growers in Iles-de-la-Madeleine rely mostly on Bassin du Havre-Aubert for spat supply. This small (-3 km^) and shallow 426 Abstmcrs. 2002 Annual Meeting. April 14-IS. 2002 National Shellfisheries Association. Mystic. Connecticut (max. depth = 3.5 m) basin have only a restricted opening to the sea and mussels spawn earlier than in any other local areas while larval growth is rapid. Thus, spat reaches sleeving size in early September. These mussels have a higher mean heterozygosity than all other local stocks and they show rapid growth and higher re- sistance to stress (including summer mortalites). This basin is ded- icated only to spat collection and no grow-out is allowed. An unknown but possibly important harvest of wild mussels by local residents arose questions about the status of this stock which pro- duces larvae for spat collection. In a 1997 survey, the low abun- dance of young mussels added worry about the future of this population. Surveys were repeated in 1999 and 2001. A very lim- ited recruitment was observed in 1999 while large numbers of adults were moved to a new area possibly by the action of currents. However, the overall population remained stable at about 1 1 mil- lion individuals. In 2001. the area covered with mussels expanded considerably and recruitment was abundant. The near future of this population, and thus spat collection, seems not at risk in terms of potential for larvae production. PREDATOR INDUCED PHENOTYPIC RESPONSE BY THE COMMON PERIWINKLE [LITTORINA LITTOREA). Jason P. Breves,* RWU Box 5327, Roger Williams University, One Old Ferry Road. Bristol. RI 02809; Andrew E. Tate, Center for Economic and Environmental Development. Roger Williams University, Bristol, RI 02809. An organism's phenotype depends upon both genetics and en- vironmental influences. The relative importance of these factors is of great importance to the field of evolutionary biology. Pheno- typic plasticity, the ability to provide a single generational re- sponse to an environmental pressure, has been used to describe this relationship between genetics, the environment and phenotype. Northern Yellow Periwinkles (Liuorina obtiisata) exhibit intraspe- cific variance in shell thickness when exposed to predation by Green crabs (Caniniis nniciuis). This response is believed to give Periwinkles a competitive advantage compared to other prey not exhibiting phenotypic plasticity. It is unknown if the Common Periwinkle {Littorina liltcrca) can alter shell morphology when exposed to predatory stress. This study examines the morphologi- cal response of Common Periwinkles to the threat of Green crab predation. Periwinkles (n = 96) were assigned in equal numbers to one of three treatment means. Mass, operculum diameter, and shell length, width and thickness were compared among Peri- winkles exposed to ambient seawater (negative control), seawater containing crab effluent, or seawater with crabs and their effluent. Initial data analysis indicates differences in growth rates among the treatments. Periwinkles exposed to crabs and their effluent grew more rapidly than those in the other treatments, suggesting that rapid growth may be a response to the threat of predation. CONTINUOUS IN VITRO CULTURE OF PERKINSUS AT- LANTICUS, PARASITE OF THE CARPET SHELL CLAM TAPES DECUSSATUS. Sandra M. Casas* and Antonio Vil- lalba, Centro de Investigacions Marinas. Xunta de Galicia. aptdo. 13, E-36620 Vilanova de Arousa, Spain; Jerome F. La Peyre, Cooperative Aquatic Animal Health Research Program. Depart- ment of Veterinary Science, Louisiana State University Agricul- tural Center, Baton Rouge, LA 70803: Kimberly S. Reeee, De- partment Virginia Institute of Marine Science, Gloucester Point. VA 23062; Carlos Azevedo, Department of Cellular Biology, In- stitute of Biomedical Sciences, University of Oporto. P-4050 Porto, Portugal. Continuous //; vitro cultures of the clam Tapes deciissaliis para- site Perkinsus cukmikits were established from infected clam gill fragments, infected clam haemolymph and parasite hypnospores isolated from infected clam gill fragments incubated in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atUmticus zoospores. The highest success rate in establishing continuous cultures was obtained with cultures initi- ated from hypnospores ( 100%), followed by cultures initiated from gill fragments (93%) and from haemolymph (30%). The source of parasite also influenced the time taken to establish cultures and the size of cultured cells. In viim proliferation of parasites was mostly by vegetative multiplication. Zoosporulation yielding motile bi- flagellated zoospores was observed in low proportion (3I mm) is reached. We have focused mainly on the potential of this population to provide an annual harvest of .'> million clams. It should not be a problem considering its >26.^ million individuals of L'i-4.'i mm and high recruitment. Further, the low predator abundance and the absence of pathologies (e.g. neoplasia) suggest it should not suffer heavy losses. The small hydraulic device used to resuspend the top sur- face layer and ease collection seems not to have negative impacts. The large size of these clams should minimize predation and net protection over seeded areas could possibly be avoided. Work has been initiated to optimize storage of large quantities of clams during the extended periods between harvesting and subsequent seeding. Experiments were also initiated to define the optimal seeding period and optimal density according to shell length. LOSS OF EYE PIGMENTATION IN TWO GAMMA- RIDEAN AMPHIPODS FROM THE BIOSPHERE. Vania R. Coelho, Columbia University. Biosphere 2 Center. Oracle, AZ K562,^; Jeffrey D. Shields,* Virginia Institute of Marine Science. Gloucester Point, VA 23062. During an ecological assessment of amphipod populations in the Biosphere 2, several specimens of Ceradocus rubromaculatus and Leiicothoe spinicarpa were observed with alterations in their eye pigmentation. For C. rubromaculatus. 1768 specimens were examined and 64 (%) presented with some degree of alteration. For L spinicarpa. 150 specimens were examined and 91 (%) presented with some degree of alteration. Grossly, the eyes appeared either completely white with the individual retinas unpigmented, or the eyes were partially pigmented with pigmentation loss surrounding a pigmented core within the ommatidia. Histologically, the optic nerves showed a range of pathologies from minor losses of pig- mentation, especially in the centrally-located ommatidia, to com- plete loss of pigmentation. In all cases, there was little to no damage associated with the optic nerves or the ommatidia. There was also no indication of increased infiltration of hemocytes into the optic nerve complex or the ommatidia. Pigment cells can be negatively affected by contaminants or by the lack of phytopig- nients in the crustacean diet. For example, crustaceans lack the ability to synthesize carotenoids and must obtain them from their diets. We speculate that the loss of pigmentation in the eyes of amphipods from the Biosphere may be caused by the lack of certain dietary phytopigments. SHELL REPAIR RATES IN SURGICALLY DAMAGED VALVES OF THE BLUE MUSSEL {MYTILUS EDULIS) AND THE RIBBED MUSSEL {GEVKENSIA DEMISSA) FROM NEW JERSEY. Tricia L. Cranmer, Department of Ma- rine and Environmental Studies. University of San Diego. San Diego, CA 921 10; Daniela Zima,* Richard Stockton College of New Jersey. Pomona. NJ 08240; Richard R. Alexander, Depart- ment of Geological and Marine Sciences. Rider University, Lawrenceville. NJ 08648. The blue mussel Mytilus eduUs and the ribbed mussel Ceiiken- sia cleiiiissa were collected (n = 120) from the salt mashes of Tuckerton, New Jersey. Equal numbers of specimens (n = \5) were notched at. or had holes drilled near, the posterior or ventral margin of the valves of each species. These shell regions bear scars from shell-crushing crabs and holes drilled by moonsnails. Repair progresses through four stages: 1 ) tissue plugging the shell gap or hole, 2) extension of the periostracum over exposed tissue, 3) calcification beneath the periostracum, and 4) valve-thickening, pigmentation, and expression of a ny ornamentation. Shell repair was completed between two to seven weeks. Average repair rates are statistically significantly faster for epibyssate M. edulis relative to the endobyssate C demissa (Mann-Whitney U test: p < 0.05) pooled for all types and location of shell damage. Correspond- ingly, field surveys show that shell repair frequency is greater among blue mussels (19% of all specimens) than ribbed mussels (109f of all specimens). Mantle tissue repaired posterior shell notches faster than the ventral margin in M. edulis. although shell repair rate is statistically indistinguishable between ventrally and posteriorly notched shells of G. demissa. Correspondingly, field surveys reveal that 96"^ of all shell repairs in M. edulis are located near the posterior margin. Mortality before completion of shell repair was significantly greater for experimentally drilled vs. notched shells of either species. Mantle tissue was damaged by penetrative shell-drilling, but not inevitably in valve margin break- OVER-WINTERING OF CRASSOSTREA ARIAKENSIS IN LAND-BASED SYSTEMS IN VIRGINIA. Alan J. Erskine and Standish K. Allen, Jr., Aquaculture Genetics and Breeding Tech- nology Center, Virginia Institute of Marine Science, Gloucester Point, VA 23062. Decimation of the Crassostrea viri^inica industry in Virginia has been well documented. Recent interest has turned to non- native C. ariakeiisis culture for experimentation. We hypothesized that it was beneficial, biosecure and cost effective for industry to over-winter C ariakeiisis inside their hatcheries. Triploid C. ari- akeiisis were deployed in raceway and upweller systems at four sites in Chesapeake Bay from November 2000 to April 2001. Three size classes, 4-6mm. 6-8mm and 8-1 2mm, were deployed in replicate at a maximum can'ying capacity of 2000 grams total wet weight. Twenty-five random oysters were sampled monthly 428 Ahstnicts. 2002 Annual Meetuig. April 14-18. 2002 National Shelltisheries Association, Mystic, Connecticut and shucked wet weights and hinge to lip shell height measure- ments were recorded. A low salinity site ( 10-I2ppt) on the bayside of Chesapeake Bay and a mid salinity site (22-25ppt) on the sea- side had the highest growth at 30 mm and 3 grams. This experi- ment shows that over-wintering juvenile C. ariakensis could be incorporated as a step in the technology of non-native oyster cul- ture. IRRADIATION OF OYSTER PRIMARY CELL CULTURES WITH ULTRAVIOLET-LIGHT TO ELIMINATE BACTE- RIAL AND PROTOZOAL CONTAMINANTS. Chwan-Hong Foo* and Jerome F. La Peyre, Cooperative Aquatic Animal Health Research Program. Department of Veterinary Science. Louisiana Agricultural Experiment Station. Baton Rouge. LA 70803. Microbial contamination of oyster primary cell cultures is an obstacle to cell line development. Perkinsus uinnnus and Vihrii) vulnificus are two prevalent contaminants of oyster cell cultures. Studies have shown that ultraviolet-light (UV) effectively kills certain aquatic protozoa and bacteria. Our objective was to develop a procedure to eliminate P. marinus and V. vulnificus from primary cell cultures by UV irradiation, while retaining the viability of oyster cells. Oyster heart cells. P. marinus and V. vulnificus cul- tures were irradiated with 50.000, 100.000, 201.000. and 402,000 (jLJoules/cm- of UV light. Two days post-irradiation, oyster cell viability was determined by measuring their metabolic activity using the MTS/PMS assay. Perkinsus marinus growth. 2, 4 and 6 days after irradiation, and V. vulnificus growth, 3, 6. and 24 hours post-iiTadiation. were both assessed by measuring turbidity and metabolic activity (MTS/PMS assay). Results indicated that oyster cells viability decreased significantly (P < 0.0001 ) with increasing UV energy levels. Peikinsus marinus growth on days 2 and 4 were significantly inhibited by all UV levels, while growth on day 6 was inhibited with UV levels above 100,000 p.joules/cm-. Vibrio vulnificus growth. 3 and 6 hours post-irradiation, was inhibited (P < 0.05) by all UV levels, but growth rebounded 24 hours after irradiation. This study suggests that UV irradiation has a very limited ability to eliminate P. marinus or V. vulnificus without negatively affecting oyster cell primary cultures. IN VITRO EFFECTS OF HEAVY METALS AND ATRA- ZINE ON PACIFIC OYSTER. CRASSOSTREA GIGAS, HAEMOCYTES. B. Gagnaire,'^ H. Thomas-Guyon,' and T. Renault.- 'Universite de La Rochelle, Laboratoire de Biologie et Environment Marin (LBEMl. avenue Michel Crepeau. 17042 La Rochelle; -Ifremer La Tremblade. Laboratoire de Genetique el Pathologic (LGP). Ronce-les-Bains. 17390 La Tremblade. In the last decades shellfish culture has developed in a signifi- cant way around the world. However, culture areas are often sub- jected to recurring pollutions. The recrudescent use of herbicides in agriculture, including atra/iiie. implies pollutant transfer to- wards the aquatic environment in estuarine areas. Moreover, in- dustrial wastes are sources of heavy metal contamination. It is appropriate to consider the harmful effects of pollutants in marine species, particularly in cultured molluscs. Bivalves, including mus- sels and oysters, have been suggested as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate contaminants within their tissues. The development of techniques allowing analysis of the effects of such compounds on bivalve biology may lead to monitoring of pollutant transfer in estuarine areas. In this context, the effects of atra/ine and the effects of various heavy metals on defence mecha- nisms were analysed in Pacific oysters. CrassDstrea gigas. Pollut- ant effects were tested //; vilro on oyster haemocytes. Cell viability and activities were monitored by flow cytometry. Enzymatic phe- noloxidase-like activity was also evaluated by spectrophotometry. Atrazine induced no effect on oyster haemocytes under the con- ditions tested. On the contrary, mercury caused a significant mor- tality of haemocytes maintained //; vilro. Aminopeptidase and phe- noloxidase-like activities were also modified in the presence of this pollutant. KEY WORDS: Pacific oyster. Crassostrea gigas. haemocytes. tlow cy- tometry, heavy metals, atrazine. phenoloxidase, cellular activities, toxicity INDUCIBLE ANTIBACTERIAL ACTIVITY IN OYSTER {CRASSOSTREA VIRGINICA) HEMOLYMPH. Holly A. Ge- froh, Matthew J. Jenny, and Ryan B. Carnegie. Program in Marine Biomedicine and Environmental Sciences; and Kevin L. Sehey. Department of Pharmacology. Medical Universitv of South Carolina. 171 Ashley Avenue. Charieston. SC 29425; Robert W. Chapman. South Carolina Department of Natural Resources. 217 Ft. Johnson Rd., P O Box 12559, Charleston, SC 29422. Oyster (Crassostrea virginica) tissues resist infection by ma- rine bacteria, but the basis of this is unclear. Bacteria invading internal spaces are quickly agglutinated and phagocytosed by hemocytes; however, areas where hemocytes are not active (e.g., epithelial surfaces) also resist heavy colonization. Oyster lysozyme kills bacteria, but is thought to be only weakly effective against the most common marine species (Gram-negatives). Soluble bacteri- olytic peptides found in many organisms, including the mussel Mvlilus edulis. may supplement phagocytosis and the activity of lysozyme in oysters as well. Our objectives were to determine whether or not anti-Gram- negative bacterial activity could be induced in the hemolymph of C. virginica. and if so, to isolate and characterize its source. Li- popolysaccharide (LPS), laminarin. or dH,0 control was injected into the adductor muscles of wild C. virginica (N = 6 oysters/ treatment) collected locally. Hemolymph was collected from the National Shellfisheries Association, Mvstic, Connecticut Abstracls. 2002 Annual Meeting, April 14-18. 2002 -il^ adductor muscles at 12 and 24 h and pooled by time interval and treatment type. Soluble protein from cell-free hemolymph plasma was desalted using a C,^ column, eluted with 509<- acetonitrile. dried down and reconstituted in dH,0. and spotted on a plate lawn of Esclu'richid coli. Plasma protein samples from LPS-challenged ( 1 2 and 24 h post-challenge) and laminurin-challenged (24 h) oys- ters inhibited E. coli growth, while plasma protein from unchal- lenged oysters did not. The induced antibacterial activity passed through a 1(1- kilodalton (kDa) cutoff membrane. Further separation by re\ersed phase high performance liquid chromatography (HPLC) and analysis by matrix-assisted laser desorption ionization mass spec- trometry (MALDI-MS) revealed several peptides around 3-7 and 9.5 kDa to be candidates for its source. These candidate peptides are currently being sequenced. (November), mussel densities had dropped to .50 to 1 15 m~" on the six original reefs but size-frequency plots showed an average in- crease in shell length of 5 mm in the largest size class of mussels over the first 3 months, indicating good individual growth rates. Recruitment into the smallest size classes also occurred on some reefs. Using the same sampling protocol, initial mussel densities on the three new reefs ranged from 430 to 1376 m". Future work will include continued monitoring of all nine reefs, construction of abutting saltmarsh. and environmental monitorinc. CONSTRUCTING SHELLFISH REEFS IN A POLLUTED. URBAN ESTUARY: SCIENTISTS JOIN WITH THE COM- MUNITY TO WORK FOR A COMMON GOAL. Jennifer Greene,* Raymond (irizzle. David Burdiclv. and Larry Ward. Jackson Estuarine Laboratory. University of New Hampshire. Durham, NH 03824: Ann Reid, Great Bay Coast Watch. Sea Grant Cooperative Extension. Durham. NH 03824. South Mill Pond, a 7-hectare estuarine embayment in the City of Portsmouth. New Hampshire has been receiving combined sewer overflows since the late 1800s. A tide gate controls water flow into and out of the Pond and there is a road causeway with box culverts that separates the Pond into inner and outer sections. The City has a major sewer re-construction project underway that will, when completed, reduce inputs of sanitary sewer wastes. Scientists joined with the City, students from local schools, and local volunteers to begin ecological restoration of the Pond in 2001. Six shellfish "mini-reefs" (each 2 x 5 m in surface area) were constructed in May using mussels (Mytiiits ediilis) trans- planted from a nearby natural reef. Mortalities as high as 80% occurred on the three reefs in the inner pond within the first 2 months, likely as a result of elevated temperatures caused by ex- tended closure of the broken tide gate during June and July. Hence, three additional mussel reefs were constructed in the inner pond in October 2001. Oysters iCnissostrea virginicci) remotely set at Jackson Estuarine Laboratory were added as 3-month old spat to the three new reefs in the inner pond and the three original reefs in the outer pond in October, resulting in six mussel/oyster reefs. Eighth grade students participated in construction of the mussel reefs and third graders participated with the oysters. Community volunteers also participated in reef construction and post- construction monitoring. Three quadrat (0.16 nr) samples per reef showed initial mean mussel densities on the six original reefs constructed in May ranged from 150 to 930 m"-. After 6 months THE EASTERN OYSTER AS AN INDICATOR SPECIES TO ESTABLISH RESTORATION TARGETS IN SOUTHWEST FLORIDA ESTUARIES. Rashel V. Grindberg.* Erin C. Rasnake, Michael Savarese, and Aswani K. Volety. Florida Gulf Coast University. College of Arts & Sciences. 10501 FGCU Blvd South. Ft. Myers. FL 33965. Water management practices within Southwest Florida have drastically altered natural water quality conditions within estuaries. The ecological and physiological responses of oysters. Crassostrea virginica, were compared among pristine (Blackwater River), semi-altered (Henderson Creek) and altered (Faka-Union canal) estuaries with historically similar hydrologic conditions. In the Faka-Union estuary, a system that receives excessive freshwater during the rainy season, the distribution of reefs, the regions of maximum living density, and the foci of maximum productivity are displaced seaward relative to pristine estuaries. Henderson Creek, an estuary receiving pulses of nutrients and freshwater due to weir design, has oyster populations with higher mean produc- tivities and higher living densities. While upstream locations in all three estuaries have lower disease prevalence among adult oysters, juveniles experience heavy mortality due to freshwater releases. Overall growth rates are higher at upstream locations. Spat recruit- ment occurred between June and October in all estuaries. Faka-Union and Henderson Creek estuaries are scheduled for restoration. The patterns of oyster distribution and physiology will help establish target restoration conditions and provide a founda- tion for the monitoring of restoration effectiveness in Southwest Florida estuaries. Henderson Creek is presently undergoing resto- ration through the retrofitting of its upstream weir to permit dy- nainic control of freshwater release. Sheet flow should be restored to Faka-Union in order to re-establish salinity levels favorable for maximum reef development, living density, oyster growth, recruit- ment, and productivity. Changes in oyster ecological and physi- ological conditions will be monitored over subsequent years to 430 Ahslnicts. 2002 Annual Meeting. April 14-18. 2002 National Shellfisheries Association. Mvstic. Connecticut LONG-TERM CHANGES IN INTERTIDAL OYSTER REEFS IN A FLORIDA LAGOON POTENTIALLY CAUSED BY BOATING ACTIVITIES: AN ANALYSIS OF AERIAL PHOTOGRAPHS FROM 1943-2000. Raymond Grizzle* and Jamie Adam.s, Jackson Estuarine Laboratory. Uni- versity of New Hampshire. Durham, NH 03824; Linda Walters, Department of Biology. Uni\ersity of Central Florida. Orlando. FL 32816. Previous research in the late 1990s had shown that some inter- tidal oyster (Crassostrea virgiiiica) reefs in that pt)rtion of the Mosquito Lagoon within the Canaveral National Seashore. Florida had dead margins consisting of mounded up, disarticulated shells. It was hypothesized that boating activities were the cause of the damage because all the reefs were adjacent to major boating chan- nels. We characterized the history of the appearance of dead mar- gins (and other reef changes) using aerial photographs taken be- tween 1943 and 2000. Imagery analyzed included prints (black & white, color, or color infrared) from 1943, 1951, 1963, 1975. 1988. and 1995, and digital imagery from 2000 (USGS 1:12,000 digital ortho-quarterquads). at scales from 1:6,000 to 1:24.000. Prints were scanned at a resolution sufficient to yield 1-m pixels. After scanning, each set of images was referenced to the year 2000 imagery using ArcView and Arclnfo CIS software. All reefs found to have dead margins based on 1995 and 2000 aerials were visited in November 2001 to confirm the presence and extent of dead areas. This provided a general ground-truthing for the "signature" (a highly reflective, light-colored area adjacent to darker-colored live reef) to be used to detect the appearance of dead margins in the historical aerials. The earliest appearance of dead margins was in the 1975 aerials on reefs adjacent to the intracoastal waterway (ICW), a major boating channel. The total number and areal extent of dead margins increased with time. Our current preliminary es- timate is that between 1 0 and 20% of the reefs in the Seashore have been damaged. The most dramatic changes have occuired in reefs along the ICW. .some apparently migrating away from the channel as much as 50 m and with empty shells mounded up a meter above the high water line. This historical analysis provides strong (al- though only correlative) evidence that boating activity has had dramatically detrimental effects on some oyster reefs in the study area. Ongoing studies are aimed at further testing this hypothesis and elucidating the actual mechanisms involved. AN OYSTER (CRASSOSTREA VIRGINICA) REEF RESTO- RATION EXPERIMENT IN NEW HAMPSHIRE INVOLV- ING CROSBREED STOCK AND NATIVE TRANSPLANTS. Raymond Grizzle, Jennifer Greene, and Stephen Jones. Jackson Estuarine Laboratory. University of New Hampshire, Durham. NH: Mark Luckenbach and Roger Mann, Virginia Institute of Marine Science, Gloucester Point. VA. The first documented MSX epizootic in New Hampshire oc- cuiTed in 1995. resulting in dramatic declines in oyster abundances on some reefs. The present project is the first experimental scale effort in the State aimed at development of a long-term program to mitigate the effects of disease. In 1999, studies were initiated in the Salmon Falls River to: (1) characterize a disease-decimated reef, (2) restore portions of the reef using spat from CROSBreed stock (an MSX and dermo-resistant line) and transplanting of native oysters, and (3) determine the effects of the restored reef on water quality. Here we report on the first two objectives. Diver surveys, tonging, and underwater videography indicated the overall bottom area probably dominated by oysters before 1995 extended along the main channel a total distance of 350 m with an average width of 15 m. Quadrat sampling in 2000 and 2001 showed this area was numerically dominated by the ribbed mussel {Geukensia demissa) with a mean density of 40 m"; oysters were 20 m", and the blue mussel {Mytilns edulis) 5 nr. Maximum, mean horizontal free- stream tlow speed measured over portions of two tidal cycles with an acoustic Doppler velocimeter was 32 cm s~'. In May 2000, approximately 230 bushels of native oysters, dredged from the Piscataqua River about 1 km south of the study site, were depos- ited in an area measuring 10 x 30 m on the natural reef bottom. Spat from CROSBreed stock were remotely set in June of 2000. held in bags for 4 months, then transferred to a 10 x 20 m area on the natural reef In October 2001. after I year of development, the CROSBreed reef area had a mean density of 226 oysters m", compared to 90 m~ on the native transplant reef, and 36 m" on the natural reef The CROSBreed oysters have shown good growth, increasing from a mean size of 26.8 mm shell height when put out in October 2000 to 48.6 mm in October 2001. and no measurable mortality. Natural spat set in 2000 was greatest on the CROSBreed reef suggesting a "minimum threshold density" for successful natural recruitment to restored reef areas. WHAT CERTAIN I9TH AND EARLY 20TH CENTURY NAVIGATIONAL AND SPECIAL PURPOSE SURVEY CHARTS REVEAL ABOUT CHANGES IN THE OYSTER REEF MORPHOLOGY OF THE LOWER CHESAPEAKE BAY. William J. Hargis, Jr.. Helen E. Woods,* Rebecca Aren- son, Sbaron De«ing. .Arman Kaltayev, Elizabeth .Mountz, Marcia R. Bernian, and Dexter S. Haven, Center for Coastal Resources Management. Virginia Institute of Marine Science. Gloucester Point. VA 23062. The decline of commercial oyster production in the lower James River is reflected in the reduction of oyster-reef dimensions over time. Employing navigational hydrographic survey charts made by the U.S. Coast Survey in 1854-55. 1871-73. and 1940s and oyster survey charts made in 1878 and 1909 by the USCGS and the old U.S. Fish Commission, we examine changes in the dimensions of the natural oyster reefs of the lower James River. Chesapeake Bay. and discuss the likely cause of those changes. National Shellfisheries Association. Mystic. Connecticut Abstracrs. 2002 Annual Meeting. April 14-18. 2002 431 MORPHOLOGY OF A CHESAPEAKE BAY OYSTER REEF SYSTEM IN 1871-1873. William J. Hargis. Jr., Helen E. Woods,* Rebecca Arenson, Elizabeth Mountz, Marcia R. Berman, and Sharon Dewing. Center for Coastal Resources Man- agement, Virginia Instuute of Marine Science. Gloucester Point. VA 23062. During research on oyster reef evolution in the Chesapeake Bay it became apparent that those once prominent benthic features must ha\e intluenced their sunounding significantly. To investi- gate the extent of that influence on the geomorphology. hydrody- namics, and other ecologically important features, we developed a 3 dimensional presentation of the once highly productive oyster reef system of the James River estuary. Charts of the study are drawn from soundings made by the U.S. Coast Survey in 1871-73 were employed. Soundings were digitized on a Numonics 2200 digitizing tablet interfaced with Arclnfo running in a UNIX oper- ating environment. The coordinate grids were projected to NAD27. A 3-D TIN (Triangulated Irregular Network) model gen- erated the picture of the bottom of the study area extant in 1871- 73. The 3-D presentation, center-piece of our presentation, clearly depicts the oyster reef system of 1871-73. Extending into the intertidal and acting as a inassive system of weirs and baffles, the upthrusting reefs (Hargis. In Press) must have affected deposition, scouring and erosion, not only near field but more distantly, even into the shallows and adjacent shorelines. Undoubtedly, they in- fluenced biological processes too. GEOGRAPHIC VARIATION IN NUCLEAR GENES OF THE EASTERN OYSTER CRASSOSTREA VIRGINICA. Cindi A. Hoover* and Patrick M. Gaffney, Graduate College of Marine Studies. University of Delaware. Lewes. DE 19958. Studies of genomic DNA in the eastern oyster, Crassostreci virginica, have uncovered genetic discontinuities in population structure. Like most benthic marine invertebrates. C. virginica has a planktonic larval stage, which is in principle capable of wide- spread dispersal. If extensive dispersal occurs, one would expect to observe genetic homogeneity across the geographic range of the species. However, studies have found geographic genetic hetero- geneity in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) of C. virginica. The break in mtDNA has prompted the division of the C. virginica into Atlantic and Gulf coast subpopu- lations. However, unlike mtDNA. the degree of nDNA differen- tiation is still poorly known. This study examines nDNA from Atlantic and Gulf coast oyster populations for genetic polymor- phisms using restriction endonuclease fragment length (RFLP) analysis, denaturing gradient gel electrophoresis (DGGE). and di- rect sequencing. Nuclear primers are currently being screened for polymorphisms in a panel of organisms representing the geo- graphic range from Canada to Mexico. This work will improve on previous research by examining additional nuclear markers and will help clarify the question of large-scale variation between Gulf and Atlantic coast oyster populations as well as shed light on smaller-scale regional patterns of variation. CHARACTERIZATION AND MANIPULATION OF SEX STEROIDS AND VITELLOGENIN IN FRESHWATER MUSSELS. Nicola J. Kernaghan'' and pjleen Monck, Univer- sity of Florida. College of Veterinary Medicine. Gainesville. PL; Carla Wieser and Timothy S. Gross, USGS/BRD/FIorida Carib- bean Science Center. Gainesville. FL. The characterization and manipulation of sex steroids and vi- tellogenin in freshwater mussels, is critical for the development of artificial culture procedures and the evaluation of reproductive health of populations. The current study included an evaluation of reproductive cycles and vitellogenin in Ellipiio Inickleyi. In addi- tion, female Elliprio hnckleyi and Lanipsilis teres were exposed to exogenous estradiol to induce spawning. Body tissues were col- lected from adult mussels and standard RIA procedures were uti- lized for androgen, and estrogen analyses. Vitellogenin, an egg yolk protein produced under the influence of sex steroids, was determined using an indirect method developed by Blaise et al. ( 1999). Sex steroid concentrations were found to be closely cor- related to reproductive activities and spawning. Tissue concentra- tions of both vitellogenin and estrogen were significantly elevated following exposure to exogenous estradiol. Histological examina- tion of gonad tissue also indicated changes in reproductive status. The development of these procedures for use with freshwater mus- sel species will be critical to the elucidation of potential habitat and contaminant effects on reproductive function, as well as the culture of endangered species. A SPECIFIC ASSOCIATION OF DOCOSAHEXAENOIC FATTY ACID WITH CARDIOLIPINS OF SOME MARINE BIVALVES. Edouard Kraffe.* Philippe Soudant. Yanic Marty, and Nelly Kervarec, Universite de Bretagne Occidentale. UMR 6521 and UMR 6539, BP 809 29200 Brest. France: Pierre Guenot CRMPO. Universite de Haute Bretagne. 35042 Rennes cedex. France. A cardiolipin (CL) class, also termed diphosphatidylglycerol. was isolated by high performance liquid chromatography from lipid extracts of Pecten maximus, Crassostrea gigas and Mytiliis edidis, and characterized by analytical and spectroscopic methods. The fatty acid (FA) composition of this CL represents a specific association with docosahexaenoic acid [22:6(n-3). DHA). which accounted for more than 90'7f w/w of the total FA of this class. The structural determination by nuclear magnetic resonance spectrom- etry and positive ion electrospray mass spectrometry verified a configuration having four identical 22:6(n-3) molecules. This spe- cific composition is different from those already reported in other eukaryotes. Indeed. CL acyl chains reported in the literature are mainly composed of monounsaturated or diunsaturated chains w ith 432 Abstracts. 2002 Annual Meeting. April 14-IS. 2002 National Shellt'isheries Association, Mystic, Connecticut 16 or 18 carbon atoms. The DHA-enriched CL may reflect a specific adaptation ni bivalves that enhances the strtictural and functional mechanisms of biomembranes in response to environ- mental variations (temperature, salinity, emersion). HEMOCYTE-MEDIATED DEFENSE RESPONSES OF THE LOBSTER HOMARUS AMERICANUS. Brenda S. Kraus.* Amy E. Beaven, and Robert S. Anderson, Chesapeake Biologi- cal Laboratory, Uni\ersity of Maryland Center for Environmental Science, P. O. Box 38. Solomons. MD 20688. Lobster hemocytes held in primary culture avidly phagocytosed untreated yeast cells labeled with tluorescein succinimidyl ester. However, serum-treated yeast cells were less readily ingested by the hemocytes. Serum agglutinins can serve to facilitate phagocy- tosis of certain foreign particles (opsonization). Although anti- yeast agglutinins were present in lobster serum they seemed to inhibit phagocytosis, perhaps by blocking sites involved with rec- ognition by hemocytes. Bacterial agglutinins have also been mea- sured in lobster sera; their role as opsonins is under study. After appropriate stimulation by phagocytosis or membrane perturba- tion, hemocytes can produce cytoto.xic reactive o.xygen species (ROS) which contribute to host defenses by destroying microor- ganisms. Luminol-augmented chemiluminescence (CL) was used to quantify ROS generation by lobster hemocytes. Phorbol myristate acetate (PMA), a protein kinase C activator, was shown to elicit ROS in hemocytes withdraw n from healthy lobsters, based on hemolymph protein concentration. However, phagocytosis of Listonella atiguillarum infrequently (-25%) produced a ROS ac- tivity in PMA-responding animals. Opsonization of L anguiUuiuin with serum did not enhance CL; this was not unexpected because no anti-Z.. augudlaniiu agglutinin activity was detected in lobster serum. Studies related to the role of agglutinins in recognition and effector mechanisms of lobster hemocytes using other biotic and abiotic test particles are in progress. POTENTIAL ELIMINATION OF THE PROTOZOAN PATHOGEN PERKINSl'S MARINVS FROM EASTERN OYSTERS BY FRESHET EVENTS. Megan K. G. La Peyre,* U.S.G.S. Louisiana Cooperative Research Unit, School of For- estry, Wildlife and Fisheries. Louisiana State University. Baton Rouge, LA 70803; Amy D. Nickens and Jerome F. La Peyre, Cooperative Aquatic .Animal Health Research Program, Depart- ment of Veterinary Science, Louisiana State University Agricul- tural Center, Baton Rouge, LA 70803. Environmental conditions have long been held to be critical controls on host-parasite interactions but little attention has been paid to the effects of short-term events on host-parasite interac- tions. We tested the hypothesis that freshet events may be detri- mental to P. marinus while ha\ina only minimal impacts on C. virginica survival. Research based predominantly on environmen- tal averages has led to the consensus that enxironmental conditions such as salinity and temperature control P. iiuiriiius infection in C. virginica. In contrast, salinity records from the Gulf coast indicate that there is considerable variation in salinity, and several studies have suggested that freshet events may be related to low P. mari- nus infection intensities of oysters in certain areas. In order to determine the effects of freshets on C. virginica and P. marinus. we initiated a controlled laboratory experiment. In April, July and December, 2001, oysters collected from Grand Isle, LA were di- vided equally between a control system (maintained at 20 ppt) and a treatment system (salinity lowered from 20 ppt to 0-1 ppt over 48 hours). Thirty oysters were sampled weekly from each tank and infection intensities determined. Oyster mortality was monitored daily. Treatment oysters did have reduced P. marinus body burden, but actual success was highly dependent on the season. Crassos- trea virginica was not adsersely affected by freshet events in cool temperature, spring and w inter experiments, but experienced high mortality during a summer freshet event. Since most freshet events occur in winter-spring months, the use of controlled freshwater events might present a potential management tool for reducing P. marinus infection in oysters. A FIELD INVESTIGATION OF THE EFFECTS OF V- NOTCHING ON THE HEALTH AND SUSCEPTIBILITY TO INFECTION OF OVIGEROUS FEMALE AMERICAN LOBSTERS. .lean Lavallee,* AVC Lobster Science Centre. Uni- versity of Prince Edward Island. 550 University Avenue. Char- lottetown. PE, Canada, CIA 4P3; Donald J. Rainnie, AVC Inc., 550 University Avenue, Charlottetown, PE, Canada, CI. A 4P3. V-notching is a fishery management practice consisting of marking ovigerous lobsters by punching a V shaped notch in the tail before returning it to the seawater. This study investigated some of the possible effects of V-notching on the health of oviger- ous lobsters during the 2000 fall fishing season on Prince Edward Island. One hundred and thirty ovigerous lobsters were physically examined and assigned to two treatment groups. Hemolymph samples were collected from 15 lobsters from each treatment group and analysed for total protein (TP), total hemocyte counts (THC) and for the presence oi Aerococcus viridans. Anopluyoides liacmaplnia and Vibrio spp. Lobsters were housed individually in cages containing 2, 3 or 4 compartments. Lobsters from the treated group were V-notched immediately prior to returning the cages to the sea bottom. Monitoring for mortality, and vigor status was conducted at least weekly. Hemolymph was taken on 15 lobsters of both treatment groups for TP and THC at the mid-point in the study. At study termination (55 days), all lobsters were re- examined and hemolymph was sampled for determination of TP, THC and presence of A. viridans. .An. Iiaemapltda and Vibrio spp. Additionally, 2 V-notched lobsters were sampled on Days 1, 3, 6, 13, 27 & 55 for presence of A. viridans. .An. hacmopliila and Viiirio spp. and for histological assessment of the wound. Mortality dur- National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 433 ing the course of the study was limited to one lobster, and reflected an act of cannibalism. There was no significant difference ^p > 0.05) between the two treatment groups for any parameter moni- tored. Histological examination of the V-notch wound revealed that the wounds appeared well sealed off to the environment by the rapid infiltration of hemocytes by 24 hours. A PRELIMINARY LINKAGE MAP FOR THE PACIFIC OYSTER CRASSOSTREA GIGAS. CONSTRUCTED WITH RAPD AND AFLP MARKERS. Li Li,* Institute of Oceanology. Chinese Academy of Science. 7 Nanhai Road. Qingdao. Shandong 266071. China; Ximing Guo, Haskin Shellfish Research Labora- tory. Institute of Coastal and Marine Sciences. Rutgers University. 6959 Miller Avenue. Port Norris. NJ 08349. The development of genetic markers and linkage maps is an important step toward the identification and potential improvement of commercially important traits in oysters. The construction of a linkage map requires a large number of molecular markers, which have been a challenge in oysters. Microsatellites are probably the best markers for linkage mapping, but they are expensive to de- velop and not readily available to many labs. Therefore, we de- veloped relatively inexpensive markers such as the random ampli- fied polymorphic DNA (RAPD I and amplified fragment length polymorphism (AFLP I markers for linkage mapping in the Pacific oyster Crassostrea gigas Thunberg. Selected markers were used for linkage mapping in a reference family with 75 progenies. The reference family was a backcross of an interstrain hybrid to one of the parental strain. ((Miyagi x Hiroshima) x Miyagi). In this study. 1 10 RAPD primers were screened with the parents and t"ive prog- enies, and 25 primers with at least one reliable and segregating band were selected for mapping analysis. The 25 primers gener- ated 259 bands, of which 102 (39%) were polymorphic in the two parents. The number of segregating RAPD markers was 41 in the mother and 1 7 in the father. Using ten AFLP primer combinations. 802 peaks were obtained, of which 253 (32%) are polymorphic. The number of segregating AFLP markers was 81 in the mother and 50 in the father. A preliminary female linkage map was con- structed with 122 RAPD and AFLP markers covering all ten chro- mosomes, and details of the map will be presented at the meeting. GONADAL MATURATION OF TRIPLOID SCALLOPS ARGOPECTEN PURPURATUS LAMARCK, 1819. Karin B. Lohrmann,* Elisabeth von Brand, and Cristian Gallardo, Uni- versidad Catolica del Norte. Facultad de Ciencias del Mar. Cas. 117, Coquimbo. Chile. It is expected that in triploid organisms the energy normally used for reproduction would be allocated to growth. However, not all triploid molluscs are completely sterile; and in some cases even gametes are produced. The aim of this study was to assess the gonadal development in the native scallop Argopecten purpiimtiis induced to triploidy. A. purpimiuis is a functional hermaphrodite, the male gonad being creamy-white, located proximal to the foot, and the distal female gonad is bright orange-red. They were induced to triploidy with 6-dimethylaniinopurine (6-DMAP). Treated ( = induced) and control scallops were processed for histology using routine meth- ods. At the age of 1 1 months, when the control scallops were mature, some treated scallops had a gonad which showed a uni- form brown colour. These were true triploids as evaluated through chromosomal counts. They showed the tendency of reducing the female gonad, only few pre-vitelogenic oocytes were observed, in otherwise empty acini. The male gonad was relatively more de- veloped, but no gametes further than secondary spermatocytes or early spermatids with no flagella were detected. DETECTION OF THE WHITE SPOT SYNDROME VIRUS (WSSV) IN CARCINOLOGIC FAUN ASSOCIATED TO SHRIMP CULTURE OF SINALOA, MEXICO, USING POLYMERASE CHAIN REACTION (PCR) AND /,V SITV H\ BRIDIZATION. Bortolini R. Jose Luis and Torres G. M. Pilar, Lab. Invertebrados. Fac. Ciencias, Universidad Nacional Autonomade Mexico. A. P. 70-371. Mexico D. F. 04510. Mexico; Montoya R. Leobardo, Lab. Virologfa. Centre de Investigacion en Alimentos y Desarrollo. U. Mazatlan. A. P. 711. Mazatlan. Sinaloa C. P. 82010. Mexico. One of the most important sanity problems in penaeid shrimp populations of trade important in the world, is originated by virus infection. Recently in our country was detected the White Spot Syndrome Virus (WSSV), that produce high mortality in the popu- lation of cultured shrimps, mainly in the states of Nayarit, Sinaloa and Sonora. For this research, we collected samples in summer of 2001. The samples were from different species associated to cul- ture farms, (Litopenaeus vannamei. L. stylirostris. Calliiwctes sapidus. Uca sp. and copepods). The copepods were homogenized and proceced in integral form by Polymerase Chain Reaction (PCR) probe, one hemolymph sample was taken from each of the different species to which was done the PCR getting the result of sequence in acrilamide, and having the corresponding marks. The samples for processing by //) situ hybridization, were fixed in Davidson's solution for 48 hours and then in OH 70. The histo- logical cuts were done 5 fj.m of thickness and were put on posi- tively slides. By in situ hybridization, infected tissues of samples of Catlinectes sapidus and Litopenaeus vannamei were easily dis- tinguished. The presence of the blue precipitations it was present in branchial and connective tissues and gut subcuticular epithe- lium. In the other species of carcinological faun was negative by in situ hybridization probe. 434 Ahstnwis. 2002 Annual Meeting, April 14-1«, 2002 National Shellfisheries Association. Mystic. Connecticut FACTORS, RISKS AND SIGNIFICANCE OF EMERGENT NEOPLASIA DISEASES IN CULTURED AND WILD SOFT- SHELL CLAMS (V/)4 ARESARIA) IN ATLANTIC CANADA. Gregory MacCalluni,* Jeffery Davidson, and Garth Arsenault. Atlantic Veterinary College. University of Prince Ed- ward Island. .^^50 University Ave., Charlottetovvn, PEI, CIA 4P3; Sharon McGladdery and Michelle Maillet, Department of Fish- eries and Oceans Canada, Gulf Fisheries Centre, .U3 University Ave.. PC Box 5030. Moncton. NB. EIC 9B6; Neil MacNair. PEI Department of Fisheries. Aquaculture and Environment, 1 1 Kent St.. Charlottetovvn. PEI. CIA 7N8. In 1999. mortalities of soft-shell clams {Mya arenaria) caused by or associated with haemic neoplasia occurred at several sites around Prince Edward Island (PEI) and in Richibucto. New Bruns- wick (NB). Haemic neoplasia has been well documented in bi- valves (clams, mussels and oysters) worldwide since the early 1970"s. The cause(s) of haemic neoplasia is/are unknown. They have been linked to infectious triggers (neoplastic cells per se or a viral vector), anthropogenic carcinogens (e.g.. polychlorinated hi- phenyls) and changing natural conditions (e.g.. abnormally high water temperatures). The most urgent question, from an environ- mental and clam production perspective, is whether the neoplasia is infective or non-infective. The objectives of this study are to: i) examine the transmissi- bility of this disease; ii) determine the geographic and seasonal distribution of haemic neoplasia in soft-shell clams from PEI (in- cluding a study to relate disease prevalence to five PEI clam popu- lations); iii) examine common environmental variables between affected and unaffected sites on PEI. NB. and Nova Scotia (e.g.. temperature, bottom-type, terrestrial run-off. human activities/ input); iv) determine whether or not sediment exposure affects emergence of haemic neoplasia; and v) determine whether or not clams which have survived haemic neoplasia have developed a resistance to the disease which can be passed onto their offspring. IDENTITY OF UNITED STATES MOLLUSK PRODUC- TION DECLINES IN THE I900S. Clyde L. MacKenzie, Jr.,* James J. Howard Marine Sciences Laboratory. Northeast Fisheries Science Center. 74 Magruder Road. Highlands. NJ 07732. In the 1900s. the historical declines in landings of estuarine mollusks along the east coast of the United States rarely were caused by overfishing, that led to permanent sharp declines in production of their larvae. Instead, habitat degration including ad- verse algal blooms, domestic pollution which led to closure of production beds, poor markets, and diseases have been the main causes. The oyster. Crassostrea virginica. landings, mainly, suf- fered from poor markets, and oyster habitats were despoiled by siltation and by dredgers removing shells while oysters were being harvested, and finally by diseases. Oysters companies sold their shells to the poultry industry and for hardening roads rather than spreading them on beds to produce more oysters. Northern quahog. MerceiHiria mcnciiaiia. landings declined due to the narrowing of bay openings, adverse algal blooms, and bed closures due to do- mestic pollution. Soft clam, Mya arenaria. landings declined due to destruction of beds, bed closures due to domestic pollution, diseases, and high temperatures. Bay scallop. Argopecteii irradi- aihs. landings declined due to adverse algal blooms, changes in bay openings, and losses of eelgrass. Zostera marina. Attributing the declines to overfishing leads resource managers away from the actual causes and delays habitat restoration. DEVELOPMENT OF A PCR-BASED ASSAY FOR DETEC- TION OF THE .FOD-ASSOCIATED ROSEOBACTER. Aaron P. Maloy* and Katherine J. Boettcher, Departinent of Biochem- istry. Microbiology and Molecular Biology, University of Maine, Orono. ME 04469; Bruce J. Barber, School of Marine Sciences, University of Maine. Orono. ME 04469. Juvenile Oyster Disease (JOD) has resulted in substantial losses of cultured Eastern oysters. C/a.s.vo.sT/cd virginica. in the north- eastern United States. Despite management strategies utilizing se- lected lines and early deployment of hatchery-produced seed, JOD continues to occur annually in Maine's Damariscotta River. Fur- ther, during the past two years, mortalities have also been docu- mented in three previously unaffected areas throughout Maine. In all years and locations, affected animals were extensively colo- nized by a novel species of marine a-proteobacteria (in the Roseo- Ixicter clade). We are cuirently evaluating a PCR-based diagnostic assay for detection of the JOD-associated Riiseobacter. This assay uses specific primers to amplify the approximately 1300 base-pair internal transcribed spacer (ITS) region between the Roseolnicler I6S and 23S rRNA genes. Direct amplifications from suspended cells are successful at concentrations as low as 1.000 cells per reaction. From sequence data and restriction length fragment poly- morphism analyses, two genetic signatures are distinguishable. One is characteristic of the 1997-98 isolates, while the other is characteristic of the 2000-01 isolates. Thus PCR amplification followed by restriction enzyme digestion provides data regarding both the presence of. and specific genotype of. the JOD-associated Roseolnutcr. It is expected that this assay will be invaluable as a screening and diagnostic tool, and for regional management efforts to control the spread of JOD. PRELIMINARY STUDY ABOUT FEEDING ECOLOGY OF THE ROCK LOBSTER, PANVLIRUS HOMARUS, LIN- NAEUS, 1785, AT IRANIAN SEASHORES OF OMAN SEA. Nassrin Mashaii, Offshore Fisheries Research Centre. Chabahar. Iran. Preliminary aspects of feeding ecology of the rock lobster, Pannlirus Iwmarus. specimens collected by diving from January 1 999 to November 2000 w as considered. Stomach contents were National Shellfisheries Association. Mystic. Connecticut Abstnicls. 2002 Annual Meeting. April 14-18. 2002 4.^3 recorded by using the point method. Fi and Pi of different prey items were measured. Monthly and seasonally frequencies of empty stomachs and different preys were compared using Chi- square tests. Monthly changes of macrobenthic communities sampled by diving were studied. Ivlev index used for comparing the importance of items between stomach contents and mac- robenthics. Pearson correlation coefficient of different prey items against some hydrological factors was obtained. Spearman corre- lation coefficient was used for correlation between different preys against size groups, sexes and ovigerous females. EVALUATION OF THE STRUCTURE AND FUNCTION OF A CREATED BLUE MUSSEL {MYTILUS EDULIS) REEF. Sean McDermott, National Marine Fisheries Service. 1 Black- burn Dr.. Gloucester. MA 01930; David Burdick, Raymond Grizzle, and Jennifer Greene. Jackson Estuarine Laboratory. University of New Hampshire, 85 Adams Point Rd.. Durham. NH 03824. In May 2001 . blue mussels {Myiilia eiliilis) were collected from a donor site and transplanted into South Mill Pond, Portsmouth. NH. as part of a broad scale project to restore a degraded tidal salt pond. A total of six mussel reefs were established in two locations: three at the inner South Mill Pond (ISMP) and three at the outer South Mill Pond (OSMP). Two general parameters were used to evaluate the structure and function of the created reefs: ( 1 1 reef population dynamics (survivorship, size frequency distribution, density, and movement) assessed mussel response to transplanting and local pond conditions and (2) faunal utilization to evaluate the establishment of ecological functions for living marine resources (LMR). Mussel population dynamics were sampled once per month (June. July. August) using a random sample protocol. Mus- sels initially responded negatively to pond conditions (poor water quality; minimal tidal exchange), resulting in high mortality. Smaller mussels had a greater survival ratio as noted in a decreased average shell length. Survival and average shell length increased after water quality and tidal flow improved. Fish community dy- namics were evaluated in reefs and reference areas through the summer (June. July. August). Reef areas had greater species di- versity per sampling effort than reference areas under slow water conditions. Apletes qiuulracus and Meiiidiu meiiiclia were most common in reef areas. Fundulus heierocHuis and M. menidia were most common in reference areas. The constructed mussel reefs functioned as habitat for LMR\s immediately, providing shelter and forage for small fish. Further assessment of utilization by other LMR's (invertebrates) is required to properly evaluate the func- tions and value of the created mussel reef GENETIC MONITORING OF OYSTER STOCK EN- HANCEMENT IN THE CHOPTANK RIVER, CHESA- PEAKE BAY. Coren A. Milbury and Patrick M. Gaffney, Graduate College of Marine Studies. University of Delaware. Lewes. DE 19958. The spread of parasitic diseases (primarily MSX and Dermo). in conjunction with overharvesting. has led to the rapid decline of Eastern oyster (Crassostrea virginica) populations. Regional variation in disease resistance may be u,seful in restoration efforts. In collaboration with the University of Maryland Horn Point Labo- ratory, we have assessed the success of recent enhancement efforts within the Chesapeake Bay using genetic markers. C. virginica exhibits regionally diagnostic 16s mitochondrial DNA haplotype profiles (North Atlantic, South Adantic, and Gulf Coast). In 1997 oysters from Louisiana broodstock were planted in the Choptank River. Maryland. The presence of newly settled spat with the Gulf Coast haplotype in the Choptank River confirms the survival and propagation of the Louisiana broodstock. An automated mini- sequencing technique (Pyrosequencing Inc.) was used to determine the mitochondrial haplotypes of oyster spat collected throughout the Choptank River. This rapid mass screening method revealed that 95'7f of spat collected were of the North Atlantic haplotype and approximately 59c were South Atlantic. Of 4538 spat screened, four (0. 1 '^ ) possessed the Gulf Coast haplotype. The use of these genetic markers has enabled us to assess the survival, propagation, and dispersal of the Louisiana oyster stock within the Choptank Ri\er. Chesapeake Bay. PROGRESS IN THE BIOLOGICAL CONTROL OF ZEBRA MUSSELS WITH MICROBIAL TOXIN. Daniel P. Molloy,* Denise A. Mayer, Michael J. Gaylo, Kathleen T. Presti, Alex- ander Y. Karatayev, and Lyubov E. Burlakova, Division of Research & Collections, New York State Museum, Albany, NY 1 2230. Recent progress in the development of bacterial strain CL0145A as a biocontrol agent of zebra mussels, Dreissena spp.. is reviewed. Strain CL0145A is a North American isolate of Pseudomonas fliiorescens. a ubiquitous, soil-water. Gram-negative bacterium, and a U.S. patent for its use for zebra mussel control has recently been issued. Pseiidomoiias fliiorescens is not a para- sitic species, and histological analysis indicates that mussels die from a biotoxin associated with strain CL0145A cells, not from infection. Therefore, future commercial products based on this microbe could contain only dead cells, further reducing enxiron- mental concerns. When zebra mussels ingest strain CL0145A cells, the biotoxin specifically destroys their digestive gland tis- sues. All zebra mussel sizes tested to date (ca. 1-30 mm long) are susceptible to kill by strain CL0145A, and high mussel mortality is achievable at all temperatures examined (range. 5-27°C). Static cultures currently produce cells of the highest toxicity, but shaking culture protocols are being developed in order to proceed to large- 436 Ahsimcts. 2(102 Annual Meeting. April 14-18. 2002 National Shelltisheries Association, Mystic, Connecticut scale t'eirnentation production. Very small-scale trials, designed to simulate tlow-through conditions, have been conducted in tempo- rarily-installed pipes within a hydropower facility, and they have confirmed that exposui-e to bacterial cells for 2 days at 23°C achieves high mussel kill. Evidence of nontarget safety has been demonstrated in laboratory and mesocosms trials. Current studies are focusing on biotoxin identification, fermentation scale-up, and identification of the key biotic and abiotic factors needed to maxi- mize zebra mussel mortality. CHARACTERIZATION OF A PARASITIC AMOEBA IN THE AMERICAN LOBSTER BY MOLECULAR SYSTEM- ATICS. Thomas E. Mullen, Jr. and Salvatore Frasca, Jr., De- partment of Pathobiology and Veterinary Science. College of Ag- riculture and Natural Resources. University of Connecticut, 61 North Eagleville Road. U-3089. Storrs, CT 06269-3089. Past mass mortality events of Long Island Sound (LIS) lobster (Homanis americamis) have been associated with a number of potential etiologies, one of which is neurologic infection by a parasitic amoeba. Histopathologic examinations of nerves and gan- glia revealed tissue invasion by an amoeba, with and without at- tendant hemocytic infiltrates. This amoeba possessed a small, round, secondary nucleus differentially stained using the Feulgen technique. Transmission electron microscopic examination of ner- vous tissue confirmed the presence of this nucleus-like organelle, or Nebenkoiper, a consistent feature of members belonging to the genus Paramoeba Schaudinn, 1896. Previous efforts to culture parasitic Paramoeba spp. and recent attempts to culture the lobster amoeba in vitro have been unsuccessful. Molecular data for the family Paramoebidae is ab.sent, and the lack of such information contributes to the ambiguity associated with classification ot these organisms. In this study we propose to characterize the rDNA of this parasitic Paramoeba by determining the molecular systemat- ics of potentially related lobose amoeba. We have sequenced the small subunit (SSU) rRNA gene of twelve previously identified organisms that have morphologic (light microscopic and ultra- structural) similarity to the amoeba infecting lobster. This SSU rDNA has provided the data necessary to describe the current systematics of the Order Euamoebida using bioinformatic com- puter methods. Based on this rDNA sequence data, species of Neoparamoeba and Korotnovella occupy a separate clade between Vannella spp and the clade classically representing the Class Lo- bosa (Acanthamoeba spp and the leptomyxid amoebae). In addi- tion, such rDNA nucleotide sequence data has allowed for identi- fication of variable and conserved sequences that could be ex- ploited for the purpose of amplifying SSU rDNA from the parasitic amoeba in lobster tissue. The rDNA sequence of this amoeba is expected to allow for molecular characterization using molecular evolution methods and provides the critical sequence elements necessary to develop primers and probes for future polymerase chain reaction (PCR) and in situ hybridization-based (ISH) diag- nostic tests to delect the parasite in lobster tissue. These tests will be important to histopathologic and molecular diagnostics integral to future health sinvcillance programs. SEASONAL CHANGES IN CELL PROLIFERATION OF OYSTER TISSUES. Kim-Lien T. Nguyen and Jerome F. La Peyre,* Cooperative Aquatic Animal Health Research Program. Department of Veterinary Science. Louisiana State University Ag- riciiltural Center. Baton Rouge. LA 70803; Terrence R. Tiersch, Aquaculture Research Station. Louisiana Agricultural Experiment Station. Louisiana State University Agricultural Center. Baton Rouge. LA 70820. Identification of mitotically active tissues should assist devel- opment of cell lines from oysters and other bivalve molluscs. Except for evaluation of embryonic tissues, limited consideration has been given to identification of optimal tissue types and time of year to establish oyster cell lines. Thus, cell proliferation in tissues of the eastern oyster, Crassoslrea virginica. was evaluated monthly for one year by an immunohistochemical assay for pro- liferating nuclear antigen (PCNA), an endogenous marker of cell proliferation that is evolutionarily conserved and present in all active phases of the cell cycle (Gl, S, G2, M). A commercial monoclonal antibody to PCNA (PC 10) was used to calculate a labeling index (percentage of labeled nuclei for 1000 nuclei counted) at 400x magnification for each tissue. We found highest proliferation in somatic tissues (labial palps > digestive diverticula and stomach epithelium > gills > mantle) in late fall to early spring (November-April in southern Louisiana). Cell proliferation ceased in these tissues in late Spring while proliferation in maturing go- nads persisted. After spawning, proliferation resumed in somatic tissues for a month or two before gonad proliferation resumed in late summer. This agrees with earlier observations that cell pro- liferation in bivalves is highly variable throughout the year. DEVELOPMENT OF A MEDIUM TO INDUCE HYPNO- SPORE FORMATION AND ZOOSPORULATION OF PER- klNSUS MARINUS. Amy D. Nickens and Jerome F. La Peyre,* Cooperative Aquatic Animal Health Research Program. Department of Veterinary Science. Louisiana State University Ag- ricultural Center. Baton Rouge. LA 70803; Sandra M. Casas. Centro de Investigacions Marifias. Xunta de Galicia. aptdo. 13. E-36620 Vilanova de Arousa. Spam. The difficulty in inducing zoosporulation of P. nuiriniis hinders our ability to study zoospores. We recently developed a medium to induce hypnospore formation and zoosporulation of P. marimis. This medium was formulated in 3 steps by determining the effects of various solutions. indi\ idually and in combination, on the size, viability and zoosporulation of cultured parasites. The solutions tested included fluid thioglycollate medium (FTM) components (i.e., yeast extract, casein hydrolysate, dextrose, cystine, sodium National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 437 thioglycollate. agar) in step 1 ; solutions of amino acids, carbohy- drates, lipids, vitamins or nucleotides in step 2; and media supple- ments (lactalbumin hydrolysate. Hyprep 4601 . pancreatin. Ex-cyte VLE. egg yolk, oyster lysate) in step 3. Solutions tested at each step which had significant positive effects on the enlargement, viability, and/or zoosporulation of cultured parasites were included in a medium for testing all solutions of subsequent steps. The size (35 + 13 (i-m. N = 100). viability (95%, N = 400) and zoosporu- lation (35%, N = 400) of parasites incubated in our final medium for 6 days were significantly greater than the enlargement ( 11 ± 2 p.m), viability (309}-) and zoosporulation (O'/r ) of parasites incu- bated in the FTM for 1 week. The initial mean parasite size was 4 ± 1 jxm (N = 100). Interestingly, enlargement and viability of parasites incubated in our medium was similar to the enlargement (35 ± 9 [xml and viability (95%) of parasites incubated in oyster lysate. Zoosporulation (12%) of parasites incubated in oyster ly- sate was however significantly lower. The availability of P. mari- nus zoospores for study will enable investigations on the role of this cell staee in P. marinus life cvcle. THE EFFECTS OF MARTEILIA SYDNEY! ON THE HOST DEFENSE RESPONSES OF THE SYDNEY ROCK OYS- TER, SACCOSTREA GLOMERATA. Rodney Peters* and David Raftos, Marine Biology Laboratory. Mucquarie University. Sydney. Australia 2109. Marteilia sydneyi (paramyxean parasite) is the causative agent of QX disease in Saccostrea glomerata, which has mortality rates of 90 to 98%. Outbreaks of QX disease have reduced oyster farm- ing in some of the major oyster producing estuaries in New South Wales, Australia to critical levels. Once, farmers had a productive period of 2 years during which they could grow out, or "fatten," the oyster to a reasonable market size. Now this period lasts only from April/May to December of the same year in the QX contami- nated rivers. Invertebrates, according to all available evidence, lack antibod- ies and lymphocytes. However, they are still capable of mounting highly efficient cellular and humoral immune responses. Our premise is that these immune responses must be either evaded or overpowered by M. sydneyi to initiate QX disease. Specifically. our investigations have centered on proteolytic cascades, such as the prophenoloxidase (proPO) system, that in other species are closely associated with host defense. The prophenoloxidase cas- cade catalyzes the hydroxylation of tyrosine to dopa and the oxi- dation of dopa to dopaquinone. finally forming the pigment mela- nin. A number of intermediates in this pathway are cytotoxic or bacteriostatic, and melanin itself contributes to defense by encap- sulating foreign material. Data presented here describes an outbreak of QX disease on the Georges River, NSW during 2000/2001. A strict congelation was evident between infection intensity and prophenoloxidase activity. ProPO activities declined rapidly at precisely the time that infec- tion was established. The data supports the contention that proPO activity is specifically suppressed by M. sydneyi in order to estab- lish infection. POPULATION BIOLOGY OF MELONGENID WHELKS IN THE INTERTIDAL ZONE IN WASSAW SOUND, GEOR- GIA. Alan J. Power,* Mary Sweeney-Reeves, Todd C. Recicar, Dodie M, Thompson, and Randal L, Walker. Marine Extension Service. Shellfish Research and Aquaculture Laboratory, Univer- sity of Georgia. 20 Ocean Science Circle. Savannah, GA 3141 1- 1011. Four species of whelk (family Melongenidae) are found within the coastal waters of Georgia: the knobbed whelk. Busycon carica: the channeled whelk, Busycotypus canalicukitus: the lightning whelk, Busycon cantrarium; and the pear whelk, Busycotypus spi- ratus. These whelks are commercially harvested using trawls and are also taken intertidally by oystermen. clammers, and sport fish- ermen. To date the whelk fishery remains one of the most eco- nomically successful molluscan fisheries in the state. There is, however, a lack of fundamental biological information to allow for sound management decisions regarding the sustainability of the fishery. Consequently, the present study was initiated to examine the temporal variability in the abundance and population structure of each species in the intertidal zone in Wassaw Sound. Georgia. Whelks were collected at low tide from six locations, on a seasonal basis, over an annual period. Prior to releasing, all were tagged with an identifying label, measured (shell length, shell width, total wet weight), and sexed. Size frequency distributions, sex ratios, and sexua! dimorphism in terms of body size were determined. Growth rates and seasonal migration patterns were examined by recapturing previously tagged specimens. The implications of these results for the whelk fishery in Georgia are discussed. A RAPID METHOD FOR ASSESSING STRESS IN THE AMERICAN LOBSTER USING A HAND HELD GLUCOM- ETER. Deanna L. Prince, Robert Bayer.* Christina Congle- ton. Shannon Colby. Danielle LaVine. Danielle Volmuth, Kat- rina Brooks. Margaret Berry, and William Congleton. Depart- ment of Animal Sciences, and School of Marine Studies, University of Maine. Orono. ME 04469; John Vetelino, Depart- ment of Electrical and Computer Engineering. University of Maine. Orono. ME 04469. A number of parameters were measured in lobsters exposed to temperature stress and anoxia from air exposure. Measurements included electrical resistance across the tail membrane, resistance across the body from mouth to anus. pH. and hemolymph calcium, magnesium, and glucose. Hemolymph glucose appeared to be the best indicator of stress as a result of an increase in hyperglycemic honiione. Based on these preliminary findings, a rapid technique was developed to measure hemolymph glucose levels using a com- 438 Abstracts. 2002 Annual Meetmg, April 14-18, 2002 National Shellfishenes Association, Mystic, Connecticut niercial hand held gluconieter available from any pharmacy. The techniL|ue insolves centrilugation of the samples and placing the serum on a disposable strip, which is read by the gluconieter. In a field sampling using this technique lobsters from areas that tended not to survive well in shipping and storage showed elevated blood glucose. ROUTES OF HEMATODINIUM SP. TRANSMISSION INTO BLUE CRABS. Michael Sheppard.* Florian Rambow, Marc E. Frischer, and Richard F. Lee. Skidaway Institute of Ocean- ography. Savannah. GA 3141 1. Heinatodiniiim sp. is a parasitic dinoflagellate which causes mass mortality of blue crabs duruig infection peaks in late spring and fall in salt marsh estuaries of coastal Georgia. The life cycle of Heiiiatodiniitin sp. in blue crabs involves several different stages including dinospores. prespores, trophonts and plasmodia [Shields. J.D.. Ann. Rev. Fish. Dis. 4:241-271 (1994)]. A series of studies were carried out to detemiine the source and possible routes of transmission of Hfiiuitodinium sp. into blue crabs in the Wassaw Sound estuary system. Both histological and a recently developed molecular diagnostic techniques, including a quantitative real-time PCR method, were used to quantify Heiiuitddiimiiii sp. in crabs and in estuarine water samples. Three possible routes of transmission were investigated in these studies; consumption of infected tissues, injection of hemolymph containing Hcimandiiuiim sp.. and expo- sure to estuarine water and sediments where infected crabs were found. Hematodinium disease was transmitted by all of these routes, but the most effective route was the transmission to healthy uninfected crabs after feeding on infected tissues from the diseased blue crabs. In several experiments there was a lack of transmission of Hcimilddiiiiiiiii sp. into crabs from water found to contain Hc- nuiiodiiiiiiin. which we speculate was due to lack of infectious forms in the water, e.g. dinospores. Partially supported by Georgia Sea Grant. THE RELATIONSHIP BETWEEN BLACK SPOT DISEASE AND LIMB-LOSS IN CANCER PAGURUS FROM THE SHETLAND ISLANDS, SCOTLAND. Shelly M. L. Tallack, North Atlantic Fisheries College. Scalloway. Shetland Islands. ZEI OTS, UK. The edible crab. Cancer pai;iirtis. is a heav ily exploited crus- tacean resource throughout the UK. including the peripherally lo- cated Shetland Islands. Black spot disease is the primary infection recorded in the local population and is believed to be most preva- lent in specimens with reduced immune systems, resulting from. for example, injury. The confrontational, minority fishing activitv of removing only claws from this species in offshore Shetland waters has led to concerns regarding a possible increase in the number of severely injured crabs. Shetland's infection rates were compared with other UK studies. Black spot disease rates were higher in Shetland than in studies on populations in Norfolk (Ayres & Edwards, 19X2) and Wales (Davies. 1999). but lower than findings from Ireland (Vogan et al., 1999). A positive relationship v\'as shown between crab size and black spot disease. Greater infection severity was associated with later intermoult stages. Sex differences were evident with the pro- portion of infected individuals being higher in males (25.61'^) than in females (I2.78'y{-). Finally, limb-loss and crab iniury were positively correlated with infection. It cannot be determined whether black spot disease rates in Shetland are linked to claw tlshing activity rates. However, as vulnerability to black spot disease may be increased through in- jury, findings from the cuirent study in addition to earlier research, imply the need for handling techniques which minimize severe limb-loss and injury in discarded crabs. OYSTER GRAZING ON TOXIC AND NON-TOXIC PSEUDO-NITZSCHIA AND THALASSISIORA WEISFLOG- GII, AND DITYIAM BRIGHTWELUI. Anne Thessen,*' - Q. Dortch,' T. M. Soniat," and G.J. Doucette,' 'Louisiana Univer- sities Marine Consortium. 8124 Hwy 56 Chauvin. LA 70344; "Bi- ology Department. Nicholls State University. Thibodaux. LA 70310; 'Marine Biotoxins Program. NOAA/NOS. Charleston LAB. 219 Fort Johnson Rd, Charleston. SC 29412. Pseudo-nit: schio spp. are chain-forming diatoms that some- times produce donioic acid, a potent neurotoxin that causes Am- nesic Shellfish Poisoning (ASP). Despite high abundances of Pscndo-nitzschia over Louisiana oyster (Crasscislrea virginicu) beds, there have been no documented cases of ASP. Two possible explanations are that oysters cannot feed on long, pointed chains or they discriiuinate against toxic cells while grazing. Short-term (<2 hr) grazing experiments were conducted with non-toxic P. pseitdodelicatissima. toxic P. niuhisencs (22-87 |jim/cell. depend- ing on species and clone; 4 cells/chain median chain length). Tlialassisiora weisfloggii (15-23 pm). and Dityluiii hrii;htucllii (70-140 |jLm). Oysters (73 to 85 mm) were collected in the field, maintained on flowing ambient seawater. and then starved for 48 hours. Cultures were added at approxiniatelv 10'' cells/liter to con- tainers with individual oysters and to controls with no oysters or killed oysters. During experiments grazing was monitored by mea- suring decreases in //( vivo fluorescence, but cell counts and vol- umes were also measured. Oysters grazed rapidly on all diatoms and cells appeared in feces within I hour. Grazing rates, based on fluorescence, were lower on both types of Pseudo-nitzschui than for the other diatoms, but those differences may not be sustained when srazins; is based on cell volume/carbon. National Shellfisheries Association. Mystic. Connecticut Abstracts. 2002 Annual Meeting. April 14-18. 2002 4.^9 HISTORIC EASTERN OYSTER (CRASSOSTREA VIR- GINICA) DREDGE AND PATENT TONG SURVEYS IN THE MARYLAND PORTION OF THE CHESAPEAKE BAY. Jessica M. Vanisko.* Man Chrisfnian, and Kennedy T. Paynter. University of Maryland, College Park. MD 20742; Stephen J. Jordan, Maryland Department of Natural Resources (MD-DNR). Cooperative Oxford Laboratory. 904 S. Moiris Street. Oxford. MD 21654. The distribution of the Eastern oyster. Crassostrea virgiiiica. in the Chesapeake Bay remains spatially variable. The densities often range from 0 to 230 oysters per m". The conversion of historic oyster surveys to spatial tiles (CIS) has provided the means to examine the potential relationships between oyster biomass and spat settlement, as well as past distributions of oysters in spatially complex ways. Extensive patent tong sampling was conducted by the Maryland Department of Natural Resources (MD-DNR) during two surveys from 1975 to 1979. and again from 1989 to 1995. Data were compared to determine if significant changes in the density of spat, small, and market oysters occurred during the time period between the two surveys. Changes in oyster density in Maryland can be attributed to environmental disturbances and the impacts of disease. Oyster biomass. as calculated from the MD-DNR dredge survey, was paired with patent tong samples in an effort to test for. among other things, a relationship between oyster biomass and spat settlement on several spatial scales. A significant relationship between biomass and spat settlement could not be established with the data available, however data from current sampling regimes are being analyzed for relationships between oyster density, disease incidence, and/or spat settlement. HEMATODINIUM INFECTION IN BLUE CRABS, SPIDER CRABS AND STONE CRABS. Anna Walker,* Department of Pathology. Mercer University School of Medine. Macon. GA 31207; Michael Sheppard, Richard F. Lee, and Marc Frischer. Skidaway Institute of Oceanography, Savannah, GA 3141 1. Hematodinium sp, is a histoinvasive parasitic dinoflagellate. We report heavy infections of Hematodinium sp. in blue crabs {Calliuectes sapidus) and spider crabs {Lihinia emarginata) col- lected during late fall in a Georgia estuary. The prevalence of Hematodinium sp. in blue crabs and spider crabs ranged from 20 to 80% at different sampling stations. Prevalence oi Hematodinium sp. in stone crabs {Menippe mercenaria) from this estuary was only 5% (n = 20). Both hemolymph and tissues were examined microscopically and by molecular techniques. Most infected crabs had a high intensity of infection (15 to 90% of the hemolymph cells were parasite cells). A sequence analysis from the 18S rRNA gene ( 1682 bp) of Hematodinium sp. from spider crabs and stone crabs showed 99.6 and y9,5'/f sequence similarity, respectively, to Hematodinium sp. from blue crabs. Infected crabs had interstitial infiltrates of parasites in all tissues, but most notably in gills, hepatopancreas, cardiac and striated muscle. Focal muscle necrosis was present in heavy infections. Following injection of infected hemolymph. tissues of healthy crabs demonstrated an influx of granular hemocytes with encapsulations. SHELL MOVEMENT AND JUVENILE SURVIVAL OF THE OYSTER CRASSOSTREA VIRGINICA ON INTER- TIDAL REEFS ADJACENT TO WATERS WITH INTENSE BOATING ACTIVITY IN THE INDIAN RIVER LAGOON, FLORIDA. Linda Walters,* Kevin Johnson, Lisa M. Wall, and Neysa Martinez, Department of Biology, University of Central Florida. 4000 Central Florida Blvd.. Orlando. FL 32816; Ray Grizzle, Jackson Estuarine Laboratory. University of New Hamp- shire. Durham. NH 03824. Resulting from anthropogenic influences such as boat wakes or natural disturbance events, reefs of the eastern oyster Crassostiea virginica in the Indian River Lagoon have been declining in size in recent years. Additionally, dead margins (i.e. mounds of tightly packed, disarticulated shells extending above the high tide line) are commonly found adjacent to boating channels. To determine the impact of boating activity on: 1 ) shell movements, and 2) juvenile oyster survival, experiments were run on eight oyster reefs in Mosquito Lagoon, the northernmost region of the Indian River Lagoon. Florida. Four reefs had large dead margins, while the other four monitored reefs had no dead margins. To measure shell movement, 25 pre-weighed, oyster shells were deployed within 0.25m" quadrants on the exposed, middle and protected regions of each reef and dispersal of indiv iduals was assessed weekly for 8 weeks. Boat activity adjacent to the eight reefs was also recorded during the eight-week trials. There was a positive correlation be- tween shell dispersal in the exposed regions of reefs and the av- erage number of boats per hour that passed by the study reefs. Additionally, shells that weighed less than 50 grams and those which exceeded 100 grams experienced the least amount of overall movement, and the protected sides of the reefs experienced the least amount of shell movement, even for those reefs with dead zones. To determine the impact of shell movement on juvenile oyster survival, 1-cm diameter clay mimics were attached to 20 shells at the exposed, middle and protected regions on all eight reefs. At weekly intervals for the first 4 weeks of the 8- week trial, damage to the clay was recorded and used as a proxy to estimate damage to C. virginica at this vulnerable stage in its life-history. DEVELOPMENT AND TESTING OF A SIMPLE FIELD SYSTEM FOR MONITORING MUSSEL SHELL GAPE SIZE. Win Watson, Steve Jury, Jennifer Wishinski, Dan O'Grady, Walter Golet, Darren Scopel, Heidi Pye, and Chris Rillahan, Zoology Department and Center for Marine Biology, University of New Hampshire, Durham, NH 03824. Mussels and other bivalves are known to vary their shell gape size in response to changes in various environmental conditions such as salinity, current or the presence of contaminants. In fact. 440 Ahstivcr\. 2002 Annual Meeting. April 1 4- IX. 2002 National Shellfisheries Association. Mvstic. Connecticut several research groups have developed biomonitoring systems based on measuring the shell status of freshwater bivalves. The purpose of our project was to develop and test a simple and rela- tively inexpensive system for measuring the shell gape size of estuarine and sahwater mussels (Mytilus edulis) in their natural habitat. Battery-powered Hall-Effect sensors were used to detect the gape size of mussels and their output, in volts, was logged at 10 sec intervals onto a HOBO datalogger. The datalogger, batteries and customized electronics were placed in a waterproof case inside a buoy that communicated with mussels on the bottom via a wa- terproof cable. Monitoring systems were deployed at 6 different locations in the Great Bay Estuary. NH and along the N.H. coast- line. Data obtained was examined to determine if changes in gape size were correlated with changing tides, light levels and/or am- bient concentrations of heavy metal contaminants. Laboratory studies, under more controlled conditions, were used to determine their response thresholds to these same stimuli. While the system developed proved very sensitive and reliable, the complex factors influencing the gape size of mussels made it difficult to defini- tively explain all the variations in gape size recorded from mussels in their natural habitat. This study was supported by CICEET and Gulf Watch grants to W.H.W. THE NATIONAL SHELLFISHERIES ASSOCIATION The National Shellfisheries Association (NSA) is an international organization of scientists, manage- ment officials and members of industry that is deeply concerned and dedicated to the formulation of ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man- agement of shellfish resources. The Association has a membership of more than 1000 from all parts of the USA, Canada and 18 other nations; the Association strongly encourages graduate students' mem- bership and participation. 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E-mail; sandra.shumway@uconn.edu or sandrashumway@hotmail.com Membership information may be obtained from the Editor or the Treasurer using the form in the Journal. Institutional subscribers should send requests to; Journal of Shellfish Re- search. P.O. Box 465. Hanover, PA 17331. ""99 Erratum Abstracts of technical papers presented at the 5th Internatumal Cunt'erence on Shellfish Restoration. Nanaimo B.C.. Canada. September 1 S-20. 2001 ^0' Abstracts of technical papers presented at the 55th Annual Meeting of the Pacific Coast Oyster Growers Association & National Shellfisheries Association. Silverdale. Washington. September 20-22, 2001 -^21 Abstracts of technical papers presented at the 22nd Annual Meeting of the Milford Aquaculture Seminar. Milford. Connecticut. February 25-27. 2002 ^~^ Abstracts of technical papers presented at the y4th Annual Meeting of the National Shellfisheries Association. Mystic. Connecticut. April 14-18. 2002 ^57 COVER PHOTO: Carpet shell clam. Tapes deaissatus (Linnaeus. 1758). The species is of Atlantic-Mediterranean origin and is distributed along the Euro-African coast from England to Senegal and throughout the Mediterranean with penctiation in the Red Sea. While the introduced short-necked clam or striped venus (Tapes philippinanim) has almost superseded the carpet clam in Italian waters. T. decussalus is grown commercially in Sardinia. Photo courtesy of Consorzio Promo/ione Prodotti Ittici. Italy. The Journal of Shellfish Research is indexed in the following: Science Citation Index". Sci Search'". Research Alert"'. Current Contents®/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current Advances in Ecological Sciences. Deep Sea Research and Oceanographic Literature Review. Environmental Periodicals Bibliography. Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts. Huayong Que and Standish K. Allen, Jr. Hybridization of tetraploid and diploid Ciassostrea gigas (Thunberg) with diploid C. ariakensis (FujitaJ 137 Pedro Saucedo, Carmen Rodrigtiez-Jaramillo, and Mario Monteforte Microscopic anatomy of gonadal tissue and specialized storage cells associated with oogenesis and spermatogenesis in the calafia mother-of-pearl oyster. Pinctada mazatlanica (Bivalvia: Pteriidae) I'l-S Alan J. Power and Randal L. Walker Growth and gametogenic cycle of the blood ark, Anadara oralis ( Bruguiere, 1 789) in coastal Georgia 157 J. A. Rodriguez-Valencia, and F. Caballero-Alegria Temporal tluctuations (1989-1999) in the populations oi Haliotis fulgens and H. cornigata (Gastropoda: Haliotidae), at Islas San Benito (Baja California, Mexico) 163 M. Kruatrachue, P. Laimek, C. Wanichanon, V. Linthong, P. Sretarugsa, E. S. Upatham, and P. Sobhon Development of the nerve ganglia of abalone, Haliotis asinina Linnaeus 173 Ingrid Bahamondes-Rojas and Marta Bretos Induction of spawning and early development in Fissurella picia (Mollusca: Archaeogastropoda) from southern Chile. 185 Ludwig C. A. Naegel and Chris J. Cooksey Tyrian purple from marine muricids. especially from Plicopurpura pansa (Gould. 1 S.^?) 193 Dominique Sud. Jean-Marc Poncet, Armelle Saihi, Jean-Marc Lebel, Dominique Doumenc, and Eve Boucaud-Camou A cytological study of the mantle edge of Haliotis tuherculala L. (Mollusca. Gastropoda) in relation to shell structure. 201 Getian Wu, Kangsen Mai, Beiping Tan. and Wei Zhu Dietary biotin requirement of juvenile abalone. Haliotis discus hamuli Ino 211 Nicholas G. Elliott, Jason Bartlett, Brad Evans, and Neville A. Sweijd Identification of southern hemisphere abalone {Haliotis) species by PCR-RFLP analysis of mitochondrial DNA 219 Alberto de Jesus Navarrete Distribution and abundance of Slrombtis costatiis (Gmelin. 1791) larvae and adults at the biosphere reserve; Banco Chinchorro, Quintana Roo. Mexico 227 O. R. Chaparro, A. E. Soto, and C. E. Bertran Velar characteristics and feeding capacity of encapsulated and pelagic larvae of Crepidida fecimda Gallardo, 1979 (Gastropods, Calyptraeidae) -^^ Oscar Efrain Holgitin Quiiiones and Jesiis Emilio Michel-Morfin Distribution, density, and length-weight relationship of Chiton arliciilatus Sowerby. 1832 (Mollusca-Polyplacophora) on Isla Socorro. Revillagigedo Archipelago. Mexico 239 Bealriz Novoa, Carolina Tafalla, Angel Giierra, and Antonio Figueras Cellular immunological parameters of the octopus. Octopus vulgaris 243 H.-Jorg Urban and Jose M. Riascos V. Estimating gonado-somatic indices in bivalves with fused gonads 249 Arnold G. Eversole and Yavuz Mazlum Comparative fecundity of three Procamharus species -55 D. J. Macintosh, J. L. Overton, and H. V. T. Thu Confirmation of two common mud crab species (genus Scylla) in the mangrove ecosystem of the Mekong Delta, '*S9 Vietnam " Biplob Das, Yusuf Sharif Ahmed Khan, Khan Towhid Osman, Pranab Das, and Md. Nurul Amin Physico-chemical changes in acid sulfate soil during semi intensive culture of Penacus monodon fabricius. in cleared mangrove areas of The Chakaria Sundarbans. Bangladesh 267 Craig S. Lawrence, Noel M. Morrissy, Philip E. Vercoe, Ian H. Williams, and Yuk W. Cheng Physical, chemical, and biological variation among freshwater crayfish {Cherax albidus Clark. 1936) research ponds . . 273 Fernando Negrete-Soto, Enrique Lozano-Alvarez, and Patricia Briones-Fourzdn Population dynamics of the spiny lobster Panulirus guttatus (Latreille) in a coral reef on the Mexican Caribbean 279 Roy Melville-Smith and Yuk Wing Cheng Responses of migrating western rock lobsters Panulirus cvgniis (George, 1962) to two different tagging release J 289 procedures A. Battison, R. Cawthorn, B. Horney, and A. Mackenzie Mushroom tyrosinase as a control material for phenoloxidase assays used in the assessment of crustacean "health" .... 295 The Journal of Shellfish Research is indexed in the following: Science Citation Index®, Sci Search®, Research Alert®, Current Contents*/Agriculture, Biology and Environmental Sciences, Biological Abstracts, Chemical Abstracts. Nutrition Abstracts, Current Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography, Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts. CONTENTS CONTINUED ON PREVIOUS PAGE JOURNAL OF SHP:LLF1SH RESEARCH Vol. 21, No. 1 Jt'NE 2002 CONTENTS Kenneth Chew Honored Life Member: John B. Glude 1 Craig L. Appleyard and Joseph T. DeAlteris Growth of the northern quahog, Mercenciria inercenaria. in an experimental-scale upweller 3 Bassem Allam, Kathryn A. Ashton-Alcox, and Susan E. Ford Flow eytometrie measurement of hemocyle viability and phagocytic activity in the clam, Riuiuapcs philippiiniium 13 M. J. Stewart and R. G. Creese Transplants of intertidal shellfish for enhancement of depleted populations; prelinimary trials with the New Zealand 21 little neck clam Edward Fahy, Maria Lyons Alcantara, Mark Norman. Ronan Browne. Vincent Roantree. and Nick Pfeiffer Mortalities in Eiisis aniiatus (Jeffreys) (Solenacea) in western Ireland 29 Michael L. Zettler Ecological and morphological features of the bivalve /\,v/ssiiliis Gould. 1850: II. Field observations Anthony S. Anderson. A. Lelania Bilodeau. Matthew R. Gilg, and Thomas J. Hilbish Routes of introduction of the Mediterranean mussel (Myliliis galtoprovincialis) to Puget Sound and Hood Canal 75 Julie A. Maguire, Maurice O'Donoghue. Stuart Jenkins, Andrew Brand, and Gavin M. Buniell Temporal and spatial variability in dredging induced stress in the great scallop Peclen iiuaiiniis (L.) 81 Marcel Frechette and Gaetan Daigle Reduced growth of Iceland scallops Chlamxs islaiulica (O.F. Muller) cultured near the bottom: a modelling study of 87 alternative hypotheses Rosalio Maldonado-Amparo and Ana M. Ibarra Ultrastructural characteristics of spermatogenesis in diploid and triploid catarina scallop (Argopecten ventricosus Sowerby II. 1 842 ) ^^ Marcial Villalejo-Fuerte, Marcial Arellano-Martinez. Bertha P. Ceballos-Vdzquez, and Federico Garcia-Dominguez Reproductive cycle of Spondylus a(/c//e'r carpenter. 1857 (Bivalvia: Spondylidae) in the "Bahia de Loreto" National Park. Gulf of California. Mexico '^-^ Young Jin Chang, Min-Do Huh, Myung-Joo Oh, and Yoshio Sugawara Baculovirus-like particles in epithelial cell of digestive diverticula of the scallop. Patinopecten vessoensis 109 /. .S(//!i7a, N. A. Stokes, R. Smolowitz, R. C. Karney, and E. M. Burreson Haplosporidimn coshde (seaside organism), a parasite of the eastern oyster, is present in Long Island Sound 113 Kwang-Sik Choi, Kyung-Il Park, Ki-Wan Lee, and Kazumi Matsnoka Infection intensity, prevalence, and histopathology of Pcrkiiisiis sp. in the Manila clam. Rmlilapcs plulippmanim. in , , n I ' "9 Isahaya Bay. Japan Pedro Saucedo, Hie Racotta, Humberto Villarreal, and Mario Monteforte Seasonal changes in the histological and biochemical profile of the gonad, digestive gland, and muscle of the calatia mother-of-pearl oyster, Pinctada mazatkmica (Hanley, 1856) associated with gametogenesis 127 CONTENTS CONTINUED ON INSIDE BACK COVER JOURNAL OF SHELLFISH RESEARCH VOLUME 21, NUMBER 2 DECEMBER 2002 The Journal of Shellfish Research (formerly Proceedings of the National Shellfisheries Association) is the official publication of the National Shellfisheries Association Editor Sandra E. Shumway Department of Marine Sciences University of Connecticut Groton, CT 06340 Standish K. Allen, Jr. (2002) Aquaculture Genetics and Breeding Technology Center Virginia Institute of Marine Science College of William and Mary P.O. Box 1346 Gloucester Point. Virginia 23062 Shirley Baker (2004) University of Florida Department of Fisheries and Aquatic Sciences 7922 NW 71"' Street Gainesville. Florida 32653-3071 Brian Beal (2004) University of Maine 9 O'Brien Avenue Machias. Maine 04634 Peter Beninger (2003) Laboratoire de Biologic Marine Faculte des Sciences Universite de Nantes BP 92208 44322 Nantes Cedex 3. France Andrew Boghen (2003) Department of Biology University of Moncton Moncton, New Brunswick Canada El A 3E9 Neil Bourne (2003) Fisheries and Oceans Pacific Biological Station Nanaimo, British Columbia Canada V9T 6N7 Andrew R. Brand (2003) University of Liverpool Port Erin Marine Laboratory Port Erin, Isle of Man IM9 6JA United Kingdom EDITORIAL BOARD Eugene Buneson (2003) Virginia Institute of Marine Science P.O. Box 1346 Rt. 1208 Create Road College of William and Mary Gloucester Point, Virginia 23062 Peter Cook (2002) Department of Zoology University of Cape Town Rondebosch 77(J0 Cape Town, South Africa Simon Cragg (2002) Institute of Marine Sciences University of Portsmouth Ferry Road Portsmouth P04 9LY United Kingdom Leroy Creswell (2003) University of Florida/Sea Grant 8400 Picos Road, Suite 101 Fort Pierce, Florida 34945-3045 Lou D'Abramo (2002) Mississippi State University Department of Wildlife and Fisheries Box 9690 Mississippi State, Mississippi 39762 Christopher V. Davis (2004) Pemaquid Oyster Company, Inc. P.O. Box 302 1957 Friendship Road Waldoboro, Maine 04572 Ralph Elston (2003) Aqua Technics/Pacific Shellfish Institute 455 West Bell Street Sequim, Washington 98382 Susan E. Ford (2002) Rutgers University Haskin Shellfish Research Laboratory 6959 Miller Avenue Port Norris, New Jersey 08349 Journal of Shellfish Research Volume 21, Number 2 ISSN: 0730-8000 December 2002 www.shellfish.org/pubs/jsr.htm Raymond Grizzle (2003) Jackson Estuarine Laboratory Durham, New Hampshire 03824 Karolyn Mueller Hansen (2004) 1524 Barley Circle Knoxville, TN 37922 Mark Luckenbach (2003) Virginia Institute of Marine Science Eastern Shore Lab P.O. Box 350 Wachapreague, Virginia 23480 Bruce MacDonald (2002) Department of Biology University of New Brunswick Saint John. New Brunswick Canada E2L 4L5 Roger Mann (2002) Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Islay D. Marsden (2002) Department of Zoology Canterbury University Christchurch, New Zealand Tom Soniat (2002) Biology Department NichoUs State University Thibodaux, Louisiana 70310 J. Evan Ward (2002) Department of Marine Sciences University of Connecticut 1080 Shennecossett Road Groton, Connecticut 06340-6097 Gary Wikfors (2002) NOAA/NMFS Rogers Avenue Milford. Connecticut 06460 Juurnal oj Shellfish Research. Vol. 21. No. 2. 441^44. 20U2. 3=- CD IN MEMORIIIM HAROLD HALEY HASKIN 1915-2002 Hal Haskin died on June 23. 2002 at the place he loved best — his Cape Shore cottage on Delaware Bay. Mo.st of his 87 years-since he spent the summer after his junior year at Rutgers working on oyster drills — had been devoted to teaching and research on the marine environment, and Delaware Bay was its focus. Hal's "Honored Life Member Biography" appeared in 1*^)99 in Volume 18(2) of the Journal of Shellfish Research. Here we reflect more on his character and recount memories of the man. Hal was bom in 1915 at Niagara Fall, NY to George and Laura Haley, the second of three children. Three years later, the children were orphaned when both parents and a grandmother died within a week of each other in the 1918 flu pandemic. A family friend, Frederick Haskin. adopted Hal — an unusual arrangement, particularly for the time, because Fred Haskin was a bachelor. Haskin was a pipefitter, a job that forced him to travel around the country, so he lodged young Hal with a retired farm family living in southern New Jersey, near the DuPont chemical plant where he sometimes worked. The daughter of the family, whom Hal knew as Aunt Jenny, supervised the home and became his de facto mother. Later, Aunt Jenny and Fred Haskin married so that Jenny officially became the mother that she had unofficially been throughout most of his childhood. It wasn't until many years later that Hal became reacquainted with his many Haley relatives still living around Niagara Falls and for the first time was called "Uncle Hal". Always a good student and with an unusually strong work ethic. Hal graduated from Rutgers College in 1936. the first student to do so with a perfect grade score. It was at Rutgers that he came under the tutelage of Thurlow Nelson and began his life-long fascination with oysters. During summers spent investigating the predatory oyster drills, Hal observed that the snails preferred young oysters to older ones. Curious about mechanisms of chcnioattraction. he entered Harvard to work with John Welsh on this phenomenon, but he spent his first summer as a graduate student at the Bermuda Biological Station studying lobster neurohormones. He grew a beard, which came in red, was introduced to sailing, and acquired a taste for his research subjects. Hal switched to algal physiology for his PhD dissertation, which he carried out under the supervision of the oceanographer Alfred Redfield. His work included the development of a method for estimating chlorophyll concentrations using spectrophotometry — a precursor to the present-day Strickland and Parson's method. At Harvard. Hal supported himself as a dorm proctor and a teaching assistant. He was a natural teacher who excelled at hands-on instruction in both the laboratory and the field. Upon receiving his PhD in 1941, Hal entered the US Army and spent the next 5 years training troops and supervising units guarding the coast from Long Island to the Virginia Capes. He returned to Rutgers as an assistant profes.sor in the Department of Zoology in 1 946 and began developing a research program devoted to marine bivalves. His early work involved culturing and rearing of hard clams on a grant from Campbell's Soup Company. In 1 950. however, he succeeded his mentor Thurlow Nelson as head of the Oyster Investigation Laboratory in the New Jersey Agricultural Experiment Station, and for many years thereafter devoted most of his research efforts to protecting and enhancing the oyster resource of Delaware Bay. Although he later became deeply involved in acquiring data on, and developing a management plan for, offshore surf clams, his heart and mind never strayed far from oysters and Delaware Bay. From his early efforts to institute harvest limits on the depleted natural seed oyster beds, through the devastation brought by the MSX parasite, to providing management data when populations later rebounded, Hal considered the Bay's oysters to be his personal responsibility. He brought to this and other tasks a unique combination of intelligence, scientific integrity, and the ability to work with people from all sides of a problem. He never shirked from controversy, but he was always armed with reams of data, which he carried in whiskey boxes in the back of his car, to backup his point of view. Hal took pains to make the data understandable to both the regulators and the fishermen. He spoke his mind, albeit diplomatically, and he rarely lost a battle. 441 442 Ford et al. One ongoing stuggle, which he inherited from Thurlow Nelson, was maintaining freshwater flow into the Delaware estuary. In this battle against diversions and dams, he had the enthusiastic participation of his wife. Peg. whom he had met when she was a recent graduate of Smith College and he was a graduate student at Harvard. The two of them eventually became a formidable team fighting for rational water management in New Jersey, particularly concerning those policies affecting Delaware Bay and its oyster populations. Although he ran a large research laboratory. Hal devoted much of his time to teaching. As a young assistant professor, he taught mainly undergraduate courses in general biology, limnology, animal physiology, and invertebrate zoology. Somewhat later, he developed graduate courses in coastal oceanography, estuarine ecology, and malacology. Most of the graduate students in the Rutgers Zoology Department took at least one of these courses at some point in their academic careers — and never forgot them. Hal did not confine his teaching to the classroom, nor did he limit it to official students. He delighted in showing anyone the anatomy of a shucked oyster or talking about the intricacies of an oyster community. His audience might be a businessman or an oyster grower — it didn't matter — his enthusiasm captivated them all. Anyone who was associated with Hal in any capacity always wound up considering himself or herself a student. And Hal. himself, never stopped being a .student. Hal retired in 1984 but he remained active long thereafter. During his nearly 50-year career, he touched many people — students, colleagues, university and government officials, and shellfish producers. More than 200 of them gathered at the Haskin Shellfish Laboratory on September 28, 2002 to remember him. People came from all along the eastern seaboard and from as far west as Colorado to tell stories, share memories, renew acquaintances, look at old photographs, and eat good food — including freshly shucked oysters farmed at the Cape Shore near the Haskin cottage. His first and last graduate students were there, as were a host of students in between. They recalled how taking one of his courses had been the stimulus to go into marine science or shellfish biology. Several remarked on how tough a questioner he was during thesis defenses and how his criticisms were right on the mark, sometimes requiring a return to the bench to do a critical experiment. Although Hal was remembered most for applied research that directly benefited the shellfish industry, his intellectual curiosity was profound. He insisted that applied research conform to the standards of basic research. Further, how could one provide sound scientific advice for management without understanding fundamental biological and ecological principals? Woe to any student or colleague who failed to convince Hal that he or she knew the basic scientific principles underlying a concept. Students remembered his Saturday courses and that he worked well into the night on Friday, and often into the early morning hours of Saturday, preparing. The classes officially ran from 8 in the morning to 5 in the evening, but leaving at 5 was viewed with a highly disapproving eye. Several students remarked on his indifference to physical discomfort on the field trips — and that he was equally indifferent to discomfort of the students. The discomfort usually took the form of "cold and wet'" or "seasick", but on one occasion it was much more senous. On an oceanography class field trip. Hal lacerated his hand severely on the pulley wheel of an outboard motor that he was attempting to start. The deep cut ran along the base of his fingers and down the outside of his palm. Flapping fingers indicated severe damage to his hand, but he merely wrapped it in a handkerchief and was fully prepared to continue the trip. At the overwhelming insistence of his students, however, he reluctantly canceled the excursion in favor of a trip to the hospital and several hours of microsurgery to rejoin the tendons leading into two fingers. Neighbors, who grew up with the Haskin children at the Cape Shore, and whose fathers were businessmen and auto mechanics, couldn't figure out what Hal did for a living. He was like a kid in an adult body who actually got paid for mucking about on the tide flats playing with oysters. But he could answer all the questions they had about the organisms they found in the bay and along the shore. Hal was an advocate of simplicity. He was not interested in "building an empire" because he felt that he would then have to spend all his time supporting and defending it — certainly much less stimulating to him than the hands-on field and lab activities that he relished. Colleagues and students remembered him as a quiet, low-keyed person who was a good listener. He was thoughtful and one knew that he had truly reflected upon a question or an issue before he gave advice. One of the fondest memories, shared by nearly all the students, was of the biannual "tray moves". These events took place each spring when trays of selectively bred oysters had to be moved from winter quarters, where they were protected from ice damage, onto the tidal flats in front of the Cape Shore Laboratory where they were exposed to disease pressure and could be easily tended. Each fall, the reverse move took place. All able hands — students, faculty, technicians, friends — were marshaled for the event because the oysters in each tray — and there were often more than 100 trays — had to be counted, samples removed, and the oysters had to be placed in freshly prepared trays. The weather was usually cold and miserable, and ones fingers quickly became numb. Tying lines on the trays so that they could be hung in the marina that served as winter quarters was especiall\ challenging under these conditions. Hal would check every single knot (four per tray, half-hitches, not bowlines) and retie most, while being silently cursed by the small group of students standing around on the dock — cold. wet. tired, and anticipating the reward that awaited them at the Haskin cottage where Peg was preparing the traditional turkey dinner and a fire was blazing in the wood stove. Fifteen, twenty, occasionally up to thirty famished people would arrive. The first order of business was the preparation and distribution of gin and tonics, and if Hal made them, they contained plenty of gin. People pitched in to set the table, make the salad, and cook the peas, but certain tasks were Hal's own prt>vince: making the gravy, mashing the potatoes, and carving the turkey. These jobs had to be performed in a very specific fashion, with Hal describing to anyone within earshot his way to successful gravy or mashed potatoes. The evening ended with homemade (by Peg) pie — cherry, pumpkin, blueberry, and pecan — along with ice cream, coffee and tea. and very muted memories of the earlier discomforts. The tray moves were planned to end on Saturday night when the turkey dinner was held. Sunday morning one could sleep late, but not so late as to miss the "flapper" breakfast — again at the Haskin cottage with Hal busy making blueberry pancakes. The tray moves eventually became very popular events that attracted ex-students and often, their friends, back to the Cape Shore for a weekend of work and good fun. Hal's publication record was modest and belies his research accomplishments. He was totally disinterested in maintaining a CV. In fact, it was difficult to locate citations, among his own files, to list in this remembrance. His exacting standards applied to writing — both style and content — and to his own as well as that of others. He had difficulty with the concept of publishing a paper that didn't tell a In Memorium: Harold Haley Haskin 443 complete story, and he usually telt that he needed more data than he had. This was particularly true for field studies, even though his data sets extended for years, and. in some cases, for decades. Although Hal always analyzed the results and used them in meetings or to advise management agencies, formal reporting was less interesting than starting a new project or going on a field expedition. Students recalled late night sessions in which everyone pitched in to help copy and assemble reports at the very last minute — typically well after the deadline. Nevertheless, Hal had little difficulty obtaining funds to run his laboratory because grant managers knew the quality of the data would be high — when they eventually received it. When he wasn't working, which often seemed like never, Hal had three great recreational passions: woodcutting, sailing, and growing lilacs. For years. Hal and Peg heated their house in southern New Jersey and their Cape Shore cottage with wood stoves. Their house in Piscataway had multiple fireplaces. Hal always carried a chain saw and splitting wedges in the trunk of his car in case he had an hour or two free to cut wood. Friends recalled that he cut down only dead or dying trees and knew exactly when they should be cut to maximize dryness and minimize decay. The principal Haskin sailboat was a 17 ft Thistle that was anchored on the tidal flats in front of the summer cottage, and sailed as often as possible at high tide with Hal at the helm and a boat full of family, friends, and students from the Cape Shore Laboratory, just down the beach. Annual beach parties were a chance for sailboat races between the Thistle and any other sailboat that could be pressed into service. More than 100 lilac bushes representing 70 different varieties, formed a fragrant hedge around the Piscataway house. Hal knew all the varietal names and delighted in escorting visitors around the hedge, "introducing" them to each plant and pointing out its unique characteristics. Each May when the lilacs bloomed, he would bring buckets full of blossoms into the lab to distribute among the staff and faculty. He stored them in the cold room, which temporarily at least, smelled delightfully of lilacs rather than aged oysters. The memorial service included tributes from representatives of numerous agencies, organizations, and institutions that Hal served, including the National Shelltlsheries Association. He joined NSA in the late 1940s and rarely missed a meeting for almost 50 years. He was Vice President in 1966-67. and President during 1967-69. He became an Honored Life Member in 1979 and. most precious to him. was given the David Wallace Award in 1984. In addition to Peg, Hal is survived by five children, Kathleen Haskin of New York City. Jean Haley of North Kingstown, RI, Elizabeth Haskin of Cape May Court House, NJ, Frederick James Haskin of Piscataway, NJ, and Mary D. Haskin of Washington, DC: and four grandchildren. Harold Haskin II. a student at Rowan University, Allegra and Elijah Penny, of Washington. DC. and William Schroer of Cape May Court House. NJ. Hal. alone and with Peg, received numerous awards over the years (see Kraeuter and Ford. 1999. J. Shellfish Res. 18: 337-339), but one of the finest was presented at the memorial service: The New Jersey Nature Conservancy named a portion of its Delaware Bayshores Tract near the Cape Shore cottage "The Harold and Margaret Haskin Nature Preserve." It was an entirely fitting honor for a lifetime of dedicated service to environmental teaching and preservation. Susan Ford John Kraeuter Walter Canzonier Haskin Shellfish Research Laboratory Port Norris. New Jersey PUBLICATIONS Welsh. J. H. and H. H. Haskin. 1939. Chemical mediation in crustaceans. IM. Acetylcholine and autotomv in Pcinilisrlws aniuilus (Gibbes). Biol. Bull. 76:405^15. Haskin. H. H. 1942. A spectrophotometric method for the analysis of chloroplast pigments. J. Biol. Chem. 144:149-160. H.iskin. H, H. 1949. Growth studies on the quahaug. Venus mercenaria. Proc. Nat'l. Shellfish. Assoc. 1949:67-75. Haskin. H. H. 1950. Selection of food by the common oyster drill Urosalpiiu cinerea (Say). Proc. Nat'l. Shellfish. Assoc, 1950:62-68. Huskin. H H. 1952 Further growth studies on the quahaug, Venus mercenaria. Proc. Nat'l. Shellfish. Assoc. 1952:181-187. Haskin, H. H. 1954. Age determination in molluscs. Trans. N.Y. Acad. Sn. 16: 300-304. Pomeroy, L. R. and H. H. Haskin. 1954. The uptake and utilization of phosphate ions from sea water by the American oyster. Crassostrea virginica (Gmelin). Biol. Bull. 107:123-129. Pomeroy, L. R.. H. H. Haskin and R. A. Ragotzkie. 1956. Observations on dinotlagellate blooms. Limol. Oceanog. 1:56-60. Dean. D. and H. H. Haskin. 1964. Benthic repopulation of the Raritan River estuary following pollution abatement. Limnol. Oceanog. 9:551-563. Haskin. H. H. 1964. The distribution of oyster larvae. Proc. Symposium on Exp. Marine Ecology. Occasional Pub. #2. Rhode Island Univ. Grad. School of Oceanography, pp. 76-80. Haskin. H. H., W. J. Canzonier & J. L. Myhre. 1965, The history of MSX on Delaware Bay oyster grounds, 1957-65. Amer. Malacol. Union Reports /or 7965 32:20-21. Haskin, H. H., L. A. Stauber& J. A. Mackin. 1966. Mincliinia nelsoni n. sp. (Haplosporida. Haplosporidiidae): causative agent of the Delaware Bay oyster epizootic. Science 153:1414-1416. Hamwi, A. and H. H. Haskin. 1969. Oxygen consumption and pumping rates in the hard clam Mercenaria mercenaria: a direct method. Science. 163:823-824. Hidu. H. and H. H. Haskin. 1971. Setting of the Amencan oyster related to environmental factors and larval behavior. Proc. Nat'l. Shellfish. Assoc. 61:.V5-.50. Van Winkle. W., S. Y. Feng, and H. H. Haskin. 1976. Effect of temperature and salinity on the extension of siphons by Mercenaria mercenaria. J. Fish. Res. Brd. Can. 33:1540-1546. 444 Ford et al. Douglass. W. R. & H. H. Haskin. 1976. Oyster-MSX interactions: alterations in hemolymph enzyme activities in Crassostrea virf>iiiicci during the course o( Minchinia nelsoni disease development. J. Invertehi: Puthol. 27:317-323. Hidu. H. and H.H. Haskin. 1978. Swimming speeds of oyster larvae Crassosuea virj^inica in different salinities and temperatures. Estuuries 1:252-255. Haskin. H. H. & S. E. Ford. 1979. Development of resistance to Minchinia nehoni (MSX) mortality in laboratory-reared and native oyster stocks in Delaware Bay. Mar. Fisheries Rev. 41:54-63. Ford. S. E. & H. H. Haskin. 1982. History and epizooliology of Haph>spi>ruliiini nehnni (MSX). an oyster pathogen, in Delaware Bay. 1957-1980. J. Inverlebr. Pathol. 40:118-141. Haskin. H. H. & S. E. Ford. 1982. Haplnspnridiinn nelsoni (MSX) on Delaware Bay seed oyster beds: a host-parasite relationship along a salinity gradient. / Inveriehi. Pathol. 40:388-405. Haskin. H. H.. R. A. Lut/ & C. E. Epifanio. 1983. Benthos (Shellfish). Chap. 13. In: J. H. Sharp, (ed). The Delaware Estuary: Research as Background tor Estuarine Management and Development. University of Delaware College of Marine Studies and New Jersey Marine Sciences Consortium. Lewes. Delaware, pp. 183-207. Haskin, H. H. & S. E. Ford. 1983. Quantitative effects of MS.X disease {Haplnsporuhnni nelsoni) on production of the New Jersey oyster beds in Delaware Bay. USA. Proceedings of ICES. CM I983/Gen:7/Mini-Syinp. Goteborg. Sweden. October. 1983. 20 p. Haskin. H. H. & S. E. Ford. 1986. Breeding for disease resistance in molluscs. Proceedings of EIFAC/FAO Symposium on Selection. Hybridization and Genetic Engineering in Aquaculture. EIFAC/86/Symp. R27. Bordeaux. France. May, 1986. 25 p. Haskin, H. H. & S. E. Ford. 1987. Breeding for di.sease resistance in molluscs. In: K. Tiews, (ed). Proceedings World Symposium on Selection. Hybridization, and Genetic Engineering in Aquaculture, Bordeaux 27-30 May, 1986. Vol. II, Heenemann Verlagsgesellschaft, Berlin, pp. 431-441. Ford. S. E. & H. H. Haskin. 1987. Infection and mortality patterns in strains of oysters Crassostrea \irf;inica selected for resistance to the parasite Haplosporiclinm nelsoni (MSX). / Parasilol. 73:368-376. Ford. S. E. & H. H. Haskin. 1988. Comparison of in vitro salinity tolerance of the oyster parasite Haplosporidinm nelsoni (MSX) and hemocytes from the host. Crassostrea virginica. Comp. Biochem. Physiol. 90A:I83-I87. Ford. S. E. & H. H. Haskin. 1988. Management strategies for MSX iHaplospmidinm nelsoni) disea.se in eastern oysters. In: W. S. Fisher, (ed). Disease Processes in Marine Bivalve Molluscs. 18. American Fisheries Society. Bethesda, MD. pp. 249-256. Haskin, H. H. & J. D. Andrews. 1988. Uncertainties and speculations about the life cycle of the eastern oyster pathogen Haplosporuhiim nelsoni iMSX). In: W. S. Fisher, (ed). Disease Processes in Marine Bivalve Molluscs. 18, American Fisheries Society, Bethesda, MD. pp. 5-22. Barber. B. J.. S. E. Ford & H. H. Haskin. 1988. Effects of the parasite MSX [Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy metabolism. I. Condition index and relative fecundity. J. Shellfish Res. 7:25-31. Barber. B. J.. S. E. Ford & H. H. Haskin. 1988. Effects of the parasite MSX [Huplosporidinni nelsoni) on oyster [Crassostrea virginica) energy metabolism. U. Tissue biochemical composition. Comp. Biochem. Physiol. 91A:603-608. Vrijenhoek. R. C. S. E. Ford & H. H. Haskin. 1990. Maintenance of heterozygosity during selective breeding of oysters for resistance to MSX disease. J. Hcrerf(0'8I:418-+23. Ford, S. E.. A. J. Figueras & H. H. Haskin. 1990. Inlluence of selective breeding, geographic origin, and disease on gametogenesis and sex ratios of oysters. Crassostrea virginica. exposed to the parasite Haplosporidium nelsoni (MSX). Aquaculture 87:285301. Hillman, R. E., S. E. Ford & H. H. Haskin. 1990. Minchinia teredinis n. sp. (Balanosporida. Haplosporidiidae). a parasite of teredinid shipworms. J. Protozoal. 37:364-368. Kraeuter. J. N.. S. R. Fegley. S. E. Ford & H. H. Haskin. 1993. Delaware Bay oyster populations: effects of seed movement, harvesting, and diseases. In: R. F. Dame. (ed). Bivalve Filter Feeders in Estuarine and Coastal Ecosystem Processes. G 33, Springer-Verlag. Berlin, pp. 531533. Dittman. D. E.. S. E. Ford & H. H. Haskin. 1998. Growth patterns in oysters from different estuaries. Mar Biol. 132:461-469. JoKnial of Shellfish Rc.winh. Vol. 21. No. 2. 44.'i-t46. 2002. Jay Donald Andrews Honored Life Member Jay Andrews, affectionately known as -Andy" by his many friends and colleagues, is widely respected for his fLindamental research on the ecology of the major oyster pathogens in the Chesapeake Bay, Haphsporidium iwl.soni and Perkinsus munnus. Less well known, but equally important, are his 22-y dataset on oyster spatfal! patterns in the Chesapeake Bay and the disease management strategies he developed for industry. Andy was bom on September 9. 1916 in Bloom, Kansas. He grew up on a wheat and cattle farm in western Kansas south of Dodge City during the Depression and drought of the 1930s. Andy attended high school in Bloom and was the top student in a class of 10. Studies were apparently easy for him and he spent much time reading Zane Gray wild-west stories and following baseball scores and players. When Andy graduated from high school in 1934, the nation was in full depression, and the western plains were in a severe ten-year drought that was making farmmg very difficult, if not impossible. Lack of moisture prevented raising wheat or fodder for cattle. Over time, Andy remembers, pastures of the family farm were filled with annual weeds, prickly pear cactus, and an explosion ot jackrabbits. After graduation, Andy's father took him to a bank in Dodge City, borrowed $100 and sent him to Kansas State College in Manhattan. College opened a new world for Andy, and he worked hard. In addition to classes, he worked 40 hours each month in the state 4-H Club for 23 cents an hour and participated in the ROTC. which paid $15 a month. He was designated the top agricultural student and obtained a degree in agriculture in 1 938, but farming in western Kansas was not an attractive proposition for Andy. Instead, he began graduate study at the University of Wisconsin in Madison, where he obtained a teaching assistantship in biology. He earned a salary ot $600 a year and had money for extracurricular activities for the first time. His love of opera developed during these years. Andy earned an M.S. in 1940 and continued on for his Ph.D. Andy's graduate studies were intenupted by World War II; he was called up in January 1941 and spent four years in the infantry. He saw action in the South Pacific at the battle of Leyte Gulf and the battle of Bataan and survived multiple kamikaze attacks on his troop ship. In 1946, he returned to Madison to finish his degree, which he earned in 1947. In the spring of 1946, Andy and fellow student. Willard Van Engel, attended a fisheries meeting in St. Louis, where they met the Director of the Virginia Fisheries Laboratory in Yorktown, VA (the precursor of VIMS), who offered them jobs that they both accepted. Van Engel was requested to work on blue crabs, and Andy was asked to concentrate on oysters. A few months later, he was advised by a visiting professor from Yale not to work on oysters, because there was already a large literature and "everything had been done already." Thankfully, Andy ignored the advice. When Andy and "Van" arrived in Virginia in the fall of 1947, they learned that the three scientists who had been at the laboratory had left that summer for positions at Texas A&M. They had been working with mussels on some compound important to the war effort. The three scientists were John Mackin, Sewell Hopkins, and Winston Menzel. 44.5 446 BURRESON Shortly after he an'ived in Virginia. Andy began monitoring oyster spatt'all patterns in the tributaries. After years of monitoring, it became clear that setting patterns could be easily separated into two distinctive types. The large tributaries and the Chesapeake Bay proper require very large stocks of brood oysters to acquire regular spatfall because of the large tidal exchange and flushing. Only the upper James River estuary met this requirement and that was because of its special circulation patterns and the large beds of transplanted oysters in the lower James being grown by private industry. Such other large rivers as the York, Rappahannock, and Potomac never had enough broodstock to produce regular spatfall. but occasional large sets did occur. The small tributaries, with low runoff, exhibit a completely different setting pattein than the large tributaries. These coastal plain subestuaries have moderate annual setting patterns, even though oyster populations are low because of high larval retention caused by the winding channels and shallow flats. Uncharacteristi- cally, Andy never published these data on spatfall patterns. Beginning in 1950. Andy tried to persuade managers in Virginia to use the small estuaries to produce seed oysters for transplantation to larger tributaries, but this advice was ignored until long after he retired. When the reef restoration strategy was initiated in Virginia in 1996. the first few reconstructed oyster reefs were placed in these small tributaries because of the larval retention patterns that Andy had documented. Andy's research on oyster diseases began when John Mackin visited VIMS from Texas to determine if PerkiiiMis nuiriiuis (Dermo disease) was present in Chesapeake Bay oysters. The discovery of this pathogen in an area lacking oil drilling confirmed that oyster mortality in the Gulf of Mexico was the result of P. marinus. not the oil indu.stry. Andy produced fundamental papers on the epizootiology of Dermo disease during the 19.')0s, and his 1988 review on the pathogen is a classic paper still widely read and cited. When Haplospondium nelsoni (MSX disease) appeared in the Chesapeake Bay in 1959. Andy immediately began conducting research on this organism and produced many seminal papers on the ecology of this pathogen as well as on Haplosporidium coslale. One of Andy's most important traits was that he published his results in a timely manner. In 1960, Andy initiated a monitoring program for H. nelsoni at VIMS using imported susceptible oysters. This program continues to the present day and provides a 42-year database on H. nelsoni prevalence and intensity that has proved invaluable for examining climate effects on pathogen abundance. Because of his broad training and experience and his inquisitive nature, Andy was very insightful. For example, he was the first to hypothesize, in 1980, that H. nelsoni was an introduced pathogen. It wasn't until 2000 that molecular data were obtained that support his supposition. Andy worked hard and expected hard work of others. You arrived on time, and you didn't leave early. Andy loved the rigors of fieldwork and scoffed at suggestions for an easier way to do things. He hauled oyster trays by hand and cleaned them by throwing countless buckets of water that he dipped by hand. After such trips he returned to the campus at VIMS wet. co\ered \\ ith mud. shirtless, in shorts, and barefoot. He surely startled more than one unsuspecting graduate student. Andy is internationally recognized for his fundamental research on oyster diseases, but he was also a long-time educator. During his career at VIMS he taught Ichthyology. Field Biology, Taxonomy and Systematics, Marine Ecology and Biologic Oceanography. Andy was a capable and energetic volleyball player. In the "interesting" lunch time volleyball games at VFL/VIMS he played with gusto, spiking near net-balls down the throats of opposing players with great vigor and glee. Road trips with Andy were always an experience. Once, while driving north for a meeting, Andy spied a walnut tree along the road. He pulled over and took an old pair of coveralls out of the trunk. He tied off the end of each leg, walked over to the tree and began filling the legs w ith w ainuts that had fallen to the ground, commenting that he hoped we would not be shot by the owner. Andy is an avid vegetable gardener and has a large plot near VIMS. He can still be seen hauling countless old milk jugs full of water in his old truck during drought periods and undoubtedly still cursing the varmints that take half his crop. Andy is a long-time member of the National Shellfisheries Association and served as President and Editor. He was elected Honored Life Member in 198.^. In 1998, he received the David Wallace Award in recognition of his career-long efforts to use science for the betterment of shellfish management and a sustainable industry. Eugene Burreson Gloucester Point, Virginia JoiiriHil of Shellfish Research. Vol. 21, No. 2, 447-448, 2002, Neil F. Bourne Honored Life Member Neil was bom August 1 1. 1929 in London. Ontario, Canada. He began his elementary school education there, and it was in London, mainly through the influence of his father, that he became interested in natural history and particularly bird watching, a hobby and passion that have continued to the present. It was this interest in natural history that eventually led him to become a biologist. The family moved to Hamilton, Ontario in 19,^9, where he completed his elementary and high school education. By the time he moved to Hamilton, he had over 150 bird species on his life list! He entered McMaster University in Hamilton in 1948 and graduated with a BSc in biology in 1952, In 1953, he completed a MSc degree at McMaster under an Ontario Research Scholarship and in 195.^-54 spent a year in Germany, where he undertook studies in limnology at the Freshwater Institute in Ploen and marine biology at the University of Kiel, He returned to Canada in 1954, entered the University of Toronto, and graduated from there with a PhD in 1959, His thesis was entitled. "Determination of carbon transfer from Chlorella vulgaris Beyerinck to Daphnia luagiui Strauss using radioactive carbon (C14) as a tracer," Three years of his studies were supported by National Research Council of Canada scholarships. On completion of his PhD in March 1959. Neil joined the staff of the Fisheries Research Board of Canada at the Biologic Station in St. Andrews, New Brunswick working under the direction of the late Dr, J, C, Medcof. a former Honorary Member of NSA, At St, Andrews, he was in charge of the sea scallop investigation that included investigations of the basic biology and ecology of sea scallops, dynamics of sea scallop populations on Georges Bank and in the Bay of Fundy. scallop gear efficiency trials, and exploratory work to locate sea scallop resources in other areas along Canada's Atlantic coast. While at St. Andrews, Neil spent two weeks at Dr. V. Loosanoff s laboratory in Milford. Connecticut studying bivalve breeding technology. Thus began a life-long commitment to shellfish aquaculture. He used knowledge gained at the Milford laboratory to begin initial attempts to culture sea scallops and was successful in conditioning and spawning adults and raising larvae to the mature stage. In 1965, Neil transferred to the Pacific Biologic Station in Nanaimo, British Columbia, first working for the Fisheries Research Board of Canada and later with the Department of Fisheries and Oceans, Initially, he worked with the late Dr, Dan B, Quayle. another former Honored Member of NSA. and remained at the Pacific Biologic Station until his retirement. During his career at the Pacific Biologic Station, Neil undertook research studies on a wide range of molluscan subjects, including the basic biology of several Pacific coast molluscan species, population studies of commercially important clams, harvesting gear efficiency, and studies to develop techniques to culture several species. He developed techniques to predict Pacific oyster spatfall accurately for the industry, a service he continues to provide. He undertook extensive surveys of bivalve resources in B,C, and. in particular, followed the dispersal of Manila clams northward along the coast. As a result of these studies, a fishery for Manila clams became established in the central coast of B,C. As a result of previous work and the influence of his stay at the Milford laboratory. Neil remained intensely interested in bivalve culture and continued studies in this field. Initial studies were undertaken to investigate the potential of butter and littleneck clams and 447 448 Chew abalone. He undertook studies with Manila clam culture that were important in establishing the Manila clam culture industrv that exists in B.C. today. From 1981 to 1991 Neil led a program to investigate the feasibility of scallop culture in B.C. Several species were studied, but the decision was made to work with the Japanese scallop. Mizuhopecteii yessoensis. and methods were developed to produce juveniles in a hatchery and raise them to commercial size within a period of two years. Much of the information culminated in the publication. A Maiuuil for Scallop Cidtnic in British Coliimhia. which continues to be used by many to the present day. Results of the work led to a private company building a scallop hatchery and beginning commercial scallop culture in B.C. He continues to serve as an advisor to this industry. Neil has been active in foreign aid work, assisting at Universities and has been involved with several scientific societies. He worked for the Canadian International Development Agency for 1 1/2 years in Fiji, where he served as Director of a Fisheries Training Program at the University of the South Pacific in Suva. He undertook an assessment of the Cuban oyster industry for the Department of Fisheries and Oceans and advised on a joint Canada-Cuba program to expand oyster culture in Cuba. He worked for the United Nations Food and Agriculture Organization in the Peoples" Republic of China and continues to serve as a volunteer ad\ isor to the Canadian Executive Service Organization. He served on the Aquatic Resources Subcommittee of the Science Council of British Columbia from 1987-2002 and was chairman from 1991 to 2002. He is an affiliate professor at the University of Washington. School of Fisheries and has been a committee member for over 20 graduate students, mostly at the University of Washington, but also at the University of Victoria, the University of British Columbia, and Simon Fraser University. Neil served on the executive boards of the Canadian Society of Zoologists, the World Aquaculture Society, the Aquaculture Association of Canada, serving as President of the latter Society from 1987-1988. He joined the National Shelltlsheries Association (NSAl in 1961 and has been active in the affairs of the society since then. He served on the Executive Committee of the Association from 1974-198.\ including tenure as President in 1981-1982; he was the first Canadian to serve as President of NSA. He has been a member of the editorial board of the Joiiniul of Shellfish Research for many years. He was made an Honored Life Member of the Association in 1990 for his exemplary service to NSA and the profession. In 1991 , he received the David H. Wallace award for promoting understanding, knowledge, and cooperation among industry members, the academic community, and all levels of government and for his outstanding success in bringing together shellfish scientists and industry officials for the benefit of shellfisheries. Neil received an Award of Merit from the Department of Fisheries and Oceans in 1993 for an exceptional and distinguished contribution to the effectiveness and efficiency of the Public Service and the Department, particularly for contributions to the aquaculture industry of British Columbia. Neil retired from the Department in March 1994. In May 2000, Neil was presented with an Honorary Lifetime Achievement Award from the Aquaculture Association of Canada. The selection board cited long-time service to the Association, the research community, industry, and young scientists as exemplary examples of the goals of that organization. He was the first recipient of this award. Neil lives in Nanaimo and continues to work at the Pacific Biologic Station as a volunteer Scientist Emeritus, publishing results of past and current work and encouraging young scientists in their careers. He still has time to enjoy his grandchildren, hike, work around the house, and undertake other activities. He is keenly interested in golf and is a rabid bird watcher with the distinction of having birded on all seven continents. One activity in his retirement is to compile his bird life list, he believes he has seen about 2,500 species of birds! Those who have golfed with him have noted that he never lets golf interfere with birding although the opposite has occurred on occasion. Kenneth Chew University of Washington Seattle, Washincton Janimil of Shellfi.sh ReMccucli. Vol. 21. No. 2. 444_t.S0. 2002. Victor G. Burrell Honored Life Member Dr. Victor (Vic) G. Burrell. Jr. has been a member of the National Shellfisheries Association (NSA) for over 30 years and served as its president from 1982-1983. He was born in Wilmington, NC in 1925 and served in the U.S. Navy during WW II from 1943-1946. He received a B.S. degree in English from the College of Charleston in 1949. In 196.5. after 15 years in his family's meat-packing business, he went on to earn both Master's Degree and a Ph.D. Degree in Marine Science from the College of William and Mary in 1968 and 1972, respectively. In large part, this interest may have been kindled through his experiences operating a charter boat in South Carolina and working part time in the commercial oyster industry. While a student at Virginia Institute of Marine Science (VIMS), he served as the liaison to the fishing industry, especially with oystermen. At that time, he developed a method of denaturing the red algal-derived seasonal color of oysters using the blower system. " In 1972. he left the staff of the VIMS and returned to the Carolinas as an Associate Marine Scientist with the then just established Division of Marine Resources in Charleston. He spent from 1972 to 1991 at the Marine Resources Research Institute (MRRI) of the Division of Marine Resources, South Carolina Department of Natural Resources (then the Wildlife and Marine Resources Department). He served as the Institute's Director from 1974 to 1991. "retiring" in 1991. Since then, he has continued to take an active role in South Carolina's marine resources, including co-authoring numerous scientific and lay review publications for NOAA and the DNR as an emeritus director and scientist at Fort^Johnson. In total, he has published over 65 scientific papers on zooplankton ecology, shellfish biology, and oyster culture. These include a series for NOAA in 1996-1997 entitled "The History. Present Condition, and Future of the Molluscan Fisheries of North and Central America and Europe, Volumes 1-3, with Clyde Mackenzie and others. In 2000, he authored a DNR educational report entitled "The Recreational Fishery in South Carolina: the Little River Story," reviewing the first organized offshore recreational fishing industry in South Carolina. He is also currently writing a history of South Carolina's oyster industry for publication. During his leadership at MRRI. Dr. Burrell saw the staff double to about fifty professional individuals, including eleven Ph.D-level marine scientists. In addition to his active participation in the NSA, Dr. Burrell served also as president of the Southeastern Estuarine Research Society (SEERS) and was named an honorary life member of SEERS. He served as president of the College of Charleston Alumni Association and was honored as the college's Alumnus of the Year in 1963. He also saw the development and construction ot the SC Aquarium serving on the executive board of the aquarium as a founding member. He is listed in Community Leaders of America, Personalities of the South, American Men and Women of Science. International Directory of Distinguished Leadership, Who's Who in the South and Southwest, Who's Who in America, and Men of Achievement. He recently moved to new digs and we will miss him walking along the road tipping his hat and waving his hand as he traveled daily to MRRI from his James Island house. However, we still expect to see him almost daily in his office (when he is not taking a vacation 449 450 COEN AND BOBO cruise) chatting witli everyone and being the gentleman that he is. He is an aficionado of big band swing and jazz, particularly that from the Depression era through post-war periods. We remember his enjoyment of Ken Burn's "Jazz" and discussed with him the pros and cons of Bum's choice of influential "characters." such as Louis Armstrong. He's knowledgeable enough about the music and musicians of that era to do a creditable history. He's been an active member of St. James Episcopal on James Island for many years, serving as Junior and Senior 'Warden on several occasions. Vic and his wife Katherine have four children, eight grandchildren, and four great-grandchildren. Loren D. Coen M. Yvonne Bobo MRRI SCDNR Charleston. SC 29412 Joiirmil of Shellfish Research. Vol. 21, No. 2. 451 -+52. 2002. Original works of art surround the artist and NSA Honored Life Member, Herb Hidu, at his home in Alna, Maine. Herbert Hidu Honored Life Memeber Herb has traveled an unusual and convoluted path to distuielion in American shellfisheries science. He was born in 1931, the son of a German immigrant and a 2nd generation Hungarian farmer and estate manager. The family never had the slightest inclination for academia. He spent his youth hunting, fishing, and observing the pleasantries of living on a Connecticut rich man's estate as a cottage inhabitant. He joined the army in 1951 and for 2 years was a paratrooper with the 82nd airborne division, which gave him his mindset for later years. After his hitch, his drinking buddy made him aware of Korean G.I. Bill eligibility. Entering the University of Connecticut, he majored in fisheries management and forestry (B.S., 1938). Along the way he had the opportunity to resume his parachuting paranoia, working a summer as a smokejumper at the United States Forestry Service base in Winlhrop, Washington, during which time he made four fire jumps. At UConn, Dr. Slater of the Entomology Department convinced hint to pursue a graduate degree in the biological sciences. He received a MS degree in Zoology in 1960 from Penn State University, working under the tutelage of Dr. Edwin L. Cooper on population structure of largemouth bass in a farm fish pond. Then, for some unexplained reason. Herb accepted a job in the alien science of shellfisheries biology, working for Dr. Victor Loosanoff at the U.S. Bureau of Commercial Fisheries shellfish laboratory in Milford, Connecticut. His position entailed conducting bioassays with shellfish larvae. The 3-year experience taught him the intricacies of shellfish hatchery culture and, perhaps more importantly, perseverance. Indeed, having endured the extremely confining environment created by the very intense Russian scientist Loosanoff, the world was now Herb's oyster and the oyster was now Herb's world. The Mid- Atlantic MSX oyster mortalities of the mid-1960s provided Herb an excellent avenue for advancement. Rutgers University and Dr. Harold Haskin were in great need of workers (students) who were versed in hatchery techniques. Efforts were concentrated on the production of experimental disease-resistant stocks. This became Hidu's "bread and butter" as a graduate student, where for over 3 years he begat many of Rutgers' experimental oysters stocks at the Cape May laboratory. He used the abundant larval stocks to conduct his study on laboratory behavior and field recruitment of Delaware Bay oysters. His 1967 Ph.D thesis is cited to this day as the definitive study on Delaware Bay oyster recruitment. After Rutgers. Hidu became a shellfisheries research biologist at the Chesapeake Biological Laboratory (CBL) in Solomons, Maryland. It was hoped thai he would fill the shoes and carry on the traditions of the late Francis Beavin. the "dean" of Chesapeake Bay oyster science. With four older veteran shellfisheries biologists, he pursued, for 3 years, field shellfish surveys of Chincoteague Bay and the effects of power plant effluent on the early life history stages of Chesapeake Bay oysters. He developed the natural feeding method for shellfish hatcheries in Maryland and stimulated an early commercial hatchery (Frank Wilde, Shadyside, MD). However, he was miscast in the position at CBL, which he viewed as a mixture of research and personnel management. As an advocate of hatchery production of shellfish to augment recruitment, he locked horns with the Maryland public oyster fishery. Furthennore. his lack of contact with students at the University of Maryland's College Park campus, 100 miles distant inland, created for Herb an untenable situation and he decided it was time to move on. His lifetime break came in 1970 when the University of Maine's Darling Marine Center entered the Federal Sea Grant Program with a "Coherent Areas" grant entitled "Culture of Resources in a Cold Water Marine Environment". Hidu was offered and accepted a position that required shellfish hatchery expertise and. more importantly to Herb, allowed access to excellent students who would pursue M.S. and Ph.D degrees in Ocean80. |jig STX eq lOOg"'- as previously de- scribed (Mackintosh et al. 2002). Investigation into the Use of MIST Alert for PSP in Shellfish Harvest Management and End-Product Testing MIST Alert for PSP test kits were supplied from Fisheries Research Services (FRS) to eight participants to evaluate its po- 455 456 Mackintosh and Smith "0 w "0 C/) Figure 1. Tlu- MIST Altrt'^' (ts( kit. Test kit strips consist of an absorption pad (Si, a nienihrant' striped with a mivtiire of toxin ana- lof;s (the "T" line), and an antihody detection reayent (the "C" line), a sample pad. and a conjugate pad containing the antibodies. A visible "T" line indicates absence of toxin in the sample and no line indicates the presence of toxin. The "C" line indicates that the sample fluid has sufficiently resuspended and mobilized the antibody color complex. As the level of toxin increases, the ""T" line progressively disappears. Remnant "T" lines are scored as having 50, 25, or O'r of color intensity relative to the "C" line. MIST Alert for PSP'^' — samples are consid- ered positive if the 'T" line is 50% or less the intensity of the "C" line. MIST Alert for ASP'^' — samples are considered positive if the "T" line forms only a faint shadow (i.e., less than 25%) or is completely absent. tenlial for field use. Participants comprised shellfish farmers, pro- cessors, and environmental health officers (EHOs). Each partici- pant was supplied with either 50 (farmers) or 100 (processors and EHOs) test kits and the equipment/chemicals necessary to perform acidic shellfish extraction as described by the manufacturer. Be- fore the study, each participant received training in sample extrac- tion and kit use. Subsequently, each participant assessed the pres- ence/absence of PSP toxins in shellfish extracts using the test kits. An aliquot of each shellfish extract analyzed, the test kit. and the participant's interpretation of the result were returned to FR.S. The kit result was reinterpreted by laboratory personnel and extracts reanalyzed by MIST Alert for PSP by an experienced user within the laboratory. Positive samples were further analyzed by the MBA. If this was not possible (e.g.. if the sample volume sup- plied from the participants was too small), samples were diluted 1:1 in a negative shellfish extract and retesled using MIST Alert for PSP. Assessment of MIST Alert for ASP Diirin/; Routine Monitoring Shellfish samples (500) from the 2001 Scottish shellfish- monitoring progratn were extracted in aqueous methanol. |50:50 (v/v) water: methanol] according to the extraction procedure of Quilliam et al. ( 1995) and as detailed in Hess et al. (2001 ). Extracts were concurrently analyzed by MIST Alert for ASP (Jellelt Biotek Ltd.. Nova Scotia. Canada) and HPLC (Quilliam et al. 1995). MIST Alert for ASP is peiformed as for MIST Alert for PSP. but samples are only considered positive if the "T" line is completely absent or appears only as a faint shadow (i.e., less than 25'7r) (Fig. I). The qualitative presence of ASP toxins by the test kits was compared with the quantitative analysis of these toxins (DA -i- epi DA = total amnesic shellfish toxins; AST) by HPLC categorized into five groups: not detected, the limit of detection (lod) that is about 2.5 |j.g AST g"' shellfish flesh. >lod - <6. 6 - <20. and >20 (xg AST g~' .shellfish tlesh. Reproducibility of MIST Alert for ASP Fifty aqueous methanol extiacts obtained fioni a variety of different shellfish species (mus.sels. Mytilus echdis: cockles. Ceras- idilenna ediile: queen scallops, Aqidpecten openidaris: Pacific oyster. Crassoslrca fiificis: king scallop. Pecten iini.xiiuus — whole animals, gonad, adductor, residual tissue — gut, hepatopancreas, gills) were tested in triplicate using MIST Alert for ASP to inves- tigate the reproducibility of the test kits. Extracts contained vary- ing concentrations of AST, as determined by HPLC, and were chosen to contain levels within the five categories above. RESULTS Use of MIST Alerl for PSP in Routine Shellfish Monitoring Results from 547 shellfish extiacts tested by MIST Alert for PSP were compaied with results obtained using the MBA (Table I . Fig. 2). All extracts (/; = 77) deteiniined to contain toxin coiicen- tiations above the legulatory limit (SO |jLg SIX eq 100 g"') by the MBA were also found to be positive for PSP toxins by MIST Alert for PSP. In addition, test kits gave a positive result for 91% (131/ 135) of samples in which the MBA detected toxin below the regu- latory limit (grouped as 40 - <80 and <4() p.g ST.X eq lOOg ' in Fig. 2). The kit did not detect toxin in two mussel extracts (Mytilus c'diilis). a king scallop gonad (Pecten maximiis). and a European oyster (Ostrea edidi.s) exti'act, the mussel extracts both being in the <40 (Xg STX eq 100 g"' category. The MBA did not detect toxin in 335 shellfish extracts. Using MIST Alert for PSP. 97 of these samples gave a positive result (29',r) giving an overall "false-positive"" rate of I8'7r. Of these samples, king scallop extracts (both gonad and whole animal) gave a higher proportion of MIST Alert PSP positive results (35 and 53'/r, respectively) than mussel extracts (25%), the most com- monly tested species in the PSP to\in monitoring program. Field Trials of MIST Alert for PSP Results from 259 shellfish extracts from eight field trial par- ticipants are shown in Table 2. In general, participants and FRS agreed in the positive or negative interpretation of the test kit results. In one case. Participant 8 recorded a negative result, but FRS considered it positive. A positive result was confirmed when this extract was re-analyzed by MIST Alert for PSP at FRS. How- ever, when further tested by the MBA. toxin was not detected. All but one Participant recorded positive results when using the kit. Detection of PSP and ASP Toxins by MIST Airrt™ 457 TABLE 1. Comparison of MIST Alert for the detection of PSP in shellfish extracts with the MBA. No. MBA MIST MBA MIST MBA MIST MBA MIST Tissue Type Tested gSO" + - 40 - <80 + - <4(l + - ND'' + - Pecteit maximus (G)^ 115 14 14 (1 ->-) 21 1 1') 14 0 60 21 39 Peclen maximus {V^f 126 37 37 U 39 39 0 12 12 0 38 20 18 Mvtilus edulis 239 25 25 0 20 20 0 15 13 T 179 44 135 Ostrea edulis 6 0 1 0 1 0 5 0 5 Crassostrea gigas 27 0 0 0 27 1 26 Acjuipeclen operciikiris 16 0 1 1 0 1 T 0 13 6 7 Cenisknlerma edule 9 1 1 0 1 1 0 0 7 ~t 5 Ensis. sp. 9 0 0 3 3 0 6 3 3 Total 547 77 77 0 84 82 2 51 49 -) 335 97 238 % compliance lOO'J 9i.m 96. 1 % 71.0'* ■'MBA ((j,g STX cq 100 j '' ND. not detected. '"" G, gonad. '' W. whole animal. Only one sample was found to contain levels over the regulatory limit when analyzed by the MBA. Iiilerpretation of "T" Line Intensity The majority of disagreement in results between participants and FRS were attributable to differences in "T" line intensity rather than whether a sample was considered positive or negative (Table 2). Participant 2 seemed to misinterpret kit instructions and in sotne cases, read the "C" line as a percentage of the "T"' line. Despite this, positive and negative samples were correctly identi- fied. Some trial participants attempted to read the kit too precisely; for example, rather than scoring the "T" line as 0, 25, 50, or 100% (see Fig. I ), results were reported as 5. 15, 30%. or so forth. Much of the difference in interpretation between FRS and Participants 6 and 7 was attributable to this "overinterpretation." but results were still correctly read as positive or negative. Reanalysis of Shellfish Extracts When shellfish e.xtracts were retested at FRS. there was over 90% agreement with the positive or negative result previously obtained by Participants (Table 2). On four occasions when FRS reanalyzed extracts (from Participants 3. 6, and 8) by MIST Alert for PSP. the result changed from negative (on-site) to positive (at FRSl. In one e.xtract from Participant 3, (a king scallop gonad) a low level of toxicity (3l(xg STX eq 100 g"') was detected by the MBA. In the extract from Participant 8, the MBA gave a negative result, while insufficient extract volume was available for further MBA testing from Participant 6, but when diluted and tested by MIST Alert for PSP, the result was considered negative. Two extracts from Participant 7 changed from a positive to negative score when reanalyzed by the kit, but because the "T" line intensity was close to 50% (see Fig. 1 ), the response was considered diffi- cult to interpret. Previous studies using HPLC showed that PSP toxin profiles of stored Association of Official Analytical Chemists (AOAC) acidic shellfish extracts do not change significantly over a five-day pe- riod (data not shown). It was, therefore, considered that during transportation of shellfish extracts from participants to FRS toxin levels remained unchanged. Use of MIST Alert for ASP in Routine Shellfish Monitoring MIST Alert for ASP was used to test 500 shellfish extracts, and the results were compared with the HPLC analysis routinely used in the ASP toxin shellfish-monitoring program (Table 3 and Fig. 3). All samples over the regulatory limit (20 fjig AST g~'), as determined by HPLC, were scored positive by MIST Alert for ASP in = 162). In samples in = 1 14) that were below the regulatory limit but above the HPLC lod (2.5 |jig AST g"' ), MIST Alert for ASP detected toxin in 93 (81.5%). The kit also detected toxin in 10.6% (13/123) of samples at the HPLC lod and a further sample (king scallop gonad) that was negative by HPLC. Of 39 samples failing into the range >lod - <6 [x-g AST g"' 24 yielded a positive result by the kit (61.5%). Of the commonly tested species, mussels yielded the fewest positive results at the lod. Reproducibility of MIST Alert for ASP Overall, 90% of samples tested gave three identical "T" line responses (Table 4). In samples wherein toxin was not detected, at ^^B Peclen mammus - Gonad I 1 Peclen maximus ■ Whole animal " ?! tr, Eu3MyMus edulis c I i Other species Toxicrty (pg STX eq 100 g ') Figure 2, Percentage agreement between the MBA and MIST Alert for PSP results for shellfish as determined by the MBA. 458 Mackintosh and Smith TABLE 2. Summary of results obtained using MIST Alert for the detection of PSP toxins in shellfish by field trial participants and FRS. No. Kits Participant I sed No. Samples Sho«ini; Disagrt'cmt-nt in +VC/-M' lntt'rpretatl(tn Between FRS and Participants No. Samples Shnwinji Disaureement In "I" Line hiterpretation BelHeen FRS and Participants I 13 15 74 4 45 0 5 20 0 6 34 0 50 14 0 1 9 16 No. Samples Siiowing Disagreement in +ve/-ve Interpretation Between FRS and Participants when Reanalyzed by MIST at FRS No. Samples Showinj; l>isaj;reement in "T" Fine Interpretation Bet'.seen F R,S and Participants Wlien Reana!>/.ed by MIST at FRS ■' W. whole animal: G. gonad; R, residual tissue; A, adductor muscle. " NA, Not apphcable. 0 No. Positive Tissue Type of Samples Positive Samples" 7 Pn un iiutAUims IW, G. R) 3 Pecten maximus (G, Wl 9 PfLlcn maximus (W, G) 0 NA" 1 Pecten maximus (W) 10 Pecten maximus (W. G. Ai 14 Mxtilus cdulis. Ensis spp.. Aquipecten opercularis. s .\f\nlus I'lhilis the lod or >20 |xg AST g^' by HPLC. tlie kit gave three identical responses in agreement with HPLC results. Eight extracts in the range >l()d - <6 \xg AST g' ' gave three identical responses — two negative and six positive. The remaining two samples each gave two identical responses — one, two negatives, and a positive; and the other two positives and a negative. Ten samples in the 6 - <20 jxg AST g"' category all gave three positive results, but only seven samples gave three identical results (Table 4). DISCUSSION MIST Alert for PSP in Roiiline Monitoring Using MIST Alert for PSP, a positive result was recorded for all shellfish extracts, from a wide range of tissue types, known to contain >80 |ji.g STX eq 100 g"' as determined by the MBA. indicating that shellfish considered a threat to public health would be detected by this method during routine PSP toxin monitoring in Scotland (Table I. Fig. 2). In addition, approximately 97^^ of samples containing toxins below the regulatory limit (by the MBA) were also found to be positive by MIST Alert for PSP. suggesting the kit has a similar sensitivity to the MBA (about 33 |jLg STX eq 100 g"'). an important factor when considering its potential use. MIST Alert for PSP failed to detect toxin in four samples in which the MBA detected toxin below the regulatory limit (33. 38. 47. and 60 |xg STX eq lOOg-'). MIST Alert for PSP detects the amount of toxin based on the ability of the antibody mixture on the test kits to detect different STX analogs. It has been reported that the test kit is less sensitive to neo-STX derivatives and requires levels close to the regulatory limit to give a positive result (Laycock et al. 2001). Differing sensitivities of MIST Alert for PSP to different toxin profiles may also account for the observed "false- positive" results (i.e.. MIST Alert recorded a positive result; whereas, the MBA indicated a negative result) observed in this study. For example. TABLE 3. Comparison of MIST Alert for the detection of ASP in shellfish extracts with HPLC. MIST MIST MIST MIST MIST No. Tested HPLC 20 HPLC 6-<20 HPLC >lod - <6 HPLC HPLC Tissue Type + - + - + - lod + - ND" + - Pecten maximus (G)*' 121 18 18 55 50 5 18 12 6 23 6 17 7 1 6 Pecten maximus (R)^ 64 54 .'^4 5 5 0 T 1 0 3 0 3 0 Pecten maximus (W)'' KM 86 86 13 12 1 1 1 0 1 0 1 0 Pecten maximus (A)" 3(1 1 1 1 1 0 11 8 3 18 1 16 5 0 5 Mytilus edulis 12S 3 3 1 1 0 5 0 5 54 2 52 65 0 6.S Aquipecten opercularis 17 0 0 1 1 0 12 3 9 4 0 4 Ostrea edulis 12 0 0 0 5 0 5 7 0 7 Crassostrea gigas 14 0 0 0 4 0 4 10 0 10 Cerastoilerma edide 7 0 0 1 (1 1 3 0 3 3 0 3 Total 500 162 162 0 75 69 6 39 24 15 123 13 110 101 1 100 % compliance 1009; 92^* 61.5% 10.6'7r 99.0<7c ' ND, Not detected; ''G, gonad; '•R, residual tis.sue; ""W, Whole animals; "A, adductor muscle. Detection of PSP and ASP Toxins by MIST Alert^^' 459 TABLE 4. Summary of the reproducibililv (>f triplicate analyses of shellfish extracts using MIST for ASP. Percentage of Number of Percentage of HPIX' Samples Giving Samples Giving Samples Giving (pg AST g"' Three identical Three Identical Two Identical Shellfish Flesh) Responses Responses Responses ND' lUU 10/10 lod" 100 10/10 >Iog - <6 80 8/10 (6 positive. 2 negative) 20 6-<20 70 7/10 30 320 100 10/10 Total 90 45/50 10 ' ND. Not detected. '' led. limit of detection (about 2.5 |jLg AST g"'). extracts containing high levels of low-toxicity toxins (e.g.. some C toxins) (Oshima 1995) may yield a negative result by the MBA but a positive result by MIST Alert for PSP (Laycock et al. 2001 ). This may be considered an advantage of the kit. because these toxins can be readily converted to other more potent PSP toxins during shellfish consumption. Shortcomings of the MBA must be taken into account when considering inconsistent results. The MBA is known to underestimate toxicity with an associated error of 207c {b09c near the detection limit) (Park et al. 1986). Use of MIST Alert for PSP in the Field Results from data obtained by shellfish farmers, processors, and EHOs show very little difference in the interpretation of posi- tive or negative results obtained by these lay persons with less experience of the test kits than staff at the routine monitoring laboratory (FRS). Similarly, in-house (FRS) and on-site (Partici- pants) results from shellfish extracts were, for the most part, com- parable (Table 2). In this study, variability in the interpretation of the "T" line response was observed. Previous trials (Mackintosh et al. 2002) also demonstrated variation in interpreting the "T" line response of MIST Alert for PSP by novice users. This trial sug- gests that this may not be a serious problem, because positive and negative samples were still correctly identified, and the manufac- ^^Mpecfen maxfmus - Gonad i///lPeclen maumus - Residual Pecten maximus - Whole animal I 1 PprtRn wattmus - Adductor muscle LLJJJ Myri'us edu/is Other species Toxicity (pg AST g^) Figure 3. Percentage of samples found to be positive by MIST Alert for ASP when compared with HPIX' results. turer advises that if doubt exists because of "T" line intensity, a positive result should be recorded. It is also considered that this variable will be reduced with more extensive use of the kit. Importantly, an extract determined to contain over 80 jxg STX eq 100 g"' by the MBA was correctly identified by a participant. A further positive extract that was not retested by the MBA (at- tiibutable to insufficient sample volume) was diluted 1:1 in a nega- tive shellfish extract and retested using MIST Alert at FRS. This extract produced a negative response, suggesting toxicity in this saiTiple was <80 jxg STX eq 100 g"'. although this would have been dependent on the toxin profile of the extract. Overall, even when considering inisinterpretation of instruc- tions (e.g.. recording of the "T" line intensity), participants cor- rectly identified positive and negative samples. Previously, we have shown that novice users can make ertors in both use and interpretation (Mackintosh et al. 2002). emphasizing that careful attention to the instructions and interpretation of the results is essential. MIST Alert for ASP in Routine Monitoring MIST Alert for ASP detected toxin in all samples containing the regulatory limit of ASP toxin (20 jjig AST g"') and above, with good reproducibility observed between test kits (Table 3, Table 4, Fig. 3). Toxin was also detected in a high percentage (82%) of extracts containing between the regulatory limit and the lod of the HPLC method used. The manufacturer of MIST Alert for ASP reports a detection limit of between 8 and 12 p,g AST g" (Jellett et al. in press); our results show that the kit will detect toxin at lower levels, particularly in king scallops. In Scotland, the con- tamination of king scallops with ASP toxins has adversely affected the offshore scallop fishery (Campbell et al. 2001, Gallacher et al. 2001); whereas, other shellfish species remain relatively unaf- fected. Results from routine monitoring of Scottish king scallops demonstrate that the proportion of scallops that currently do not contain ASP toxin as determined by HPLC is about 30% (J. Petrie pers. comm.). This implies that the majority of scallop extracts tested by MIST Alert for ASP will give a positive result, regardless of whether the scallops contain toxin below the regulatory limit, and would still be marketable. Overall, results from this study indicate that MIST Alert for ASP is currently too sensitive to screen out low- toxicity king scallop samples in routine monitoring programs, but it inay be suitable for other shellfish species (e.g.. mussels). However, the requirement for an alternative method to the current HPLC tech- nique for ASP toxin detection in shellfish is not considered para- mount, because the ethical concerns surrounding the MBA do not need to be considered. It has been reported that MIST Alert for ASP and PSP can be pertbrmed using the same extract; that is, AGAC extract (sample homogenate extracted in O.IN HCl). Be- fore this study, AOAC acidic extracts were examined by MIST Alert for ASP and PSP (data not shown). It was found that, unless these extracts were used immediately, they were unsuitable for ASP testing because of the instability of DA in HCl, suggesting a single extraction protocol would not be feasible. In addition, acidic extracts are unsuitable for HPLC analysis if further investigation were required. CONCLUSION In conclusion, the MIST Alert for PSP has been successfully used dunng two monitoring seasons (Mackintosh et al. 2002) to 460 Mackintosh and Smith determine the presence/absence of PSP toxins in Scottish shellfish. The test Ivits confirmed the presence of PSP toxins in samples considered to contain toxin at and above the regulatory limit (80 |a.g STX eq 100 g"'). MIST Alert for PSP could be used in moni- toring programs as a screen to eliminate negative and low PSP toxicity samples before use of the MBA for quantitative analysis of positive samples. For example, if MIST Alert for PSP had been used for this purpose during the study period in 2001. there would have been a reduction of 439f of the MBAs. Funhermore. field trial results indicate that the PSP kit has the potential to be used by shellfish farmers as a shellfish harvest management tool and by EHOs and processors in end product testing. Results suggest that MIST Alert for ASP is too sensitive to use as a screen in the Scottish ASP toxin-monitoring program and in end-product testing for king scallops. A higher detection limit would be desirable to reduce the high number of MIST Alert for ASP positives observed at toxin levels <6 [Lg AST g"' by HPLC. In contrast, it is possible that the kit may be used as a screen for ASP toxins in other shellfish species and this should be studied further. ACKNOWLEDGMENTS The authors thank Susan Gallacher for extensive advice. God- frey Howard. John Turriff. Joyce Petrie. Margaret McCann. Nigel Brown, and Alasdair Scott for the pro\ ision of samples and tech- nical assistance. We also thank Joanne Jellett for advice and in- formation. This work was funded by the Food Standards Agency, UK. Grant No. B04006. LITERATURE CITED Campbell. D. A.. M. S. Kelly. M. Busman. C. J. Bulch. E. Wiggins. P. D. R. Mcieller. L. Morton. P. Hess & S. E. Shumway 2001. Amnesic shellfish poisoning in the king scallop. Peclen imiximiis. from the west coast of Scotland. / Shellfish Res. 20:7,s-84. Gallacher. S.. G. Howard. P. Hess. E. Macdonald. M. C. Kelly. L. A. Bates. N. Brown. M. MacKen/ie. P. Gillibrand & W. R. Turrell. 2001. The occurrence of amnesic shellfish poisons in shellfish from Scottish wa- ters. In: G. M. Hallegraeff. S. I. Blackburn. C. J. Bolch & R. J. Lewis, editors. Harmful algal blooms 2000. Inlergovemmental Oceanographic Commrssion of UNESCO, pp. 30-33 Gallacher. S.. F. Macintosh. A. M. Shanks. S. O'Neill. I. Riddoch & F. G. Howard. 1998. Monitoring for paralytic shellfish poisons in Scodand and progress in research to replace the use of the mouse bioassay. J. Shellfish Res. I7:1647-16.'il, Garthwaite. I. 2000. Keeping shellfish safe to eat: a brief review of shell- fish toxins and methods for their detection. Trends Food Sci. Tech. 11:23.^-244. Garthwaite. 1.. K. M. Ross, C. O. Miles, R. P. Hansen, D. Foster. A. L. Wilkins & N. R. Towers. 1998. Polyclonal antibodies to domoic acid and their use in immunoa,s,says for domoic acid in seawater and shell- fish. Nar. To.xins. 6:93-104. Hess, P.. S. Gallacher, L. A. Bates & N. Brown. 2001. Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid, in shellfish from Scotland by liquid chromatography and mass spectrom- etry. / AOAC III!. 84:1-11. Jellett. J. F..R. L. Roberts. M. V Laycock. M. A. Quilliam & R. E. Barrett, (in press). Detection of paralytic shellfish poisoning (PSP) toxins in shellfish hssue usmg MIST Alert"*', a new rapid test in parallel with the regulatory AOAC mouse bioassay. Toxicon. Laycock. M. V., J. F. Jellett. E. R. Belland. P. C. Bishop. B. L. Theriault. A. L. Russell-TaUrie. M. A. Quilliam. A. D. Cembella & R. C. Rich- ards. 2001. MIST Alert'^'; A rapid assay for paralytic shellfish poi- soning toxins. In: G. M. Hallegraeff S. I. Blackburn. C. J. Bolch & R. J. Lewis, editors. Harmful algal blooms 2000. Intergovernmenlal Oceanographic Commission of UNESCO, pp. 254-2?6 Llewellyn. L. E.. J. Doyle. J. Jellett. R. Barrett. C. Alison. C. Bentz & M. Quilliam. 2001. Measurement of paralytic shellfish toxins in moUuscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assay with mouse bioassay. HPLC analysis, and a commercially available cell culture assay. Food .\ddil. Contain. 18: 970-980. Mackintosh. F.. S. Gallacher. A. M. Shanks & E. A. Smith. 2002. Assess- ment of MIST Alert'^'. a commercial qualitative assay for the detection of paralytic shellfish poisoning toxins in bivalve mollusks. J. AOAC Inf. 85:632-641. Oshima, Y. 1995. Post-column derivatization HPLC methods for paralytic shellfish poisons. In: G. M. Hallegraeff. D. M. Anderson & A. D. Cembella. editors. Manual on harmful marine microalgae. IOC Manu- als and Guides No. 33, UNESCO. Paris, pp. 391-396 Park. D. L.. W. N. Adams, S. L. Graham & R. C. Jackson. 1986. Variability of mouse bioassay for determination of paralytic shellfish poisoning toxins. J. AOAC Int. 69:547-550. Quilliam. M. A.. M. Xie & W. R. Hardstaff. 1995. Rapid extraction and cleanup for liquid chromatographic determination of domoic acid in unsalted seafood. J. AOAC Int. 78:543-554. Schantz. E. J., E. F. McFarren. M. L. Schafer & K. H. Lewis. 1958. Purified shellfish poison for bioassay standardisation. / AOAC Int. 41:160-168. Wright. J. L. C. R. K. Boyd. A. S. W. DeFretais. M. Falk. R. A. Foxall. W. D. Jamieson, M. V. Laycock. A. W. McCulloch. A. G. Mclness. P. Odense, V. P. Pathak. M. A. Quilliam. M. A. Raggan, P. G. Sim. P. Thibault. J. A. Walter. M. Gilgan. D. J. A. Richard & D. Dwar. 1989. Identification of domoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Cun. J. Cheni. 67:481—190, Journal of Shellfish Rcsennh. Vol. 21. No. 2. 461-463. 2002. DIARRHETIC SHELLFISH POISONING ASSOCIATED WITH PROROCENTRUM LIMA (DINOPHYCEAE) IN PATAGONIAN GULFS (ARGENTINA) ANA MARIA GAYOSO,' * STACIE DOVER," STEVE MORTON,^ MARK BUSMAN.' PETER MOELLER.- VANESA K. FULCO,' AND LUCIE MARANDA' 'Ceiitro Niicional Patagonico, Consejo Nacional de Investigaciones Cienti'ficas \ Tecnicas. B. Brown s/n, 9120 Puerto Madryn. Chubut, Argentina: 'Marine Biotoxin Program USDOC/NOAA, 219 Fort Johnson Road, Charleston. South Carolina 29412-91 10: Graduate School of Oceanography. University of Rhode Island. Narragansett. Rhode Island 02882-1 1 97 ABSTRACT A serious diarrhetic shellfish poisoning (DSP) intoxication caused by the consumption of mussels harvested in the Gulfs of San Jose and Nuevo. Patagonia, Argentina occurred in March (autumn) 1999. This was the first observation of DSP toxins along the Argentine coast. Cells of the dinoflagellate Prorocemrum lima (Ehrenberg) Dodge were found in water samples, as were epiphytes upon macroalgae and in the stomach contents of the mussels Aidacomya aim (Molina) and Mytilus ediilis platensis (d'Orbigny). Extracts from both mussel species were positive for DSP-like activity using the nuorimetric phosphatase inhibition assay. When the extracts were analyzed using liquid chromatography and tandem mass spectrometry, peaks of okadaic acid or dinophysisloxin I (DTX-1 ) were not observed in the nonhydrolized samples. DTX-1 was detected in hydrolized extracts of Mytilus MdAulaLomya. The Myiilus extract contained 21.2 ng DTX-1 per gram of the whole tissue: the Aulacomya extract contained 94.0 ng DTX-1 per gram. KEY WORDS: Argentina; diarrhetic shellfish poisoning; okadaic acid: Prorocentrum lima, toxic dinoflagellate INTRODUCTION Harmful algal blooms have increased in geographic distribu- tion, magnitude, and frequency during the last decades (Anderson 1989: Smayda 1990; Hallegraeff 1993). Diarrhetic shellfish poi- soning (DSP), a severe gastrointestinal disturbance following the consumption of shellfish, is also an increasing problem. DSP re- sults from the accumulation, in shellfish tissues, of polyether tox- ins produced by dinoflagellates belonging to the genera Dinophx- sis and Prorocentrum (Wright & Cembella 1998 and references therein). In his 1993 review, Hallegraeff recognized only the coast of Chile as being affected by DSP in South America. Fenari et al. (1993) and Mendez and Ferrari (1994) later reported DSP inci- dents occurring in Uruguay. Proencj'a and Rorig (1995) and Proeni;a et al. (1998) documented DSP occurrences in southern Brazil. To date, only species of Dinopliysis have been the causative agents of DSP cases reported from South America (Lembeye et al. 1993, Lembeye et al. 1996. Proen^a et al. 1998, Uribe et al. 2001 ). Since 1995, oceanographic research along the North- Patagonian coast of Argentina has included phytoplankton inves- tigation, microscopical observations of stomach content of mus- sels, and analysis of physical and meteorological data. Because of recurrent blooms of another toxic dinoflagellate, Alexandrium tamarense, in the area (Esteves et al. 1992, Caireto et al. 1998, Gayoso 2001), monitoring programs are conducted only for para- lytic shellfish poisoning toxins in shellfish (mouse bioassay ) by the local (Chubut) government, who is responsible tor imposing bans on shellfish harvest. Although the known DSP producers, Dino- pliysis acuminata and Prorocentrum lima, are present (Esteves et al. 1992, Santinelli et al. 1994, Gayoso 2001), monitoring pro- grams for DSP are not currently conducted in Argentina. On March 20. 1999. shellfish harvested in the Gulfs of San Jose and Nuevo (Chubut) were served at a social event despite voiced concerns from one of us (A.M.G.) that toxic dinoflagellates other than Alexandrium tamarense were potentially present in the water column and in the shellfish digestive glands. More than 40 people *Corresponding author. E-mail: gayosolScenpat.edu.ar suffered from gastrointestinal disorders after consuming the shell- fish. Physicians from the Dr. Andres Isola Public Hospital reported symptoms of diarrhea, nausea, and abdominal pain, consistent with the DSP syndrome. After the incident, samples were collected from the two north-patagonian gulfs for phytoplankton and epi- phytic dinoflagellate identification, chemical analysis of mussel tissues, and microscopic examination of the stomach content of mussels was undertaken to document the causative toxin(s) and the responsible organisms. This work reports on the first recognized episode of diarrhetic shellfish poisoning on the Argentine coast. MATERIALS AND METHODS Water samples at two depths (surface and bottom) with a 2.5 L Van Dom bottle and vertical and oblique hauls with a 25 (j.m mesh plankton net were taken at two stations: Puerto Madryn. situated on the west coast of Golfo Nuevo (42°46'S, 65°02'W) and San Roman, located on the north coast of Golfo San jose (42M5'S, 64'15'W). Dominant macroalgae and mussels [Aulacomya aira and Mytilus edulis platensis) were manually collected from the bottom (ca. 18 m depth) at each station by scuba divers. The samples were collected twice a month from March to June 1999. The identification of species was made using the net samples, a light microscope provided by Noniarsky illumination, and a scanning electron microscope. For quantitative analysis, sub- samples (from the bottle samples) were settled in a chamber from a cylinder of 50 mL and counted with an inverted microscope. The species of macroalgae were analyzed separately, subsamples (2-6 g wet weight) were placed in a tube containing a solution of formaldehide-filtered seawater, the suspended epiphytic di- noflagellate populations were examined and their abundance, ex- pressed as cells per gram wet weight of macroalga, was estimated from counts using a 1 niL Sedwick Rafter chamber. The mussel stomach contents were analized within 4 h of collection, the stom- ach content of at least six mussels was extracted with a Pasteur pipette, the contents were pooled, diluted with filtered seawater, and observed on a slide using a coinpound light microscope. Mi- croalgae present were identified and their relative abundance was estimated. 461 462 Gayoso et al. Toxin Analysis Toxin analysis was performed on the following two mussel samples: (1) Aulacomya atra, which were collected in Golfo Nuevo on March 18, 1999; and (2) Mytihis edulis platensis. which were collected in Golfo San Jose on March 25. 1999. Both were frozen whole until the time of analysis. Sample Extraction For each species, 5 g wet weight of whole mussels were ho- mogenized in 80% methanol tor 2 min using a Polytron. The resulting extract was filtered through a GHB glass fiber filter and the filtrate brought to a volume of 5 mL. To convert all metabolites to the parent okadaic acid (OA) or dinophysistoxin-1 (DTX-l ). 2.5 mL of each sample were hydroly/ed by heating at 76''C for 40 min after addition of 250 jxL of 2.5 N NaOH (Mountfort et al. 1999). Therefore, for each species two separate samples were analyzed for DSP-toxins. Protein Phosphatase Inhibition Assay The prolciii phosphatase inhibition assay was performed in a 96 well format using the procedure of Vieytes et al. ( 1997). The assay tests the ability of OA standard or unknown sample to inhibit activity of purified protein phosphatase 2A against a niiorimetric substrate, 6.8-difluro-4-methylum belliferyl phosphate (DiFMUP). All samples were diluted in reaction buffer (50 iiiM Tris HCl pH 7.0, 0.1 mM CaCU) at least 4-fold to reduce the methanol con- centration to s5% in the assay. This step was to eliminate inhi- bition of the enzyme by methanol. For the assay, 10 p.L of a 1 : 1 dilution of 40 mM NiCl, and 1 mL/mL bovine serum albumin. 17 (J.L of reaction buffer. 33 p-L samples and standards (0.03-10 nM OA; NRC, Ottawa Canada) and 200 (jlL of purified PP2A enzyme (Upstate Biotechnology, Lake Placid, NY) were added to duplicate wells of a 96 well plate (Costar, Corning, NY). To start the reac- tion, 120 m DiFMUP (10 mM; Sigma, St. Louis, MO) was then added to each well and the reaction allowed to proceed for 1.5 h at 37"C, followed by 30 min at 4°C. Protein phosphatase activity was determined by fluorescence (360 nni excitation; 460 nm emission) in the wells using a tluorimetric plate reader (Fluoslar. BMG Labo- ratory Technologies. Durham, NC). OA-like activity in the sample was quantified relative to the standard curve. The detection limit of the fluorimetric phosphatase inhibition assay was approximately 1 X 10"'" M okadaic acid equivalents. Liquid Chromatiinraphy and Tandem Mass Spectromelry (LC-MS/MS) Samples that displayed protein phosphatase inhibition activity were analyzed by LC-MS/MS using a Finnigan LCQ mass spec- trometer. The methanolic extracts were injected on a C18 column (Zorbax 2.1 x 150 mm) and eluted with a gradient of 50 to 95% methanol/water containing 0. 1% TFA at a How rate of 0.2 niL/min. A splitter device was used to direct 10% of the column effluent to the electrospray source. The mass spectrometer was operated in positive ion mode. Toxins were analyzed by trapping |M-t-Nal-i- species for each toxin and conducting selected ion monitoring experiments for distinctive fragment ions from the collisionally activated dissociation of the trapped parent ions. Chromatographic traces were acquired for the detection of the fragment ions and undissociated parent ions. The limits of OA and DTX-l detection were approximately I x 10"' and 1 x 10"'* M. respectively, as measured by LC-MS/MS. RESULTS P. lima (Fig. 1) was unequivocally identified in net samples from the gulfs of San Jose and Nuevo. The two main thecal plates (valves) of the specimens found were obovate (length 33-38 jjim, width 22-24.2 (j.m, n = 10) and cellular morphologic features agreed with the descriptions given by Faust ( 1991 ) and McLachlan et al. (1997, as Exuviaella lima). A row of conspicuous marginal pores, scattered valve pores, and valve center free of pores were observed. The species was registered in water samples (cell den- sities less than 100 cells L"') on March 18. April 13 and June 29. 1999 ( 16.5-18 'C). Its occurrence coincided with the autiunn phy- toplankton peak in which Rhizosolenia setigera Brightwell. Chaetoceros citrvisetiis Cleve. C. diadema (Ehrenberg) Gran, C. didymus Ehrenberg, and Pseudo-nilzschia pimgens (Grunow ex Cleve) Hasle were most abundant, and A. tamarensc (Lebour) Balech was also present. Dominant macroalgae in the gulfs San Jose and Nuevo, Dictyota dichotoma (Hudson) Lamouroux (Phaeophyceae),/4/iornc/H'i/m/i//r<»/«/»/)( (J. Agardh) Baldock. Ce- ramiiim rubnim (Hudson) C. Agardh (Rhodophyceae). and Ulva rigida (C. Agardh) Thuret (Chlorophyceae) showed an epiphytic assemblage of diatoms and dinotlagellates that included P. lima. Cell densities of P. lima varied from approximately 750 cells per g wet weight of macroalga at station San Roman to 1 782 cells per g wet weight at station Puerto Madryn. Intact cells (with protoplasm) of P. lima were observed in the stomach contents of the mussels on March 18, May 18, and June 29. 1999. During the study period, the diet of A. arra and M. edulis platcii.si.s consisted mainly of diatoms, with benthic forms outnum- bering pelagic species. Prorocentniin micans. benthic pennate dia- toms. Puralia sulcata, and Thalassiosira spp. were the most abun- dant organisms found in the mussel .stomachs. Extracts of both mussel species were positive for DSP-like activity using the tluorimetric phosphatase inhibition assay. The Mylilus sample contained 33.5 ng of OA-equivalent activity per gram of whole tissue, whereas the Aulacomya sample contained 150 ng/g. The difference between hydrolized and nonhydroli/ed samples using this assay were not significant. Extracts of the nonhydrolized samples analyzed using LC-MS/ MS did not exhibit peaks corresponding to okadaic acid or dino- Figure 1. DSP IN Argentina 463 physistoxin-1. However, dinophysistoxiii-1 was detected in the Mxtiliis and Aidaaniiya extracts that hud been hydrolized. yielding 21.2 ng DTX-l/g whole tissue and 94.0 ng/g. respectively. DISCUSSION Dinoplivsis acuminata is a frequent component of the phy- toplankton in the area; its abundance can reach up to 1 .9 x 10' cells L"' (Gayoso 2001 ). However, no known gastrointestinal disorders have accompanied its presence. The episode of diarrhea following shellfish consumption observed in March 1999 appears to be re- lated to the presence off. lima. It is an important component of the epiphytic community associated with the dominant macroalgae in the gulfs. The species was also present in the mussel stomach contents and in the water samples. Not only was P. lima present in mussel digestive glands, but DSP toxin activity was also detected in M. edidis platensis and A. alia, strengthening the causal relationship between P. lima and the gastrointestinal disorders of March 1999. If toxin regulations such as those implemented in the European Union had been in effect, the shellfish harvest would have been halted; most EU countries tolerate no DSP toxins above detection levels (Shumway et al. 1995). LC-MS, a highly selective and sensitive method for detection of DSP toxins (Quilliam & Wright 1995), showed a peak corre- sponding to DTX- 1 in hydrolyzed samples of M. edulis platensis and A. alia, whereas neither OA nor dinophysistoxin was observed in nonhydrolyzed extracts. This finding is indicative of the esteri- fied nature of DTX-1 within the mussels; esterified DSP toxins (also called DTX-3 complex! are acyl derivatives that also possess toxic activity (Quilliam & Wright 1995). Upon sample hydrolysis, the esterified toxin converts to the active parent compound. Es- terified DTX- 1 is found to be the main DSP toxin in two other shellfish, the scallop Patinopecten yessoensis and the mussel Myti- lus fialliiprovincialis (Suzuki & Mitsuya 2001 ). A significant dif- ference was found in the toxin concentration of the two hydrolyzed samples: 21.2 ng DTX-l/g of whole Mytiliis tissues versus 94.0 ng/g for Aulacomya. This finding may be explained by the time of collection (1 week apart for the two species), different specific feeding and digestion behavior, and location of the two populations. ACKNOWLEDGMENTS The authors thank M.L. Piriz for the taxonomical identification of macroalgae. This study was funded by the Agenda de Pro- mocion CientiTica y Tecnologica, Repiiblica Argentina, PICT/98 010.^709 and supported by funds from NOAA National Ocean Service. REFERENCES Anderson, D. M. 1989. Toxic algal blooms and red tides: a global per- spective. In: T. Okaichi. D. M. Anderson & T. Nemoto, editors. Red tides: biology, environmental science and toxicology. New York: Elsevier, pp. 11-16. Carreto, J. 1., N. G. Montoya. A. D. Cucchi Colleoni & R. Akselman. 1998. Alexandriiim lamarense blooms and shellfish toxicity in the .Argentine Sea: a retrospective view. In: B. Reguera. J. Blanco. M. Fernandez & T. Wyatt, editors. Harmful algae. Xunta de Galicia and Inlergovem- mental Commission of UNESCO, pp. 131-134. Esteves. J. L.. N. Santinelli, V. Sastre, R. Diaz & O. Rivas. 1992. A toxic dinoflagellate bloom and PSP production associated with upwelling in Golfo Nuevo. Palagonia Aigenlina. Hydrobiologia 242:1 15-122. Faust. M. A. 1991. Morphology of ciguatera-causing Prorocenlruin lima (Pyrrophyta) from widely differing sites. / Phycol. 27:642-648. Ferrari, G.. S. M. Mendez & A. Brazeiro. 1993. Dinophysis acuminata asociada a registro de toxina diarreica de molu.scos bivalvos de Uru- guay. Montevideo: Decimo Simposio Cientffico Tecnologico de la CTMFM. pp. 63-64. Gayoso, A. M. 2001. Observations on Alexandriiim lamarense (Lebour) Balech and other dinoflagellate populations in Golfo Nuevo. Patagonia (Argentina). J. Plankton Res. 23:463^68. Hallegraeff, G. M. 1993. A review of harmful algal blooms and their apparent global increase. Pliycologia 32:79-99. Lenibeye, G., T. Yasumoto, J. Zhao & R. Fernandez. 1993. DSP outbreak in Chilean fjords. In: T. J. Smayda & I. Shimizu. editors. Toxic phy- toplankton blooms in the Sea. Amsterdam: Elsevier, pp. 525-529. Lembeye. G.. A. Clement. C. Molinet. N. Marcos. A. Sfeir & X. Rojas. 1996. Monitoreo de Alexandriiim catenella y Dinophysis acuta en X y XI regiones. Resumenes VI Symposium de Algas marinas chilenas y IV encuentro de Microalgologos. Puerto Montt, Chile: Instituto de Fo- mento Pesquero, p. 65 MacLachlan, J. L., G. T. Boalch & R. Jahn. 1997. Reinstatement of the genus Exuviaella (Dinophyceae. Prorocentrophycidae) and an assess- ment of Prorocenlriim lima. Pliycologia 36:38—16. Mendez, S. & G. Ferrari. 1994. Control de floraciones algales nocivas en aguas uruguayas. IOC Workshop Report No lOI. Anex 111. Paris: UNESCO, pp. 37-40. Mountfort, D. O., G. Kennedy, I. Garthwaite. M. Quilliam. P. Truman & D. J. Hannah. 1999. Evaluation of the fluorometric phosphatase inhi- bition assay in the determination of okadaic acid in mussels. Toxicon 37:909-922. Proen(;a, L. & L. Riirig. 1995. Mussel production and toxic algal blooms in Santa Catarina State, southern Brazil. IOC/UNESCO Hannful Algal News 12/13. p. 5. Proenga, L., F. Schmitt. T. Costa & L. Rorig. 1998. Just a diarrhea? Evidence of diarrhetic shellfish poisoning in Santa Catarina. Brazil. Ciencia e Ciduira 50:458-162. Quilliam, M. A. & J. L. C. Wright. 1995. Methods for diarrhetic shellfish poisons. In: G. M. Hallegraeff, D. M. Anderson & A. D. Cembella, editors. Manual on hannful marine microalgae, IOC Manuals and Guides No .V^. UNESCO, pp. 95-1 II. Santinelli. N., G. Gaille & A. Lettieri. 1994. Harmful algae toxicity along north patagonian coast. IOC-UNESCO Harmful .Algae News 9:6. Shumway. S. E.. H. P. Van Egmond. J. W. Hurst & L. L. Bean. 1995. Management of shellfish resources. In: G. M. Hallegraeff D. M. Anderson & A. D. Cembella. editors. Mimual on harmful marine microal- gae, IOC Manuals and Guides No 33. UNESCO. Paris, pp. 433-161. Smayda. T. J. 1990. Novel and nuisance phytoplankton blooms in the sea: evidence for a global epidemic. In: E. Graneli, B. Sundstrom, L. Edier & D. M. Anderson, editors. Toxic marine phytoplankton. New York: Elsevier, pp. 29—40. Suzuki, T. & T. Mitsuya. 2001. Comparison of dinophysistoxin- 1 and esterified dinophysistoxin- 1 (dinophysistoxin-3) contents in the scallop Patinopecten vessoensis and the mussel Mxlilns galloprovincialis. Toxicon 39:905-908. Uribe. J. C, C. Garci'a, M. Rivas & N. Lagos. 2001. First report of diar- rhetic shellfish toxins in Magellanic Fjords. Soiiiliern Chile. J. Shellfish Res. 20:69-74. Vieytes, M. R., O. I. Fontal, F. Leira, J. M. Vieites & L. M. Botana. 1998. A fluorescent microplate assay for diarrhetic shellfish toxins. In: B. Reguera, J. Blanco, M. Fernandez & T. Wyatt, editors. Harmful algae. Xunta de Galicia and Intergovernmental Commission of UNESCO, pp. 533-536. Wright. J. L. C. & A. D. Cembella. 1998. Ecophysiology and biosynthesis of polyether marine biotoxins. In: D. M. Anderson, A. D. Cembella & G. M. Hallegraeff editors. Physiological ecology of harmful algal blooms. NATO ASl Series. Serie G, vol. 41. Beriin: Springer-Verlag, pp. 427-451. Journal of Shellfish Research. Vol. 21. No. 2. 465-469. 2002. REDUCING NEUROTOXIC SHELLFISH POISON (NSP) IN PACIFIC OYSTERS (CRASSOSTREA GIGAS) TO LEVELS BELOW 20 MOUSE UNITS • 100 G"' GRAHAM C. FLETCHER,' BRENDA E. HAY," AND MARGARET F. SCOTT' 'New Zealand Institute foi' Crop & Food Research Limited. Private Ba.ti 92169. Auckland. New Zealand: 'AquaBio Consultants Ltd.. P.O. Box 560. Shortland St. Auckland. New Zealand: ^New Zealand Institute for Crop & Food Research Limited. Private Bag 11030. Pahuerston North, New Zealand ABSTRACT Pacific oysters previously exposed to the algae Karenia hreve. which produces neurotoxic shellfish poisoning (NSP). were held in experimental depuration tanks for up to 5 days prior to determmation of their NSP levels by mouse bioassay. Although depuration reduced NSP levels in the oysters to around the regulatory limit (20 mouse units • 100 g"'). levels below the regulatory limit could not be assured at temperatures of 1 5'C and 20=C and salinities of 24'tf and iA"c with the standard bioassay used. Detoxification appeared less effective at 1 2 'C. The use of an in-line filter to remove free algae or ozone to eliminate algae and toxins did not enhance detoxification. Use of a biological filter at I2°C in the tanks or feeding the oysters with Isochiysas galhana also did not assure NSP levels below the regulatory limit. Exaniinafion of NSP distribution in the shellfish tissues revealed that during depuration, the percentage of this lipid soluble toxin associated with the organs of the digestive system (gills and digestive viscera) decreased while levels increased in high lipid tissues, such as the gonads. We postulate that NSP is difficult to eliminate from oysters because it is incorporated into the lipid stores of the animal. KEY WORDS: neuriitoxic shellfish poison. Pacific oyster. Cra.s.'io.sirea liii^as. detoxification INTRODUCTION Although the uptake of algal toxins by bivalve shellfish is a problem worldwide for .shellfish producers, to date there have been very few successful strategies to mitigate the effects of toxic algal blooms. Current controls rely on growers monitoring toxin levels in shellfish and ceasing harvesting when levels exceed prescribed limits. This is in contrast to the situation when shellfish are ex- posed to bacterial pathogens. In this case growers may have the options of either relaying (moving shellfish to unaffected areas and harvesting again when the shellfish have naturally purified them- selves after a period of weeks) or processing the shelltlsh in land- based depuration tanks where they will be cleared from bacterial pathogens within a few days (Roderick 1994). Similar strategies for the detoxification of shellfish from algal toxins are not com- mercially available. The uptake and elimination of three of the four main algal toxin groups affecting shellfish have been investigated in laboratory studies: paralytic shellfish poison (PSP) (Bricelj & Shumway 1998), diarrhetic shellfish poison (DSP) (Cembella et al. 1998) and amnesic shellfish poison (ASP) (Wohlgeschaffen et al. 1992. Whyte et al. 1995). These studies suggest that land-based detoxi- fication only appears to be feasible for ASP. Relaying to eliminate toxins could be developed but further research is required and there would be risks of introducing toxic algae to unaffected areas. Little work has been i^eported on detoxification of shellfish af- fected by the fourth toxin group, neurotoxic shellfish poisoning (NSP). Despite promising work that showed that NSP toxins were susceptible to oxidation in ozonated seawater (Schneider & Rod- rick 1995) little work on the toxins within shelltlsh has been pub- lished (Fletcher et al. 1998). NSP occurs when people eat shellfish that have been subjected to dinotlagellate blooms composed of Karenia (syn. Gymnodinium) species. These blooms commonly occur in the Gulf of Mexico where the causative alga is Karenia breve (syn. Gymnndiiiiiiin breve. Prychodiseiis brevis). One out- break, caused by a K. /);cr(--like species, has been recorded in New Zealand (Chang 1995). We have previously reported the results of six experiments designed to determine whether Pacific oysters {Crassostrea i^igas) might be successfully detoxified of NSP tox- ins in a depuration system (Fletcher et al. 1998). We found that, although mean NSP levels could be reduced to levels below the regulatory limit of 20 mouse units • 100 g"', levels below this limit could not be assured in individual tests using the regulatory method (APHA 1970). This was regardless of whether ozone was u.sed in the seawater. We now report on four experiments designed to confirm the previous results, investigate why levels below 20 mouse units were not consistently achieved and to evaluate other detoxification methods that may result in additional decreases in NSP levels. MATERIALS AND METHODS K. hreve cells were grown in a medium derived from the GP medium of Loeblich and Smith (1968). The original GP medium contained 85% seawater, but because a higher percentage of sea- water gave better growth rates for K. breve (unpublished data) we modified the GP medium in this study to the following composi- tion: seawater (I L), KNO, (200 mg in 2 mL H,0), vitamin so- lution ( 1 mL), trace element solution (5 mL), and soil solution (5 niL). This medium was autoclaved (12rC. 15 min). and then K,HP04 (8.7 mg in 4 mL autoclaved) was added aseptically. The vitamin solution contained thiamin HCl ( 100 mg), vitamin B,, (0.1 mg). biotin (0.2 mg) and H^O ( 100 mL). The trace element solu- tion contained Na.EDTA (6 g), FeClj.eH^O (0.29 g), H^BO, (6.85 g), MnCK.4H,0 (0.86 g). ZnCK (0.06 g), CoCl_..6H,0 (0.026 g). and H,0 (I L) adjusted to pH 7.9 with NaOH. The soil solution was the supernatant after filtering (Whatman No. 2 filter) a mixture of soil ( 1 kg) and H,0 (2 L) that had been autoclaved ( 12rC, 60 min). Oysters were supplied with A', breve cells at rates of 10 to 25 X 10" cells.oyster"' (Table 1 ) and left for 24 h to ingest the cells and take up the NSP toxin, as described previously (Fletcher et al. 1998). This feeding regime gave initial toxin levels from 29.6 to 70.7 mouse units • 100 g"' (Table 2). Four detoxification experiments (designated Experiinents 1 to 4) were carried out in the experimental tanks described previously (Fletcher et al. 1998). Briefly the tanks consisted of two closed systems: one of four 50 L tanks plumbed in parallel to a tempera- 466 Fletcher et al. TABLE 1. Experimental conditions and NSP levels for uptake and detoxification of NSP from Pacific oysters. Oysters Detoxification Treatment Uptake A. breve Detoxification Conditions Experiment Weight' Lipid' Water Number (S.E.r (S.E.I Start Date Number Supplied Treatment Temp. Salinity Filter 1 12.14 (0.22) 1.79 (O.OS) 3/4/97 U 25 U.V. 15 24 lb 10 u.v. 15 24 Ic 10 U.V. 15 24 2 15.91 (0.56) 2.18 (0.19) 4/10/97 1 10 Ozone 20 34 5 3 16.29 (0.74) 3.94 (0.11) 8/27/97 3a 18 U.V. 20 34 5 3b 18 Biofilter 12 34 Bio 4 10.81 (0.14) 3.17 (0.09) 10/20/97 4a 23 U.V. 12 34 4b 23 U.V.-" 12 34 4c 23 Biofilter 12 34 Bio 4d 0 U.V. 12 34 ' Mean drained wet weight of soft tissues for all oysters in the experiment, - (S.E.) — Figures in parentheses are the standard errors of the means. ^ Mean lipid levels for all oysters in the experiment expressed as a percentage of the drained wet weight of soft tissue. ■* Fed hochrysiis during the experiment (days 2 and 3). ture controlled sump and the other of a single 50 L tank niaintained at the same temperature. Both systems had in-line 5 \i.m cartridge filters (FilterPure 5PW10. Contamination Control. Auckland) and ultraviolet lamps (Steriflo 369P, Contamination Control, Auck- land) available. Ozone could be supplied to tanks 1-4 by a corona discharge generator (CDIOOO. Novozone. Auckland) using oxygen as the source gas. When applied, water was ozonated using an electronic controller to provide a reading of 350 ± 20 mv (redox electrode MC241Pt. Radiometer) in the shellfish tanks, which con- verts to an Eh of 549 mv using the correction factor of -t- 1 99 mv applicable to the Ag/AgCI. KCI reference electrode (Vogel 1961 ). Additionally, in Experiments 3b and 4c, oysters were placed in a 1000-L biofilter tank ( 12 ± 0.5°C) where recirculating water was purified by passing it through a sand and shell-based biologically active filter. Ozone and ammonia levels in the seawater were measured using Palintest Photometer 5000 methods (Anon. 1994) and salin- ity was determined using a reft-actometer (Atagol. NSP levels in the oysters were measured using the APHA method ( APHA 1970). Each NSP test was carried out on at least 100 g drained meats from 10-12 oysters. One or two such pooled samples were tested from each treatment on each testing occasion with the two samples being taken from separate tanks when the treatment was spread over more than one tank (details of timing and numbers of samples taken are recorded in Fig. 1 ). The NSP method can detect toxin levels down to about 10 mouse units • 100 g"', depending on the size of mouse used ( 1 8-22 g). Where toxicity was observed but the relevant mice did not die, the toxin level was scored at half the minimum detectable level. Crude lipid levels were determined from the weight of lipid extracted in the ether extraction during the NSP extraction procedure. The experimental conditions under which the oysters were made toxic and maintained during depuration are presented in Table I. The depuration conditions in Experiments I and 2 were designed to complement the half factorial design used in previous work (Fletcher et al. 1998), increasing the replication for each TABLE 2. Fitted exponential curves. NSP Levels (m.u. • 100 g-') Parameters (standard errors)' Regression Coefficient Treatment Start (S.E.)- End (S.E.)' a b r (R') la 60.3(21.0) 15.0 (3,2) lb 62.5(11.9) 23,7(7.1) Ic 70.7 (3.5) 24.5 (3.9) la.b.c 22.97 (3.49) 41.52(6.04) 0.116(0.226) 0.715 2 54.4(1,2) 12.8 (1.4) 13.36(1.20) 41.04(2.45) 0.065 (0.080) 0.976 3a 29.6 (5.5) 12.0(0.5) 11.77(4.89) 17.78(6.24) 0.329 (2.408) 0.859 3b 29.6(5.5) 15.2 (3,2) 0,00(157,14) 29.73(156.45) 0.880 (0.813) 0.710 4a 51.9(0.02) 24.7 (6,3) 26,13(6,73) 25.19(10.62) 0.343 (0.414) 0,426 4b 51.9(0.02) 23,2 (4,4) 0,00(58,22) 48.57(55,71) 0.823 (0.299) 0.640 4c 51.9(0.02) 31,6(2.0) 31.95(2.54) 19.95 (4.76) 0.000(0.312) 0.787 ' Parameters fitted to equation I: Y = « + b.r^ with the standard error of the parameter in parentheses. - Mean NSP level of the 1-2 samples taken at the start of the experiment. •' Mean NSP levels of all samples taken from Days 3. 4, and 5, Reducing Neurotoxic Shellhsh Poison 467 Experiment 1 H 1 1- Expertment 3 ■^il' ^ Experiment 2 Gill r=^ Palps ^ ) ^^ Gonad \i_y ' Digestive Experiment 4 i5?:=_§— Detoxification Time (days) Detoxitication Time (days) Figure 1. Detoxification progress. Kach point represents one NSP analysis of a pooled sample of 1(> to 12 oysters. Curves represent the best fit to Kq. 1: V = a + b.r^. Pic diagrams represent the distribution of NSP in various tissues (as in Table 3) at the indicated times. dotted line = regulatory limit (20 mouse units • g"'). Experiment I: Oysters in experimental detoxification tanks with 15 C seawater at 24'7f salinity disinfected with U.V. light, (a) D toxin level 1, (b) A toxin level 2. (cl • toxin level 3. Experiment 2: • Oysters in experimental detoxification tanks with 20 C seawater at 34% salinity disinfected by ozone. Experiment 3: Detoxification with U.V. (20°C) or Biofilter (12°C). • (al Oysters in experimental detoxification tanks with 20''C seawater of 34'/, salinity disinfected with U.V. light. V (b) Oysters in biofilter tanks with 12 C seawater at 34% salinity. Experiment 4: Detoxification with U.V., (12 C), feeding with ho- chnsus or not. or biofilter ( 12 C). • (a) Oysters in experimen- tal detoxification tanks with 12°C seawater of 34% disinfected with V.X. light. O (b) Oysters in experimental detoxification tanks with 12 C seawater of 34% disinfected with U.V. light fed with Iso- chrystis galbana. V (c) Oysters in biofilter tanks with 12 C seawater at 34% salinity. ♦ (d) Oysters with no initial toxin. factor (ozone or ultraviolet light sterilization, in-line t'lltration to 5 |xm or no filtration, water temperatures of 15°C or 20'-'C and sa- linities of 247tr or 33%r). Also, in Experiment 1, three batches of oysters were separately fed different levels of K. breve (Treatments la, lb. and Ic). and then subjected to identical detoxification con- ditions. Experiment 3 compared UV sterilization at 20°C (Treat- ment 3a) with the use of the biofilter at 12°C (Treatment 3b). Experiment 4 also compared UV sterilization (Treatment 4a) with the use of the biofilter (Treatment 4c) but all oysters were held at 12°C. Additionally. (Treatment 4b) oysters were held in the same conditions as Treatment 4a except that they were fed with 10 million cells Isnchrysiis gathana per oyster on days 2 and 3. Treat- ment 4d was a control where oysters that were not toxic were placed in the same tanks as Treatment 4a oysters and tested at the end of the experiment (after 5 days) to see whether detectable levels of toxin had been accumulated during the trial. Nonlinear regression analysis was used to fit exponentially de- clining detoxification curves of the form: Y = fl -I- h.i (1) where Y was the mean NSP level for a treatment on a given day and X was the number of days since the beginning of the experi- ment. The parameter a is the asymptote of the curve (the level to which NSP approaches after some large number of days); a ■¥ b is the initial NSP level; and r is related to the speed of approach to the asymptote. The model was constrained with r < 1 and a > 0. Accumulated analysis of variance was used to compare the fitted curves. Analysis of variance (ANOVA) was applied to the mean NSP levels after day 2 (exclusive) and on parameter a in Equation I to determine whether any of the experimental factors had statis- tically significant {P < 0.05) effects on the outcome of detoxifi- cation for 6 experiments providing a balanced set of factors (Runs 1 to 4 of previously published work (Fletcher et al. 1998). with Experiments 1 and 2 of the current work). Residual Maximum Likelihood (REML) statistics were similarly applied to compare the effect of treatments in all experiments. At the end of Experiment 2 (day 4) and at the beginning and end (days 0 and 5) of Experiment 3a. the distribution of toxin in various tissues was determined as well as the NSP levels in two pooled samples of 10 oysters. To determine toxin distnbution. 100 oysters were dissected [see Quayle (1969) for a description of C. gigas anatomy] into mantle (up to the junction between the mantle and gill, adductor muscle, gill, labial palps, gonad (i.e.. the white colored tissue surrounding the digestive organs) and digestive tis- sues (remaining visceral mass). The tissues from the 100 oysters were pooled, tested for NSP, and the results were related to the total NSP level found in the pooled samples of the 10 whole oysters. RESULTS The NSP results for the 4 detoxification experiments are pre- sented in Figure 1 and Table 2. Initial toxin levels ranged from 29.6 (Experiment 3) to 70,7 (Experiment 1 Treatment c) mouse units g"' with considerable variation in levels recorded from iden- tical condiuons of uptake (Table 1). As observed previously (Fletcher et al. 1998), there was a period of rapid detoxification followed by a period of no significant changes in toxin levels (from Days 3 to 5 inclusive). The results of fitting these data to Eq. I are shown in Table 2. As separating the 3 batches of shellfish in Experiment 1 did not improve the goodness of fit the NSP results were analyzed as a single treatment. Statistical analyses of Experi- ments 3 and 4 did not demonstrate significant differences in NSP levels between treatments, with or without an in-line biofilter or with or without feeding with /. galbana. Treatments with an in-line biofilter did not result in increased ammonia levels in the water with time while treatments in the smaller tanks did. as reported previously (Fletcher et al. 1998). Adding the extra replication of factors provided in Experiments 1 and 2 to the previously reported (Fletcher et al. 1998) half factorial experiment (Runs 1^) effec- tively reduced the confounding effect of shellfish weight and ini- tial NSP level from the analysis and allowed valid ANOVA. How- ever, this still showed no significant differences between the four factors evaluated; temperature (1 5°C, 20°C), salinity (24%c. 34%r), in-line filter (5 \xm. absent), disinfection agent (UV, ozone). Fur- ther. REML analysis of all the experiments also showed no sig- nificant effects (P > 0.05), although detoxification from treatments at 12°C was possibly less effective than from those at 15°C (F < 0.10). Results from the analyses of different tissues in Experiment 2 and Experiment 3 Treatment a are presented in Table 3. Immedi- ately after toxification (Day 0. Experiment 3), the toxin was con- centrated in organs associated with ingestion and digestion (gills, palps, digestive tissues) while at the end of the detoxification 468 Fletcher et al. TABLE 3. Distribution of toxin in ovster tissues. Tissue Weight Lipid NSP NSP Experimem 3a. Day 0 mantle 20.9 3.74 <10.0 II aiiiiuclor 13.1 0.38 <10.0 0 gill 18.3 1.66 17.9 14 palps 11.2 5.27 14.6 7 gonad 22.8 4.98 < 1 ()-( 1 0 digestive 13.7 5 131 78 Experiment 3a. Day 5 mantle 30.3 3.3 <10.0 tl adductor 12.3 0.39 <1(1,0 0 gill 16.3 2.04 -) digestive 10,7 4.3K 57.9 66 ' Weighted contribution of the tissue to total amount of toxin for all tissues. experiments (Day 4 of Experiment 2 and Day 5 of Experiment 3) the remaining toxin was located in the palps, digestive tissues and gonads, all tissues with high levels of lipid. DISCUSSION The fit of data to Eq. I (as indicated by the R" statistic) ranged from a very good fit for Experiment 2 to poor for Experiment 4 Treattnent a. However, the poor fits can be explained by the large variability between results from pooled samples of identically treated oysters on a given day rather than the inappropriateness of the exponential model. Similar results were found when fitting the previously published data (Fletcher et al. 1998) from Runs I to 6 with the R- statistic ranging from 0.565 to 0.999 and the a param- eter from 5.21 to 18.29. In New Zealand's only confirmed NSP event, NSP stopped being recorded in shellfish (including Pacific oysters) shortly after the causative organism disappeared from the water column (Chang et al. 1995). In an open system where fresh seawater was continually suppMed to the oysters it could be ex- pected that parameter a in Eq. 1 would be /.cro (i.e., given suffi- cient time the oysters would completely purify themselves of the toxin as happened in the wild). In fitting the curves this only proved true for two treatments: Experiment 3 Treattnent b where the oysters were in large tanks filtered through a biofilter and Experiment 4 Treatment b where oysters were fed with algae dur- ing the detoxification experiment. The levels of NSP after 3-5 days for these two treatments were no lower than other experi- ments, so detoxification was not more rapid in these treatments, but the value of 0 for parameter a suggests that complete detoxi- fication would occur with time. The overall results suggest that the closed tank system is fundamcnlally limited in its ability to totally detoxify oysters of NSP. Practical requirements for bacterial depuration of shellfish al- low (hat salinity can vary from ambient by up to 20"^^ without affecting performance (e.g.. Interstate Shellfish Sanitation Confer- ence 1997). In our work, reducing salinity from an ambient of 34 to 247cc (30% reduction) did not have a significant effect on toxin loss, so a similar parameter could be applied for NSP detoxifica- tion. Previous work showed that bacterial depuration of Pacific oysters was more effective at 15°C-20°C than at temperatures below I4°C (Fletcher et al. 1991 ). The current work confirms that a similar pattern applies for NSP detoxification in this species with some evidence of reduced detoxification at 12°C, but no differ- ences in detoxification effectiveness at 15^'C and 20°C. Inclusion of an in-line 5 |jim filter did not improve the effectiveness of detoxification, suggesting that uptake of NSP associated with par- ticles in the water (e.g., whole algal cells) is not important in this system. As the products of ozonating seawater break down NSP toxins (Schneider & Rodrick 1995) it was expected that NSP detoxification would be more effective for oysters in ozonated water. However, the results did not show any such effect, probably due to the relatively low levels of ozone we were able to add to the water, levels thai produced a change in redox potential in seawater from the ambient of around 450 to 550 mv. This level was used because, in preliminary trials. Pacific oysters were observed to close their shells and cease pumping al redox potentials of over 600 mv. Thus, ozonation did not appear to be beneficial in reduc- ing NSP toxin levels within oysters. In the initial work, we observed that the levels of ammonia in the water increased substantially during the detoxification experi- ments. We postulated that these levels of ammonia and other meta- bolic byproducts might have inhibited oyster function, thereby preventing detoxification after the finst 2 days of the experiments. Holding oysters in the larger tank where water was filtered by the biological filter in Experiments 3 and 4 was designed to test this. Although there was no build-up of ammonia in this tank, there was still no significant detoxirication after the first 2 days (Fig. 1 1 and rates and levels of detoxification were not significantly different to those oysters in the experimental tanks without the biological fil- ter. Similarly, feeding the oysters did not result in any marked improvement in detoxification. Although these treatments may have slightly improved detoxification, the differences were not sufficient to assure the production of oysters below the regulatory level within the 5-day period. The location of toxins in the different oyster tissues goes some way towards explaining the failure to achieve complete detoxifi- cation. The locations of the NSP toxin at the start of Experiment 3 support the hypothesis that the toxin is associated with algal par- ticles that are in the process of being ingested or digested. How- ever, during the detoxification period the toxin was eliminaled from the gills and appeared in the gonad tissues. After detoxifica- tion, although the total levels of NSP were reduced, the remaining toxin only appeared in high lipid tissues at the end of Experiments 2 and 3. This suggests that while some of the algal particles may have been passed out in feces others have been digested and some of the lipid-soluble toxin has been transferred to the animals" lipid stores. Subsequent detoxification, therefore, will be a much slower process, reliant on the oysters" need to metabolize their lipid stores. The considerable variability in toxin levels in oysters held un- der identical conditions is of concern. The method we used is the one accepted by regulatory authorities and the laboratory is ap- proved under FDA protocols for this test. Although considerable variability is expected when testing individual shellfish (e.g., Scotti et al. 1983, White et al. 1993), pooling 10 to 12 oysters Reducing Neurotoxic Shellfish Poison 469 should limit the effects of oyster-to-oyster variability on the re- sults. In some experiments (e.g.. Experiment 2) the results of rep- licate samples were acceptably close, but in others using the same test procedures (e.g.. Experiment 1 ) wide variances were observed. The control in Experiment 4 Treatment d was designed to test whether some of this variability might be due to the uptake of toxins excreted by other oysters. However, this did not appear to be the case, as the non-toxic oysters in this treatment did not accumulate toxin to detectable levels. The observed variability is critical in a regulatory environment where the test is usually car- ried out on a single pooled sample of 10 to 12 oysters from a given area. For example, in Experiment 1 Treatment c on Day 4 one sample was below the regulatory liinit while another was well above (Fig. la). Due to the variability in results, an argument could be made that it would be better to base the regulatory limit on the mean of a number of samples rather than a single pooled sample. In this case many of our experiments would have met the regula- tory limit. The extra testing required to obtain mean values would place an extra cost on the industry when the current regulatory limit has been effecti\e in preventing NSP illness. There is little knowledge of the human dose-response curve for NSP but a 100-g sample is a reasonable reflection of an average serving for a con- sumer. CONCLUSIONS The levels of NSP in Pacific oysters were reduced to levels near the regulatory limit of 20 mouse units • 100 g"' in 2-3 days when held at temperatures between 15°C and 20°C and salinities be- tween 24^;f and 349m. However, no treatment consistently pro- duced levels below the regulatory limit within the 3-day trials. This failure can be explained by the hypothesis that toxins migrate from the readily accessible surfaces of digestive tissues and are incor- porated into the animals" lipid stores. The level of variability en- countered with the standard APHA test procedures suggests that they need to be reviewed to confirm their suitability for assuring safe levels of NSP toxins in shellfish. ACKNOWLEDGMENTS This work was funded by the New Zealand Foundation for Research. Science and Technology. Contract CO2406. We are grateful to G Summers for technical assistance and to J F Potter for statistical analyses. LITERATURE CITED Anon. 1994. The Palintest System. Tyne & Wear, England: Palintest Ltd. APHA. 1970. Recommended Procedures for the Examination of .Sea Water and Shellfish. 4th ed. Washington: The American Public Health Asso- ciation, Inc. Bricelj, V. M. & S. E. Shumway. 1998. An overview of the kinetics of PSP toxin transfer in bivalve molluscs. In: B. Reguera, J. Blanco, M. L. Fernandez & T. Wyatt. editors. Harmful Algae. Xunta de Galacia and Intergovernmental Oceanographic Commission of UNESCO, Vigo. pp. 431-436. Cembella, A. D., A. G. Bauder & M. A. Quilliam. 1998. Uptake and detoxification kinetics and compartmentalization of DSP toxins in mol- luscan shellfish. In: Abstracts and Director)', Second International Con- ference on Molluscan Shellfish Safety, 17-21 November 1997. Iloilo City. Philippines: Department of Science and Technology, p. 8. Chang. P. H. 1995. The first records of Gymnodinium sp. nov. (cf breve) (Dinophyceae) and other harmful phytoplankton species in the early 1993 blooms in New Zealand. In: P. Lassus, G. Arzul, E. Erard-Le Denn, P. Gentien & C. Marcaillou-Le Baut. editors. Harmful Marine Algal Blooms — Proceedings of the Sixth International Conference on Toxic Marine Phytoplankton, October 1993. Nantes. France. Lavoiser, London, pp. 27-32. Chang. F. H.. L. MacKenzie. D. Till, D. Hannah & L. Rhodes. 1995. The first toxic shellfish outbreaks and the associated phytoplankton blooms in early 1993 in New Zealand. In: P. Lassus, G. Arzul. E. Erard-Le Denn. P. Gentien & C. Marcaillou-Le Baut. editors. Harmful Marine Algal Blooms — Proceedings of the Sixth International Conference on Toxic Marine Phytoplankton. October 1993. Nantes, France. Lavoiser, London, pp. 145-151. Fletcher, G. C, B. E. Hay & M. G. Scott. 1998. Detoxifying Pacific oysters [Cnissostreii gigas) from the neurotoxic shellfish poison (NSP) pro- duced by Gymnoiiinitim breve. J. Shellfish Res. 17:1637. Fletcher, G. C, P. D. Scotti & B. E. Hay. 1991. The depuration of Pacific oysters {Crassostrea gigas). In: W. S. Otwell, G. E. Rodrick & R. E. Martin, editors. Molluscan Shellfish Depuration. Boca Raton: CRC Press, pp. 227-237. Interstate Shellfish Sanitation Conference. 1997. National Shellfish Sani- tation Program Guide for the Control of Molluscan Shellfish. 1997 Revised ed. Washington: Food and Drug Administration. Loeblich. A. R. & V. E. Smith. 1968. Chloroplast pigments of the marine dinotlagellale Gyrodinium resplendens. Lipids 3:5-13. Quayle, D. B. 1969. Pacific Oyster Cuhure in British Columbia. Ottawa: Fisheries Research Board of Canada. Roderick, G. E. 1994. Depuration and relaying of molluscan shellfish. In: C. R. Hackney & M. D. Pierson. editors. Environmental Indicators and Shellfish Safety. New York: Chapman & Hall. pp. 331-363. Schneider. K. R. & G. E. Rodrick. 1995. The use of ozone to degrade Gymnodinium breve toxins. In: R. Poggi. & J.-Y. Le Gall, editors. Shellfish Depuration. Second International Conference on Shellfish Depuration. Rennes 6-8 April. 1992. IFREMER. Plouzan pp. 277-289. Scotti, P. D.. G. C. Fletcher. D. H. Buisson &. S. Fredencksen. 1983. Virus depuration of the Pacific oyster [Crassostrea gigas) in New Zealand. New Zealand J. Seience 26:9-13. Vogel, A. I. 1961 . A Text-book of Quantitative Inorganic Analysis Includ- ing Elementary Instrumental Analysis. 3rd edition. London: Longmans. White, A. W.. S. E. Shumway. i. Nassif & D. K. Whittaker. 1993. Varia- tion in levels of paralytic shellfish toxins among individual shellfish. In: T. J. Smayda & Y. Shimizu. editors. Toxic Phytoplankton Blooms in the Sea. Proceedings of the 5th International Conference on Toxic Marine Phytoplankton, Newport, RI, 28 October-I November 1991. Amsterdam: Elsevier Science Publishers, pp. 441^446. Whyte, J. N. C, N. G, Ginther & L. D. Townsend. 1995. Accumulation and depuration of domoic acid by the mussel. Myriliis califomianiis. In: P. Lassus. G. Arzul. E. Erard-Le Denn, P. Gentien & C. Marcaillou-Le Baut. editors. Harmful Marine Algal Blooms — Proceedings of the Sixth International Conference on Toxic Marine Phytoplankton, Octo- ber 1993. Nantes, France. Lavoiser, London, pp. 531-537. Wohlgeschaffen G. D.. K. H. Mann. D. V. Subba Rao & R. PocklingUm. 1992. Dynamics of the phycotoxin domoic acid: accumulation and excretion in two commercially important bi\al\'es. / Appl. Phycol. 4:297-310. Journal oj Slu'llfisli Rescaivh. Vol. 21. Nii. 2, 47I-+77, 2002. RECIRCULATION OF DINOFLAGELLATE CYSTS BY THE MUSSEL, MYTILUS EDULIS L., AT AN AQUACULTURE SITE CONTAMINATED BY ALEXANDRIUM FUNDYENSE (LEBOUR) BALECH F. M. HARPER,* E. A. HATFIELD, AND R. J. THOMPSON Ocetin Sciences Centre, Memorial University of NewfounJUuul. St. Jolin's. NewfoiindUnul. Canada AlC 5S7 ABSTRACT Holding suspension-feeding bivalves at an aquaculture site may facilitate the maintenance of toxic dinotlagellate populations by concentrating transient vegetative cells or resuspended cysts. To examine the role of the mussel. Mytilus eiliilis. in recirculating cysts within an aquaculture site contaminated with the dinotlagellate Alexandrium fundyense. sediment cores and fecal samples were collected in September and October 1996. In the interim period, a bloom oi A. fundyense vegetative cells began. Mussels egested similar concentrations of dinotlagellate cysts (Scrippsiella sp., A. fundyense. and an unknown Grey species) regardless of the location of the mussel sock in the site, or the position of the mussel in the water column. In September, more putative A. ostcnfeldii cysts were egested in feces collected from the bottom of two socks than in those from the top. One sock was located at greater depths near a barrier island and the other in a shallow northeastern cove. Within each dinotlagellate species, there were no significant differences between cyst concentrations in sediment throughout the site, the exception being the high concentrations in September of putative A. oslenfeldii beneath the sock located near a barrier island (182 cysts-cm"^). Post-bloom, there were significantly fewer A. fundyense cysts in the sediment underlying the sock near a barrier island. In contrast, there were significantly more putative A. oslenfeldii cysts in the sediment in the shallow northeastern cove (580 cysts-cm"'). The daily replenishment rate of A. oslenfeldii cysts in bottom sediments by mussel fecal deposition was estimated as 2 x lO'^ cysts m"" d"'. or about 8%. This may be a considerable contribution to the maintenance of this dinotlagellate species in a mussel aquaculture site, but further studies are required to compare other inputs and outputs of cysts to establish the relative importance of bivalve aquaculture. KEY WORDS: dinofiagellate. Alexandiiuni. cysts, mussel, aquaculture. PSP INTRODUCTION Accumulations of resting cysts (or hypnozygotes) of benthic dinotlage Hates have been observed in various marine environ- ments: offshore trenches and depressions, fjords, and the shallow coastal embayments often selected for shellfish aquaculture opera- tions (Dale 1976. Dale et al. 1978. Anderson & Morel 1979, White & Lewis 1982). Certain locations that accumulate cysts as a result of selective deposition due to hydrographic and sedimentary pro- cesses can act as "seed beds", producing motile cells that then initiate blooms (Steidinger 1975, Anderson & Morel 1979, Tyler et al. 1982). More than SO species of marine dinotlagellates are known to produce cysts (Matsuoka et al. 1989). including species of the toxic d\noi\digt\\aXt Alexandrium (Halim) Balech (formerly classified as Gonyaidax spp. or the Protogonyaulax cateiwlla/ tamarensis species complex). In many parts of the world, A. taina- rense (Lebour) Balech has been responsible for incidents of para- lytic shellfish poisoning (PSP: see review by Hullegraeff 1993). PSP is a .serious public health risk that threatens the commercial harvest of both wild and cultured bivalve populations. Potent neu- rotoxins can accumulate in bivalves through ingestion of vegeta- tive cells of toxic dinotlagellates during suspension feeding (Shumway et al. 1987, Shumway 1990). Cysts formed at the end of Alexandrium blooms have also been implicated as vectors of PSP toxin transfer to bivalves (Dale et al. 1978, White & Lewis 1982). There has been some debate concerning the toxicity of the cysts relative to the motile vegetative cell (Dale et al. 1978. Yentsch & Mague 1979, White & Lewis 1982, Cembella et al. 1990), although there is a consensus that A. tamarense cysts con- tain PSP toxins. The abundance of A. fundyense (Lebour) Balech cysts in sediments has been positively coiTelated with maximum- *Correspondmg author. Present address: Department of Biology. Dalhou- sie University Halifax. Nova Scotia. Canada. B3H 4J1 recorded levels of PSP toxins in the blue mussel, Mytilus edulis (Schwinghamer et al. 1994). Mussels ingest A. tamarense cysts and pass them in fecal pellets (Anderson 1984), but whether bi- valves can extract the toxins from cysts and assimilate them re- mains to be established. Bivalves inhabiting areas with bottom sediments contaminated with toxic cysts cannot avoid encountering these cells during re- suspension events. Dense concentrations tif suspension-feeding bi- valves in areas such as aquaculture sites may facilitate the main- tenance of a seed bed of toxic dinotlagellate hypnozygotes by concentrating transient vegetative cells or resuspended cysts and depositing these cells onto the underlying sediment in the form of pseudofeces or feces. Mussel aquaculture is an expanding industry in Newfoundland: production has increased from 320 tonnes in 1991 to over 1700 tonnes in 1999 (Statistics Canada 1999). Surveys of coastal sedi- ments around the Province have revealed contamination by A. fundyense cysts in many areas (McKenzie 1993, McKciizie 1994. Schwinghamer et al. 1994). One former mussel farm, located in Barred Island Cove, Notre Dame Bay, is permanently closed to aquaculture due to persistent PSP contamination, and has been the focus of ongoing research (Fig. 1 ). Sediment analysis at this site, a cove with two barrier islands and a shallow sill, has revealed A. fundyense cyst concentrations ranging from 20 to > 1,000 cysts-cm"^ (McKenzie 1993, McKenzie 1994, Schwinghamer et al. 1994). This site experiences frequent sediment resuspension as the result of strong winds. The objective of this study was to examine the role of the mussel, M. edulis. in recirculating dinotlagellate cysts and main- taining Alexandrium populations within an aquaculture site. The dinotlagellate cyst composition of feces collected from mussels from the tops and bottoms of socks was determined to test the hypothesis that mussels suspended further away from the contami- nated sediment egest fewer cysts. To facilitate comparisons be- 471 472 Harpi-:r i-t al. 49°30'25" -- E i 4.0. 5.6- ■+■ ■+■ ■+■ ■+■ -+- Sock 1 Sock 2 Sock 3 Sock 4 Sock 5 Figure 2. Location of mussel long-lines ( — I and socks ( ♦ ) at the study site. Barred Island Cove, in Notre Dame Bay, Newloundland. Lengths of s(Kks and sediment sampling depths are depicted for specified socks (insert). tween different sampling locations in the site, the concentrations of cysts in the sediment beneath the mussel socks were determined. The biodeposition rate of dinotlagellate cysts in fecal pellets was then calculated to estimate the rate at which cultured mussels returned resuspended cysts to the sediment surface. MATERIALS AND METHODS ColUctum and Examiiiatiiin nf Mussel Fecal pellets Mussels were sampled in .September and October 1996 from the tops and bottoms of mussel socks previously established at the study site. Barred Island Cove, Notre Dame Bay, Newfoundland (see Fig. 1 ). Wind speed and tide stage, factors that could induce heavier sediment resuspension, were similar for the two sampling periods. Sediment traps deployed in September and retrieved in late October contained Alcxtintlrimii cells in various stages of en- cystment, suggesting a mid- to late-October bloom (McKenzie et al. 1998). Socks (4.6-ni length) were selected based on results of earlier studies conducted at this site, which demonstrated that the highest cyst concentrations occuired in the shallow sediment al the northeastern edge of the cove (McKenzie 1994, Schwinghamer et al. 1994). Socks 1 and 2 were located near the barrier islands; .sock.s 3-5 were situated in the noitheastern cove (see Fig. 1 ). On September 24, 1996. five mussels (Mytihts edulis. mean shell length 6.^.5 mm ± 10.2 SD) were collected from the top and five from the bottom of each of socks I, 2. and 4. Two species of mussels. M. edulis and M. rrossidus. co-e.xist in Newfoundland; those used in this study were identified as M. edulis ba.sed on size and morphologic characteristics (Freeman et al. 1992). Further- more, over 95% of mussels in this part of the coast of Newfound- land are M. edulis according to genetic markers used by Innes and Thompson (unpublished data). Mussels were transported on ice (8 h) to the Ocean Sciences Center. Logy Bay; no mortalities occuired. On arrival, mussels were scrubbed tree of epibionts and placed in individual l-L con- tainers with 700 niL UV-sterilized, filtered (1.0 |j.m) seawater (FSW) at 15"'C. Mussels were removed after a 15 h incubation period and the fecal pellets carefully collected and stored in the dark at 4°C. No preservative was necessary, as low storage tem- perature (4°C) and darkness do not stimulate germination of cysts (Perez et al. 1998). Preliminary trials indicated that the gut reten- tion time of dinotlagellate cysts was approximately 9 h. therefore 15 h was considered adequate time for complete digestion and/or egestion; Scarratt et al. ( 199.^) proposed that 12 h would be suf- ficient time to purge cysts from mussels. Since the mussels re- mained closed during transport, there was no egestion of feces. Prolonged retention of the material within the digestive tract dur- ing transport did not appear to affect the egestion of intact di- notlagellate resting cysts, although some cysts may have been digested so that the values for cysts egested may be underesti- mates. On October 29. four mussels (mean shell length 4.^.6 mm ± 6.7 SD) were sampled from the top and bottom of each of socks 1 . 2. .^. and 5. Fecal pellets were collected on-site: after being scrubbed free of epibionts, individual mussels were placed in plexiglass cylinders (diameter = 70 mm; height = 75 mm) filled with sur- face water. Cylinders were sealed with a rubber stopper. lea\ ing an air pocket, and submerged for 19 h at 7'-C. Fecal material was then collected and transpoiled to the laboratory for processing. No pseudofeces production was ever observed. Fecal contents were disaggregated by repeated aspiration through a micropipette tip, followed by gentle sonication for thirty minutes in an ice-water ultrasonic bath (Cole Parmer 8851). In preliminary testing of methods for disaggregating fecal pellets, gentle sonication in the bath did not destroy any cysts and resulted in a homogeneous suspension of fecal material (Harper 1997). Sonication by microprobe and by ultrasonic bath has no significant negative effects on germination of A. tunuirense cysts collected from sediment (Perez et al. 1998). Cysts in each sample were counted (triplicate samples) with an Utermiihl settling chamber. Dinotlagellate cysts were identified and enumerated by direct cell counting using phase and epitluorescence microscopy. Although this approach is laborious and time-consuming, it yields the most accurate information about the composition of the phytoplankton and the condition of the cells (Sakshaug 1990). An electronic particle counter could not be used for enumeration of the cells in the fecal material as the cysts of each species were approximately the same size (40 |j.iti diameter) and shape, and thus cysts of any given species could not be distinguished from those of other spe- cies. To facilitate comparisons between mussels from different lo- cations and of different sizes, cyst abundance values were ex- pressed per mg total dry weight (DW) and per mg ash-free dry weight (AFDW) of feces. Duplicate samples of 0.7-1.2 niL ho- Recirculation oh Dinoflagellate Cysts by Mussels 473 moaeneous fecal suspension were retained on preweighed. pre- combusted (450 "C) Whatman GF/C filters (25 mm) under gentle vacuum, then rinsed with 10 mL 3% ammonium formate to re- move salts. Filters were oven-dried to constant dry weight at 60°C, then combusted at 450°C for 12 h. cooled in a desiccator and icweighed to determine the AFDW of feces. Weights were deter- mined within 1 |xg with a Mettler UM3 microbalance. Collection, Conceittration, and Exaoiinotion of Cysts jroni Sediment On each of the sampling dates, four sediment cores were col- lected by hand by SCUBA divers within a 1-m" area beneath mussel socks I— f in September and socks 1-5 in October. The sediment was composed of small rocks, fine gravel and silt. The top 6-8 cm of sediment and 2-3 cm of overlying water was col- lected using a 50-mL polycarbonate centrifuge tube (cross- sectional area 5.92 cm") with the conical tip removed (McKenzie 1993. McKenzie 1994; McKenzie & Schwinghamer 1994). Cores were transported on ice in the dark to the laboratory where the top 3 cm of sediment and the overlying water were transfen'ed to clean 50-mL tubes, then stored in the dark, unpreserved. at 4°C. Di- noflagellate cysts were enumerated in the top 3 cm of sediment rather than just in the tlocculent surface layer (Anderson & Wall 1978. Dale et ul. 1978. Lewis et al. 1979). As many Alexcmdiium tamarense (Conyaulax excavata) cysts can be present in the top 3^ cm of sediment as are found at the surface (White & Lewis 1982). To ensure adequate dispersal of detritus, sediment samples were sonicated with two bursts (each 45 sec, power level 4) of a Branson 250 Probe Sonifier. Samples were then fractionated to separate and concentrate dinoflagellate cysts following the den- sity-gradient technique of Schwinghamer et al. (1991). In brief, after sonication the sediment was sieved through 80- and 20-p.m Nite.\ nylon sieves, retaining the material on the 20-jji,m sieve. A step gradient was then formed by slowly injecting "light" and "dense" solutions of a non-toxic, aqueous colloidal silica suspen- sion (Nalco 1060), made isosmotic with seawater (salinity 329(0 using sucrose, beneath the sieved cyst suspension in a 50-mL centrifuge tube. The tubes were then centrifuged at 1,500 g for 30 niin at 4°C and the concentrated layer of dinoflagellate cysts at the interface between the light and dense layers removed for exami- nation. The dinoflagellate cyst composition of each sediment core was determined in triplicate by examining the concentrated cyst layer with phase and epifluorescence microscopy. Each count was per- formed on 100-200 |j.L of the homogenous cyst slurry, the volume settled for each core being adjusted to yield a total of 100-3(J0 cysts per slide (Venrick 1978. with reference to Lund et al. 1958). Results were expressed as the number of cysts-cm"'' sediment. Dinoflagellate Cysts in Feces and Sediment The cysts of four dinotlagellate species were enumerated in fecal and sediment samples: Scrippsielta sp. (Stein) Loeblich. Al- i:\anclriiiiii fuinlyciise. and two unknown species. Cysts were iden- tified using morphologic characteristics: the shape of the cyst body and its ornamentation, cyst size and diameter, wall structure, and the presence of food reserves and pigmented bodies (Matsuoka & Fukuyo 1995). The first unidentified form resembled the resting stage of A. ostenfcldii (Paulsen) Balech et Tangen. a toxic di- notlagellate that has been observed in the Gulf of St. Lawrence (Levasseur et al. 1996). and in Nova Scotian coastal enibayments (Cembella et al. 1998. Cembella et al. 2000). These cysts matched the description of the A. nsiciifcUlii cyst provided by MacKenzie et al. (1996); a spherical cell (-40 [j.m diameter) containing one or two red-brown pigmented granules and areas of condensed, golden-brown pigmentation radiating from the center of the cell towards the periphery. Identification of the vegetative cells that emerged from these cysts was not possible, as repeated attempts at establishing cultures were unsuccessful, but these vegetative cells had the typical gonyaulax form (Hallegraeff 1995), although they did not survive more than 48 h. These cysts are referred to here as putative A. ostenfcldii pending further germination studies. The second unidentified cyst is referred to as the Grey cyst because of its appearance in phase microscopy. It is possible that these cysts were partially digested A. fimdyense cysts as they were approximately the same dimensions and clearly contained a red pigmented body, but all were tapered at one end, in contrast with the two rounded ends of cylindrical A. fundyciisc cysts. Grey cysts did not germinate under cullurc conditions and currently remain unklentitled. Statistical Analyses The concentrations of the dinoflagellate cysts in feces from mussels collected from the top and bottom of each sock were analyzed by two-way analysis of variance with the sock and the position of mussel on the sock as fixed factors and the individual mussel as the unit of analysis. Separate analyses were performed for the abundance of each dinotlagellate species present in mussel feces, expressed per mg DW and per mg AFDW feces. September and October data were treated separately as only socks I and 2 were sampled in both months. The concentrations of the four species of dinoflagellate cysts in the sediment beneath the socks were analyzed by one-way analysis of variance with the sock as the fixed factor. Analyses were per- formed separately for September and October data, and foi' each cyst type, expressed as the total number present-cm"'' sediment. A two-way analysis of variance was performed to compare pre- and post-bloom concentrations of the cysts in the sediment beneath socks 1—1. Month and sock were fixed factors in the analysis; the individual cores were the experimental units. Where the F value exceeded the critical value at P = 0.05. planned comparisons were made using Fisher's LSD method (Mil- liken & Johnson 1984). The criterion for statistical significance in all analyses was P < 0.05. RESULTS Concentrations of Dinoflagellate Cysts in Mussel Feces The concentrations of dinoflagellate cysts egested were highly variable between individual mussels (Fig. 2, Fig. 3). In September (see Fig. 2), the concentrations of Sciippsiella cysts egested were low. ranging from 0-142 cysts-mg"' DW and 0-98 cystsmg"' AFDW feces. Few Alexandrium fimdyense cysts were egested, from 0-28 cystsmg"' DW and 0-846 cystsmg"' AFDW feces. Concentratit)ns of the unknown grey morphotype were also low, ranging from 0-71 cysts-nig"' DW and 0—174 cysts-mg"' AFDW feces. There were no significant differences in the concentrations of Scrippsielta sp., Alexandrium fimdyense. and Grey cysts be- tween mussel feces collected from the tops and bottoms of socks. More putative Alexandrium ostenfeldii cysts were found in the mussel feces collected in September (see Fig. 2) than any of the 474 Harper et al. 800 '01I600 1 1400 1 1200 - §"1000 - ^ 800 o 1-T 1-B 2-T 2-B 4-T 4-B Location of mussels (Sock - Position) Figure 2. Concentrations ot dinotlagellate cysts egested in mussel feces collected from the tops (T) and bottoms (B) of socks in September. Values are standardized per mg dry weight of feces and per mg ash- free dry weight. Each bar is the mean ± standard error Ih = 5). The asterisk (*) indicates signillcant differences (/' 9 0.1)5) between the concentrations of a dinollagcllate species in feces collected from the top and bottom of a specific sock. other dinoflagellate cyst types quantified. Putative A. ostenfehUi concentrations varied from 10 to 846 cystsmg"' DW and 18 to 2,190 cystsmg"' AFDW feces. Significantly more of these cysts were found in mussel feces collected from the bottom of sock 4 than from the top (P = 0.0001 for values expressed in terms of DW; P = 0.004 for AFDW) and from the bottom of sock 2 than from the top {P = 0.039 for DW). Sock 4 was located in the northeastern cove; the bottom of the sock was only 0.6 m above the sediment. Socks 1 and 2 were located near the bairier islands and were further from the bottom. 1.9 and 2.4 m from the sediment respectively (see Fig. 1). In October (see Fig. .^), there were no significant differences in the concentrations of any of the dinoflagellate species between fecal samples from mussels collected from the tops and bottoms of the socks. The concentrations of Scrippsiella sp. egested were similar to those found in September, ranging from 0-90 cystsmg"' DW and 0-216 cystsmg"' AFDW feces (see Fig. 3). Alexandniim fundyense cysts concentrations in the mussel feces remained low. varying from 0-170 cystsmg"' DW and 0 to 181 cysts-mg"' AFDW feces. Concentrations of putative A. ostenfeldii cysts ege- sted ranged from 0-130 cysts-mg"' DW and 0 to 325 cysts-mg"' AFDW feces. Grey cyst concentrations were also low; consistent with amounts egested in September, ranging from 0 to 130 cystsmg"' DW and 0 to 325 cystsmg"' AFDW feces. The error in counting cysts in the fecal suspension was esti- mated from five replicate counts of a single sample containing 494 cysts (putative A. ostenfeldii). The coefficient of variation (CV) was 18% mg"' DW and mg"' AFDW feces. The error in this method is comparable to the 20% CV reported by Ishikawa and Taniguchi (1994) for enumeration of cysts of Scrippsiella sp. in sediment. The counting error (CV) for the sediment cores was 14%, estimated by five replicate counts of putative A. ostenfeldii cysts in one sediment core that contained 156 cystscm"''. Concenlratiuns of Dinuflagellate Cysts in Sediment Beneath Mussel Socks On both sampling dates, concentrations of Scrippsiella sp.. Al- e.mndriiiin fundyense and Grey cysts did not differ significantly within each species between sediment samples collected through- out the site {P > 0.05, Fig. 4). In September. Scrippsiella sp. cysts numerically dominated the sediment with a mean concentration of 596 cystscm"' (±335.6 SD) compared with the mean A. fundyense concentration of 89 cysts-cm""* (±88.7 SD). Grey cysts were scarce (mean 4 cystscm"' ±6.8 SD). In October, mean Scrippsiella sp. concentrations were 306 cystscm"' (±144.2 SD), A. fundyense mean concentrations were 142 cystscm"'' (±137.3 SD). and very few Grey cysts were found (mean 0.6 cysts-cm""" ±1.3 SD). In September, there were significantly more putative A. osten- feldii cysts in the sediment beneath sock 1 than elsewhere in the site (P = 0.008). In October, however, there were no significant differences in putative A. ostenfeldii cyst concentrations in the sediment beneath the mussel socks (P > 0.05). Sediment trap data collected in October as part of a concunenl study (McKenzie et al. 1998) suggested that an A. fundyense bloom began during mid- to late-October. Post-bloom, there were significantly fewer A. fundyense cysts-cm"' beneath sock 1 than 1-B 2-T 2-B 3-T 3-B 5-T Location of mussels (Sock - Position) Figure i. Concentrations of dinoflagellate cysts egested in mussel feces collected from the tops (Tl and bottoms (B) of socks in October, Values arc standardized per mg dry weight of feces and per mg ash-free dry weight. Each bar is the mean ± standard error (/( = 4|. Legend as in Figure 2. Recirculation of Dinoflagellate Cysts by Mussels 475 Figure 4. Concentrations of dinoflagellate cysts per cm' in the sedi- ment beneath mussel socks sampled in September and October. Sock numbers refer to designations on Figure 1. Each bar is the mean ± standard error (/; = 3|. The asterisk (*) indicates a significant differ- ence between the concentrations of putative A.oslenfeldii cysts beneath sock 1 and concentrations beneath socks 2-4 in September (P = 0.008). Legend as in Figure 2. elsewhere (P = 0.001). In contrast, there was significantly more putative A. ostenfeldii cysts beneath socks 3 and 4 in October than there were in September {P = 0.006 and P = 0.02, respectively). DISCUSSION Significance of Mussel Position in the Water Column with Respect to Cyst Contamination Vertical repositioning of mussels in the water column may limit their exposure to toxic dinoflagellate cells (Desbiens et al. 1990). To reduce PSP contamination, Desbiens and Cembella (1993) placed mussels near the bottom of the water column (13-15 m deep) during a bloom of vegetative Alexandrium excavatum cells and raised the mussels close to the surface when A. excavatum was concentrated near the bottom of the water column. Mussels placed near the bottom were the least toxic for most of the exposure period. In this study, we hypothesized that mussels suspended close to the sediment would egest more dinoflagellate cysts than mussels suspended near the top of the water column. We found that this depended on the location of the mussel sock in the site and the sampling time. In September, mussels collected from the bottom of socks near a barrier island and in the northeastern cove of a former aquaculture site at Barred Island Cove. Newfoundland, egested more putative A. ostenfeldii cysts than mussels collected from the top of the socks. In October, however, similar numbers of A. ostenfeldii cysts were egested regardless of the proximity of the mussels to the underlying sediment and their location in the site. No differences were found in either sampling date within the con- centrations of Scrippsiella spp., A. fundyense or Grey cysts in feces from mussels collected from the tops and bottoms of the socks. It is not possible to establish a direct relationship between the concentration of cysts beneath a mussel sock and the concentra- tions in feces egested by the overlying mussels. Mussels may have been ingesting cysts from other sources (e.g.. horizontal advection. newly formed cysts in the water column resulting from encysting vegetative cells), or cells may have encysted within the digestive tract. For example. Grey cysts were numerous in mussel feces collected in October, but few were present in the underlying sedi- ment. Samples from the water column are needed to determine the source of the egested cysts, but it is clear that suspending cultures near the top of the water column does not reduce the exposure of mussels to cysts at this site. The depth of sediment erosion may be a factor in the resus- pension of dinoflagellate cysts, depending on the depth distribution of the cysts of each species. White and Lewis (1982) demonstrated that there are as many A. tamarense (Gonyaulax excavata) cysts in the top 3 cm of sediment as in the flocculent surface layer, but it is not known whether there are vertical gradients in the abundance of other types of cysts within the sediment. There is no clear relationship between the density of dinoflagellate cysts directly beneath the mussel socks and the number of cysts passing through the overlying mussels. Distribution of Dinoflagellate Cysts in Bottom Sediments Concentrations oi Scrippsiella sp.. Alexandrium fundyense and Grey cysts did not differ significantly within each species through- out Barred Island Cove. Sediment core samples collected in No- vember and December of 1992 showed the highest concentration oi A. fundyense (1,130 cysts-cm""*) in the shallow sediment on the eastern edge of the site (McKenzie & Schwinghamer 1994). In October 1993, the highest concentration of A. fundyense (118 cysts-cm"^) was detected near the barrier islands (McKenzie 1994). In September 1996. the highest concentration oi A. fundy- ense cysts was found beneath sock 1, near the barrier islands (371 cysts-cm"''); in October, the highest concentration was found in the northeastern edge of the cove beneath sock 3 (214 cysts-cm""*). Similarly, the highest concentrations of putative A. ostenfeldii were found in the sediment beneath sock 1 in September (182 cysts-cm""*) and beneath sock 3 in October (580 cysts-cm"'). The distribution and concentrations of dinoflagellate cysts in the sedi- ment are cleariy variable both temporally and spatially within this site. Between September and October 1996, concentrations of A. fimdyense cysts either did not change or significantly decreased in sediments beneath the mussel socks samples. In contrast, there were significantly more putative A. ostenfeldii cysts beneath socks in the northeastern cove in October than there were in September. In the interim, a bloom of Alexandrium vegetative cells began (McKenzie et al. 1998). Although this bloom was attributed to a proliferation of A. fundyense cells, it is possible that it may have been caused by both A. fundyense and A. ostenfeldii blooming concurrently, triggered by the same environmental conditions; A. ostenfeldii does not tend to form monospecific blooms (Cembella et al. 1998). Vegetative cells of A. fundyense and A. ostenfeldii arc difficult to distinguish under the light microscope, the method used by McKenzie et al. (1998). In material fixed in Lugofs iodine, Hansen et al. (1992) were unable to distinguish between the two species. 476 Harper et al. Blooms of motile, cyst-forming dinoflagellate species can in- crease cyst deposition in the underlying sediments. For cysts to accumulate, the deposition rate must exceed the rates of loss through regeneration, excystment and transport (Cembella et al. 1988). In this study, we did not observe a post-bloom increase in cyst concentrations of A. fuiidyense in the surface layer of the sediment, but there was an increase in putative A. ostenfeklii that may have been the result of transport of cysts within the site or encysting of vegetative cells. Vegetative cells were still in the process of encysting in the water column, as various encystment stages of A. fiindyense were observed in sediment trap samples (McKen/ie et al. 1998). Previous studies at this site enumerated only those cysts that appeared exactly like A. fiDidxi'iise (McKen/ie 1993. McKenzie 1994. McKenzie & Schwinghamer 1994). In 1996, putative A. ostenfeldii cysts were as abundant as A. fiindyense in the sediment. The vegetative cells of A. ostenfeldii have been documented in the St. Lawrence Estuary and Gulf (Levasseur et al. 1996) and in coastal Nova Scotia (Cembella et al. 1998). This species has not been recorded in Newfoundland, possibly because it is difficult to identify. Maximum concentrations of ,4. fiindyense (371 cy.sts-cm"'') and putative A. ostenfeldii cysts (580 cysts-cm"'') in the sediment from this site are comparable with values from other identified cyst beds in eastern Canada. Sediments in the lower St. Lawrence estuary can contain 400-1., "it )()+ A. e.xcavaliim cysts-cnr\ depending on the time of the year (Cembella et al. 1988, Cembella 1990). The southern Bay of Fundy. particularly to the east and northeast of Grand Manan Island, is rich in A. fiindy- ense (G. excavata) cysts, ranging from 2,000-8.000 cystscm"' sediment (White & Lewis 1982). In Newfoundland, sediments at Harbour Grace, Conception Bay, contain between 30-150 A. fiiiuhense (G. excavata) cysts-cm"' (White & While 1985). Estimated Biodepositioii Rate of Cysts from Suspended Mussel Cultures The replenishment rate of bottom sediments by putative A. ostenfeldii cysts egested by mussels can be estimated from the rate of biodeposition, the process by which feces and pseudofeces settle to the bottom (Haven & Morales-Alamo 1966). Since this study was not designed to investigate the biodeposition rates of di- noflagellate cysts, mussels were not continuously exposed to the cells of interest. Values from the following calculations later may. therefore, underestimate the actual rates of biodeposition since the mussels collected in this study were isolated from the seston and were not permitted to feed continuously. Over the 15 h incubation period in September, mussels (n = 30) egested an average of 333 putative A. ostenfeldii cysts-mg~' DW feces. In October, mussels (n = 32) egested an average of 2.4 cysts-mg"' DW feces over 19 h. Navarro (1983, as cited in Na- varro & Thompson 1997) reported that biodeposition rates of -53 mm shell length Mytihis cliileiisis ranged from 10-38 mg DW-musseT' d"'. Using an intermediate value of 20 mg DW-musseP' d"'. the biodeposition rate of putative A. ostenfeldii cysts by an individual mussel was 6,660 cysts-mussel"' d"' in September and 47 cysts-mussel"' d"' in October. Comparison of these values with the concentrations of cysts in the bottom sedi- ments requires information on the stocking density of mussels. In Newfoundland, the average mussel farm has an estimated 30.9 mussels-m"" bottom (C. Couturier, pers. comm.), therefore in Sep- tember approximately 2.1 x 10^ putative A. ostenfeldii cystsm"" d"' were transfened to the sediment in the form of biodeposits. In October, approximately 1 .5 x 10"" cysts-m"" d ' were deposited on the bottom. Mean concentrations of putati\e A. ostenfeldii cysts in the sedi- ment, expressed as numbers-cm"" integrated through the upper 3 cm. were 2.55 x 10'' cysts-m"" in September and 4.26 x 10'' cysts-m"" in October. Approximately 8% of the putative A. osten- feldii cysts in the sediment were being replenished daily in Sep- tember by fecal pellet deposition from the overlying mussel stocks. The replenishment rate in October was considerably lower, less than 1% of the cysts in the sediment being deposited in the form of mussel feces. The egestion of putative A. ostenfeldii hypnozygotes by M. ediilis in feces provides a mechanism for the recycling of di- noflagellate cysts to the sediment. An approximate daily replen- ishment rate of 8'/(- may be a considerable contribution to the maintenance of cyst populations, and could alter the composition of the sediment over time. Before the contribution of mussel aqua- culture to dinoflagellate cyst recycling within a site can be deter- mined, the number of cysts in biodeposits must be compared with other inputs and outputs of cysts, such as horizontal ad\ection or natural sedimentation during blooins. Future research should in- clude the determination of concentrations of cysts and vegetative cells in the water column of aquaculture sites and comparison with appropriate reference areas. The hydrographic characteristics of each site must also be considered as these can clearly play an important role in distribution of cysts. ACKNOWLEDGMENTS The authors thank the O.S.C. field services unit and D. Mc- Kenzie for sample collection and transport, C.H. McKenzie and M. Riehl for technical support, and H. Chen, D. Schneider and W. Blanchard for statistical advice. The manuscript was improved by comments from S. Shumway and two anonymous reviewers. 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REPRODUCTIVE CYCLE OF THE CHOLGA PALETA, ATRINA SEMINVDA LAMARCK, 1819 (BIVALVIA: PINNIDAE) FROM NORTHERN PATAGONIA, ARGENTINA RODRIGO CASPAR SORIA,* MARCELA SUSANA PASCUAL, AND VICTOR HUGO FERNANDEZ CARTES Laboratorio y Criadero de Mohiscos Bivuhos. Instltiito de Biologia Marina y Pesquera Alte. Slonii. CC 104. (8520) San Antonio Oesle. Rio Nei^ro. Argi'mina ABSTRACT Alrina seminuda (Bivalvia:Pinnidae) is distributed naturally along the Atlantic coast of America from North Carolina to the Valdes Peninsula. At the San Mati'as gulf (North Patagonia), its southern distributional limit, it inhabits subtidal areas of fine and coarse sandy bottoms, on depths ranging from 4 to 30 m. The reproductive cycle of a population of A. seminuda was studied from October 1999 to October 2000. Histologic samples of gonad tissue were obtained from adult individuals collected at Piedras Coloradas (40 '53'S. eS'WWl. Six stages of gonad development were described on the basis of histo-morphologic characteristics: indifferent, early active, developing, mature, spawning, and spent. This study indicates that A. seminuda exhibits an annual reproductive cycle. Gametogenesis starts at the begmnmg of autumn and is prolonged during the winter. A high synchronism in maturation and spawning occurs at November and December. The spent stage begins at mid-summer and concludes at the end of that season. Muscle index (Ml) fluctuates inversely to the gonad index (Gl) along the reproductive season, suggesting that this species uses reserves stored in the adductor muscle for gonadic maturation. A. senumida is a gonochonc stable species that exhibits, on a 1.05% of Ihe individuals, two types of functional hermaphroditism KFA' WORDS: bivalves. Pinnidae. Atrina seminuda. reproducti\'e cycle INTRODUCTION Species belonging to the family Pinnidae are commonly known as pen-shell, hacha, rompechinchorro, wing-shell, razor-shell, and locally as cholga paleta. They are distributed along sheltered sites of warm and temperate seas, at depths up to thirty meters. The cholga paleta, Atrina seminuda Lamarck 1819, is a mem- ber of the Pinnidae family and is the only Pinnidae present at the Patagonian coast. This species is distributed from North Carolina. United States, to the San Jose Gulf. Argentina (Scarabino 1977, Lodeiros et al. 1999). A. .'teminuda has been cited by the synonyms Pinna seminuda. Pinna listeri. and Atrina (Sen-alrinat seminuda (Scarabino 1977). At the San Mati'as Gulf (40°40' S, 63°30' W) (Fig. I) this species inhabits sandy bottoms at depths ranging from five to thirty meters. The cholga paleta lives in a vertical position with the anterior portion of its body deeply buried on the substrata, to which it is attached by large and strong byssal threads. The biology of A. seminuda had not been studied until present. However, several studies have dealt with other members of the family Pinnidae, focusing reproductive biology (Noguera & Gomez-Aguirre 1972, Coronel 1981, Yongqiang & Xiang 1988, de Gaulejac et al. 1995a, de Gaulejac et al. 1995b, Maeda- Martinez el al. (unpublished manuscript), Rodriguez-Jaramillo et al. 2001. Ceballos-Vazquez et al. 2000). ecology and population structure (Butler & Brewster 1979, Keough 1984, Butler 1987, Warwick et al. 1997. Richardson et al. 1999), fisheries, and aqua- culture (Sung & Myong 1984. Cendejas et al. 1985, Bishui et al. 1987, Yoo & Yoo 1984, Yoo et al. 1988. Maeda-Martinez et al. 1996, Reynoso-Granados et al. 1996, Guo et al. 1999). Studies on reproductive biology and gonadic cycle are impor- tant tools in fisheries and aquaculture management of valuable commercial species. Several methods can be used to estimate the reproductive cycle in bivalves (gonadal macroscopic appearance ♦Corresponding author. E-mail: rgskua@yahoo.com. ar or frottis, dynamics of embryos and larvae in the plankton, timing of larval recruitment, physiologic indexes behavior, etc) being the histologic study of gonadal tissues the most reliable method. In this study we describe the reproductive cycle of Atrina sem- inuda from Northwest San Mati'as Gulf (Patagonia, Argentina) through histologic analyses and the role of the adductor muscle as a storage organ of reserves used in gonad maturation. MATERIALS AND METHODS Samples were collected at Piedras Coloradas (40°53' S. 65°04' W), a site located on open waters of the NW coast of the San Mati'as gulf (see Fig. I ). The sampling site has bare bottom, com- posed of coarse sand and gravel, covered with sparse bivalve shell fragments. Tidal current speed is 0.3-0.5 m.s^'. and tides are semidiurnal (average height: 6.31 m) (Servicio de Hidrografia Na- val 1994). Monthly average sea surface temperature fluctuates between 10.4°C in August, and 19.8°C in February, and salinity varies between 34.19ff and 35.1%r (Fernandez 1989). Sampling was carried out from October 1999 to October 2000. on a monthly basis with the exception of the period October to February when samples were collected fotlnightly. Individuals were randomly collected by diving along a straight transect, at depths ranging from 8-12 m. Surface seawater temperature was recorded at each sampling date. Seventy individuals of sizes ranging from 150-250 mm total length were selected from the sampled lot for histologic treatment and to estimate muscle index (Ml). Histologic Treatment The visceral mass of each individual was separated from the adductor muscle. A section of gonadal tissue (approximately 1 cm') from the posterior-dorsal area was excised from each indi- vidual. Each tissue section was fixed in Davidson's solution, stored in 70% alcohol and dehydrated in consecutive baths of ethanol and 1:1 mix of 1007c. ethanol and xylene. Tissue samples were embedded in paraffin (56/58°C). Five to seven |j,m sections 479 480 SORIA ET AL. San Antonio Bay 41° S- San Matias Gulf 65'^' W [ South V Amenca Las Gnitas Beach Piedras Coloradas Beach San Matias Gulf- - - -la Study Area Figure 1. Location of the study area at the Northwest coast of the San Matias (lulf. (Patagonia) Argentina. were made with rotary mierotonie and stained with hematoxiliii and eosin. Analysis of the Repriidiiilire Cycle Histologic sections were examined under light luicroscope. Sex was determined for each animal. A scale of gonadal maturity was developed using as a basis a scale of maturity stages described for other Pinnidae, namely At- riiui ntaura (Maeda-Marti'nez et al. unpublished manuscript) and Fiwui nigosa (Noguera & Gomez-Aguirre 1972, Coronel 1981, Ceballos-Vazquez et al. 2000). Relative frequencies of gonadal developmental stages were obtained throughout the annual cycle. Sex ratio was estimated during the period over which mature and spawning individuals appeared. A Chi-sqiuuc lest was used to assess differences from the 1:1 sex ratio. Frequencies Distribuliun anil Mean Size of Oocytes Oocyte size was determined from histologic sections using an eyepiece graticule calibrated with a stage micrometer (x400). Five to eight females were randomly selected from the lot sampled during each collection date, and at least 100 oocytes (among those with a clearly visible nucleoli) per female were measured through the longest axis, transverse to the nucleus, hidividuals with scarce oocytes and extensive phagocytosis (spent and indifferent indi- viduals) were not analyzed, following the criteria recommended by Grant and Tyler (1983a. 1983b). Gonad Index A gonad index (GI) that represents the reproductive activity was estimated using a numerical grading system based on the maturity stages de.scribed from each monthly collection data (Hef- fernan et al. 1989). Three categories were established on the basis of gonad development: I = Indifferent (I) + Spent (VI), 2 = Early Active (II) + Developing (III), and 3 = Mature (IV) + Spawning (V). The GI was estimated by multiplying the number of individu- als belonging to each category by the category score (1 to 3 ). then summing the values and dividing the resulting value by the total number of animals in the sample. Muscle Index Dry weight of both posterior adductor muscle and remaining soft tissues from 40 individuals on each collection date was ob- tained using a drying chamber (85^C) until constant weight was reached (-72 h). Muscle index was estimated as MI = (adductor muscle weight/soft tissue weight) x 100. Mean values were cal- culated for each collection date. RESULTS Sex Ratio A total of 378 individuals were studied (mean shell length = 194.37 mm; SD = 21.49 mm) of which. 175 were females (46.3%). 124 were males (32.87r). 4 were hermaphrodites ( 1 .Q5''r ) and 75 (19.8%) could not be sexed (Fig. 2). Sex ratio during the spawning season (1.15 females: 1 male, n = 90) did not differ significantly (P s 0.05) from the expected 1:1 ratio. Histologic Analyses Macroscopic and histologic analysis of gonadal tissues, al- lowed the authors to establish the following stages according to the characteristics exhibited during development. Indifferent No evidence of gametic development, acini small with total absence of gametes. Connective tissue is abundant. In this stage it is not possible to determine sex (Fig. 3). Externally the gonad is brownish, watery, and flaccid. Females Early Active: Acini irregular in shape containing oogonias, giving place to pievitcllogenic pear shaped oocytes (average = 7.6 \xm SD = 3.2 |xm) attached to the walls of acini. Inteifollicle connective tissue decreasing (Fig. 4A). Macroscopically. gametogenic activity is evidenced due to color and swelling changes. Gonad acquires a pale pink color in the region next to the disiestive eland. Reproductive Cycle of Atrina seminuda 481 100% 0% I S^ 8^ B"" r5 ^ ^ jH :'s,i*-:, .•i.-^;*^-» .y.-i^j^ifv- ' •••■ ,.•• > J ^ .-V ■•>•■.--. • ■■ "■ • - ".: Figure 3. Photomicrograph of gonadal stages: indifferent stage; scale bar = 50 urn. tending towards the posterior region. Gonadal tissue becomes or- ange in color. Mature: Acini are distended and fulfilled with ripe polygonal shaped oocytes (average = 45.6 p.m; sd = 23.7 |j.m); some oo- cytes remain attached to the walls of acini. Intrafollicle area is reduced to a minimum. The boundary of acini is not distinguish- able. Connective tissue is reduced or it is absent (see Fig. 4C). Gonad reaches its largest area, gonadal tissue is swollen and is red colored. Spawning: Acini partially spawned containing few oocytes that expand and lose the polygonal shape. Phagocytes are present. Little connective tissue is present. A new proliferation of oocytes attached to the walls of acini can occur (see Fig. 4D). Externally the gonad exhibits red color, begins to decrease and loses swelling. Spent: Acini are collapsed as a result of oocyte release. Re- sidual oocytes are irregular in shape; are free in the lumen and begin to be absorbed. Phagocytes proliferate during this st;ige. Connective tissue abundant. A new proliferation of oocytes can occur attached to acini walls (see Fig. 4E). Macroscopically. the gonadal tissue shows a pink-red color and watery aspect. 482 SORIA ET AL. - «^ ^••. ^ :';i ■ O <» -> '■ S if -f^.-';^! Figure 4. Photomicrographs of gonadul stages ol .1. seminiida. Females: ( \l earl> acti\e, (B) de\eloping, (C) mature, (1)1 spawning (E) spent: and males: iFl earl> active, ((i) developing, (H) mature, ll) spawning and (J) spent: scale bar = 50 jim. Reproductive Cycle of Atrina seminuda 483 Males Early Stage: Several primary germinal cells are attached to the wall of the acini. A variable quantity of spermatogonia is located along the internal wall of the acini arranged in bands of several cells. Connective tissue is decreasing (see Fig. 4F). Externally, this stage is evident due to color change and swell- ing of gonadal tissue. It acquires a grizzly white color. Developing: Spermatogenesis exhibits a centripetal evolution from the internal wall towards the lumen. Spermatocytes and sper- matids are free and tilling the lumen. Connective tissue continues decreasing as gamete accumulation proceeds (see Fig. 4G). Gonadal tissue grows invading the digestive gland, and extends towards the anterior area. Gonadal tissue acquires a whitish color. Mature: Acini are distended, filled with spermatozoa with their tails pointing towards the acini lumen. Spermatocytes and spermatides conform a thick layer against the follicular walls. Connective tissue is absent. Boundaries between acini are not dis- tinguished (see Fig. 4H). Gonadal tissue is swelling and has a white color. Spawning: Acini are partially empty, showing a marked de- crease in the number of spermatozoa. Connective tissue is increas- ing (see Fig. 41). Externally, gonadal tissue begins to loose size and swelling, and has a grizzly color. Spent: Acini collapsed with few residual spermatozoa, con- nective tissue is abundant. No evidence of active gametogenesis. Phagocytes proliferate during this stage (see Fig. 4J). Gonadal tissue shows a watery aspect and a whitish-grizzly color. Hermaphrodites A. seminudci exhibits two types of functional hermaphrodites, both equally represented. Type 1; Gonads exhibit separated female and male acini (Fig. 5A). Externally, it is possible to distinguish male gonadal tissue and clear reddish female "patches". Type 2: Female and male gametes arranged inside the same acinus. Oocytes area located at the periphery of the acinus and spermatozoa in the center of the acinus (see Fig. 5B). This type was not detected macroscopically during sampling. The individu- als exhibit the appearance of a typical female gonad. Seasonal Change of Reproductive Cycle During the period of study, superficial water temperature ex- hibited its highest value (21.2°C) in January 2000 and the lowest value (9.7°C) on September 2000 (Fig. 6). The annual reproductive cycle of the total population of A. seminuda is summarized in Fig. 7a. In October 1999 only early active (12.5%) and developing individuals (87.5%) are repre- sented. The highest proportion of mature individuals is found in late November (82%). sharply decreasing by middle December. Spawning peaks by the end of December (84%) while, in the following months, individuals at spent and indifferent stages gradually increase. From the end of February to March, the ma- jority of the population is in the undifferentiated stage (72%). Gonad differentiation begins at middle March involving a small Fifiure 5. Photoniicrodraphs of hermaphroditic individuals of .4. sem- inuda. i.X) type 1: separate male and female acini, (Bl type 2: male and female gametes in the same acini bar = 5(1 nm. portion of the population. Intense gametogenic activity is evi- denced through the winter with individuals showing early active and, in lower proportion, developing gonads. By October 2000, as opposed to the former year, the majority of the population has developing gonads (60%) (see Fig. 7a). Females In October 1999 all females show developing gonads. The highest proportion of mature individuals is found at the end of November (86%), time at which spawning begins. Spawning peaks in December (86%) and extends through the summer involving low numbers of individuals (10% in January). Gonad proliferation begins in March, with gonads in early active and developing stages represented until October (see Fig. 7b). Males In October male gonads are mainly at the developing stage (75%). during November and December gonads at the stages de- veloping, mature and spawning coexist in the population. Evacu- ation begins in November and. at the end of this month. 78% of mature individuals are detected. Spawning mainly occurs in De- cember, even when spawning extends along the summer until May. Spent individuals are found from January to April. Gonads development begins on April and proceeds throughout the winter and spring months (see Fig. 7c). Frequencies Distribution and Mean Size of Oocytes In October 1999 mean oocyte diameter (MOD) was 27.07 (xm (s 11.63 |jim). During November and December oocytes grow. 484 SORIA ET AL. a H Figure 6. Surface seawater temperature recorded at each sampling date at Piedras Coloradas. showing the maximal MOD on November 26 {45.63 |jim; s = 23.74). In early December oocytes ranging 35-65 ^im decrease in niiiii- bers, while oocytes of sizes ranging 5-35 jxm, increase. MOD decreases at this time, reaching 29.47 jjim (s = 15.84). By the end of December the pattern inverts and the MOD again increases reaching 41.52 fxm (s = 12 \xm) on December 24. In January and February 2000 the mode represented by 35-65 |jim oocytes have diminished markedly and simultaneously a new mode of prolifer- ating oocytes arises in individuals showing spawning and spent gonads. This new proliferation is reflected by an abrupt decline in MOD values that reach 16.11 fxm (s = 12.65) in January, and 14.4 p.m (s = 12.55) in February. This new summer proliferation does not seem to culminate in a spawning event due to the fact that these oocytes do not reach the typical mature oocyte size in the season (Fig. 8). By the end of February there is a total absence of oocytes in the gonad. At the beginning of March, MOD shows the minimal values of the annual cycle (mean = 8.08 |xm: s = 3.98), and the low dispersion of the data shows the debut of gametogenic activity (see Fig. 8). Gonad Index Gonad index exhibits a marked seasonal pattern (Fig. 9). GI increases at mid spring reaching its highest values by the end of November and December as a result of the higher numbers of mature and spawning individuals. Minimal is found by the end of February when most individuals have undifferentiated gonads. Gl increases again in March indicating the debut of gametogenesis (see Fig. 9). Muscle Index MI also exhibits a seasonal pattern, showing an increase of 73.6'/f from the end of November, when it has its lower value (20.64%), to August when it reaches its highest value (35.8%) (see Fig. 9). The MI shows, during the reproductive season (October to February), the opposite pattern to the GI. decreasing whilst this increases. This behavior is held until March when both indexes increase and remain stable during the winter months and the debut of spring. DISCUSSION A. semiiuula exhibits an annual pattern of reproduction at the Northwest of San Mati'as Gulf. Gametogenesis initiates at the be- ginning of autumn, when suiface seawater temperature ( = 19 "O begins to decrease. A high synchronism in maturity and spawning is recorded. Spawning takes place during the end of spring (No- vember to December) when seawater temperature is around 20°C. By the end of December, most of the population was spawning or had already evacuated their gonads. Females exhibit a higher syn- chronism in maturation and spawning than males. Oogenesis be- gins earlier in autumn and ends earlier in summer than spermato- genesis. This fact suggests that the small amount of undifferenti- ated individuals found from June to October can be mainly attributed to males whose spent stage is displaced on time com- pared with females. The analysis of oocyte size supported and facilitated the inter- pretation of the reproductive cycle of A. seminuda. Oocyte prolif- eration and growth could be easily identified, suggesting that the first cohort (December oocytes) is released; meanwhile the new- cohort of oocytes that proliferates during spawning or in spent individuals (February oocytes) does not reach maturity and is probably not released. These summer oocytes are most likely re- absorbed. Gametogenesis is defined by exogenous (mainly temperature and food) and endogenous factors and the relation among them shapes the pattern of each reproductive cycle (Sastry 1979). A. iiiciiira from La Paz. Mexico, behaves as many tropical and sub- tropical species, maturing during the warmer months and spawning when temperature decreases (Maeda-Marti'nez et al. unpublished manuscript). On the other hand. P. iiii;i>sa. from Bahi'a Con- cepcion. Mexico show the inverse pattern, with reproductive ac- tivity occurrmg during the warmer months (March to November) and reproductive inactivity in winter (November to February) (Ce- ballos-Vazquez et al. 2000). Both species show a protracted re- productive period. A. seminuda shows a similar reproductive pat- tern to that ai A. pevtiiuihi japonicci from Yoja Bay, Japan (Yoo & Yoo 1984). which shows a protracted developing period and a shorter spawning season that coincides with the warmer months. Reproductive Cycle oe Atr/na seminvda 485 100% ^ 80% H > o U ^ 60% u O g °" 40% (a) 20% - 0% -I"— ^' ■■■■ S ^ !S S5 lii^ T T T T T T T T T 100% 1 □ indifferent |»Ml mature early active mSM developing spawning ^ spent Figure 7. Reproductive cycle of A. seminuda at Piedras Coloradas. Relative frequencies of gonadal stages from October 1999 to October 2000. (a) Total population, (b) females, and (c) males. 486 SORIA ET AL. ^ 80 60 40 20 >. 60 S 40 3 u 80 60 40 I I ll. 80 - (a) 7-Oct-99 N=4 n=400 x=27.07 s(l=11.6 ■ I ■ ^*^' 80 60 H 40 20 0 L"M 25 45 65 (d) lO-Dec-99 N=5 n=503 \-=29.47 stl=15.8 (e) 24-Dec-99 N=8 n=751 x=41.52 sd=12.01 80 60 40 20 0 X (c) 26-Nov-OO N=5 n=500 x=45.6 40 .d=23.7 ^^, 80 60 0 80 60 40 20 0 80 60 40 20 0 1 (g) 3-Feb-OO N=7 n=300 \-14.44 stl=12.5 (h) 14-Mar-()0 N=5 n=135 x=8.08 s(J=3.9 (i) 16-Apr-OO N=5 n=607 x=10.63 s (1=7.03 0) 26-May-OO N=6 n=606 x=15.04 s(l=13.3 25 45 65 Oocyte diameter 80 60 40 20 80 60 40 20 I l.ll. 0) 3-Aug-OO N=8 n=802 x=23.16 s(l=16.9 il.ii (m) 19-Sept-OO N=6 n=597 x=15.55 sd=15.5 xl 1 1 1« (n) 20-Oct-OO N=8 n=800 s=27.86 sd=15.6 25 45 65 Figure 8. Frequency (%) distribution of oocyte diameter ((jm). N = number of females: n = number of oocytes; x = mean oocyte diameter. This pattern is also shared by other Northern Patagonian bivalve species, as Amiantis purpurata (Morsan 2000). Aequipecten tehu- elcluis (Narvarte 2001). Mytiliis ediilis platcnsis (Trancart 1978) and Ostiea ptwkluma (Morriconi & Calvo 1979). The adductor muscle of ,4. seminudu (localK called "callo") shows marked seasonal changes becoming light in weight and watery after spawning. The Ml sharply decreases during the start of the spawning season, suggesting that this specie uses for repro- duction the reserves stored in the adductor muscle. As the propor- tion ot non-spawned individuals in the population decreases, the Reproductive Cycle of Atrina seminuda 487 -Q- MI O- GI 40 35 30 MI 25 20 15 1 ..0-.. •©■''' •Q /" ■^^--E] . ■ \ ,©■ \ ^ /^~'^^ ©' \ V -Fl — J V .,,....-e- ,©■ ■0----O 1 1 1 1 1 1 1 1 1 1 1 2 GI §^ ON o o § o o o o o o o o © © © © § © © o fs o o 2 ^ © 2 ON © o 1-H 1-H 1-H 1—1 1-^ o o © o o © t 00 © § o Figure 9. Variation of O = mean gonad index (GI), and D = muscle index (MI) from October 1999 to October 2000. MI initiates a gradual increase. This relation among the MI and the reproductive cycle has not been described for other members of the Pinnidae. Maeda-Marti'nez et al. (unpublished manuscript) found no relation between the MI and the reproductive cycle for Atrina Duiiirii. a warm temperate sea species that has a different repro- ductive behavior than 4. seminuda. Pelecypods exhibit a wide variation in the expression of their sexuality ranging from strictly gonochoric species to those that are invariably functional hermaphrodites (Sastry 1979). Hermaphro- dite individuals are often found in normal gonochoric species. The cholga paleta/1. seminuda is a stable gonochorist (Giese & Pearse 1974) that shows a low frequency of hermaphrodites (1.05%). The most common form of hermaphroditism in pelecypods is "type 1" where separate female acini and male acini occur in the same individual (Sastry 1979). although the occurrence of both sexes in the same acini can also exist. The cholga paleta shows both types of functional hermaphroditism. In the case of "type 2" hermaphroditism, the male products are arranged in the lumen and the oocytes are arranged on the walls of acini, suggesting a protan- dric pattern of gonad development. Ceballos-Vazquez et al. (20(J0) documented 20.99f of her- maphroditism in P. lugosa. and Butler (1987) reported Q.V/c of hermaphrodites in P. bicolor from Australia. Coronel ( 1981 ) de- scribed for P. rugosa a similar "type 2" hermaphroditism as the described in our study. Hermaphroditism "type 1" was reported in Aulacomya aler (Tortorelli 1987). Mytitus califomianus (Young 1941. Young 1945) and Mytilus edulis (Lubet 1959). Hermaphrodites have not been reported for cogeneric species A. maura (Maeda-Marti'nez et al. unpublished manuscript), and A. pccunaui japanica (Yongqiang & Xiang 1987). ACKNOWLEDGMENTS We are grateful to Inslituto de Biologi'a Marina y Pesquera Alte. 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THE PHYSIOLOGICAL ECOLOGY OF BLACK-RIBBED MUSSELS, SEPTIFER VIRGATUS (WIEGMANN) (BIVALVIA: MYTILIDAE) ON A SUB-TROPICAL WAVE-EXPOSED SHORE IN HONG KONG QIN-FENG GAO, KA-KEI MAK, AND SIU-GIN CHEUNG* Department of Biology and Chemistry. City Universit}- of Hong Kong. Tat Chee Avenue. Hong Kong SAR. China ABSTRACT Oxygen consumption and ammonium excretion by Septifer virgahis were measured in situ on an exposed rocky shore montlily from January to December 1999. Condition index and reproductive cycle were determined simultaneously. Spawning was diphasic, the first phase being recorded in spring (March to April). The second spawning was more prolonged (July to October), but less intense than the first. Oxygen consumption was temperature-dependent and demonstrated a seasonal pattern with a peak being recorded from summer to early autumn and with lowest values in winter. Seasonal variation in excretion rate was small with a major peak in summer, possibly associated with post-reproductive stress and high temperatures. 0;N ratio reflected energy metabolism and utilization with a peak in spring and early summer (>30) when growth and gametogenesis were resumed. In late summer, spawning was initiated and temperatures rose high and became lethal. 0:N decreased to minimum values. KEY WORDS: Septifer. oxygen consumption, excretion. 0:N ratio, mussels INTRODUCTION Numerous studies have been reported on the relationship be- tween environmental variables and physiological processes of bi- valves and corresponding mechanisms whereby the organisms are functionally acclimated to their environments (Bayne 1976. Gos- ling 1992, Dame 1993). Among various factors, exogenous ones such as food, temperature, and salinity, and endogenous ones such as size, age, and reproductive activity are imponant in controlling an individual's physiological processes (Bayne 1976, Bayne & Widdows 1978, Bayne & Newell 1983, Huang & Newell 2002). Representatives of the Mytiiidae are highly successful coloniz- ers of intertidal habitats, playing a significant role in energy flow and nutrient cycling due to their dense populations and filter- feeding patterns (Smaal & Prins 1993. Gili & Coma 1998). Among various mytilids. northern temperate/boreal species of Mytiliis have received most attention (Seed & Suchanek 1992). Black- ribbed tiiussels, Septifer virgatiis (Wiegmann) are distributed throughout the Indo West-Pacific and dominate the mid-littoral zone, forming a continuous band on exposed rocky shores includ- ing Hong Kong (Iwasaki 1995. Morton 1995). As the ecological equivalent of Mvtihis ediilis and M. galloprovincialis on northern Atlantic shores and M. califuritiamis on northwestern Pacific shores (Morton 1995), it is surprising that very little is known about this species. In Hong Kong, 5. virgatiis matures at a shell length of -15 mm and lives for 4-5 y, although older individuals, possibly up to 1 2 y of age. occur as solitary organisms lower down the shore. Two groups of narrowly spaced growth bands are de- posited annually, one during winter (December to March) and another in summer from July to October (Morton 1995, Richard- son et al. 1995). Most adult individuals remain mature year round. Spawning is divided, however, into two phases, one in spring (February to March) and another in autumn (September to Decem- ber). Such a diphasic pattern is suggested to be the result of intense stress in summer when high rock temperatures at mid-day coincide with low spring tides (Liu & Morton 1994. Morton 1995). Such conditions also cause mass mortalities of mussels, barnacles and limpets (Williams & Morritt 1995). In spite of the above research ♦Corresponding author. E-mail; bhsgche@cityu.edu. hk on S. virgatiis in Hong Kong and elsewhere (Tsuchiya 1983, Iwasaki 1995), no systetnatic data have been reported upon sea- sonal variations in physiological responses and their relationships with e.xogenous and endogenous factors. The objectives of this study are to determine the seasonal changes in physiological re- sponses (oxygen consumption, ammonium excretion) under ambi- ent teinperature and food conditions as well as body condition and reproductive cycle. The results provide interesting comparisons with data for other bivalves, particularly the ecologically equiva- lent mytilids from temperate/boreal regions. MATERIALS AND METHODS Sample Collection and Determination of Hydrographic Conditions S. virgatiis occurs as a continuous band in the higher intertidal zone of a wave-exposed shore at Cape D'Aguilar on the south- eastern extremity of Hong Kong Island. Hong Kong. About 50 individuals of S. virgatiis of shell lengths 14—66 mm were col- lected monthly from the shore from January to December 1999 (except in Novetnber when sampling was prevented by strong waves). Epibionts on the shells were removed and individuals were kept for three days in an outdoor fiberglass tank supplied with seawater pumped continuously from the study site. Food conditions of the seawater. i.e., total particulate matter (TPM: mg P' ). particulate organic matter (POM: tug P' ), particu- late inorganic matter (PIM: mg 1"') and organic content (f), were determined using the filter and ash method. Six samples of sea- water (200 ml each) were filtered through ashed and pre-weighed 25 mm glass fiber filter papers (Whatman GF/C), rinsed with isotonic ammonium acetate, dried at 90' C for 24 h, weighed to the nearest 0. 1 mg, then ashed in a muffle furnace at 450"C for 6 h and reweighed. The concentrations of TPM and PIM were thus mea- sured directly. POM was calculated by subtracting PIM from TPM and f was estimated as f = POM/TPM. Temperature (°C). dis- solved oxygen (mg I"') and salinity (7cr) were also measured dur- ing each visit. Oxygen Consumption and Nitrogen Excretion To determine oxygen consumption rate (Vo,: p-g h"' ). each S. virgatiis was placed in a separate sealed perspex chamber (300- 489 490 Gao et al. 550 ml, depending on the size of the animal). For individuals smaller than 20 mm, four to six were put in a container and the calculated oxygen consumption was divided by their number. Two empty chambers were used as controls. The sealed chambers were bathed in a fiberglass tank supplied with seawater pumped con- tinuously from the sea to reduce the effect of temperature on the respiratory activity of the contained animals. Each chamber was sealed for about 60 min, the time chosen according to a prelimi- nary study such that the oxygen concentration declined neither too excessively to affect the normal respiration, nor too slightly to accurately determine variations in oxygen concentration (Wong & Cheung 2001 ). Initial and final dissolved oxygen values were de- termined with a YSI DO meter. The oxygen consumption rate was ineasured as |xg h~' after correction with the control. Ammonium excretion rate (V^: fxg NH^-N h"') was deter- mined using the phenolhypochlorite method (Parsons et al. 1984). Individual S. virgatiis were maintained in separate glass beakers filled with 200 ml filtered seawater (GF/C) for 1 h and the initial and final concentration of ammonium measured. Similar to the oxygen consumption rate measurement, 4-6 individuals smaller than 20 mm were used and the calculated ammonium excretion rate divided by the number in each beaker. Two beakers without animals served as controls. Oxygen consumption and ammonium excretion rates were transformed to atomic equivalents and the ratio of oxygen to ni- trogen (0:N ratio) computed. Condition Index and Size-Slandardization After determining respiration and excretion rates, the shell length of each individual was measured with vernier calipers to the nearest 0.01 mm. Body tissues were then dissected out and soma and gonad dried separately at 80°C to constant weight. Tissue dry weight was measured by an electronic balance to the nearest 0. 1 mg. Body condition was represented by the condition index (CI) calculated from tissue (TDW: mg) and shell (SHOW: g) dry weights according to the equation CI = TDW/SHDW (Brown & Hartwick 1988). Dry gonosomalic index (DGSI), representing the reproductive cycle, was calculated as the ratio of gonad dry weight (GDW: mg) to total tissue dry weight (TDW: mg), i.e., DGSI = GDW/TDW (Lee 1985). Respiration and nitrogen excretion rates were plotted against shell length and body weight following the allometric equation, Y = aX , where Y is the physiological parameters, X is body size, and a and b are coefficients. To facilitate comparisons of physi- ological rates in different seasons, physiological rates were size- standardized according to the following equation: Ys = (Ss/Sq)'' X Y„ (Strychar & MacDonald 1999) Where, Y and S were the physiological parameters and bivalve body size, respectively and standard and observed measurements refer to the subscripts 's" and "o", respectively. Analysis of covari- ance (ANCOVA) indicated that the slopes of the monthly allo- metric equations were unequal, (i.e., no all-year pooled slope might be regressed) b, therefore, was the coefficient in the above allometric model derived from monthly data (Packard & Board- man 1987). Physiological parameters were frequently standardized with body weight. One defect of body weight, however, was its seasonal flexibility compared with shell length, especially when large varia- tions in the condition index occur (Navarro et al. 1996), to which the current study conformed. Moreover, it is widely accepted that ventilation rates, one of the factors regulating respiration, is de- pendent on ctenidial area, which is highly correlated with shell length (Jones et al. 1992. Hughes 1969; Bayne et al. 1976). We, therefore, used shell length instead of tissue weight to standardize the physiological parameters (Iglesias et al. 1996, Labarta et al. 1997). Physiological rates were standardized to a 40 mm indi- vidual of S. virgaliis. the dominant size at the study site. Statistical Procedures To obtain functional relationships between physiological re- sponses and environmental conditions, a set of regression equa- tions was fitted to experimental data, following standard least- squares procedures. Regression analyses were performed by simple linear and non-linear procedures, depending on the most appropriate function to be fitted in each case (Zar 1999). Multiple regression analysis was conducted when physiological parameters were correlated with more than one environmental condition, and the collinearity between independents was tested with coUinearity statistics of SPSS measured with tolerance value and condition index (Belsley et al. 1980. SPSS Inc. 1999a, SPSS Inc. 1999b). Independent(s) highly correlated with others was (were) elimi- nated from the independent list and the regressive model was reconstructed until all the intercorrelations between the indepen- dent variables were removed. Residuals were also analyzed to check normality, constant variance of predicted dependents and other necessary assumptions of the regression model. Data were transformed if necessary to meet the regressive requisites, (i.e., normality of datum distribution and homogeneity of variances). Analysis of variance (ANOVA) or analysis of covariance (ANCOVA) was used, depending on whether covariance existed or not, for comparisons among the groups of data. Prior to analysis, raw data were diagnosed for normality of distribution and homo- geneity of variances using the Kolmogorov-Smirnov test and Lev- ene test, respectively. All statistical procedures were performed with software SPSS, release 9.0 (SPSS Inc. 1999a. SPSS Inc. 1999b, SPSS Inc. 1999c). RESULTS Hydrography Seasonal variations in hydrographic parameters including sea- water temperature, dissolved oxygen, salinity and seston charac- teristics are presented in Table 1. Temperature underwent seasonal variations with high values (~29°C) being obtained in summer, decreasing gradually to low ones (-17'C) in winter. Dissolved oxygen level was quite constant throughout the year (except in December 2000) and was centered around 6.5 mg 1"'. Salinity was fully marine throughout the year except in summer (June to Au- gust! when rainfall was highest and value fell to 269J(. Monthly changes in food concentration (TPM) were small but an excep- tionally high value was obtained in January, which was six times that o'l the lowest value (5.1 I mg P') obtained in June and was probably caused by strong winter monsoon winds and consequent strong waves (Morton & Morton 1983). The value of f varied from 0.20 in April to 0.50 in October with higher values being obtained in summer to autumn (June to December). DGSI and CI Temporal variations in total tissue, soma, and gonad dry weight of a 40 mm mussel are shown in Figure 1 and DGSI and CI in Physiological Ecology of Septifer virgatus 491 TABLE 1. Seasonal variations in seston characteristics (±SD) including TPM (mg T'), POM (nig T'), PIM (mg I"'), and f. and temperature, dissolved oxygen (DO I and salinity from January to December 1999. TPM POM PIM Temperature DO .Salinity Month (mgl"') (mg r') (mgr') f ("C) (mg r') (%o) Jan 33.82 ±1.33 7.74 ±0.14 26.08 ± 1.42 0.23 ±0.01 17.1 5.90 33.6 Feb 9.07 ±1.58 2.05 ± 0.40 7.02 ± 1.29 0.26 ± 0.04 18.2 7.60 33.8 Mar 5.74 ± 0.65 1.13±0.14 4.61 ±0.56 0.23 ± 0.03 22.1 7.07 34,0 Apr 7.67 ± 1.79 1.48 + 0.29 6.19 ± 1.51 0.20 ± 0.02 24.4 6.99 33.8 May 9.47 ± 3.23 1.96±0.18 7.51 ±3.08 0.23 ± 0.06 25.5 6.13 33.7 Jun 5.11 ±0.39 1.85 ±0.17 3.26 ± 0.39 0.37 ± 0.04 28.9 6.47 24.5 Jul 5.67 ±0.91 2.12 + 0.46 3.55 ± 0.42 0.38 ± 0.05 29.6 6.30 27.4 Aug 6.75+0.91 2.99 ±0.79 3.76 ±0.91 0.44 + 0.09 28.6 6.87 27.0 Sep 6.47 ± 1.10 2.84 ±0.97 3.63 ± 0.57 0.44 ± 0.07 28.0 5.61 32.5 Oct 6.89 ±1.32 3.36 ±0.75 3.52 ± 0,79 0.50 ± 0.05 25,7 6.25 32.0 Dec 7.2 1 ± 0.73 2.59 ± 0.29 4.62 ± 0.57 0.35 + 0.05 17.6 9.90 33.0 Annual mean 9.47 ± 8.05 2.76 ± L79 6.71 ±6.50 0.33 ±0,11 24,3 ± 4.5 6,81 ±1,13 31.3 ±3.3 Figure 2. Soma dry weight remained constant throughout the year with a single peak in February. Two peaks, however, were iden- tified for the gonad with a major one occurring from February to March and a smaller but extended one from May to September. Seasonal changes in the reproductive cycle were represented by DGSI for which two peaks were identified, one in winter and the other in summer (Fig. 2). DGSI was positively correlated with CI (Pearson correlation coefficient r = 0.442. /; = 402, P < 0.001). CI was also correlated with both temperature (Pearson correlation coefficient, r = -0.265, n = 402. P < O.OOI ) and TPM (r = 0.054, „ = 402, P = 0.276) but not f (r = 0.039, /; = 402. P = 0.436). Oxygen Consumption, Ammonium Excretion, and Oxygen: Nitrogen (0:N) Ratio Allometric equations relating oxygen consumption (Vo,) and shell length are presented in Table 2. Vo^ was significantly cor- related with shell length for all months. The slopes of the regres- sions were significantly different (ANCOVA, F,|,3k() = 5.82, P< 0.001 ) and varied from 1 .30 in September to 2.68 in February. Vo, of a standard-sized (40 mm) 5. virgaliis varied significantly with time (ANOVA, DF = 10, 391, F = 34.51. P < 0.001 ) with high values in summer (May to September) and lowest one in January (Fig. 3). The regression analysis showed that Vo, was significantly Jan Feb Mar Apr May Jun Jul month Aug Sep Oct Nov Dec Figure 1. Septifer virgatus. Monthly variations (±SD) of the total tissue (TDW: mg). soma (SDW: mg), and gonad (GDW: mg) dry weight from January to December 1999. affected by temperature and the relationship between respiration rate, temperature and shell length can be described by the follow- ing equation: Vo, = 0.0177 X SL' = 529.54, P < 0.001) 0.726. F, With temperature as the covariate. salinity did not significantly affect Vo, (ANCOVA, F, 1.098. P = 0.295). There was a negative correlation between Vo, and body condition with a Pear- son correlation coefficient of -0.14 (;; = 402. P < 0.001 ). Allometric relationships relating ammonium excretion (V^) and shell length are presented in Table 3. V^, was significantly correlated with shell length for all months with the slopes of the regressions varying significantly with months (ANCOVA, F,u j^,, = 10.09, P < 0.001). The regression coefficient ranged from 1.13 in September to 2,89 in January. V^, of a standard 40 mm S. virf^aliis varied significantly with time (ANOVA, DF = 10. 391, F = 33.13, P < 0.001) with the lowest value being obtained in April and a maximum one in September (Fig. 3). As compared with Vo,, the correlation between V,^ and temperature was weak (P < 0.001) with a Pearson correlation coefficient (r) of 0.21. Using teinperature as covariate, salinity did not significantly affect Vn (ANCOVA, F, .407, P = 0.237). V^. however, was positively correlated with Vo, (r = 0.138, n = 402, P < O.OI ) and negatively with CI (r = -0.176, n = 402, P < 0.001). 0:N of a standard 40 mm S. virt^iitiis varied significantly with 160 140 120 100 Q 80 ■ 60 u.b - 0 5 - ■ h \ -•-DGSI -»-CI 0,4 : O 0,3 , a k' r 02: \ K^-i^'^^^^ 0,1 ■ 1- - L J L Np-— — ■ 0- ■ ^ , t -^A 40 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec month Figure 2. Monthly variations (±SD) of condition index (Cll and the dry gonosomatic index (DGSI) of a standard 40 mm Septifer virgatus from January to December 1999. 492 Gao et al. TABLE 2. Allometric relationships between respiration rate (\o,: fig h') and shell length (SL: mm) as well as monthly variation of V02 of a standard 40 mm SL Septifer rirgatiis. Regression Residual sum Standard Vo, Month Equation sum of square of square F value P value r square (mean ± SD) Jan Vo, = 0.0063 X SL--'"' 11.149 3.871 F..,., = 95.05 <0.001 0.74 83.2 ± 25.2 Feb Vo, = 0.0053 X SL-*" 8.274 2.098 F,.24 = 94.66 <0.001 0.80 107.3 ±34.2 Mar Vo, = 0.087 X SL' "- 9.928 1.929 F|.36 = 185.32 <0.00l 0.84 107.5 ±26.1 Apr Vo, = 0.739 X SL'-" 4.748 4.407 F,28 = 30.17 <0.001 0.52 162.7 + 65.2 May Vo, = 1.346 xSL"» 5.488 2.402 F,,. = 82.25 <0.001 0.70 228.8 ±65.3 Jun Vo, = 0.605 + SL'"' 9.453 2.089 F, ,s = 158.40 <0.001 0.82 233.0 ± 49.2 Jul Vo, = 0.062 X SL--' 20.458 8.779 F,,7 = 86.22 <0.001 0.70 240.4 ± 116.8 Aus; Vo, = 0.075 X SL- '^ 27.245 4.189 F,..u = 260.149 <0.001 0.87 251.0 ±77.5 Sep Vo, = L895 X SL"" 7.339 4.578 F,..,6 = 57.71 <0.001 0.62 243.1 ±80.2 Oct Vo, = 0.477 X SL' " 7.568 8.261 •^I.SK = 34.81 <0.001 0.48 142.8 ±62.3 Dec Vo, = 0.01 1 X SL-" 19.168 4.031 F,..,7 = 175.96 <0.001 0.83 142.5 + 46.9 time (ANOVA, DF = 10. 391. F = 9.15. P < 0.0(11 ) with hiyli values being obtained from April to August and low ones from September to March (Table 3. Fig. 4). The annual mean value of 0:N was 24.0 with highest and lowest values being 36.1 and 10.5, respectively. 0:N was significantly correlated with temperature (Pearson correlation coefficient r = 0.236. n = 402. P < 0.001) but not with salinity and food conditions. DISCUSSION Morton (1995) showed through histological studies that indi- viduals of S. virgatiis from the same site as the present study remained mature year round. Spawning, however, was limited to two periods in spring (February to March) and autumn (September to December). This matches with our data on DGSI for which the values decreased in March to April and July to October, showing that they reflect reproductive cyclicity satisfactorily. The increase in somatic growth in winter to spring was probably an artefact as the gonad in S. viifiatiis, like in other mussels, was not a discrete organ with gonoglands ramifying throughout the body. Complete separation of gonadal and somatic tissue, therefore, is impossible. This is further confirmed by the suspension of growth from De- cember to March with a winter growth ring formed (Morton 1995). Lee (1988) also found the coupling of gametogenic event and DGSI for another local mytlid, the green mussel Pcnni viridis. The bimodal pattern of spawning and recruitment demonslrated in this 400 -, 350- 300- 250- 200 • 150 ' 100 ; 50- -^SV02 1 } k -•-SVN t \ ^ r T \ ' 1 T 1 ky ^ ^ — ' / ^ \, ^ x^--r 1 L • r I Y' 1 -L i 18 16 14 < 10 ^ c 8 "O IT ■■ 6 — " ■ 4 • 2 0 Jan Feb l^ar Apr May Jun Jul Aug Sep Oct Nov Dec month Figure 3. Monthly variations (±SD) of oxygen consumption (SV,,,: ng h"') and ammonium excretion (SV^: pg h"') of a standard 40 mm Septifer rirj>alus from January to December 1999. study is common to other local mytilids (Morton 1991) and is correlated with seasonal changes in hydrography in Hong Kong of which high summer temperatures (possibly in conjunction with lowered salinities as a result of enhanced summer rain) dividing an otherwise single pattern of summer breeding into pre- and post- summer phases (Morton 1991. Morton 1995). Although DGSI showed two major peaks with similar amplitude, only one was identified for CI in February with a less obvious decrease being obtained from August to December. As growth was suspended in winter to spring (Morton 1995). a higher CI value indicated that reproductive output in spring was much higher than in summer. An extended spawning in summer may help alleviate the problem of post-reproductive stress and increase survival when high rock tem- peratures at mid-day coincide with low spring tides (Morton 1995). Similar observations were also documented for another locally dominant mussel, Penui viridis, inhabiting a sheltered harbor (Cheung 1993a). Food availability is a major factor determining gonadal growth and reproductive cycle (Bayne 1976, Newell el al. 1982, Kang et al. 2000). Food availability in this study was the lowest when the gonad was developing from April to July (Table 1). It was, how- ever, also the time for body growth that was diphasic, with spring growth occuiTing from March/April to June and autumn growth from September/October to December/January (Morton 1995). A trade-off for limited resources between growth and reproduction may help explain a lower reproductive output in summer than autumn/winter when food availability was highest (October to February). The oxygen consumption rate of S. virgiitus demonstrated a seasonal pattern with peaks recorded from summer to early autumn and low values in winter. A similar .seasonal pattern has been documented for a number of bivalves (Bayne & Newell 1983, Cheung 1993b, Hummel et al. 2000, Huang & Newell 2002) with temperature, food availability, and reproductive condition being the major determining factors (Babarro et al. 2000). This study indicated that oxygen consumption in 5. vigaliis was most signifi- cantly affected by temperature. The effect of food availability was insignificant, although numerous reports have demonstrated that oxygen consumption was reduced under low values of food quality (Babarro et al. 2000) as the result of a lowering of digestive ac- tivity and erowth (Bavne & Widdows 1978, Bavne et al. 1989). Physiological Ecology of Septifek virgatvs 493 TABLE 3. Allometric relationships between NHj-N excretion rate (\\,: pg h ') and shell length (SL: mm) as Htll as monthly variation of excretion rate (S\\) and 0:N ratio of a standard 411 mm Septifer virgatus. Regression Residual SV^ Month Equation sum of square sum of square F V jlue P value r square (mean ± SD) 0:N Jan Vm = 0.000155 X SL-'*^ 14.215 11.624 F,„ = 40.36 <0.001 0.55 7.39 ± 3.08 14.38 ±20. 11 Feb Vn = 0.000209 X SL- ^" 8.990 9.511 F,,24 = 22.68 <0.001 0.49 7.24 ± 3.70 17.82+ 13.43 Mar Vn = 0.000456 X SL- " 17.532 20.885 F,,. = 30.26 <0.001 0.46 7.05 ± 4.32 22.61+21.76 Apr V^, = 0.0259 X SL' '" 4.390 5.488 F,.2B = 22.40 <0.001 0.44 4.73 ± 2.05 36.10 ±19.09 May Vn = 0.0270 X SL' ■•' 6.008 2.402 F,.,6 = 90.23 <0.001 0.71 5.80+ 1.66 34.50 ±15.63 Jun Vn = O.OllSxSL'" 10.847 4.359 F,„ = 87.10 <0.001 0.71 7.13 ±2.42 32.03 ± 13.73 Jul Vn = 0.00307 X SL-"" 18.248 5.531 F,,7 = 122.07 <0.001 0.76 7.29+2.91 34.88 + 32.92 Aug Vn = 0.00391 X SL-" 25.270 12.492 Fl,40 = 80.92 <0.001 0.67 10.53 ±4.96 29.92 ± 30.37 Sep v>, = 0.245 X SL'" 5.520 5.946 F,,,, = 33.42 <0.001 0.48 16.93 ±6.26 14.90 ±9.50 Oct Vn = 0.00365 X SL- -" 15.892 4.290 F,,,s = 140.76 <0.001 0.79 12.96 ±4.51 10.52 ±5.04 Dec Vn = 0.0295 X SL' " 6.710 1.791 F,,7 = 138.62 <0.001 0.79 7.91 ± 1.68 16.04 ±4.58 Such a discrepancy may be due to small niictuations In food avail- ability in this study when particulate organic matter was show n to vary from 1.13 to 3.36 mg P' (except in January). Significant correlation between reproductive activity and oxygen consumption have been reported by a number of authors. Bayne and Widdows (1978) obtained a significant correlation between oxygen con- sumption rate and gametogenic index and not with temperature tor Mvtilus edulis. Similar results were reported for the cockles Cenis- todemm edule by Newell and Bayne ( 1 980), whereas other studies have reported significant correlation between oxygen consumption and temperature and reproductive period (De Vooys 1976. Iglesias & Navarro 1991, Smaal et al. 1997). As the gametogenesis of S. virgatus covaried with temperature and growth was resumed in spring, it is difficult to distinguish the relative importance of tem- perature, growth and reproductive activity in determining the oxy- gen consumption rate in spring. Oxygen consumption rate, how- ever, was lower in winter when the gonads were developing. Unlike oxygen consumption, the correlation between excretion rate and temperature was weak. A number of workers have docu- mented a close relationship between excretion rate and reproduc- tive cycle (Bayne & Scullard 1977. Bayne & Widdows 1978. Smaal et al. 1997, Babarro et al. 2000). Such correlations were suggested to be the result of a heavy reliance on protein catabolism for energy when mussels are reproductively ripe and have low ■ 80 ■70 •60 ■ 50 .40° 30 ' 20 10 0 Jan Feb Mar Apr t^ay Jun Jul Aug Sep Oct Nov Dec month Figure 4. Relationships between temporal variations (±SD| in DGSI and 0:N ratio of a standard 40 mm Septifer virgatus from .lanuary to December 1999. 0.4-, -♦-DGSI " 0.35- r^^ y -•-0:N 0,3 ■ \ , / N k ■ 0.25- L\ r/ \ ^ i - ; 1 " \ A- ' < \ ^\X 0.15] ■■ / ^ ^ ^ k 1 0,1 - \^ ^ X" ■ , 0.05- 0 - ^^•"^"^ glycogen content (Bayne & Scullard 1977). Spawning started in August when excretion rate increased abruptly. As glycogen stores are low during that time, energy for maintenance was derived from substantial protein and ammonium production increases. This is further confirmed by the coupled oscillation between DGSI and 0:N ratio from June to December (Fig. 4, see later). The problem of post-reproductive stress is aggravated by high rock temperatures at midday coinciding with low spring tides (Morton 1993). Such stresses also resulted in high mortality of liinpets (Williams & Morritt 1995), barnacles and 5. \irv,atus (Liu & Morton 1994). Temperature started to decrease in October and there was a sus- pension of spawning and a resumption of growth; excretion rate also started to decrease and 0:N ratio increased. A low 0:N ratio (<20) has been used as an indicator of nutri- tional stress for marine bivalves (Bayne et al. 1985) as it shows an increased reliance on protein as a catabolic substrate rather than carbohydrates and lipids (Huang & Newell 2002). It may also be related to the greater demand for dietary carbon than nitrogen so as to preserve carbon based energy for utilization in seasons when food resources are low (Kreeger 1993, Huang & Newell 2002). High 0:N ratios (>30) coupled with gametogenesis and body growth in spring (April to June), resource demands for gametoge- nesis, therefore, were mostly derived from temporal nutrition ac- quisition instead of body reserves. As stress became intense in summer, growth was suspended and spawning was initiated and there was a heavy reliance on protein catabolism for energy re- sulting in a low 0:N ratio (Fig. 4). When growth and gametoge- nesis resumed in autumn as temperatures decreased, the 0:N ratio increased gradually and peaked in spring. A number of studies have demonstrated the significance of reproductive activity to oxygen consumption, ammonia excretion and 0:N ratio for species with a well-defined reproductive cycle such as Mytilus edulis (Bayne & Widdows 1978. Widdows et al. 1984. Smaal et al. 1997) and Mytilus galloprovincialis (Navarro et al. 1991, Babarro et al. 2000). Being a subtropical species with a diphasic spawning pattern, seasonal variations in energy metabo- lism and utilization by S. virgatus are satisfactorily reflected in 0:N ratio and are closely related to reproductive cycle, in addition to environmental influences such as temperature and food avail- ability. Although oxygen consumption is most significantly af- fected by temperature, excretion rate is mainly influenced by 494 Gao et al. growth and nutritional stress associated with intense heat and re- productive activity. ACKNOWLEDGMENTS The worl< described in this study was substanlially supported by a grant t'roni the Research Grants Council of the Hong Kong Special Administrative Region. China (CityU Project No. 9040279). 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Aquaculture 193:123-137. Zar. J. H. 1999. Biostatistical Analysis. New Jersey: Prentice-Hall. 663 pp. Jouniul of Shellfish Research. Vol. 21. No. 2. 497-501. 2002. MICROALGAL FOOD OF THE RIBBED MUSSEL AULACOMYA ATRA (MOLINA, 1782) IN GOLFO NLEVO (PATAGONIA, ARGENTINA) NESTOR FERNANDO CTOCCO* AND ANA MARIA GAYOSO Centra Nacional Patagonia). CONICET. Boulevard G. Brown s/n. 9120 Puerto Mudryn. Chuhut. Argentina ABSTRACT StoniaLli contents ol the ribbed mussel Auhuomya alia and phytoplanklon samples from the west coast of Golfo Nuevo (Chubut Province. Argentina) were analyzed monthly between August 1997 and December 1998. Twenty-five taxa of microalgae ranging from 9 to IM (xm in size were found in stomach contents along with other miscellaneous items. A marked .seasonal variation of microalgal food was not observed in Aiilacomya atra. Diatoms such as Paralia siikata and Thalassiimra sp. were observed year round. Variation in the benthic to total species ratio revealed that during most of the year benthic and pelagic microalgae contributed equally to the diet of the ribbed mussel. In contrast, during dinotlagellate blooms, as occurred during late spring and summer, dinotlagellate cells dominated the ribbed mussel stomach contents. This findmg is of special importance in relation to the recurrent blooms of the PSP causative agent, Alexamlniim lamarense. in the Golfo Nuevo. KEY WORDS: ribbed mussel. Aiilaconmi aim. food resources, phytoplankton. Southwest Atlantic. Patagonia INTRODUCTION Mussels are suspension feeders. Quality and abundance of food affects growth rate, gonad development and survival of bivalve mollusks (Bayne & Widdows 1978, Newell et al. 1982, Berg & Newell 1986). It is generally assumed that suspension-feeding spe- cies rely on phytoplankton as their main source of energy. Yet it has been demonstrated that benthic species can also play an im- portant role in the feeding ecology of various bivalves (Mikulich & Tsikhon-Lukanina 1981. Tsikohn-Lukanina 1982). In fact, it is possible to find benthic and pelagic food species equally repre- sented in the guts of bivalves from shallow waters (Shumway et al. 1987). However, several authors have pointed out that food selec- tion occurs within in bivalves (Shumway et al. 1985, Sidari et al. 1998). The ribbed mussel. Aiilacomya atra (often incorrectly cited as Aiilacoima aier according to Cazzaniga. 1994) (Molina. 1782). locally named cholga, supports shellfisheries in the northern Pat- agonian gulfs of Southwest Atlantic (Ciocco et al. 1998). It is one of the most common bivalve species around the coasts of southern South America (Fig. I), ranging northwards to San Mati'as Gulf (41°S) on the Atlantic and to Peril along the Pacific (I5°S). The quantity and quality of food available is a major limiting resource for suspension feeding organisms (MacDonald & Thomp- son 1985. MacDonald & Thompson 1986, Chauvaud et al. 1998). Until now information on the specific food items used by AiiUi- comva atra was only available for the Chilean coasts (Guzman & Campodonico 1975, Osorio et al. 1982). This is the first report on feeding in Aiilacomya atra along the Argentina coast. Golfo Nuevo, situated at 43°S on the Patagonian coast of Ar- gentina (Fig. 1 ), is a rough elliptical, semi-enclosed body of water in contact with the Southwestern Atlantic Ocean through a 17-km wide strait. Phytoplankton is dominated by diatoms and dinotlagel- lates (Gayoso 2001 ). Recurrent blooms of the toxic dinotTagellate, Ale.xandrium tamarense (Lebour) Balech. have been documented, resulting in human health hazard (Esteves et al. 1992. Ciocco 1995, Gayoso 2001 ). Other harmful dinofiagellate species includ- ing Proroceiilnim lima (Fhrenberg) Dodge (Gayoso & Ciocco 2001) and Diiioplivsis acuminata Claparede & Lachmann have also been found in the area adding potential risks for human health and marine resources. In this study we describe the stomach con- tents of ribbed mussels in order to: (1) characterize the nature of the food items: (2) assess seasonal variation of food items; and (3) provide preliminary information about the relationships between microalgal species in water samples and algal cells in the stomachs of the ribbed mussel. wc^^-'i 100 10 V-.-. X. r ( ry" c V" ro . ^ CD r CJ O o L V-' f c 03 10 20 30 40 90 80 50 40 30 ?0 50 *Corresponding author. E-mail: cioccofe'cenpat.edu.ar 70 60 Figure 1, Map of South America showinj; dislriljution «X Aulacomya ultra (gray circltsl and location of sampling area (arrow). 497 498 ClOCCO AND GaYOSO a s Z 3 ~ a -f 03 — Q " o - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + _^ M3 •o ON r*-i ^ 1 1 vr, o r^i '/") 1 " — ' r- m t £ m m •O 1 a. m On ■i ■— ^ 1 JZ ^ CO w 8 ac d. — :S CQ s a. i£ S^ S a ^2 2 d. -2 ■2 o Q ^ c^ "C ■^. 5^ ^j ,c a ^ U O" Q Q. — o _ o — r^, — 5=, oj — ;; f -J- 00 CQ CQ ^ <^ ^ — — Tf <^l — ■ VO I I op -S: S oa ^ ^ fi I ^ "" P Cu £ n-i -- _ m a 5.S .y s s .2 S- e ^3 o o -5 X ~ S " 6 K -s Q. '^ ~ • S" -F '^ 2 o. D- 2 .j 5 S S 2; :? S; (£ 5 R ^ 11 :i 2 S g S 35 ^tnz Figure 2. Cell number variation of diatoms (A) and dinoflagellales (B) in tiie water samples of Golfo Nuevo and ratio of benthic species over total species of Aulocomya altra guts in each sampling dale (C), phytoplankton community. The average ratio of benthic species to total species numbers from 6 stomachs per sampling date (Fig. 2C) widely varied throughout the year; it varied from less than 0. 1 in summer (February 1997) and late spring (November to December 1998), during Prorocentrum micans and Alexandriwn tamarense blooms, to a ratio of almost 1 in May 1998. Percentages between 0.4 to 0.5 were commonly registered (Fig. 2C). Moreover, some of the more representative phytoplankton taxa and algal groups, such as Chaetoceros spp., Skeletonema costatum and phytoflagellates, were never found in the stomach contents. DISCUSSION The size range of the food items detected in the stomach con- tents in this study (9-23 1 ixm) is similar to that reported for A. atra 500 ClOCCO AND GaYOSO from Arica, Chile (21-270 |j,m, exceptionally up to 725 ixm; Oso- rio et al. 1982) and other species such as Myrilus eiliilis Linnaeus (10-250 (j.m; Newell & Shuniway 1993) and inshore Placopecten mageUanicus (GmelinK8-240 \i.m: Shumway et al. 1987) from the Gulf of Maine. A wider size range of food items were reported in offshore Placopecten iinigclUinicus (10-350 (jlui; .Shumway et al. 1987) and Patinopecten yessoensis (Jay)(9-950 |jLni; Mikulich & Tsikhon-Lukanina 1981). Silicotlagellates. foraminifera, and spe- cially tintinnids were other items observed in ribbed mussel stom- achs in this study. The three groups are also miscellaneous items reported in A. arm from Arica. Chile (together with abundant detritus and other items; Osorio et al. 1982) and gut contents of other bivalves such as Clihiinxs varia (Linnaeus) (foraminifera. Hunt 1925). Placopecten ntagellanicus (silicotlagellates, Shum- way et al. 1987), Aequipecten tehuelclms (d"Orbigny)(silico- tlagellates, Vernet de Hall 1977), Mytilus echilis (silicoflagellates. Newell et al. 1989) and Mytilus galloprovincialis Lamarck (sili- coflagellates, Sidari et al. 1998). Two diatom peaks are observed as a recurrent feature of the annual phytoplankton cycle in Golfo Nuevo (Gayoso 2(MI ). This common characteristic was not reflected in the annual variation of microalgal food of the ribbed mussel. Among the most represen- tative species of the diatom bloom. Skeletonema costatum. Clia- etoceros socialis. C.curvisetus. C.diademu, C. didynuis and Pseiido-nhzchia piingens. only the latest, was observed in the ribbed mussel gut (Table I ). Absence of dominant pianktonic mi- croalgae has been reported for other bivalve species such as the absence of Chaetoceros spp. in Placopecten mageUanicus (Shum- way et al. 1987) and S. costatum in Aequipecten tehuelclms from Golfo San Jose, Argentina (Vernet de Hall 1977). These cases suggest a probable selective feeding capacity as indicated for oth- ers bivalves (Field 1922. Vernet de Hall 1977, Shumway et al. 1985. Shumway et al. 1987. Sidari et al. 1998), The absence of phytoflagellates in A. atra stomach contents may be explained by quick digestion of some small (mainly <10 (xni in diameter) algal species ("Shumway et al. 1987). reduced retention efficiency of particles less than 7 |jim (MLihlenberg & Riisgard 1978) and/or difficulties in identification of micro-flagellates in bivalve stomach contents due to the fragility of these small cells (Vernet de Hall 1977). A marked seasonal variation was not observed in the microal- gal food of Aulac(miya atra. Diatoms such as Paialia sulcata and Thalassiosira sp. were observed year round. Variation in the ratio of benthic over total species demonstrated that during most of the year benthic and pelagic microalgae contributed equally to the diet of the ribbed mussel in the Golfo Nuevo. In contrast, during di- noflagellate blooms, as occurred during late spring and summer, the percentage of benthic species dropped to less than 10% (Fig. 2C). The fact that P. micans and A. tamarense were found mostly as intact cells in the stomachs suggests, however, that they are not assimilated by the ribbed mussels. The capacity of concentrate large amounts of dinoflagellates was also observed in Mytilus gal- loprovincialis during a bloom of Dinophysis (Sidari et al. 1998). In A. atra from Magellanic Region (Southern Chile) Guzman and Campodonico (1975) idenlified Alexandrium catenella (Whedon & Kofoid) Balech (formerly Gonyaulax catenella) in A. atra from Punta Arenas, Chile. Lembeye (1981) reported the toxic di- noflagellates Alexandrium catenella and Diiu>pliysis acuta Ehren- berg (DSP causative agent) in stomach contents of A. atra from Chilean Magellan waters. Great densities of Alexandrium tama- rense can explain the high toxicity of the ribbed mussels when the species is blooming in the Golfo Nuevo (Esteves et al. 1992, Andrinolo et al. 1999). Toxicity values up to 1,750 |jig STXeq/100 g were reported in A. atra from Golfo Nuevo by Andrinolo et al. (1999). ACKNOWLEDGMENTS The authors thank the anonymous reviewer for the constructive criticisms that helped to improve our work. This study was funded by CONICET, Republica Argentina, PIP 4269-96. LITERATURE CITED Andrinolo. D., N. Santinelli. S. Otafio. V. Sastre & N. Lagos. 1999. Para- lytic shelltlsh toxins in mussels and Alexandrium tamarense at Valdes Peninsula. Chubut, Patagonia, Argentina: kinetics of a natural depura- tion. J.Shellfhli Res. 18:203-209. Bayne, B. L. & J. Widdows. 1978. The physiological ecology of two populations of Mytilus edulis L. Oecologia 37:137-162. Berg, J. A. & R. I. E. Newell. 1986. Temporal and spatial variations in the composition of seston available to the suspension feeder Crassostreu virginicci. Estuar. Coast. Shelf Sc. 23:375-386. Cazzaniga, N. J. 1994. A concern for grammar: the name of the magellanic mus.sel (Bivalvia: Mytilidae). Malac. Rev. 27:110. Ciocco, N. 1995. Marisqueria medianle buceo en el golfo San Jose. Plan de Manejo Integrado de la Zona Costera GEF/PNUD). Serie Informcs tecnicos 2:1-39. Ciocco, N. F., M. L. Lasta & C. Bremec. 1998. Pesquen'as de bivalves: mejilldn, vieiras (tehuelche y patagdnica) y otras especies. In: E. Bos- chi, editor. El Mar Argentine y sus recursos Pesqueros. Volume 2. Argentina: Instituto Nacional de Investigacion y Desarrollo Pesquero. INIDEP. Mar del Plata, pp. 142-166. Chauvaud. L.. G. Thouzeau & Y. M. Paulet. 1998. Effects of environmen- tal factors on the daily growth rate of Pecten ma.ximiis juveniles in the Bay of Brest. J. Exp. Mar Biol. Ecol. 227:83-1 11. Esteves. J. L., N. Santinelli, V. Sastre, R. Diaz & O. Rivas. 1992. A to.xic dinoflagellate bloom and P.SP production associated with upwelling in Golfo Nuevo.atagonia Argenlma. Hydrnbiologia 242:115-122. Field. 1. A. 1922. Biology and economic value of the sea mussel Mytilus edulis. Bull. Bur Fish. Wash. 38:127-159. Gayoso. A. M. 2001. Observations on Alexandrium tamarense (Lebour) Balech and other Dinoflagellate Populations in Golfo Nuevo. Patagonia (Argentina). / Plankton Res. 23:463-468. Gayoso, A. M. & N. F. Ciocco. 2001. Observations on Prorocentrum lima from North-Patagonian coastal waters (Argentina) a.ssociated with a human diaorrheic disease episode. Harmful Algae News 22:4. Guzman. L. & I. Campodonico. 1975. Marea roja en la region de Magal- lanes. Pub. Inst. Patagonia IChile) Ser. Mon. 9:9—14. Hunt. O. D. 1925. The food of the bottom fauna of the Plymouth fishing grounds. / Mar. Biol. As.wc. UK 13:560-599. Lembeye. G. 1981. Segunda aparicion del veneno paralitico de los mariscos (VPM) asociado a Gonyaulax catenella. en Magallanes (Chile). \9%\.An. Inst. Patagonia 12:273-276. MacDonald. B. A. & R. J. Thompson. 1985. Intluence of temperature and food availability on the ecological energetic of the giant scallop Pla- copeclen mageUanicus. 1. Growth rates of shell and somatic tissue. Mar Ecol. Prog. Ser 25:279-294. MacDonald. B. A. & R. J. Thompson. 1986. Intluence of temperature and food availability on the ecological energetic of the giant scallop Pla- copecten mageUanicus. III. Physiological ecology of the gametogenic cycle and scope for growth. Mar. Biol. 93:37-48. Mikulich. A. & Y. Tsikhon-Lukanina. 1981. Food of the scallop. Oceun- ,;/ox.v 21:633-635. MiCROALGAL FOOD OF AULACOMYA ATRA 501 Muhlenberg. F. & H. U. Riisgard. 1978. Efficiency of particle retention in 13 species of suspension-feeding bivalves. Ophelia 17:2,^9-246. Newell, R. I. E.. T. J. Hilbish. R. K. Koehn & C. J. Newell. 1982. Temporal variation in the reproductive cycle of Mytihis eilulis L. (Bivalvia. Mytilidae) from localities on the east coast of the United States. Bid/. Bull. 162:299-310. Newell. C. R.. S. E. Shumway. T. L. Cucci & R. Selvin. 1989. The effects of natural seston particle size and type on feeding rates, feeding selec- tivity and food resource availability for the mussel Mytilus eilulis L. at bottom culture sites in Maine. J. Shellfish Re.\. 8:187-196. Newell, C. R. & S. E. Shumway. 1993. Grazing of natural particulates by bivalve molluscs; a spatial and temporal perspective. In: R. F. Dame, editor. Bivalve Filter Feeders in Estuarine and Coastal Ecosystem Pro- cesses. NATO ASl Series Vol G 33. Berim Heidelberg: Sprmger- Verlag. pp. 85-148. Osorio, C, R. Iguain, B. Baric & C. Navarrete. 1982. Alimentacion natural de Aulacoiimi ciler (Molina. 1782) en Punia Paloma, Arica (Mollusca, Bivalvia. Mytilidae). Bol. Miis. Nac. His!. Nat. Chile 39:111-1 18. Shumway. S. E., T. L. Cucci. R. C. Newell & C. M. Yensel. 1985. Particle selection, ingestion and absorption in filter-feeding bivalves. J. E.\p. Mai: Biol. Ecol. 91:77-92. Shumway. S. E., R. Selvin & D. Schick. 1987. Food resources related to habitat in the scallop Placopecten magellaniciis (Gmelm. 1791): a qualitative study. J. Shellfish Res. 6:89-95. Sidari. L., P. Nichetto, S. Cok, S. Sosa. A. Tubaro, G. Honsell & R. Delia Loggia. 1998. Phvtoplankton selection by mussels, and diarrhetic shell- fish poisoning. Mar. Biol. 131:103-111. Tsikhon-Lukanina. Y. A. 1982. Food spectra of bottom molluscs. Ocean- ology 22:751-754. Vernet de Hall, V. 1977. Alimentacion de la vieyra tehuelche iChlamys tehiielcha). Internal Report. Puerto Madryn, Argentina: Centre Nacio- nal Patagonico. 26 pp. Journal of Shellfish Research. Vol. 21, No. 2. 503-5US. 2U02. TRIPLOID INDUCTION OF MYTILUS EDULIS USING 6-DIMETHYLAMINOPURINE JOHN BRAKE,' JEFFREY DAVIDSON,^* AND JONATHAN DAVIS^ ' Coastal Oregon Marine Exp. Station. Hatfield Marine Science Center. 2030 S. Marine Science Dr., Newport. Oregon 97365; -Department of Health Management. Atlantic Veterinary College, University of Prince Edward Island. 550 University Avenue. Cluirlottetown. PEL Canada. CIA 4P3: ^Baywater Inc.. 15425 Smoland Lane. Bainhridge Island. Washington 981 10 ABSTRACT The induction of tnploidy in Myiilus eduli.s. using 6-dimeth\iaminopurine (6-DMAP). was investigated as a potential method of providing high quality sterile product in Prince Edward Island (PEl), Canada. Initial results indicated induction by blocking the second polar body would require a treatment starting time of 21 min post-fertilization at lOX. Poor yields in subsequent spawns caused a re-examination of these factors by investigating the meiotic events post-fertilization. The resulting description was used to develop a series of suitable treatment time windows for testing inducing techniques. Triploids were successfully produced with the use of 6-DMAP at various concentrations and at various times post-fertilization. The optimal investigated procedure for induction of tnploidy in Mytihis ediilis was the treatment of eggs at 20°C with 400 ^JLmol/L of 6-DMAP starting at 24 min post-fertilization for a treatment time of 10 min. This treatment yielded 83.1% triploids in induced samples with a survivorship of 1.39% to the D- vehger stage. The investigated methods for producing triploid Mytilus edidis are not yet optimized to commercial levels. This work forms a basis for further work in the optimization of this technique. KEY WORDS: 6-dimethylaminopurine. meiosis. mussel, Mytilus edulis. triploid INTRODUCTION The Prince Edward Island (PEI) mussel aquaculttire industry is a large contiibutor to Canada's aquaculture sector, accounting for greater than 80% of all Canadian mussels over the past two de- cades. One area of concern to the PEI mussel aquaculture industry is the harvesting and marketing of mussels during, and just after the spawning season. Ripe mussels (near spawning) can spawn due to the stress of shipping. Also, mussels that have recently spawned have significantly reduced meat yield making them less appealing to the consuming public. In PEI. the marketing of non-spawning triploid mussels during this period would alleviate these problems and allow the industry to market a high quality product year round. Triploids are organisms with three sets of chromosomes, dif- fering from most other sexually reproducing organisms that have two sets (diploids). Triploids also do not normally undergo meio- sis, as the three sets of chromosomes cannot property synapse. Therefore triploids have poorly developed gonads and produce far fewer gametes than diploids (Allen 1988). Tnploidy is an aberrant genetic state in molluscs, and most organisins, and therefore must be produced artificially. The main objective of this study is to develop and optimize a triploid induction strategy. The important considerations for developing a triploid-inducing technique for commercial use are effectiveness and safety. 6-dimethylaminopu- rine (6-DMAP) is far less dangerous than cytochalasin B (CB) and equally effective in most cases (Desrosiers et al. 1993). therefore it was selected as the chemical induction agent for testing in this study. In molluscs, triploid induction occurs through the use of an external treatment on a fertilized egg. The goal of coinmercial triploid induction of shellfish is to produce a high percentage trip- loid cohort with high survivorship throughout the hatchery stage (Allen et al. 1986). Imperative to the induction of both a high percentage of triploids and a high survival rate in a cohort of triploid mussels is the determination of the proper time at which to *Corresponding author. Tel. E-mail: davidsonfeupei.ca -f 1-902-566-0666; Fax; -f 1-902-566-0823; begin triploidy induction, and the treatment duration. In the past, the stage of development when 50% of the eggs showed polar body formation has been used as a common cue to start triploid induction treatment in oysters (Allen et al. 1989). Although the duration of the treatment varies between hatcheries, this treatment has a finite duration, the optimal time of which is determined by the timing of meiotic events. The time, from which a triploid induction treatment may be initiated until it is no longer effective. may be referred to as the induction window. One of the objectives of this study is to determine the optimal time for triploid induction initiation in Mylihis edidis embryos. Another objective of this study is to define the induction window for Mylihis edidis using an effective concentration of 6-DMAP and a standardized set of pa- rameters. This work serves as the foundation for the future opti- iTiization of the triploid-inducing technique (using 6-DMAP) in Mytilus edidis. MATERIALS AND METHODS All experiments were conducted at the Ellerslie Shellfish Hatchery (ESH). in Ellerslie, PEI. Flow cytometry, to define ploidy level in samples, was performed at the University of Wash- ington in Seattle. Washington, and at the Whiskey Creek Shellfish Hatchery in Tillamook. Oregon. Broodstock were obtained from a mussel lease located in Lennox channel, near Lennox Island. PEI. These animals were conditioned for 6 weeks, during which time they were batch fed a mix of cultured microalgae at water changes every 2 days. Spawning was induced by a -i-IO°C thermal shock. Fertilization occurred within 2 h of gamete collection so as not to compromise gamete quality. Description of Polar Body Extrusion Timing Temperatures of 15°C and 20°C were investigated. 20°C was selected as it is an easy achievable temperature in most shellfish hatcheries, and 1 3°C was selected, as it is closer to the temperature during the natural local spawning in this species, and also common in many shellfish hatcheries. Approximately 3 million eggs were pooled from 3 females for use in each temperature treatment. Sperm was also pooled from 4 individuals for egg fertilization. 503 504 Brake et al. Figure I illustrates the sampling procedure used for ttiis experi- ment. Eggs were placed on a 20 |xm sieve within a 20 L bucket of I |jLm filtered sea water (FSW). This bucket was placed in a water bath to keep the eggs and surrounding sea water at 20°C. Eggs were sampled by raising the screen out of the water and aspirating eggs from the screen (without an excess of sea water) and imme- diately returning the screen to the bucket (Fig. 1). The aspirated eggs were immediately placed into a pre-loaded 2-ml microcen- trifuge tube containing 2*^ buffered formalin fixative. The sample was mixed well so as to fix the eggs rapidly. The eggs were sampled prior to fertilization, at fertilization, and at subsequent 3-min intervals up to 2 h post-fertilization. Simultaneous to this sampling, the same procedure was being followed using the same egg and sperm pools in a water bath of 15°C. Under light micros- copy, polar bodies in the fixed egg samples were visible using a 0.5% aceto-orcein stain (Guo 1991). For each sampled time, the number of eggs (out of the first 50 visualized on the slide) showing visible polar bodies was recorded. When this number reached 25 (50% of eggs), the target had been reached. Effect of 6-DMAP Concentration on Percent Triplvid Induction The sperm from 6 males was pooled for this experiment. The sperm was used to fertilize the pooled eggs from 4 females. The Eggs developing on a 20nm sieve in FSW. B Sieve is removed, and eggs are aspirated without FSW. c Eggs are placed in a pre-loaded microcentrifuge tube and mixed well for rapid fixation. D Sieve is placed back in FSW to allow for more egg development prior to next sampling. ^' Figure 1. Schematic of the experimental sampling procedure used in the description of pnlar body extrusion timing and the description of meiotic events. pooled eggs were counted and loaded into IL plastic beakers at a density of 100,000 eggs per beaker (100 eggs per ml of FSW). Treatments were replicated three times in this experiment and consisted of eight concentrations of 6-DMAP, at each of the two investigated temperatures. The tested 6-DMAP concentrations were similar to those used by Desrosiers et al. ( 1993) who ranged concentrations from 0 to 600 jjimol/L. The same range was se- lected for experimentation in this case as the resolution in their results gave clear indications of the effect of chemical concentra- tion on percent triploid induction. For each treatment the appro- priate amount of 6-DMAP was weighed and placed in a labeled aluminum foil packet. When a treatment was initiated the chemical was emptied into the beaker and rapidly mixed into the water until it was dissolved. Egg suspensions were mixed every few minutes throughout the treatment duration to ensure adequate chemical contact with all eggs. Water baths of 1 5 C and 20°C were used to keep egg suspensions at the proper temperature. Eggs were al- lowed to acclimate to the proper temperature for 30 min just prior to fertilization for optimal induction (Allen et al. 1989). All eggs were treated at 21 min post-fertilization for 20 min duration. At the conclusion of the treatment duration the eggs were thoroughly rinsed, placed onto a 20-|xm sieve, and resuspended into their respective treatment beakers with new FSW. The egg suspensions were allowed to incubate for 24 h, then samples were taken. Em- bryos that had grown to the D-veliger stage were collected by rinsing them onto a 64-|jim sieve. Sampled embryos were trans- ferred into a 15 ml centrifuge tube filled with FSW. These samples were used for ploidy determination. Description of Meiotic Events Eggs from 4 females were pooled for use, and sperm from 7 males was pooled for fertilization. This experiment was only per- formed on one sample of pooled fertilized eggs held at 20°C. A water temperature of 20°C was used as this was shown in the results of the description of polar body extrusion timing (the first experiment) to have more synchronous meiosis, (therefore it should be more effective than I5°C for triploid induction). Samples of eggs were taken every minute from fertilization until one hour post-fertilization. Each of the first 50 eggs observed for every sample were categorized as being in a particular stage of meiosis. The first 50 readily scored comprised the sample for each time investigated. Evaluation of Several Treatment Windows Sperm from 5 males was pooled to Icrlili/e the eggs pooled from 12 females. All treatments were standardized using a tem- perature of 20°C and a 6-DMAP concentration of 400 p.mol/L and 100 eggs/ml of FSW. The treatments investigated included differ- ent treatment starting times based on the observed meiotic events in the previous experiment. Four treatment durations of 5, 10, 15, and 20 min were investigated along with four treatment starting times of 24, 27, 30, and 33 min post-fertilization. Survivorship was assessed by taking 1 5 ml samples of each culture at day 3 and fixing them with Lugol's iodine. A survivor was later scored as a fixed D-veliger that showed food in the gut, under light micros- copy. Samples were taken for both ploidy and sur\'ivorship. RESULTS Description of Polar Body Extrusion Timing Table 1 summarizes the results. In the 20'^C group polar bodies appeared as early as 15 min post-fertilization and showed the mean Triploid Induction of Mytilus epulis 505 TABLE 1. Percent of polar body I extension in the 15 C and 20 C groups. Time (Min) 15 C ICC (1 3 6 9 12 15 18 21 24 27 2 ±2.0 30+ 10.58 38.67 ± 14.05 63 ±15.56 2.67 ± 1.15 23.33 ± 3.05 50.67 ±4.16 desired score of 50"^ polar body display at 21 min ( 50.67 Vr ±4.16) post-fertilization. Samples taken from the 15°C group showed a slower development of polar bodies, with none appearing until 1 S min post-fertilization. All samples from the 15"C group showed 50% polar body display by 27 min post-fertilization (63% ± 15.56). The I5°C group was also less synchronous, with replicates showing a standard deviation of as much as 15.56% (of eggs .showing polar bodies) between them at 21 min post-fertilization. The largest standard deviation in the 20°C group at any given time interval was 4.16%. Effect of 6-DMAP CnnceiUralioii on I'erceiil TripUiid Iiidiutioii Figure 2 is a summary of the results. In all treatments the number of live D-larvae was too low to run each replicate sepa- rately for ploidy analysis. Therefore, all three replicates for each treatment were combined for ploidy determination by flow cytom- etry. There were no triploids in the controls, however with the addition of 6-DMAP. all of the tested concentrations except the 50 p,mol/L treatment at 15°C produced triploids. In all cases except the 500-|xmol/L concentration (that had an outlier percent triploid of 9.8% and was therefore excluded) the I5''C treatments gave a lower percent triploidy than did the 20''C treatments. For both temperatures, the rate of increase in the percent of triploids in- duced decreased at concentrations higher than 300 |xmol/L. There is little increase in the percent triploids with an increase in 6-DMAP concentration beyond 500 |j.mol/L. Description of Meiotic Events Figure 3 is a summary of the results. Telophase I (the stage when polar body 1 is extruded) was visible as early as 12 min % Triploid Induction .100 90 80 70 60 50 40 30 20 10 0 / 15deg. C 20 deg. C 100 200 300 400 6-DMAP Concentration (pmol/L) 500 600 Figure 2. Percent triploid induction of Mytilus ediilis using 6-DMAP. 0 14 21 25 29 33 37 41 45 49 53 57 Time Post Fertilization (min) - - - Telophase 1 I Telophase 2 I — —Anaphase 2 Cell Cleavage Figure 3. Summarized time-related meiotic events in Mytilus edulis at 20 C. post-fertilization. Anaphase 2 (the stage that is a precursor to polar body 2 being extruded) began at 22 min post-fertilization. Telo- phase 2 (during which polar body 2 is extruded) began at a time of 27 min post-fertilization. Egg cleavage (the first mitotic embryonic cleavage) began at 44 min post-fertilization. Approximately 50% of the scored eggs were going through Anaphase 2. and 50% through Telophase 2 at 32 min post-fertilization. Evaluation of Several Treatment Windows The investigated treatment windows varied greatly in both the percentage of survivors to the D-stage (survivorship) and also in the percent triploid induced. Table 2 is a suminary of the obtained results. The control samples tested showed a 37.6% survivorship to the D-stage. with no triploids present. The highest mean treatment survivorship (9.337ri) was found in the treatment begun 24 min post-fertilization for a duration of 5 min. The lowest mean survi- vorship (0%) was found in the treatment begun at 27 min post- fertilization for duration of 20 min. The highest mean percent triploid induction (83.1%) was found in the treatment begun 24 min post-fertilization for duration of 10 min. The lowest mean percent triploid induction (0%) was found in the treatment begun at 33 min post-fertilization for duration of 15 min. Tetraploid peaks were evident in a number of these treatments from the re- sulting printout of the flow cytometry (Table 2). DISCUSSION Evaluating the Induction Window Using Meiotic Events The determination of the proper induction window is crucial to triploid induction for a number of reasons. First, the induction treatment is potentially lethal to fertilized eggs when exposure times are too long. Desrosiers et al. ( 1993) found that longer ex- posures to 6-DMAP interfered with first cleavage and resulted in developmental abnormalities, especially in Mytilus edulis. In cases where the induction comes early, although a cohort with a high percentage of triploids might be produced, the survivorship would be low, making the cohort less useful from a commercial perspec- tive. Secondly, poor timing might not only lower survivorship, but also affect the percentage of triploids that would be produced in a given cohort. If the induction window (although the proper length of time) is initiated too late, meiosis II will not be blocked and the production of mostly diploids will result. This would result in a cohort with a high percentage of diploids, and thus a lower per- 506 Braki: i-:t al. TABLK 2. Results of investigation treatment windows. Initiation Time Duration (min) (min) U 5 10 15 2(1 0 M, = 37.6 ± = 0 lo.y 24 M, = 9.33 ±5.7 M. = 1.39 ± 1.6 Ms = 0.98 ±0.8 M., = 0.28 ± 0.6 M, = 56.1 ± 17.9 M, = 83.1 ±8.8 M, = 59.3 ± 18.3 M, = 68.6 ±5.4 27 M, = 3.90 ± 2.8 M, = 0.84 ± 0.9 Ms = 0.97 + 1 .0 M., = 0 M, = 4H.0± 14.2 M, = 65.8 ± 12.2 M, = 76.5 ± 13.0 M, = 49.2 + 19.0 30 M. = 6.96 ± 2.9 M.. = 0.98 ± 1.5 M, = 3.2 ± 2.5 M, _ 0.42 ± 0.6 M, = 51.8 ±5.9 M, = 49.5 ± 6.6 M, = 11.6 ±4.6 M, = 17.2 ±5.8 33 M, = 5.58 + 2.1 M, = 2.23 ±1.6 Ms = 5.29 ± 3.0 Ms = 3.07 + 2.9 M, = 27.6 ±9.3 M, = 14.9 ±13.7 M, = 0 M, = 2.20 ±3.8 M., = mean % survivorship to the D-veliger stage (3 replicates/treatment) M, = mean % triploidy of the sampled D-veligers. * tetraploids were found in the ploidy sample. centage of triploid.s. It is important to note that not all eggs are in the same meiotic stage at any time. If the induction window begins too early, the result would be a blocking of nieiosis 1 for most of the eggs, resulting in pentaploids (5N) being produced. Pentap- loids do not survive to the D-veliger stage. This was shown to occur in Mytihis galloprovincialis by Scarpa et al. (1993). The authors exposed eggs to Img/L of cytochalasin B from 7 to 35 min after fertilization, blocking meiosis I. The resulting developing pentaploid eggs showed a subsequent inhibition of first cleavage and died. Therefore the resulting cohort would have a large per- centage of triploids at the D-veliger stage, as whatever wasn't a pentaploid would likely be a triploid (having meiosis 1 completed and being in meiosis II during treatment). This cohort would likely have a lower survivorship however, as the pentaploids would all die. The first experiment (the description of polar body extrusion timing) was the first attempt to find a starting tinie for triploidy induction with reference to the time of fertilization. It was assumed at that time that it was more critical to find the initiation time of induction, as the exact duration of the treatment could be refined in further experiments. It was also decided that both temperatures of 15''C and 20' C would be investigated as they are both common. and easily achievable working temperatures in shellfish hatcheries, and they would give some insight as to a temperature effect in the induction window. The niain objective of this experiment was to determine the time at which 50'/r of fertilized eggs developed polar bodies. Fifty percent polar body formation has been used as a common cue to start triploid induction treatment for shellfish, with treatments ending after various periods depending on the species (Baker 1996. Allen et al. 1989). It is important that most of the eggs are highly synchronous in terms of meiotic events. If most eggs are in the same stage during induction, and it is the effective stage, the result would be a higher percent triploidy. Lu (1986) found that meiotic events of the eggs are more synchronous at higher temperatures (not exceeding the physiologic limilsl. Given this finding, a higher temperature within the physiologic limit would be expected to produce more .synchronous meiotic events. This was supported by the finding that more eggs were in the same meiotic stage on average at a given sampling time in the 20°C sample compared with the 15 °C sample in the first experiment. One observation in related work was that during the prt)duction of both triploid and diploid animals for a simultaneous growth trial there was an unexpected high mortality level during repeated spawns. This initiated a reevaluation of the treatment window, particularly the induction initiation tiniing as detennined to be between 21 and 24 min post-fertilization in the description of polar body extrusion timing. One possible explanation for repeated low survivorship (less than 0.01% to the D-veliger stage) may have been that initiation of treatment to induce triploidy might have been too early. If this were the case, polar body I extrusion would be blocked in most eggs, forming pentaploids. Pentaploids do not survive to the D-veliger stage. However, any eggs that were past this stage would likely have polar body 2 extrusion blocked and thus become a triploid. The observed trend in the related growth trial spawns was in concordance with this phenomenon as the survivors of these spawns.although few were mostly triploids (all over 80% triploidy, with many over 90%). Therefore, the description of meiotic events involved a more in-depth study of the meiotic events in Mytihis editlis at 20°C. As opposed to attempting to treat at the time just after polar body I was extruded, a tinie-related description of the meiotic events was used. This strategy was shown to be successful in Geoduck clam triploid induction (Vadapolas 1999). Desrosiers et al. ( 1993) found that triploid induction treatment using 6-DMAP was effective at the metaphase I stage. In Geoduck clam induction however. Va- dapolas (1999) found that extrusion blocking with 6-DMAP just at the point where 50% of the eggs were in Anaphase 2 and 50% of the eggs were in Telophase 2 produced optimum results. The description of polar body extrusion timing and the descrip- tion of meiotic events showed similar results in terms of polar body 1 extrusion liines. The description of polar body extrusion timing illustrated that most eggs had extruded, or were in the process of extruding polar body 1 by 21-24 min post-fertilization. The description of meiotic events similarly showed that most eggs were undergoing polar body I extrusion, scored as Telophase 1, at a time of 24 min post-fertilization. The fact that both experiments Triploid Induction of Mytilus edulis 507 showed a similar time of polar body 1 extrusion infers that if the induction initialization used in the growth trial spawns was in fact early, causing mortality, it was not because the target of first polar body extrusion was improperly identified. Triploid Induction The effect of 6-DMAP concentration on percent triploid induc- tion demonstrated that 6-DMAP could be used at a number of different concentrations to induce triploidy in Mytilus edulis. Al- though 15°C was shown to be less synchronous (and thus less optimum for triploid induction) in the determination of polar body extrusion times, these results were not yet obtained at the time of the first induction experiment. In all cases the 20"C samples had a higher percentage of triploids than did the 15°C sample (Fig. 2l. This is likely due to the fact that eggs at 20°C are more synchro- nous in terms of their meiotic stage, resulting in a higher percent- age of eggs in the effective treatment stage, and a higher percent of triploids. This is in concordance with the results from the de- scription of polar body extrusion timing that showed a higher degree of meiotic synchrony in eggs at 20°C compared with 15°C. This is also supported in the literature as Lu (19861 found that meiotic events of eggs are more synchronous, and thus should give a higher percent triploid induction, at higher temperatures (not exceeding the physiologic limits). In past studies, treatments with a higher degree of meiotic synchrony allowed for more effective induction treatments as more eggs were in the target stage during treatment, resulting in a higher percent triploid and a higher sur- vivorship (Downing & Allen 1987, Allen et al.. 1989. Gerard et al. 1994). In light of these past studies, and given that the 20°C gave both a higher percent triploid induction and a higher degree of meiotic synchrony, the 20°C treatment is considered more optimal than the 15'C treatment. The evaluation of several treatment windows tested various induction windows (initiation and duration times), which could be compared with the meiotic events described previously. Survivor- ship was low in all cases. This was likely because a relatively high concentration of 6-DMAP (400 |j,mol/L) was used. A decrease in the number of normally developed D-larvae (presumed to not be capable of survival) with an increase in 6-DMAP concentration was reported by Gerard et al. (1994). In the case of the current experiment, the relatively high 6-DMAP concentration was re- quired to ensure that a suitably high percentage of triploids was produced, and was based on the results from the effect of 6-DMAP concentration on percent triploid induction (Fig. 2). Treatments initiated at 24 min post-fertilization produced both the highest percent triploid (83.1 at a duration of 10 min) and the highest survivorship (9.33 at a duration of 5 min). Taylor Resources Inc., a hatchery that commercially produces triploid Mytilus gallopro- vincialis. typically obtains 95-100'7f triploidy with lO-lS'/r sur- vivorship. It is difficult to compare these refined procedures with the results from these preliminary experiments, as much more optimization is required to reach commercial relevance. As these were preliminary experiments, a triploid percentage of greater than 50% with as high a survivorship as possible was the objective. This was based on the caveat that further optimization would improve both the percent triploid induction and survivorship. Past attempts at inducing triploidy in Mytilus edulis have given a wide range of optimal treatments, resulting in a wide range of percent triploid induction and survivorship. Beaumont and Kelly (1989) produced, at best, 25% triploid-producing eggs using heat shock alone (from 15°C to 25°C at 10 min post-fertilization for 10 min). The authors measured the percentage of eggs that were de- veloping, and the percentage of those that were developing nor- mally. They found that, at best, approximately 10% of treated eggs developed. Of those that developed, approximately 60% developed normally and would likely go on to develop as triploid embryos. One treatment had less than 5% development and less than 7% of those showed normal development. However, in the same study, it was found that CB treatinents could produce up to 60% triploid larvae in the optimal treatment group (using Img/L of CB al 5 min post-fertilization for 15 min). Again there was a significant reduc- tion in the percentage that developed, and of those, which devel- oped normally. Approximately 8% of treated eggs developed, and approximately 63% of those developed normally. The current re- sults (Table 2) illustrate some treatments that showed better in- duction performance when compared with the aforementioned study, with up to 83.1% triploid induction (the 24-10 treatment) and up to 9.33% survivorship (the 24-5 treatment) to the D-veliger stage. Yamamoto and Sugawara ( 1988) were able to produce up to 97.4% triploidy by the use of heat shock alone (from 20°C to 32°C at 20 min post-fertilization for 10 min). and found apparently no negative effect upon survival rates. However, these results are considerably higher than those produced by Beaumont and Kelly (1989). As well. Yamamoto and Sugawara (1988) report yields much higher than those found in these experiments that showed results slightly higher than those of Beaumont and Kelly (1989). Yamamoto and Sugawara ( 1988) gave high yields with heat shock alone despite the fact that 6-DMAP is generally accepted as being a more effective triploid inducer than temperature alone (Desro- siers et al. 1993). Yamamoto and Sugawara (1988) also found 2-22% triploids in their controls that they attribute to handling stress. No other known triploid shellfish work has shown triploids in their controls. In the Chilean blue mussel. Mytilus chilensis. Toro and Sastre (1995) found an optimum induction treatment using heat alone (from 18°C to 32°C at 10 min post-fertilization for 10 min) to induce 51%^ triploid with 29%: survival to an age of 15 days. The optimum percent triploid induction in their study was lower than in these experiments, the survival rate was much higher however. Scarpa et al. (1994) compared six methods to induce triploidy directly (not including 6-DMAP) and found that cytocha- lasin B (CB) was the most effective at inducing triploidy in Mytilus gatloprovinciidis. obtaining 86%^ triploidy to the D-veliger stage. These authors also found that, averaged between their two trials. 71.6%r survived the CB treatment. These reported results are higher than the results of this study, however, as mentioned pre- viously, we excluded CB from our trials due its carcinogenic ef- fects. Although percent triploid induction in this study is comparably high (over 50%f ) in some treatments when compared with many shellfish studied (Beaumont & Fairbrother. 1991 ), survivorship is lower than expected. Future optimization of the triploid induction method should focus on increasing survivorship while not com- promising percent triploid induction. Improved hatchery tech- niques and husbandry may also increase survivorship, but cannot help to increase the percent triploidy. When considering a com- mercial technique, a high percentage of triploids in a cohort are not the only consideration in an induction technique. It has to be balanced with finding a method that produces a high survivorship of the triploids to remain economically feasible. One further noteworthy result is the incidental production of tetraploid mussel larvae during some of the treatments in the 508 Brake et al. evaluation of several treatment windows (Table 2). Six different treatment combinations produced some measurable number of tet- raploids. There may be commercial significance in these findings. Triploids can be produced at a rate of lOO'^r with a high survivor- ship, and without using chemicals by mating a tetraploid to a diploid (Guo et al. 1996). Therefore, if tetraploids could success- fully be produced and reared, they might become commercially important animals in the production of triploid mussels. Scarpa et al. ( 199.^) found that tetraploidy could be induced in MytUiis gal- loprovincUdis by suppression of both polar body 1 and 2. This may have been the mechanism for the production of the tetraploids found in these results. However, when these tetraploid-inducing treatments are compared with Figure 3. a pattern that suggests another mechanism is evident. All tetraploid producing treatments but the 24-15 treatment overlap cell cleavage. Theoretically, if an inhibition treatment overlaps cell cleavage, the chromosomes could divide into two sets but the cell would not cleave. The result will be one cell with double the diploid number of chromosomes, and subsequent mitotic divisions will result in tetraploid larvae (Beaumont & Fairbrother 1991 ). This has been shown as a viable method of tetraploid induction. Guo et al. (1994) attempted to produce tetraploids by inhibiting mitotic cell cleavage with heat shocks, producing up to 45% in one case, however no tetraploids in their study survived past the D-stage of development. Future work on triploid production of Mytilus edidis should focus on the production of tetraploid broodstock used to produce 1007f triploid larvae without the use of chemicals. Many commercial triploid Pacific oysters are now produced using this method (Chew 2000). It has been demonstrated that using the puromycin analogue 6-DMAP at various concentrations can produce triploid mussels. At the conclusion of this research the optimum investigated pro- cedure for induction of triploidy in Mytilus edulis was to treat eggs at 20°C with 400 (jimol/L of 6-DMAP starting at 24 min post- fertilization for a treatment time of 10 min. This treatment yielded 83.1% triploids in induced samples with a survivorship of 1.39% to the D-veliger stage. The current investigated methods for pro- ducing triploid Mytilus edulis are not yet optimized to commercial levels. These findings are a basis for further work in the optimi- zation of this technique. ACKNOWLEDGMENTS The authors thank Garth Arsenault for technical support throughout this study. We also wish to thank Benoit Eudeline for assistance with How cytometry throughout the project. Funding for this project was provided by a grant from the National Research Council (NRC) through its Industrial Research Assistance Pro- gram (IRAP). the PEI Aquaculture and Fisheries Research Initia- tive, the Prince Edward Island Aquaculture Alliance (PEIAA). and the PEI Department of Fisheries. Aquaculture and Environment. Allen. S. K. 1988. Triploid oysters ensure year-round supply 31:58-63. Allen, S. K., S. L. Downmg. J. Chailoti & J. H. Beattie. 1986. Chemically and pressure-induced triploidy in the Pacific oyster Cicissostrea gigas. Aquaculture 57:359-379. Allen. S. K., S. L. Downing & K. K. Chew. 1989. Hatchery Manual for Producing Triploid Oysters. Seattle: University of Washington Press, 6 pp. Baker, B. 1996, Building a better oyster. Bioscience 46:240-244. Beaumont. A. R. & K. S. Kelly. 1989. Production and growth of triploid MmiIus cilulis larvae. ,/. Exp. Mar. Biol. Ecol. 132:69-84. Beaumont. A. R. & J. E. Fairbrother. 1991. Ploidy manipulation in mol- luscan shellfish: a review. / Shellfish Res. 10:1-18. Chew, K. K. 2000. Update on Triploid Pacific Oysters. Aquaculture 26: 87-89. Desrosiers. R. R.. A. Gerard. J. Peignon, Y. Naciri. L. Defresne. J. Mo- rasse, C Ledu, P. Phelipot. P. Guerrier & P. Dube. 1993. A novel method to produce triploids in bivalve molluscs by the use of 6-Dim- ethylaminopurine. J. E.\p. Mar. Biol. Ecol. 170:29^3. Downing. S. L. & S. K. Allen. 1987. Induced triploidy in the Pacific oyster, Crassostreu giga.s: optimal treatments with cytochalasin B depend on temperature. Aquaculture 61:1-15. Gerard, A.. Y. Naciri. J.-M. Peignon. C. Ledu & P. Phelipot. 1994. Opti- mization of triploid induction by the use of 6-DMAP for the oyster Crassostrea gigas (Thunberg). Aquae. Fish. Manage. 25:709-719. LITERATURE CITED Ocearuis Guo. X. 1991. Studies on tetraploid induction in the Pacific oyster, Cras- sostrea gigas (Thunberg). Ph.D Thesis. Seattle: University of Wash- ington. 167 pp. Guo. X., W. K. Hershberger, K. Cooper & K. K. Chew. 1994. Tetraploid induction with meiosis 1 inhibition and cell fusion in the Pacific oyster iCras.w.ftrea gigas. Thunberg). J. Shellfish Res. 13:193-198. Guo. X.. G. A. DeBrosse & S. K. Allen. 1996. All-triploid Pacific oysters (.Crassostrea gigas Thunberg) produced by mating tetraploids and dip- loids. Aquaculture 142:149-161. Lu, J.-K. 1986. The Effects of Temperature and Salinity on Meiosis, Fer- tilization and First Mitosis of The Pacific Oyster (Crassostrea gigas) eggs. Masters Thesis. Seattle: University of Washington. 122 pp. Scarpa, J., K. T. Wada & A. Komaru. 1993. Induction of tetraploidy in mussels by suppression ot polar hody formation. Nippon Suisan Gak- kai.ihi 59:20n-202i. Scarpa. J.. J. E. Toro & K. T. Wada. 1994. Direct comparison of six methods to induce triploidy in bivalves. Aquaculture 1 19:1 19-133. Toro, J. E. & H. D. Sastre. 1995. Induced triploidy in the Chilean blue mussel, Mytilus chilensis (Hupe, 1854) and performance of triploid larvae. J. Shellfish Res. 14:161-164. Vadopalas, B. 1999. Development and optimization of triploid induction techniques in the geoduck clam, Panopea ahrupia. Masters Thesis. Seattle: University of Washington. 81 pp. Yamamoto, S. & Y. Sugawara. 1988. Induced triploidy ui the mussel, Mytilus edulis. by temperature shock. Aqucuultioe 72:21-29. Jotimal ol Shellfish Rf.saurh. Vol. 21, No. 2. 5(W-.'^I7. 2U02. MUSSEL CULTURE AND COCKLE FISHERIES IN THE NETHERLANDS: FINDING A BALANCE BETWEEN ECONOMY AND ECOLOGY PAULINE KAMERMANS* AND AAD C. SMAAL, Netherhmds Institute for Fisheries Research. Centre for Shellfish Research. P.O. Box 77. 4400 AB Yerseke. The Netherlands ABSTR.ACT 111 the Netherlands, wild stocks of mussel seed are fished and mussels are cultured on bottom plots. In addition, wild stocks of the edible cockle are dredged for harvest. Two of the areas where these activities are carried out are nature reserves. In 1993. the government implemented a policy in these reserves to ensure the conservation, protection and development of natural values and processes in which human activities should fit in. Fishing for shellfish is considered a traditional activity in these waters. Therefore, it is allowed, but under the restriction that no negative effects are caused. As a result of this policy, fishing for mussel seed and cockles is not allowed in areas with a high potential for the development of mussel beds and seagrass fields. A number of bird species are dependent on shellfish for their food requirements. Therefore, the policy makes use of a reservation system. This means that, in years when mussel and cockle stocks are low, an amount is reserved for the birds and cannot be fished. The government and shellfish industry agreed on co-management, (i.e., the fishermen are responsible for implementing the measures). This task is carried out by Producers' Oroanizations. An overview of the viewpoints of the interest groups and the role of policy makers and scientists is given. KEY WORDS: Mvtihi.s echilis. Ceruslodcnmi edule. co-management, culture, fisheries, government regulations INTRODUCTION Shellfish are an important component of the ecosystem as they filter the water and are a food source for organisms such as birds and humans (Dame 1996). The protection and restoration of shell- fish beds can contribute both to preservation of estuarine and near coastal ecosystems and to sustainable economic development of the coastal zone. In the Netherlands, six shellfish species are com- mercially e.xploiled (Smaal & Lucas 2000). Different species are harvested in different areas (Fig. 1). In the coastal zone fishing is directed at wild stocks of the trough clam Spisula siibtnmcaia. and the American razor clam Ensis directus. In the Oosterschelde es- tuary and the saline lake Grevelingen the European oyster Ostrea ediilis and the Pacific oyster Cnr^sostreci !>igii.s are cultured. Fish- ing for seed of the blue mussel Myliliis edulis takes place in the Wadden Sea and on rare occasions in the Oosterschelde estuary. The mussels are cultured on bottom plots located in the Wadden Sea in the North and the Oosterschelde in the South. Fishing on wild stocks of the edible cockle Cerastudenna ediile takes place in the Voordelta, Western Scheldt, Oosterschelde and Wadden Sea. This article describes an attempt to combine the exploitation of shellfish stocks and the protection of estuarine habitats in the Neth- erlands. To reach such a goal agreement among stakeholders is essential. A description of the mussel culture and fishing for mus- sel seed and cockles is presented. Regulations concerning the man- agement of shellfish stocks and other habitats are summed up. Furthermore, an overview of the viewpoints of the interest groups and the role of policy makers and scientists is given. METHODS Mussel Culture in the Netherlands In the Netherlands, the culture of the blue mussel Mytilus edulis depends on seed that is fished twice a year in the most western part of the Wadden Sea (see Fig. 1). Seed mussels are approximately 10- ."^0 mm in length. These mussels are transported to the bottom ♦Corresponding author. Tel.: -1-31-113-672302: Fax: -1-31-113-573477: E-mail: p.kamermansCs'rivo.wag-ur.nl culture plots, which are leased from the government. The plots are located in the Wadden Sea and the Oosterschelde (see Fig. 1 ). The mussels are left to grow to market size (>45 mm). Depending on the environmental conditions this size is reached in 1-3 y (Dijkema 1997). The mussel seed catches show large annual fiuc- tuations that reflect the variability in spat fall (Fig. 2a). Experience of the mussel growers shows that a catch of 65 x 10" kg seed mussels is needed to sustain a yearly production of 100 x 10'' kg mussels (see Fig. 2b). This relatively low conversion is caused by heavy mortality as a result of predation by birds and starfish, and losses due to storms. The mussel seed target is not always reached. In fact, during the last ten years the target was reached only once. Thus, mussels are imported from other areas such as Germany (Dijkema 1997). The Netherlands is the fourth largest blue mussel producer in the world, after China, Spain, and Denmark (FAO 20G0a, FAO 2000b). In 1999 the total landing value of mussels was Dfl 125 million = 55 million USD (Productschap Vis 2000). Almost 40'7f of the mussels are exported to Belgium. Cockle Fisheries in the Netherlands Dredging for cockles takes place in the Wadden Sea, the Voordelta, the Oosterschelde and Western Scheldt (see Fig. I). Wild stocks are fished mechanically with suction dredges. In ad- dition, a small group of fishermen makes use of rakes with nets that are deployed by hand. The majority of the cockles is not sold in their shell, but cooked and canned for transportation to Spain. Therefore, landings are expressed in meat instead of fresh weight (Fig. 3). The catches show considerable variability again caused by fluctuations in spat fall. The main harvest area is the Wadden Sea. In 1998 a large cockle stock was available, and the total value of the catch was Dfl 60 million = 27 million USD (Steins 1999). The Netherlands is the number one cockle producer in the world (FAO 2000a). Producers ' Organizations All groups involved in the mussel and cockle industry (i.e., fishermen, vessel owners and shellfish sellers) are organized in the Mussel Producers' Organization (PO Mussels) and the Cockle Pro- ducers' Organization (PO Cockles). The POs depend on voluntary 509 510 Kamermans and Smaal Figure 1. Shelirish fishing and culture areas in The Netherlands. membership. POs offer attractive facilities to its members and they have legal powers to enforce binding agreements. The POs for- mulate Shellfish Fisheries Management Plans {e.g.. Productschap Vis 2(X)0). These include yearly fish plans, which are based on results of yearly surveys that determine the amount and distribu- tion of mussel seed and cockles. In some cases (e.g.. when mussel seed is present on a sheltered location), seed is not fished in fall, but left for next spring. The Role of the Netherlands Institute for Fisheries Research Every March, since 1992. the Netherlands Institute for Fisher- ies Research (RIVO) carries out a subtidal mussel seed survey in the western part of the Wadden .Sea. This survey is conducted by order of the PO Mussels. The sampling set-up is stratified (i.e.. the sampling grid is intensified at locations where subtidal mussel beds are present). These locations are known from the previous year and qualitative assessments carried out in early spring by fishery inspectors of the Ministry of Agriculture, Nature Manage- ment and Fisheries. At every sampling point a 20-cm-wide cockle dredge is used for 150 m. The mesh size is 5 mm. For profitable seed fishery a biomass of 0.1 kg mussels per m~ is necessary (Fig. 4). The RIVO akso monitors the distribution of seedbeds and the development of mature mussel beds in the intertidal with aerial photography surveys in combination with ground truthing. Each May. since 1990. RIVO carries out a basin-wide cockle survey in the Wadden Sea. the Oosterschelde and the Western Scheldt. The survey is under contract by the Ministry of Agricul- ture. Nature Management and Fisheries and the PO Cockles. The sampling grid is stratified with a denser grid at cockle beds (Fig. 5). The location of these beds is derived from a qualitative survey that the fishemien carry out just prior to the annual RIVO survey. From the survey data the total cockle stock is determined. Dutch Regulations The Wadden Sea and the Oosterschelde do not only function as areas where shellfish fishing and culture takes place, but al.so as nature reserves. The birds, wetlands and other habitats in these areas are protected under several national and international laws such as the Ramsar Convention, the European Directive on the Conservation of Wild Birds, and the UNESCO Man and Biosphere Program. The areas used to have extensive seagrass meadows and intertidal mussel beds. Renewed development of these habi- tats is desirable. For migrating birds, the Wadden Sea and the Oosterschelde are important wintering areas or stopover sites for refueling on the flyway between the North Pole and Africa. In addition, a number of resident species, such as oystercatchers and eider ducks, are present (Rosner et al. 1993). In these protected areas human activities are possible only when they do not cause negative effects. In the early 199()s, low stocks of both cockles and mussels were present in the intertidal Mussel Culture and Cockle Fisheries in the Netherlands 1UU - 80- -1 1 — 1 target o) 60 - 1= r 40- 20 - 0- u L_JjXI^ D spring . ■ autumn 1 91/92 92/93 93/94 94/95 95/96 96/97 97/98 98/99 99/00 season ■ Import D Wadden Sea ■ Oosterschelde 180 j 160 140 120 100 80 60 40 20 0 Seasons Figure 2. Mussel seed catches and mussel landings in the Netherlands in million kg fresh weight. Targets are indicated with dotted lines. Wadden Sea (Beukema 1993). This was a result of a number of factors including reduced natural spat fall, storms and unrestricted fishing activities (Dankers 1993). At the same time, high mortality of a shellfish eating bird species was ob.served (Smit et al. 1998). Thus, the possibility of a link between bird mortality and shellfish fisheries appeared. The public awareness and social commotion caused by these events triggered the formulation of regulations to better protect the area. In 1993. the government implemented a policy for the period 1993 to 2003 to ensure the conservation, protection and develop- ment of natural values and processes in the Wadden Sea and Oost- erschelde estuary (LNV. 1993). Human activities must fit into this policy. Targets are restoring bird populations at levels of the 1980s, and promoting development of seagrass beds and 2.000- 4.000 ha of stable intertidal musselbeds (LNV 1999). Fishing for shellfish is considered a traditional activity in these waters. There- fore, it is allowed, but under the restriction that no negative effects are caused. The government makes use of co-management (i.e.. the fishermen are responsible for implementing the measures). The PO Cockles and the PO Mussels carry out this task. Consumption- sized cockles and mussels are also the preferred prey of oyster- catchers and eider ducks (Zwarts et al. 1996. Nehls & Ruth 1994). Therefore, the policy makes use of a reservation system in the shellfish fisheries. In years when mussel and cockle stocks are low. the aim of the policy is to ensure that 60% of the food requirement of birds is reserved. However, the calculations underlying the policy have been questioned by (Ens 2000). Figure 6 shows large annual fluctuations in cockle stock as a result of variability in spat fall. Since the implementation of the food reservation policy in 1993. fishing for cockles was not allowed in the Wadden Sea in 1996 and restricted to the subtidal areas in 1997. In the Ooster- schelde fishing was not allowed in 1997. 1998, 1999. and 2000. Another regulation is that fishing for mussel seed and cockles is not allowed in areas with a high potential for the development of mussel beds and seagrass fields. In 1993. 14% of the intertidal flats in the Oosterschelde and 26% of the intertidal Wadden Sea were permanently closed to shellfishing. In 1999. an additional 5% of intertidal flats in the Wadden Sea were closed (Fig. 7). The loca- tion of these areas is based on habitat maps for seagrass meadows and mussel beds. The maps are produced with CIS models that include parameters such as exposure time, wave action, sediment characteristics (Brinkman et al. 2002. de Jonge & de Jong 1999). To monitor that shellfish fishing does not take place in the closed areas all vessels are equipped with a black box. This system reg- isters the ship's position at 1-min intervals when the ships are fishing. The data are retrieved from the boxes by an independent agency and the POs give penalties to offenders. In addition to the government measures, the Shellfish Fisheries Management Plan of 512 Kamermans and Smaal 16 14 12 ^ Voordelta D Westerschelde ■ Oosterschelde D Wadden Sea I I I g •^10 O 0) b 4 2 0 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 00 01 Years Figure i. Cockle landings in the Netherlands in niilliun kg meat. the POs for the period 1993 to 2003 includes a reduction in the Evaluation Projects number of cockle vessels, a more spread out distribution of the cockle fleet, restricted fishing periods for both cockles and mussel In 1998. the effectiveness of the governmental measures was seed, and yearly fishing plans on amounts fished and fish locations evaluated for the period 1993 to 1998 in the so-called EVA I based on survey data (Produclschap Vis. 2000). project (LNV 1998. Smit et al. 1998). Due to a series of cold mussel biomass in g/m^ . -100 . 100-500 • 500 - 1000 • 1000 - 2000 • 2000 - 3500 no mussel I mussel culture plots 10 km 6^ Figure 4. Subtidal mussel seed biomass in the western Dutch Wadden Sea. Results of RIVO sur\e) in 2000. Mussel Culture and Cockle Fisheries in the Netherlands 513 cockle density in n/m^ • 0 . 1-500 • 500-1000 • 1000 - 5000 20 km Figure 5. Cockle den.sit> in the Dutch Wadden Sea. Results of RIVO cockle survey in 2000. winters with liigh shellfish mortality in this period, clear conclu- sions on effects of shellfisheries could not be drawn from the available dataset. The rate at which intertidal mussel beds devel- oped was lower than expected. Intertidal mussel spat fall occurred in 1994 only (van Stralen & Kesteloo-Hendrikse 1998). and the intertidal mussel area in the Wadden Sea is approximately 1000 ha in 2000 (Kater & den Os 2001 ) Therefore, an additional 59^ of the intertidal flats was closed based on the newly developed mussel habitat map (Brinkman et al. 2002). In 2000, the second phase of the evaluation started. In this EVA II project (Ens et al. 2000) the effects of shellfish harvests on nature in the Wadden Sea and Oosterschelde is e\alualed for the entire 10-year period from 1993 to 2003 (LNV & VW 1999). The project is based on the following policy questions (Ens et al. 2000). • What are allowable effects of shellfish harvests on mussel beds, seagrass meadows and shellfish consuming birds? • Have the measures taken so far had the expected effect? • Do we comply with the international regulations on bird pro- tection? • When effects are negative, what can we do to reduce these effects? These policy questions were translated into a number of research sub-projects. Effects of mussel fishery and culture on bird populations are studied. The effects of cockle dredging on cockle stocks; stable intertidal mussel beds, other zoobenthos, seagrass meadows, and sediment composition are studied by comparing their development in open (fished) and closed (unfished) ureas. In addition, availability of black box data gives the opportunity to further specify the fishing intensity within the open areas. Linking survey information with black box data and catch data gives in- formation on the relationship between density of shellfish, fishing intensity and yields. Another subproject checks the assumptions on which the calculation of the amount of food reserved for birds is based. What is the profitable shellfish density for birds'^ Does it matter where the food is located (both in the tidal zone and in the horizontal plane)? Which fraction of the total stock is usable by birds? And finally, the slow recovery of both seagrass meadows and mussel beds warrants refinement of the habitat maps. In this way a better selection of closed areas is possible. The results of the evaluation project will play a role in the final decision on the continuation of shellfish harvesting, which the government will take in 2003. Finding a Balance Between Economy and Ecology Cockle fishermen and mussel farmers make a living with their activities and represent the commercial economy, while nature conservationists protect birds and habitats, such as seagrass beds and mussel beds, and are concerned with ecology. Both groups agree that the Wadden Sea and Oosterschelde should not be com- pletely closed for human activities (e.g., recreation and fishing) provided that these activities do not have negative impacts. The discussions center on these impacts. Despite the policy to reserve food for shellfish eating birds, a further decline in oystercatcher population is taking place (Fig. 8). In addition, the development of intertidal mussel beds and seagrass meadows is slow. These ob- servations cause tension between the different stakeholders. The different forms of shellfish fishing and farming show different degrees of opposition between the stakeholders. Traditional fishing for cockles by hand does not cause much tension. Fishing for mussel seed in the subtidal area is starting to become an issue, as these stocks also provide food for eider ducks. The main disagree- ments are on fishing for intertidal mussel seed and mechanical cockle fishing (Piersma et al. 2001. Smaal et al. 2001. Camphuy- sen el al. 2002). 514 Kamermans and Smaal 250 200 150 <1> E c o E 100 50 0 a I ByBH food reservation 25 sublit, from 1990 riRczica^EjRc: <— og o ^ UD ti> r^ o:- -x- -i^ -— oj o f IT- -j^ r-- ■» -T^ o '- oj •■•'• ^ u"' •^' r-- o:- ix- -^ r^ f- 1-- r-- r-- r-- r-- r-- r^ o:' o:- CO o:- 05 CO CO CO co co 'n> cr- co ov m tx- ov 'X- -r^ x» o CT)CT>CT>oo^o>o>o:'a:'"X>'7>oo^cr>0"^'7^<7^<7>cr>oo:>x^coocn'7:'Cri'T>a:>o Figure 6. Cockle stock in the Dutch Wadden Sea as determined by extrapolation from various sampling campaigns in the VVaddcn Sea and the basin wide RIVO surveys from 1990 onwards. It is assumed that both birds and fishermen need a cockle density of more than 50 per m* for profitable use of the stock (grey pari of the bars). Part of the grey bar is black, which indicates the catch. From 1993 onwards 12.6 million kg meat is reserved as food for birds. This is indicated with a horizontal line. The cockle and mussel seed fishermen see an increase in re- strictions, without the expected restoration of the ecosystem. As they have already put considerable effort in a changed manage- ment (e.g., the use of fishing plans and black box systems), they are reluctant to agree to more. Shellfishermen point out that, al- though bird populations are smaller than in the eighties, they have increased since the seventies. Nature conservation groups see the continued decline of bird populations as a sign to stress the pre- cautionary principle. The definition of the precautionary principle is under debate. The con.servation groups define it as: when un- certainty on the effects of a human activity exists, do not allow it. The European Commission have extended this with a balancing of interests: measures taken on the basis of the precautionary prin- ciple need to be in relation to the chosen protection level and need to be based on research on possible costs and benefits of carrying out that measure (COM 2000). Both fishermen and environmen- talists question the calculated aniounts of food needed by the birds. Fishermen ague that the lower level of bird populations in the seventies did not coincide with lower shellfish stocks. Nature groups and ornithologists (Ens 20(K), Camphuysen 1996, Cam- phuysen et al. 2002) point out thai not the total shellfish stock, but the stock that is available for birds is of importance. This may lead to higher food reservation values. Furthermore, there is debate about the closure of intertidal areas to proniote development of mussel beds. Fishermen have the impression that trtoderate fishing improves the sediment and enhances survival of niussel seedbeds, while environmentalists are of the opinion that natural processes should not be interfered with. The images that both groups have of each other strongly influence the discussions that are part of the decision-making process (Steins 1999). Thus, securing ongoing communication between interests groups is of great importance. Identification of common interests should play a key role. In addition to the tension between fishernien and conservation- ists, there is tension between science and politics. The government wants to keep the shellfish industry and protect nature at the same time. To do this properly they want to make decisions on objective scientific grounds. The scientists are unable to reach consensus on scientific grounds for the presence or absence of a link between shellfishery activities and the recent ecosystem developments (de- cline in bird populations, and slow recovery of intertidal mussel beds and seagrass meadows). Scientists generally work on a micro scale and can be fairly certain about their conclusions, while poli- ticians need answers on a macro scale. The advice of the scientists needs to be objective and exact. However, when findings from Mussel Culture and Cockle Fisheries in the Netherlands 515 Figure 7. Location of areas that are closed for shelirish fisheries in the Dutch VVadden Sea. 250000 200000 ' 150000 100000 50000 1984 1986 1988 1990 1992 1994 1996 1998 2000 1974 1976 1978 1980 1982 Figure 8. Development of total oyster catcher population in the Dutch Wadden Sea from 1975 to 1999. Counted numbers in fall (open squares) and following winter (closed squares) are indicated. Running mean (black line) is calculated from these counts. Counts in severe winters (closed squares with arrows) are not included in mean (Ens 2002). 516 Kamermans and Smaal small scales are upgraded to larger scales the results will always have a certain degree of uncertainty. To avoid scientific conflicts. and remain credible to society, it would be better to present results in the form of ranges instead of single values (Hauge 2000). How- ever, this causes problems for the managers, as they need single values when drawing up permits. Thus, alternative ways to deal with scientific uncertainties concerning natural resources are needed. CONCLUSIONS The balance between economy and ecology is very delicate. In fact, balancing between economy and ecology is a more appropri- ate way to describe the current situation. Co-management has not brought the solutions the government hoped for. At present, two options dominate the discussion on the future of shellfisheries in the Wadden Sea and Oosterschelde. These are: • A continuation of the fisheries as before, which seems unac- ceptable to the nature conservation groups. • A buy out of the mechanical cockle fishery, which seems un- acceptable to the fishermen. The shellfish-fishermen, -farmers, and -sellers have set up a foundation called ODUS (Development Sustainable Shellfish- eries). This foundation proposes the following methods for cockle fishing and mussel farming that aim for effects that are within the restoration capacity of the system and could be more sustainable (ODUS 2001). • Selecting optimal areas for harvest may enlarge the efficiency of cockle dredges. Newly developed fishing gear may reduce the effects of the dredges on the bottom. • Management of closed areas can be dynamic instead of static. The location of closed areas could be based on annual variations according to the location of the cockle stock • Relaying of cockles may reduce mortality and enhance growth. This can be the beginning of a development from fishery to culture. • 1-ishing of unstable mussel seed beds will use seed before it is lost in storms. • Methods should be developed to avoid large seed losses on culture plots. This will enhance the yield and thus reduce the amount of mussel seed needed. • Optimization of the mussel stock on culture plots to the exploi- tation capacity of the system w ill enhance the yield. In our opinion, these promising options should receive more scientific attention. Conservation of the natural system is a com- mon interest of all stakeholders. Degradation of the system will lead to reduced biodiversity and lower resilience. Both birds and fishermen need a well functioning ecosystem that guarantees the continued presence of shellfish stocks. ACKNOWLEDGMENTS We thank Bruno Ens and Mariette Kip for critically reading an earlier version of the manuscript. LITERATURE CITED Beukema, J. J. 1993. Increa.sed mortality in alternative bivalve prey during a period when the tidal flats of the Dutch Wadden Sea were devoid of mussels. Neth. J. Sea Res. 31:39.S-406. Brinkman, A. G.. N. Dankers & M. van Stralen. 2002. An analysis of mussel bed habitats in the Dutch Wadden Sea. Heliioluiul Marine Re- search ib:59-15. Camphuysen. C. J., C. M. Berrevoels, H. J. W. M. Cremers, A. Dekinga. R. Dekker, B. J. Ens, T. M. van der Have, R. K. H. Kats, T. Kuiken. M. F. Leopold, J. van der Meer & T. Piersma. 2002. Mass mortality of common eiders (Somateria mollissima) in the Dutch Wadden Sea. win- ter 1999/2000: starvation in a commercially e.\ploited wetland of in- ternational importance. Biological Consen-ation 106:303-317. Camphuysen. K. 1996. Ecoprofiel eidereend. RIKZ 96.146x, 1-125. Den Haag. RIKZ. COM. 2000. Mededelmg van de Europese Commissie over het voorzorgsbeginsel d.d. 2 februari 2000, 1 p. Dame, R. F. 1996. Ecology of marine bivalves. An ecosystem approach. Boca Raton: CRC Press. 25A pp. Dankers, N. 1993. Integrated estuarine management — obtaining a sustain- able yield of bivalve resources while maintaining environmental qual- ity. In: R.F. Dame, editor. Bivalve filter feeders in estuarine and coastal ecosystem processes. Berlin: Springer. 479-51 1. de Jonge. V. N. & D. J. de Jong. 1999. Zeegras in de Nederlandse Wad- den/ee. werkdocument RIKZ / OS-99.808x. Dijkema, R. 1997. Molluscan fisheries and culture in The Netherlands, NOAA Technical report NMFS 129:1 15-136. Ens, B. J. 2000. Berekeningsmethodiek voedselreserverinj; Wadden/ee. Allerru-rapport 136:1-70. Ens. B. J.. R. Earners & A. C. Smaal. 2000. Onderzoeksplan EVA II. 1-67. Den Burg/ Den Haag/ Yerseke. RIKZ. Ens. B. J. 2002. What we know and what we should know about mollusc fisheries and aquacultures in the Wadden Sea. In: W. J. Wolff, K. Essink, A. Kellerman & M. A. van Leeuwe, editors. Proceedings of the lOth International Scientific Wadden Sea Symposiiini. Den Haag: Min- isterie van LNV. FAO. 2000a. Yearbook of fishery statistics, capture production 1998. Rome. Vol 86/1 FAO. 2000b. Yearbook of fishery statistics, aqaacultiirc production 1998. Rome. Vol 86/2. Hauge, K. H. 2000. Fisheries scientists' struggle for objectivity. ICES CM 2000/W:06. Kater. B. & D. den Os. 2001, Hel mosselbestand op de dioogvallende platen ui de Wadden/ee in hel voorjaar \an 2000. RIVO Rapport C0b9/01. LNV. 1993. Structuumota Zee- en Kustvisserij. Vissen naar evenwicht. Regeringsbeslissing. 109 pp. LNV. 1998. Structuumota Zee- en Kustvisserij Evaluatie Kustvisserij- beleid 1993-1997. Den Haag. Groningen. Ministerie van LNV. LNV. 1999. Beleidsbesluit schelpdiervisserij kustwateren 1999-2003. 14 pp. LNV & VW. 1999. Projectplan EVA II. I -10. Ministerie van Landbouw. Natuurbeheer en Visserij - Directie Visserij - Ministerie van Verkeer en Waterstaat - Directoraat-Generaal Rijkswaterstaat Hoofddirectie Wa- ter, Nehls. G. & M. Ruth. 1994. Eiders, mussels, and fisheries in the Wadden Sea - continuous conflicts or relaxed relations'.' Ophelia Siippl. 6:263- 278. ODUS. 2001, Sustainable shellfish industry, a vision into sustainable de- velopment of the Dutch shellfish industry suminaii/ed. Yerseke: Stich- ting ODUS. 8 pp. Picrsma. T., ,^. Koolhaas. A. Dekinga, J. J. Beukema. R. Dekker & K. Essink. 2001. Long-term indirect effects of mechanical cockle- dredging on intertidal bivalve stocks in the Wadden Sea. J. Appl. Ecol. 38:976-990. Productschap Vis. 2000. Beheersplan Schelpdiervisserij Kustwateren 1999-2003. 28 pp. Rosner, H. U., J. Frikke. H. Meltotte. M. van Rooinen. C. J. Smit & P. Sudbeck. 1993. The joint monitoring project for migratory birds in the wadden sea; report to the Trilateral Cooperation on the Protection of Mussel Culture and Cockle Fisheries in the Netherlands 517 the Wadden Sea. Wilhelmshaven; Common Wuddeii Sea Secretariat 1-16. Smaal, A. C. & L. Lucas. 2000. Regulation and monitoring of marine aquaculture in The Netherlands. / Appl. Ichtyol. 16:187-191. Smaal. A.. J. Craeymeersch. P. Kamermans & M. van Stralen. 2001. Is food shortage the cause of Eider duck mortality'.' Shellfish and crab abundance in the Dutch Wadden Sea ( 1 99+- 1 999). Wadden Sea News- letter 2001 No. 1:33-38. Smit. C. J.. N. Dankers. B. J. Ens & A. Meyboom. 1998. Birds, mussels, cockles and shellfish fishery in the Dutch Wadden Sea: how to deal with low food stocks for eiders and oystercatchers. Senckenbergia Mar- itinici 29:141-153. Steins. N. 1999. All hands on deck — an interactive perspective on complex common-pool resource management based on case studies in the coastal waters of the Isle of Wight (UK), Connemara (Ireland) and the Dutch Wadden Sea. Thesis, Wageningen Universiteit, Wageningen 225 pp. van Stralen, M. R. & J. J. Kesteloo-Hendrikse, 1998. De ontwikkeling van het kokkelbestand in de Waddenzee ( 1971-1997) en de Oosterschelde (1980-1997). RIVO rapport COO.5/98. Zwarls, L., J. T. Cayford. J. B. Hulscher, M. Kersten. P. M. Meire & P. Triplet. 1996. Prey size selection and intake rate. In: J. D. Goss- Custard, editor. Oystercatcher: from individuals to populations. Oxford University Press, Oxford, pp. 30-55, Journal of Shellfish Ri".ccinh. Vol. 21. No. 2. 519-527, 2002. SETTLEMENT OF POMATOCEROS TRIQUETER (L.) IN TWO SCOTTISH LOCHS, AND FACTORS DETERMINING ITS ABUNDANCE ON MUSSELS GROWN IN SUSPENDED CULTURE DIRK A. CAMPBELL AND MAEVE S. KELLY* Scottish Association for Marine Science. Oban. Argyll. PA37 IQA. Scotland. United Kmi^dom ABSTRACT In Scotland, some 2.000 tons of mussels {Mylilus ediilis ) are produced each year from suspended rope cultures. Mussels can act as a settlement substrate for tubeworm, Pomatoceros spp. and badly fouled mussels are devalued and may be discarded. The estimated cost to the Scottish rope-grown mussel industry is between £300,000 and £500,000 per annum. Established growers believe the problem is worsening. Collector plates were deployed at two sites in two lochs on the West Coast of Scotland to monitor Poimitoceros spp. settlement. In conjunction, in sint trials at a mussel farm site assessed tubeworm settlement on rope-grown mussels. The tubeworm P. irujiieter was found to be the prevalent species settling on the collector plates and on the mussels. The timing of peak tubeworm settlement differed between lochs hut was synchronous between sites and different depths within the same loch. Peak settlement in both lochs occurred after the highest seawater temperatures were recorded. Settlement intensity differed between lochs, sites, and depths, indicating the scale of variation in settlement withm lochs. Mussel shell size was distinguished as a significant factor influencing Pomatoceros spp. settlement as higher numbers of tubeworm settled on larger mussels. In large mussels (mean shell length 60.8 mm ± 0.70 SE) tubeworm settlement was greatest in treatments where conspecific adults were already present and higher levels of settlement were found in mussels stocked at lower densities. In small mussels (mean shell length 33.5 mm ± 1.03 SE) the initial stocking density and tubing system had no effect on tubeworm settlement. Differences in the fouling intensity between mussel stocks were attnbuted to variation m the abundance of tubeworm larvae in the water column and the size of the mussels at the time of retubing. A loot"* mortality could be expected in adult P. uiqiieter after 24.1 h and 35.4 h when exposed to air at 7°C and I3°C, respectively. In areas where tubeworm is a persistent problem consistent annual monitoring of the Pomaloceros spp. settlement is recommended as part of a management strategy to avoid heavy fouling on mussel stock. Grow-out strategies to alleviate tubeworm fouling on rope-grown mussels are discussed. KEY WORDS: Ponmtoieros. Mxlilic ciliili.s. settlement, tubeworm fouling, mussel culli\ation INTRODUCTION Calcareous tube-dwelling polychaeles from the genus Pimiato- ceros have long been considered to be primary fouling organisms (Ciisp 1965. OECD 1967). In Scottish waters, mussels are likely to be colonized by two species, Pomatoceros lamarcki (Quatrefages) and P. triqiieter (L.). P. lamarcki is an intertidal to shallow sub- tidal species, whereas P. triqiieter is generally considered subtidal. particularly adjoining deep water (Hayward & Ryland 1995). Ma- ture adults are capable of breeding all year round; however, re- productive studies from other countries suggest episodes of peak settlement over relatively short periods ( 1-3 wk) (Klockner 1976. Castric-Fey 1983). The larval phase is temperature dependent and lasts for approximately three weeks (Seagrove 1941); however, monitoring larval populations from plankton tows has proved un- satisfactory in the past because the soft-bodied larvae do not pre- serve well (Senz-Braconnot 1968). Newly settled worms can reach full tube length (20-60 mm) and sexual maturity in four months (Castric-Fey 1983) and are therefore capable of rapid colonization. However, the species distribution and settlement of P. lamarcki and P. triqiieter in Scottish lochs has not been described. The cultivation of Mytiliis ediilis L. in suspended culture in Scotland increased to approximately 2.000 tons in 2000. The Scot- tish rope-grown mussel industry maintains its competitive edge over the Irish rope-grown mussel and UK dredge industries by trading in premium-quality mussels only. Farmed mussels consti- tute an excellent substrate for the settlement of many other organ- isms, which from the perspective of the mussel farmer are collec- tively termed fouling. Suspended long line cultures are particularly *Correspondmg author. E-mail.' mke@dml.ac.uk vulnerable to fouling because they are continually submerged and considerable effort is required to clean crop for marketing (Hick- man 1992). Both barnacles and tubeworms frequently settle on mussel shells. Barnacles are not as problematic and are usually removed by the brtishes used to strip mussels from culture lines, however, tubeworm (Pomatoceros spp.) longer than 5 mm are resistant to brushing and result in badly fouled mussels being devalued or discarded. Mussels with more than 7% of the shell fouled are not considered Grade A quality. Tubeworm-fouled mus- sels are not suitable for sale as ••live-in-shell" product to retailers as the fouled shells are considered visually unattractive, the tube- worm can die before the mussel causing an offensive smell and the tubeworm can be released from the tube on cooking and appear unappetizing. In Scotland, it is considered uneconomical to pro- duce Grade B rope-grown mussels because of the higher economic investment involved compared with dredged mussel operations, and the competition for the export market from the larger Irish cultivation industry which produces primarily Grade B stock. The estimated cost of tubeworm fouling to this industry is between £300,000 and £500.000 per annum (P. Marshall SSMG) and es- tablished growers believe the problem is worsening. Previous work has focused on the competition for food re- sources between fouling suspension feeders and rope grown mus- sels (Lesser et al. 1992). However, few data exist with regard to reducing the impact of fouling organisms in cultured mussel sys- tems. A survey of Irish rope-grown mussel growers indicated the most important factors in managing tube-worm fouling on their farms were the timing of handling (delaying grading and thinning of stock until after the main tubeworm settlement), the density of mussels on the lines (outer mussels forming a barrier to settle- ment), the influence of weather and the depth of culture line (Pur- 519 520 Campbell and Kelly cell & Cross 1994). All of these factors have been identified by Scottish mussel growers as potentially being of major importance to fouling levels on their sites. The objectives of this study were to first describe tubeworm species colonizing Scottish rope-grown mussels, monitor settle- ment, and determine the timing of peak settlement in two contrast- ing lochs and at different depths, thereby ascertaining inter and intra-loch variation in settlement abundance. Second, field trials were conducted to assess the effect of mussel density, depth, re- tubing material, and the presence of adult tubeworm conspecifics on tubeworm settlement intensity in two size classes of mussels. Third, to evaluate the use of aerial immersion as a method to control tubeworm growth on mussels. The data collected provide basic information for the development of husbandry practices to alleviate tubeworm fouling. MATERIALS AND METHODS Sample Sites Two lochs. Loch Stn\en (N55"59'.W5'07') and Loch Beag (N56°53',W3°44'| with established mussel fanns, were selected as the rope-grown stock here was known to be prone to annual settle- ments of tubeworm species (Fig. 1). Loch Striven is a narrow Fjordic loch, almost 13 km in length with the third longest flushing time (12 days) of any sea loch (Edwards & Sharpies 1986). In contrast. Loch Beag is not a true fjordic loch but an elongated embayment with greater mixing and fresh water run-off (Howson et al. 1994). Timiiif; of SellkmeiU The collector strings consisted of settlement plates (a roofing slate, 40 x 23 mm) attached by cable ties to a length of 6 mm polypropylene rope, at depths of 2. 5, and S m. Before deployment, the plates were conditioned for two weeks in aquaria of sand- filtered seawater and kept in darkness to promote a predominately bacterial biofilm (Hammer et al. 2001 ) because lack of an estab- lished biofilm on slates has been shown to limit settlement (Chan & Walker 1998). Fortnightly, from mid-April to mid-December 2001, at each of two sites within Loch Striven and Loch Beag (Fig. 2), four replicate collector strings were deployed and two replicate collector strings, which had been in place for a fortnight, were recovered for tubeworm enumeration. Thus, on every sampling visit, two replicate collector strings were added to those in the water, and they remained there until completion of the monitoring program. A Tinytalk' ' II temperature logger was deployed at a depth of 5 m in each loch to record sea temperatines throughout the sampling period. All tubeworm ob,served on both sides of the 12 slates recovered from each loch, every fortnight, were enumer- ated with the aid of transparent counting grid. Pomatoceros Species Identification Tubeworms were identified from slates retrieved at the end of the experiment, which had been placed in the water during the peak settlement period. Individuals (>20 mm tube length) were randomly selected from replicate slates at each depth, site, and loch: removed from their tubes; and identified under a dissection microscope on the basis of opercular structure (Zibrowius 1968), which has been confirmed by previous genetic studies as suitable to distinguish the two species (Ekaratne et al. 1982). West Coast of Scotland Figure I. Map of the West Coast of Scotland and sampling locations al Lochs Beag and Striven. Rectangles represent areas of long line mussel cultivation. Small black squares show subsites at which collec- tor plates were deployed (mld-.\pril to mid-December 20(11). Kffecl of Mussel Size, Density, Retuhing System, and Conspecifics on Tubeworm Settlement At Loch Striven in mid-August 2001, before peak tubeworm settlement, rope-grown mussels, with <2'> of individuals fouled with tubewomi (termed "clean"), were graded to create two dis- tinct size classes with 1) large clean mussels (mean shell length 39.9 mm ± 1.03 SE, n = 50) and 2) small clean mussels (mean shell length 33.5 mm ± 1.03 SE, n = 50). Mussel cultures are allowed to establish on 8-m lengths of polypropylene rope hanging vertically. Before attachment by the byssal threads, the mussels are supported by a mesh tube. Mussels may migrate through different tubing systems at different rates and/or establish different feeding structures on the culture line, which could influence tubeworm abundance on mussels. For this experiment the clean large and small mussels were retubed using two common tubing systems: cotton tubing and plastic "pergolari" mesh, at a high and a low stocking density forming eight different treatments (Tables 1 and 2). A further group 3), large mussels (mean shell length 61.7 mm Mussels Grown in Suspended Culture 521 12000 - 10000 2.1-Stnven site 1 200 - 0 - 2.3-Beag site 1 1/5 1/6 1/7 1/8 1/9 1/10 1/11 1/12 400 200 0 - Time (months) Time (months) Figure 2. Mean Striven (Apr to and standard deviation for Pomatmeros spp. settlement counts per slate at 2, 5. and 8 m at two sites 4 km apart within Loch Dec 2001), 500 m apart in l,och Beag (note difference in scale on the Y-axis I. and seawater temperature ( C) at 5 m. ± 0.915 SE. n = 501 with >98% of individuals fouled with tube- worm, were graded and retubed using plastic '"pergolari" mesh only at a high and a low stocking density, forming a further two treatments (Tables I and 2). The mean shell length was signifi- cantly smaller in the small clean mussel treatment than in the two large mussel treatments, which were not significantly different in terms of shell length, thus allowing the inclusion of mussel shell length as a treatment effect (F = 250.2. P < 0.001, df = 2,Tukeys P < 0.05; Table 2). Each of the 10 treatments had 5 replicates that were suspended vertically and at random, approximately 40 cm apart, along the horizontal main line. The treatments were removed from the water after 16 wk (mid-Deceinber) during which peak tubeworm settle- ment had occurred and then declined to a base level. The num- ber of Pomatoceros spp. that had settled on individual mussel shells sampled from a depth of 4-5 m from each replicate was counted. TABLE L Treatment characteristics. Treatments Replicates Tubing Density (A) Large clean mussels 1 2 3 4 (B) Small clean mussels 5 6 7 8 (Cl Large fouled mussels 9 10 Cotton High Cotton Low Pergolari High Pergolari Low Cotton High Cotton Low Pegolari High Pergolari Low Pergolari High Pergolari Low 522 Campbell and Kelly TABLE 2. Shell length (mm) and stundard error (SEl and slocklnj; densities of the mussel treatments. TABLE 3. Mean and SD Pninatmeros spp. counts at peak settlement for each depth at sites in Loch Striven and Loch Beaj;. Treatment Length I mm I (/I = 50 (±S.E.) Stocking Density kg/m) High Low Large clean yLjty (±1.05) 3 Large fouled 61. 7-' (±0.92) 5 Small clean 33.5*' (±1.03) 1.75 0.875 Means with different superscripts in the same column are significantly different iP > 0.05. ANOVA and Tukeys). Mussel Stock Fouling Comparisons In early August 2001. the fouling intensity of tubeworm on rope-grown mussel stocks with varying treatment histories in Loch Striven was assessed. In Loch Striven the mussel spat generally settles from the plankton in spring (late- April to early-May), by the following spring the nius.sels are termed 1-y-olds. At intervals over the growing cycle the mussel stocks may be stripped from the culture rope, graded according to siz.e, and resuspended at a lower density. The different groups and depths examined were deter- mined by the stocks (settled in spring 1999 or 2000). which were available at the site. Tubeworm fouling intensity was compared on 1-y-old ungraded mussels ( 1-y ungraded), 1-y-old mussels that had been thinned in November 2000 (1 y Nov), and in small 2-y-old mussels that had been graded in July 2001 (2 y July). Mussels from 2. 4, 6, and 8 m depth on culture line from each stock were compared. A 1-m length of mussel culture line was removed from five replicate culture ropes at the four different depths and indi- viduals separated and washed. As a measure of mussel density the total weight of individuals per m was recorded to the nearest 0. 1 kg. Finally, the percentage of mussels with tubeworm and the number of tubeworm per mussel for a kg sample per replicate were ascertained. Aerial Immersion Time and Tubeworm Survivorship In December 2001, rope-grown mussels heavily fouled with tubeworm were collected from Loch Striven and transported in cool boxes to the laboratory aquaria. In each of two temperature- controlled rooms at 13°C and 7°C (both at 95% humidity), groups 80 70 20 10 - ■■ I Gorton Cotton - high - low ^H 3 1 — 1 4 Perg - Perg - high low Jl 2 3 4 5 No. of Settled Tube worm Depth (m) n \lean No. Pomatoceros spp per slate (±SD) Site Loch Striven Loch Beag 1 -) 1 2029-'(±552) 124" (±18.4) 5 2 4019" (±423) 389=" (±236) 8 2 4825" (+202) 222"" (±120) T 2 T 5159" (±127) 194" (±53.0) 3 -) 9124" (±10.^9) 628"" (±5.0) S -) 11711'' (±65.0) 97 r (±94.0) Figure 3, Pomatoceros spp. frequency on small mussels (%). Means with different superscripts in the same column are significantly different (P > 0.05. ANOVA and Tukeys). of five mussels with three or more adult tubeworm per mussel shell were divided among raised trays randomly placed within a con- tinuous flow seawater tank. The mussels with tubeworm were left for five days to allow recovery and acclimatization to laboratory conditions before the start of the experiment. All tubeworms were identified as P. triqueter. At time 0, all trays were removed from the seawater, excluding six replicate control trays, which remained immersed for the duration of the experiment. Six replicate trays of mussels were returned to their respective tanks after 6. 12. 18. 24. 30. and 36 h of aerial exposure at either 13T and 7'C. Forty-eight hours after each tray was returned to the seawater, the percentage of tubeworms alive on each mussel was recorded. Individuals were assumed to be dead when they did not retreat into their tubes when touched by a pin. Statistical Analysis ANOVA was used to test for significant differences among treatments and depths, the data having met the assumptions of the test. Test of association (x") was used to compare tubeworm fre- quency distributions among the treatments. The linear correlation between variables was assessed using Pearson's correlation coef- ficient (/■). Regression analysis was performed to determine the functional relationship between tubeworm survivorship {9c) and immersion time (hi. ANCOVA and Tukeys pairwise comparison was used to compare regression lines between the different treat- ments. The statistics package Minitab* version 13.1 was used for all analyses. RESULTS Throughout the experimental period, juvenile Pomatoceros spp. were the most abundant species observed on collector slates in both lochs. The distribution of settled juvenile tubeworm on the slates tended to be uneven, composed of aggregated groups with a greater proportion of individuals on the lower half of the slate. After two weeks of immersion at each site, the tube length of individuals on the slates was <3 mm. Pomatoceros spp. Species Identification All individual tubeworms examined on slates from the peak settlement period at Loch Striven and Loch Beag were identified as P. triqueter. Therefore, P. triqueter was assumed to be the most abundant of the Pomatoceros species present and, furthermore, responsible for colonization and fouling of rope-grown mussels in the lochs. Mussels Grown in Suspended Culture 523 Pomatoceros spp. Settlement The timing of peak settlement differed between lochs but was found to be synchronous between sites and over depth within the same loch. Significant differences in settlement intensity were ob- served between lochs, sites, and depths. At the Loch Beag sites two periods of peak settlement were observed (26.6.01-10.7.01 and 4.9.01-18.9.01) with the highest mean number of P. triqueter per slate (971 ± 94 SD, site 2) recorded in the later peak (Table 3 and Fig. 2). It is not known whether settlement at Loch Beag was in fact two discrete periods or one potentially more intense period disrupted by environmental conditions. A single period of peak settlement was recorded at Loch Striven (26.9.01-15.10.01); with the highest mean number of individual P. triqucler per slate (11711 ±65 SD, site 2), an order of magnitude higher than in Loch Beag (Table 3 and Fig. 2). The Loch Striven and Loch Beag later peak events occurred as summer seawater temperatures declined. In both Loch Striven and Loch Beag at peak settlement, site 2 had significantly greater settlement than site I (Loch Striven. F = 280.0, P < 0.001. df = 1; Loch Beag, F = 27.2. P < 0.02, df = 1; Tukeys, P < 0.05), indicting the scale on which P. triqueter settlement intensity can vary within lochs at sites 500 m to 4 km apart (Loch Beag and Loch Striven, respectively). In Loch Beag. no effect of depth on settlement intensity was observed at site 1. whereas at site 2. significantly greater settle- ment occurred at 5 m and 8 m than at 2 m. The effect of depth on P. triqueter settlement was more pronounced at Loch Striven. Site 1 had significantly greater settlement at 5 m and 8 m than at 2 m and at site 2 settlement increased significantly with increasing depth. Effect of Mussel Size, Density. Retubing System, and Conspecifics on Tubeworm Settlement The number oi Pomatocervs spp. settling on small mussels was found to be significantly lower than that on the large mussel treat- ments. Among the small size class treatments, the percentage of mussels with si tubeworm on the shell ranged between 21.5% and 28.4%, whereas in the large size class treatments, the percent- age of mussels with >1 tubeworm on the shell ranged between 91% and 100% (Tables 4 and 5. Fig. 3 and 4). Thus, analyses on small mussel treatments were performed separately to the large mussel treatments. Small Mussel Treatments The small mussels migrated successfully from the two tubing systems. Pomatoceros spp. were observed to have settled predomi- nately along growth checks on the mussel shell exterior with a smaller proportion having settled on the umbo and shell ligament regions. No significant difference in the mean percentage of individuals fouled was found (ANOVA. F = 1.24. P = 0.329, df = 3) and no significant differences were ob-served among frequency distribu- tion of Pomatocero.s spp. fouling between the small mussel treat- ments (four classes. 0. 1.2, 3-I-, x"' = 10.29, P = 0.328. df = 9; Table 4. Fig. 3). Large Mussel Treatments Significant differences in number of tubeworm per individual were identified between the large mussel treatments (ANOVA. F = 17.1, P < 0.001. i// = 5; Table 5). However, in all large mussel pergolari treatments, the mussels did not establish outside the tube because the mesh size proved too small to allow mussels to mi- grate through the apertures. Thus, the mussels were confined within the tube for the duration of the field trial, affecting the structure of the feeding colony and preventing comparison be- tween pergolari and cotton tubing systems with respect to tube- worm fouling. Clean cotton-tubed mussels at low density had a significantly greater number of tubeworm per mussel than clean cotton-tubed mussels stocked at high density. No significant differences were observed between clean cotton-tubed mussels at low density and clean pergolari-tubed mussels at high and low densities. No sig- nificant differences were observed between clean cotton-tubed mussels at low density and fouled pergolari mussels at high and low densities. Tubeworm numbers per individual were signifi- cantly greater in fouled pergolari-tubed mussels at high and low densities compared with clean pergolari mussels at high and low densities (Table 5). From the test of association analysis (x~), four significantly different distributions among frequency classes of tubeworm on large mussels were identified; 1 ) clean cotton-tubed and clean pergolari-tubed at high density had the greatest proportion of mus- sels with low frequencies of tubeworm and the proportion of mus- sels decreased steeply in higher tubeworm fouling classes (Fig. 4. 4.1 and 4.3); 2) clean pergolari tubed at low density had a large proportion of mussels with low frequencies of tubeworm fouling, which did not decrease as steeply over higher tubeworm fouling classes compared with distribution 1 (Fig. 4, 4.4); 3) clean cotton tubed at low density comprised of an approximately even fre- quency distribution of mussels throughout the tubeworm fouling categories (Fig. 4. 4.2); 4) fouled pergolari-tubed mussels at high and low density also consisted of an approximately even frequency distribution of tubeworm frequency classes among mussels yet had the lowest proportion of mussels in low tubeworm fouling catego- ries (Fig. 4. 4.5 and 4.6). TABLE 4. Mean (%) of mussels with Pomatoceros spp. and SE in the small mussel treatments. Treatment Tubing Density Replicates Mean % Mussels With Pomatoceros spp (±SEl CV % Cotton Cotton Pergolari Pergolari High Low High Low 22.3'' (±2.65) 28.4° (±4.82) 21.3° (±2.02) 21.5° (±1.81) 26.7 38.0 21.2 18.8 Coefficient of variation is given (CV%). Means with different superscripts in the same column are significantly different (P > 0.05. ANOVA and Tukeys). 524 Campbell and Kelly M 40 0-) 5-9 10-14 15- Pomatocews freq. classes 2 30 Z 20 0-4 5-9 10-14 15- Pomatoceros freq. classes 0-4 5-9 10-14 15- 0^4 5-9 10-14 15- Pomatoceros freq. classes 0^ 5-9 10-14 15- Pomatoceros freq. classes 3 30 Z 20 - 0^ 5-9 10-14 15- Pomatoceros freq. classes Pomatoceros freq. classes Figure 4. Pomatoceros spp. frequency classes on large mussels (100 mussels per treatment). Mussel .Slock Fouling Inlcnsily Coinparisniis The results demonstrate the wide \ariation in the percentage of mussels fouled that exists among different mussel stocks within the farm site, with the mean percentage mussels fouled among the different mussel stocks ranging between 0.7% (l-y ungraded stock) and 5.9% (l-y Nov-graded stock; Table 6). The stocks — l-y Nov, l-y ungraded, and 2-y July mussels were all significantly different with respect to levels of tubeworm fouling, with a greater percentage of mussels fouled in the l-y Nov stock and least in the l-y ungraded stock (F =55.6. P < 0.001. elf =2; Tukeys P < 0.05). Variation in fouling intensity was observed to increase as depth increased. However, no correlation between the percentage of fouled mussels and the variables depth (m) and density (mussels kg/m) among individual stocks or stocks combined was found. A negative correlation was found between depth (m) and mussels density when all treatments were combined (;■ = -0.532. P < O.OOl. n = 60). indicatmg that as depth increases mussel density alom; the culture line decreases. Mussels Grown in Suspended Cultlire 525 Treatment TABLE 5. Mean number of Pnmaloceros spp. per mussel and SE in the large mussel treatments. Fouling Clean Clean Clean Clean Fouled Fouled Tubing Cotton Cotton Pergolari Pergolari Pergolari Pergolari Density High Low High Low High Low lUO 100 100 100 100 100 Mean No. Pomaloceros spp. Per Mussel (±S.E.) 5.S1' (±0.501 10.55" (±0.84) 5.07^ (±0.38) 7.24" (±0.58) 10.10^+0.65) 10.45N+0.66) CV% 87.1 79.6 75.1 80.7 64.4 63.3 Coefficient of variation is given (CV%). Means with different superscripts in the same column are significantly different (.P < 0.05. ANOVA and Tukeys). Aerial Immersion Time and Tubewonn Surviviirship Although increasing P. irii/iieter mortality was clearly evitjent. as aerial immersion time (h) increased, no mortalities amongst the mussels occurred during the trials. No P. tnqiictcr mortalities were observed in the control treatment, which was never immersed dur- ing the experiment (Fig. 5) The interaction term (time x treatment) was significant (ANCOVA, F = 5.90. P = 0.02). indicating that the slopes. iP. triqiieier % survival rates over time) were significantly different at 7°C and 13°C, at the 95% confidence level. Adult P. irkjuerer were predicted to achieve a mean 100% inortality after 24.1 h and 35.4 h at an air temperature of 7°C and I3°C. i-espectively (Table 7). DISCUSSION P. iriqueter was the most abundant of any species settling on collector plates and the only species of tubeworm found on the mussels. This is in concordance with the observation that P. kumi- rcki occurs more frequently in intertidal and shallow turbid sub- tidal waters (Hayward & Ryland 1995) and that P. iriqueter can out-compete P. lamarcki for settlement space at depths of 13 m. although the dominance of one species over the other is thought to be dependent on climatic conditions (Castric-fey 1983). The pattern of settlement in the two Scottish lochs was consis- tent with that in other temperate populations of P. triqiieier (Klockner 1976. Castric-fey 1983), with peak settlement occurring later in the year, possibly the result of the more northerly latitude (Klockner 1976). The timing of peak settlement differed between lochs, presumably as a result of specific biologic and physical differences (Crisp 1974. Dirnberger 1990) but was synchronous within each loch and over depth. Levels of settlement differed between lochs, sites, and over depth within the same loch, giving indication of the scale on which intra-loch variation in environ- mental factors influence settlement abundance. Such monitoring should be repeated to ascertain year-to-year variation in occur- rence of peak settlement within Scottish lochs. The higher settlement levels observed at Loch Striven may result from a long flushing time combined with comparatively little fresh water input and mixing (Tett et al. 1986): consequen- tially, the larvae are retained within the loch for longer periods of time and rarely e,\perience lowered salinity, which is reported to reduce settlement (Castric-fey 1983). In addition Loch Striven has had an established mussel site (8 y). whereas the site in loch Beag was recently established (3 y) and is approaching its second har- vest. The scales of the operations are also significantly different (standing mussel stock at Loch Striven is 200 tons and Loch Beag is 30 ton). A longer period of mussel production at Loch Striven may have increased the standing population of tubeworm through the accumulation of live mussels and shell material beneath the farm, reported to increase site availability for settlement of calcar- eous polychaetes (Tenore et al. 1982. Kaspar et al. 1985). Increasing or peak seawater temperature may act as a direct or indirect cue for optimum gamete production or spawning or create optimal conditions for larval settlement and survival, resulting in discrete and intense periods of tubeworm settlement. At higher temperatures bacterial loading would also be maximal and may also generate a cue for settlement. A high bacterial density has been identified as a major cue for the settleinent response of P. lamarM (Hammer et al. 2001). and mussel farms have been shown to possess increa.sed microbial assemblages resulting from elevated levels of organic enrichment (La Rosa et al. 2001). Speculatively, the exaggerated metabolic output from mussels intensively farmed within a restricted loch environment may en- hance or promote a tubeworm larval settlement response. Natural concentrations of ammonia (NH,). a principle component of ex- cretion in bivalves, released by adult oysters can be sufficient to induce settlement of oyster larvae, particularly at highest tempera- tures when peak values for total NH,-NH4* occur (Fitt & Coon 1992). Mussels may therefore be more susceptible to tubeworm TABLE 6. Mean length of mussels (mm) with SE mean density of mussels (kg/m) with SE, mean ( -7, ) of mussels with Pomaloceros spp. and SE for each mussel stock. Treatment Length (mm) (H =50) (±.S.E.) CV^f Mean Density kg/m (±S.E.) 1 y Ungraded 1 y Nov 2 y July 37.8 (±1.45) 40.4 (±0.66) 41.7 (±0.70) 27.1 11.5 11.9 6.0 (±0.80) 4,4 (±0.20) 4.0 (±0.20) Means with different superscripts in the same column are significantly different (P < 0.05. ANOVA and Tukeys). Mean % Mussels Fouled (n = 100) (±S.E.) 0.66-'{±0.17) 5.89" (±0.51) 2.64" (±(J.,34) 526 Campbell and Kelly 100 . \ \ \ \ • 13 °C O 7"C 13 °C 80- 7°C > E 60 40- 20 - 0 - i \ i \ \ ^ \ \ 0 5 10 15 20 25 30 35 40 45 Emersion Time (hrs) Figure 5. Mean /'. triqiwler survival (%) and standard error against aerial immersion time Ih) at 13 C and 7 C after a 48-li recovery period for the linear section of the relationship. Regression lines are fitted. fouling immediately upon re-immersion after grading, for in ad- dition to offering an unprotected surface for settleinent. a pulse of excreted nitrogenous waste, accumulated during immersion and released on re-immersion, could further induce P. liic/iierer larvae to settle. Tubeworm settlement cues with respect to environmental conditions on a mussel farm warrant further research. The field trial highlighted mussel size (shell length) as an im- portant factor influencing P. thqueter abundance on farmed mus- sels because small mussels had consistently fewer tubeworms. This may be a result of the fast growth rates of smaller mussels, preventing successful tubeworm attachment. Tubeworms were not found on areas of fastest growth such as the posterior shell edge but predominately on shell growth checks and on the umbo indi- cating settlement to have occurred on regions of relatively slower growth and/or at periods of disturbance and slow growth. The density of smaller mussels established on culture lines is greater than large mussels, having smaller interstices between individuals and less of a surface area for settlement of fouling organisms. As mussels age. a change in the nature of the inussel shell texture or its biofilm may make them more attractive for tubeworm settle- ment. No effects of initial stocking density or tubing system on tubeworm settlement on small mussels were distinguished, indi- cating that size has a greater influence on settleinent than either of these two factors. Further work should determine the relationship between mussel size and susceptibility to tubeworm settlement. P. triqueier settlement was greatest in treatments with conspe- cific adults on the mussels. However, there was no difference in tubeworm settlement levels between high and low densities in the fouled mussel treatments. This suggests that the cue to settle pro- vided by conspecifics was sufficient to outweigh the effects of stocking density. Settlement on or near conspecitlc adults has ben- efits; adults derive reproductive benefits from being within aggre- gations and larvae that settle near adults benefit from choosing a habitat likely to support post larval growth (Pawlik 1992). P. lam- arcki has been shown to settle in response to chemical substances originating from the body of conspecific adults or juveniles (Chan & Walker 1998). As a result, whenever practical, individuals with fouling should be removed from mussels that are to be retubed for on growing. Tubeworm numbers were more abundant on the clean large mussels stocked at low stocking densities; this may be attributed to greater shell surface areas exposed for settlement and or a decrease in overall feeding activity in mussels at lower densities on the culture line. In areas of high tubeworm settlement, mussel lines should be stocked at optimum densities for maximal rapid growth and distributed evenly throughout the tubing. A wide variation in fouling intensity was observed among mus- sels stocks with different treatment histories at the farm site. Differences in the fouling intensity between the stocks are most likely to be attributed to the settlement intensity of tubewonii and the size of mussels at the time of retubing. The influence of depth on settlement was not as distinct along mussel culture lines when compared with the collector slates. Consequently, adjusting cul- ture line depth to control fouling may prove ineffectual. No cor- relation could be found between inussel density and degree of fouling. However, counting the number of mussels on a meter of culture rope may have been too insensitive as a measurement oi density as mussel density was not always homogenous within a meter of culture line and settlement site selection by tubeworm is influenced over small scales (Pawlik 1992). Further studies of increased precision would determine the true effect of these variables. As a result of the discrete nature of P. triqiieter settlement, mussel growers could routinely monitor tubeworm settlement and to avoid practices such as grading and returning mussels to the water in periods of intense tubeworm settlement. Monitoring settlement should be conducted specifically for each loch. Growers should monitor settlement in conjunction with physical variables such as seawater temperature and salinity specific to individual sites to reveal areas and conditions that consistently yield lower levels of tubeworm fouling, and criteria for establishing new mus- sel farm sites should include hydrographic conditions that mini- mize tubeworm fouling. Furthermore, mussel-management strate- gies should adhere to minimizing the time mussel stocks are in the water, particularly in the second growing season when indi\ iduals reach a critical size and become vulnerable to excessive tubeworm fouling. In contrast, an alternative approach to limiting tubeworm settlement might be to avoid grading in the second year, thereby TABLE 7. The regression coefficients a and p and respective .SE. Pearson's correlation coefficient (r), probahility and predicted aerial emersion times Itprid ll""ll with upper and lower 959;: confidence intervals (C.I.). to achieve l(M)'7f mortalitj for /'. triqueier at 7 C and 13 C after a 48-h recovery period. Temperature Coefficient a (±SE) Coefficient p (±SE) (h) Lower •JS'/r C.L Upper 95% C.L 7'C 13 C 24 24 116.8 (±6..'i2) 126.2 (±9.381 -4.S6 (±0.402) -3..S7 (±0.323) -0.919 {.P < 0.001) -0.940 (P < 0.001) 3.S.4 24.1 33.0 22.3 38.6 26.5 Mussels Grown in Suspended Culture 527 leaving mussels undisturbed and allowing a mixed size-class feed- ing colony to develop. Adult P. triqueter were comparatively resilient with respect to aerial immersion, with a predicted mean 100% mortality after 24.1 h and 35.4 h at an air temperature of 7°C and 13X. respectively. However, as a result of the lengths of immersion time needed to achieve 100% P. triqueter mortality, it may not be practical for growers to use aerial exposure as a method to control tubeworm growth on mussels to be retubed and harvested at a later date. Although no mussels died within the experimental period, aerial immersion over such periods of time may cause undue stress in rope-grown mussels unaccustomed to exposure, resulting in higher mortalities after grading and retubing and delayed growth. ACKNOWLEDGMENTS The authors thank David Scott of Loch Striven Mussels and Ian MacKinnon of Loch Beag Mussels for their invaluable contribu- tion towards the project. Sincerest thanks are also due to Simon Howard and Galium Cameron of Loch Striven Mussels for their support throughout. This study was funded by Highlands and Is- lands Enterprise, The Highland Council and The Crown Estate. Castric-fey, A. iy83. 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J. Exp. Mar Biol. Ecol. 140:89-105. Edwards, A. & F. Sharpies. 1986. Scottish sea lochs: a catalogue. Scottish Marine Biological Association and the Nature Conservancy Council. Dunstaffnage Marine Laboratory Library, Oban, Argyll. PA37 IQA. Ekaratne, K., A. H. Burfitt, M. W. Flowerdew & D. J. Crisp. 1982. Sepa- ration of the two Atlantic species of Pomatoceros. P. lamarckii and P. triqueter (Annelida: Serpulidae) by means of biochemical genetics. Mar Biol. 71:257-264. Fin. W. K. & S. L. Coon. 1992. Evidence for ammonia as a natural cue for recruitment of oyster larvae to oyster beds in a Georgia salt marsh. Biol. Bull 182:401-108. Hammer, J. P., G. Walker & J. W. Latchford. 2001 . Settlement of Poma- toceros lamarckii (Serpulidae) larvae on biofilmed surfaces and the effect of aerial drying. J. E.xp. Mar. Biol. Ecol 260:1 13-131. Hay ward. P. J. & J. S. Ryland. 1995. Handbook of the marine fauna of Northwest Europe. Oxford: Oxford Univ. Press, pp. 258-260. Hickman. R. W. 1992. Mussel cultivation. In: E. Gosling, editor. The mus- sel Mytilus: Ecology, physiology, genetics and culture. Developments in aquaculture and fisheries science. Vol. 25. Amsterdam; Elsevier, pp. 465-510. Howson, C. M., D. W. Conner & R. H. F. Holt. 1994. The Scottish Seal- ochs. J.N.C.C. Report, 164. Dunstaffnage Marine Laboratory Library. Oban, Argyll. PA37 IQA. Kaspar, H. F.. P. A. Gillespie. I. C. Boyer & A. L. Mackenzie. 1985. Ef- LITERATURE CITED fects of mussel aquaculture on the nitrogen cycle and benthic commu- nities in Kenepuru Sound. Marlborough Sounds. New Zealand. Mar. Biol. 85:127-1.36. Klockner, K. 1976. On the ecology of Pomatoceros triqueter (Serpulidae Polychaeta). Breeding season, choice of substratum, growth and mor- tality. Helgolander wiss. Meeresuntez 28:352—400. La Rosa. T., S. Mirto. A. Marino. V. Alon/o. T. L. Maugen & A. Ma^/ola. 2001. Heterotrophic bacteria community and pollution indicators of mussel — farm impact in the Gulf of Gaeta (Tyrrhenian Sea). Mar. Environ. Res. 52:301-321. Lesser. M. P., S. E. Shumway, T. Cucci & J. Smith. 1992. Impact of foul- ing organisms on mussel rope culture — interspecific competition for food among suspension-feeding invertebrates. J. Exp. Mar. Biol. Ecol. 165:91-102. OECD. 1967. Catalogue of main marine fouling organisms. Vol. 3: Ser- pulids. Paris: Organisation for Economic Co-operation and Develop- ment, pp. 79. Pawlik. J. R. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar Biol. Rev 30:273-335. Purcell, R. & M. Cross. 1994. Technical Report on Tubeworm Fouling. Cork: Aquaculture Development Centre. University College Cork. Ire- land. Seagrove, F. 1941 . The development of the ^erpulid Pomatoceros triqueter L. Quart. J. Micros. Sac. 82:467-540. Senz-Braconnot, E. 1968. Donnees ecologiques et biologiques sur la fixa- tion des Serpulidae dans la Rade de Villefrance-sur-mer. Vie Milieu 198:109-132, Tenore, K. F., L. F. Boyer. R. M. Cal, J. Corral. C. Garcia-Fernandez. N. Gonzalez. E. Gonzalez-Gurriaran. R. B. Hanson, J. Iglesias. M. Krom. E. Lopez-Jamar, J. Mcclain. M. M. Pamatmat. A. Perez. D. C. Rhoads, G. de Santiago. J. Tietjen. J. Westnch & H. L. Windom. 1982. Coastal upwelling in the Rias Bajas. NW Spain — contrasting the benthic regimes of the Rias de Arosa and de Muros. J. Mar. Res 40:701-772. Tett. P.. R. Gowan, B. Grantham, K. Jones & B. S. Millar. 1986. The phytoplankton ecology of the Firth of Clyde sea-lochs Striven and Fyne. Proc. R. Sac. Ed. 90B:223-238. Zibrowius, H. 1968. Etude morphologique systematique et ecologique des serpulidae (Annelidae. Polychaeta) de la region de Marseille. Reel Trav. Sm Mar Endoume 43:81-252. Journal of Shellfish Research. Vol. 21, No. 2, 529-537. 20U2. MUSSEL DREDGING: IMPACT ON EPIFAUNA IN LIMFJORDEN. DENMARK PER DOLMER* Danish Insliiiik' for Fisheries Research. Charhilleiiliiiul Castle. 2920 Charloltenlwul. Denmark ABSTRACT Species composition and population density ol epibenthos are described in two areas in Limfjorden. Denmark. Both areas covered both a mussel fishing ground and an area that has been permanently closed tor mussel dredging since 14SS. Furthermore, mussels were dredged in a part of the mussel fishing grounds in both areas four months before the investigations. The rest of the fishing grounds had not been exploited for at least four years. This study describes the short-term impact (4 mo) and long-term impact (>4 y) of mussel dredging using the permanently closed areas as controls. The data were analyzed by multivariate statistics. In both short-temi study areas significant effects of dredging were observed. A number of taxa (sponges, echinoderms, anthozoans, molluscs, crustaceans, and ascidians) had a reduced density or were not observed in fished areas four months after the fishing was ended. In one of the two long-term study areas, significant differences in species composition and density were observed between fished and closed areas, indicating that the fishery may have a long-term impact on the epibenthic community, whereas in the other long-term area no difference was observed between fished and control areas. Significant reductions in the amount of shell debris and gravel were observed in the dredged areas. The impact of the loss of these benthic stitictural compiments on ecosystem processes and functions is discu.ssed. A"£)' WORDS: long-term impact, short-term impact, fishery impact, benthic epifauna, mussel dredging, .seabed structure INTRODUCTION The use of dredges, beam-, and otter-trawls in demersal fish- eries and the development of heavier gear types with increased fishery effort have raised a growing concern on the impact at benthic ecosystems (see review by Jennings & Kaiser 1998. Hall 1999, Collie et al. 2000). Recent studies have investigated the direct impact of towed-bottom fishing gear on benthic organisms (Bergman & Hup 1992. Eleftheriou & Robertson 1992, Brylinsky & Gibson 1994. Thrush et al. 1995. Cunic & Pany 1996. Collie et al. 1997, Tuck et al. 1998. Hall-Spencer et al. 1999. Hoffmann & Dolmer 2000, Dolmer et al. 2001), the impact on seabed topogra- phy and seabed composition (Hall et al. 1990, Pranovi & Giova- nardi 1994. Kaiser & Spencer 1996. Schwinghamer et al. 1998). and the effect of suspension of bottom sediment and release of oxygen-consuming substances and nutrients (Riemann & Hoff- mann 1991, Dyekja;r et al. 1995, Pilskaln et al. 1998). Indirect effects of the fisheries include changes in the trophic structure (Babcock et al. 1999) as the reported increase in density of scav- engers feeding on injured or discarded species (Gislason 1994, Jennings & Kaiser 1998) or as changes in species behavior and species interactions (Ramsay & Kaiser 1998). Because the blue mussel is an economically and ecologically important species in many coastal areas, mussel dredging inay significantly affect the form and the function of benthic ecosys- tems. Blue mussels form tridimensional matrices of byssus-thread interattached mussels. These stabilized structures are important for assemblages of associated organisms (Tsuchiya & Nishihira 1985, 1986, Svane & Setyobudiandi 1996, Ragnarsson & Raffaelli 1999). In soft-bottom habitats, solid components such as gravel and biogenic structures such as mussel beds and shell debris sub- stantially increase substratum heterogeneity and complexity. These structures are reported to be important for invertebrates as spatial refuges from predators (Heck & Crowder 1991. Dumas & Witman 1993, Lee & Kneib 1994, Thiel & Dernedde 1994) or as a sub- stratum for settling invertebrates and sessile organisms (Witman & Suchanek 1984. Baker 1997, Lapointe & Bourget 1999). Mussel dredging may destroy mussel bed structures and remove important "E-mail: pdo(s'dfu. min.dk seabed structuring components such as shell debris and gravel. The fishery thus exerts a potential diiect impact on the ecosystem by changing benthic habitat structures and indirectly interfering with invertebrate settling processes and prey-predator interactions. In Limfjorden, Denmark, an extensive fishery for blue mussels, Mxtilus edulis. exists. In a previous study in Limfjorden. Dolmer et al. (2001 ) demonstrated that mussel dredging reduced the den- sity of invertebrates living in the bottom. In particular, the poly- chaetes were significantly reduced in number. Another investiga- tion in 1997 of the long-term impact of mussel dredging on the epifauna in an area that has been closed for mussel dredging since 1988, however, failed to show any effects of the mussel fishery (Hoffmann & Dolmer 2000). The aim of the present study was to compare the spatial cotnposition of asseinblages of epibenthic fauna on mussel fishing grounds four months after a fishery was finished, in areas that have not been fished for more than four years, and in areas that have been permanently closed since 1988 to test the short-term and long-term impacts of mussel dredging on the epifauna. The second objective of the study was to measure the impact of mussel dredging on the amount of shell debris and gravel on the seabed. MATERIAL AND METHODS The study was conducted in Limfjorden (Fig. I ). This area is a micro-tidal, eutrophic water system of interconnected enclosures in northern Denmark, supporting a high production of blue mus- sels. The mean mussel biomass in the part of the area open to mussel dredging (-750 km") is about 600.000 tons with large temporal variations (Dolmer et al. 1999), This biomass supports the largest fishery in Europe exploiting natural mussel populations. The annual mussel landing is approximately 100,000 tons or 15% of the mussel stock. The short-term and long-term impact of mussel fishing on benthic epifauna was investigated in two different areas in Limt- jorden; in Lugstor Broad, in the central part of the fjord, and in the Ager0 area, in the western part of the fjord (Fig. 1). Part of both areas has been permanently closed to mussel fishing since 1988. In L0gst0r Broad, the northern part of the area is closed and in the Agero area, the central part is closed. During April-May 1999 (4 months before the investigation) mussels were commercially dredged in the area just southwest of the closed area in L0gst0r 529 530 DOLMHR Figure 1. Map of the two study areas and their stations. The double hues indicate the separation between fished areas and areas that have been closed to mussel nshin;; since 1988. In Logstor Broad (eastern area), the area north of the double line is closed and the area south of the line is fishing ground. In the Agero area (western area), the area between the two double lines is closed to mussel fishing. The stations used in the analyses of long- and short-term impact are separated with dotted lines. (O) control stations and (•) stations with mussel dredging. Broad and in the area just north of the clcsed area in the Ager0 area. Furthermore, data from the mussel stock assessment pro- gram, where the inussel biomass at 250-,^,'>0 stations in the Lim- fjorden is described annually (Dolmer et al. 1999). indicate that that the area southeast of the permanently closed area in L0gst0r Broad and the area south of the permanently closed Ager0 area has not been fished at least since 1996. Epifauna in the Logstor Broad and the Agero areas was described in September 1999 to test whether the species composition and density differed between per- manently closed areas, areas that were fished four months before the study, and areas that had not been fished for niore than four years. Replicated stations in the area closed to fishing were con- trasted with replicated stations located in adjacent areas that were subject to harvesting practices. Shorl-Tcnn Impact (4 Mo) Stations 7-8 in Logstor Broad and stations 2 1 and 23 in the Agero area were fished by mussel dredge during April-May 1999 and significant impact on the seabed was observed when diving in September. To test the impact of dredging on the epifauna species. co]iipc)sition on the two stations in each area was contrasted with two control stations in the nearby closed areas. Stations 7 and 8 were contrasted with stations 9 and 10 in LogstOr Broad and sta- tions 21 and 25 were contrasted with stations 15 and 26 in the Agero area (Fig. 1). No signs of dredging activity were observed on the seabed when diving at the control stations. Liiiiji-Term Impact (>4 >J In the eastern part of L0gst0r Broad, the stations in the closed area (12, 13, 14) were contrasted with the stations in the area open for mussel dredging { 15, 18, 19. 20). In the Agero area, the stations in the closed area (1 . 5. 7. 10. 11) were contrasted with the stations in the area open for mussel dredging (6. 8. 9. 24). At each station, the epifauna (>1 ) cm was identified and quan- tified. Colonial species such as hydro/oans and bryo/oans were omitted from the study because of difficulties in quantifying these taxa. The sponges were included in the investigation, and here each distinct colony was classified as one individual. At each replicated station, the epifauna in 30 circles of 0.24 m" were quan- tified by use of SCUBA diving. The circles were marked with an iion ring I'andomly placed on the sea floor. The material in the last 1 0 circles was collected and the population density of blue mussels and the weights of shell debris and pebbles were measured in the laboratoi'y. The epifauna species composition and density data was tested with the PRIMER-5-package (Plymouth Routines in Multivariate Ecological Research). Before the analysis, the ring samples with- out epibenthic organisms were excluded from the datamatrix. Bray-Curtis similarity indices were calculated on 4"' root trans- formed species density data according to Clarke and Warwick (1994). The stations were plotted in a Multi-Dimensinal Scaling plot (MDS) to identify separate clusters of stations. Differences between contrasted stations were tested in two steps by ANOSIM analyses. It was tested if there were differences among replicated stations within each treatment area (fished contra closed) in each area and if differences (P < 0.01 ) were obtained then an ANOSIM analysis tested for differences in species composition between con- trasted stations on average ranked data. If no differences were observed aniong replicated stations then an ANOSIM analysis tested for differences between contrasted stations on the set of data treating each ring observation observations as separate data. The SIMPER procedure identified which taxa contributed most to the dissiiiiilaritv among contrasted stations. The significances of the differences in density of these taxa were tested with / tests. Observed differences between contrasted stations can be due to short-term and long-term impacts of mussel dredging or may be caused by environmental gradients in the study area. It was as- sumed that if an environmental gradient affected the similarity between stations there would be a positive relationship between spatial distance and similarity between stations. On a MDS plot. the stations would then form a unidirectional track with the spatial and similar close stations close to each other and most spatial distant and dissimilar station at the longest distance. To test wheth- er the similarity among stations was related to their spatial relation. Mussel Dredging 531 the MDS plots of the stations, including an indication of the sta- tions spatial relationships, was analyzed. Impact on Substratum The data on weights of shell debris and gravel at each of the short-term impact study stations were tested separately for the L0gst0r and the Ager0 Area in nested two-way ANOVAs with fishery/closed status as the first factor and the stations as the sec- ond factor — nested to the fishery/closed factor. Before the tests. the data were ///-transformed and tested for homoscedasticity (F,„,^-test). To test the relationship between diversity and substra- tum composition, a Shannon-Wiener index was calculated for each sample from the short-temi study stations. The relationships between this index and the amounts of shell debris and pebbles were analyzed by linear regression. To test the role of mussels forming a biogenic substratum important to the associated fauna, the relation between the Shannon-Wiener index and the density of mussels in each sample was analyzed by linear regressions at station 7 and 8 and 9 and 10 in L0gst0r Broad. RESULTS During the investigation, a total of 1 1 and 20 different epifauna species were recorded in L0gst0r Broad and the AgerO area, re- spectively. MDS ordinations of the stations in L0gst0r Broad and the Ager0 area showed that the stations in the short-term study areas were separated into two clusters, including stations from the fished and the closed part i)f the study areas, respectively (Fig. 2). The separation of the stations from the long-term study areas was not that clear-cut. Low oxygen concentrations (<2 mg 0-, P' ) were recorded during two weeks in August 1999 in parts of Logstor Broad. Some mortality of blue mussels was observed after this oxygen deficiency, but also other species may have been affected. Because the distribution of areas with oxygen deficiency did not overlap with the distribution of the tlshed areas, the low oxygen concentration did not interfere with the results of this study. Short- Term Impact In the two short-term study areas, no differences were observed among stations within each treatment area and the difference be- tween dredged and closed stations was tested on complete sets of data. The ANOSIM analysis showed that there were significant differences between the two dredged stations and the two control stations in both L0gst0r Broad (P = 0.001 ) and the Ager0 area (P = 0.001 ). The SIMPER procedure and ; tests indicated that a large number of species disappeared or had a reduced density in the two dredged areas, including poriferans. echinoderms. anthozoans, gastropods, crustaceans, and ascidians (Tables 1 and 2). Splitting the data into two functional groups, mobile and sessile species, indicated that the sessile species contributed to a larger part of the dissimilarity between the control and the dredged stations (51- 61%) than the mobile species, although these species still contrib- uted significantly to the dissimilarity (.30-42*). ANOSIM analy- ses on data split into functional groups showed a significant dredg- ing impact on sessile fauna both in L0gst0r Broad and in the Ager0 area (Table 3). Long-Term Impact In both L0gst0r Broad and the Agero area, significant differ- ences among stations within each treatment area (fished contra closed) were observed (Table 3). Consequently, the analysis of the (a) / ■"•"■ ~7\ 5 ~ 11^ / / 7 k* '"■ ■^ / 10 / , 1 ( 25 )i 8 ; 9J (... y / 6: .^■'6 ~^\ \ Agere 21 stress: =0.06 (b) /"io \ ■'9 \ X ^ (f •.. 18 N •* -^10 ) Logstor Br. stress=0.07 J 7) Figure 2. MDS plot of station means in the .Ajjero area (a) and l.ogstar Broad (bl. The control area stations are delineated with a solid line and stations from fished areas with a dashed line. The gray background Indicates long-term study stations and the white background short- term stations. long-term fishery impact was conducted on average rankings and re-ranked data. The ANOSIM analyses testing the species compo- sition at the stations in the fished area in the southern part of L0gst0r Broad and in the closed northern part showed that there was no difference {P = 0.771) between stations indicating that no long-term effects could be observed. In the Agero area, a signifi- cant difference (P = 0.024) between the species composition in the central closed area and the southern area where mussel dredg- ing is legal may indicate a long-term impact. The SIMPER pro- cedure and f tests on data from the Agero area indicated that a number of species disappeared or had a reduced density in the area open to mussel dredging (Table 4). The MDS of the similarity between stations and their spatial relationships (Fig. 3a and b) show no relationship between simi- larity and spatial distance in the two short-term study areas. This indicates that differences in species composition between stations are not due to an environmental gradient in the area. In the long- term study areas, some of the stations with the longest distance in-between showed a relationship between similarity and distance 532 DOLMER (stations 11-10-7 in the Agero area and stations 18- 19-20- 15 in L0gst0r Broad). The spatial distribution of the dredged part of the long-term study area in Ager0. with a central station (station 6) and three stations in a semicircle, make an analysis of environmental gradients impossible because no relation between similarity and distance can be established because the distances from station 6 to all three stations in the semicircle are equivalent. Impact on Suhslratum Amounts iif shell debris and gravel differed significantly be- tween dredged and control stations in both areas (Fig. 4a and b). In Logstor Broad, the weights of shell debris ranged from 0.5 kg m"" at station 7 and 8 in the fished area to approximately 2 kg m"" at station 9 and 10 in the control area. In the Agero area, the weights of shell debris ranged from 0 kg m"" at station 21 and 25 in the fished area to I kg m~" at station 15 in the control station. Gravel was only found at the stations in Logstor Broad ranging from 0 kg m"" at station 7 in the fished area to 0.7 kg m"" at station 9 in the closed area. The nested ANOVA analyses for both areas showed a significant effect of dredging on the weights of shell debris (P = 0.000) whereas no significant differences were observed among stations when nested to the fished/control areas (Logstor B: P = 0.467. AgerO: P = 0.215). The weights of gravel were not only significantly affected by the dredging (P = 0.000). but also by the stations {P = 0.001 ). Plotting the Shannon-Wiener diversity (H') index in the samples from Logstor Broad as a function of the amount of shell debris a significant (P < 0.01) positively correla- tion was obtained for the data from the fished area. The similar relationship hold constant in the permanently closed area (Fig. 5). As the amount of shell debris in the Agero and the weight of pebbles in Logstor broad are low at the fished stations, similar relations cannot be plotted for these data. The relationship between H' and the mussel density (Fig. 6) showed a similar pattern. A significant positively correlation (f < 0.01) was obser\ed in the area open to mussel dredging whereas in the pennanently closed area only a trend can be detected (P = 0.09) DISCUSSION To achieve a holistic sustainable management of fisheries, knowledge of the fishery impacts on the target populations and the ecosystem is required. In respect to fishery impact on ecosystems, such management has to consider both the short-term and long- term impact and the recovery time for the ecosystem to re-establish a habitat. This study describes significant differences in the species composition among stations in areas tlshed four months before sampling and control stations. A number of taxa (poriferans, echi- noderms. anthozoans. molluscs, crustaceans, and ascidians) had a reduced density or were not observed in fished areas four months after the fishing was ended. These differences included reductions in both mobile and sessile species. Differences in the species com- position were also observed between an area that had not been fished for more than four years and a closed control area. A sig- nificant reduction in shell debris and gravel was observed in dredged areas. The observed differences between stations in fished and closed areas may be due to mussel dredging but could also be caused by environmental gradients in the study area (e.g., wind exposure, water current, sediment composition, primary production). A MDS plot relating similarity and spatial distance between stations, how- ever, did not indicate any relation in the short-term areas, suggest- ing that the observed differences in species compositions are due to dredging activity and not due to environmental gradients. As the distance between the closest dredged and control stations in the short-term in Agero and Logstor Broad is 750 and 200 m, respec- tively and that the demarcation line between fished and closed area was drawn without any reference to physical conditions in the area, it is reasonable to conclude that no environmental gradients were involved. The stations used in the long-term impact analysis had a more extensive distribution, with <10 km between the most distant stations. In Logstor Broad, the dissimilarity was related to the distance between stations, indicating that a gradient probably in- fluenced the area. This environmental gradient may increase the variability in species composition, making it more difficult to de- tect long-term impact of the fishery. In the Agero area, the way the stations were distributed made the detection of an environmental gradient difficult. Consequently, it is difficult to judge whether the observed differences in species compositions were due to long- term impact of mussel dredging or to a gradient. The design of fishery impact studies is a trade-off between a robust experimental design and the amount of effort that can be invested in the study. An often-used statistical design is a BACI TABLK 1. Short-ttrm impact: species contributing to the dissimilarity {%) between fished stations 7-8 and control stations 9-10 in a closed area in Legster Broad. Mobility Dissimilarity ( % ) St 7- -8 Dredged St 9- -10 Control Mean 2SE Mean 2 SE 1 test P Corella parallelogrammu S 37 O.Ot) (1 III! 16.00 8.25 0.00** Macropodia rostrota M 14 0.00 u.oo 0.33 0.29 0.02** Cran^on crangon M 10 0.07 0. 1 3 0.60 0.46 0.03* Mvliliis cthilis S 9 15.40 9.85 1 5.00 7.70 — Sagania irogladxtes s 8 0.73 0.52 3.40 0.93 0.00** Metridiitni senile s 7 0.13 0.19 0.07 0.L3 0.56 Carcinus maena.s M 6 0.00 0.00 0.13 0.19 0.16 Mobile species .W 0.07 — 1.06 — Sessile species 61 16.26 — 34.47 — The species are separated into mobile species (M) and sessile species (S). The pooled mean density (individuals m'-) and 2 SE are given and the differences in densities between dredged and undredged areas are compared with I tests (**P < 0.025. *P < 0.05). Pooled dissimilarity contribution and mean-densities are given for two functional groups. Only species contributing more than 4% to the dissimilanty are included in the table. Mussel Dredging 533 TABLE 2. Short-term impact: species contributing to tlie dissimilarity (Vr) between fished stations 21-25 and control stations 15-26 in a closed area in the Ajjero area. St 21-25 Dredged St 15-26 Control Mobility Halichondria panicea S Asterias rubens M Sagarlia troglodytes S Teuita felina S Metridium senile S Carcinus maenas M Macropodia rostnita M Mytilus edulis S Buccinum imdatuni M Hinia reticulata M Crangon crangon M Mobile species Sessile species Dissimilarity % lb 12 11 10 6 6 6 5 42 51 Mean u.ou 0.27 0.27 0.07 0.93 0.60 0.07 0.00 0.07 3.87 1.67 6.55 1.27 2SE O.UO 0.26 0.32 0.13 0.98 0.37 0.13 0.00 0.13 0.95 0.72 Mean 0.40 1.00 2.27 0.40 2.33 1.80 0.00 0.07 0.00 7.20 1.27 11.27 5.47 2 SE 0..^7 0.44 1 .06 0.31 1.89 0.67 0.00 0.13 0.00 1.72 0.62 ( test P 0.03* 0.01*' 0.00*'' 0.05* 0.19 0.00*< 0.32 0.32 0.32 0.00*' 0.40 The species are separated into mobile species (M) and sessile species (S). The pooled mean density (individuals m"-) and 2 SE are differences in densities are tested with / tests (**P < 0.025. *P < 0.05). Pooled dissimilarity contnbution and mean-densities are given for groups. Only species contributing more than 4% to the dissimilarity are included in the table. given and the two functional Test area Short-term impact Logstor Br. Short-term impact Agero Long-term impact Logstor Br. Long-term impact Agero Test on functional groups Mobile species Logstor Br. Sessile species Logstor Br. Mobile species Agero Sessile species Agero TABLE 3. ANOSIM analyses of epifauna species composition and density. Similarity of Treatment Stations ( % ) Data for .Analysis of Fishery Impact Fishery Impact Global R 0.1-2 11^1 0.1 0.1 4-38 19-25 0.1-2 0.1-91 Complete set of data 0.224 Complete set of data 0.066 Average ranking -0.093 Average ranking 0.363 Complete set of data -0.082 Complete set of data 0.3 Complete set of data 0.016 Complete set of data 0.039 Fishery Impact Pi 9c) 0.1 0.2 77.1 2.4 94 0 36 4 The data in each area was analysed in two steps. First it was tested if there was difference among stations within each treatment area (fished contra closed), and if there were differences (P < 1%) then the analysis of the fishery impact was conducted on average rankings. If no difference was observed, then the impact analysis was conducted on complete sets of data. TABLE 4. Long-term impact: species contributing to the dissimilarity ("7f ) between fished stations (6, 8, 9, 24) and control stations (1. 5, 7, 10, II) closed area in the Agero area. Hinia reticulata Crepidula fornicata Crangon crangon Ascidiella aspersa Metridium senile Carcinus maenas Dissimilarity (%) 28 22 11 7 6 5 Dredged Control Mean 2SE Mean 2SE / test P 3.90 0.66 13.20 1.71 0.00** 0.00 0.00 62.40 20.98 1.53 0.41 3.79 0.79 0.00** 0.00 0.00 5.28 2.50 0.01** 0.10 0.11 2.32 0.70 0.00** 0.67 0.27 2.51 0.5S 0.29 The mean density (individuals m"') and 2 SE are given and the differences in densities are tested with ; tests {'• contributing more than 4% to the dissimilanty are included in the table. *P < 0.025. *P < 0.05). Only species 534 DOLMER (a) 5,.. , 7 24 : ••••1 10 ■■' ■■■ y 25 \ ■■•\6 -•'■•• „. 8 ... 9 11 2/-'' \ Agero 21 stress=0.06 (b) .15 14.:-iV; 20 >/'..'.^V""''3 19 12 ■^ \. 10 18/ Logstor Br. stress=0.07 8 Figure 3. MDS plot ol station means in the Agero area (a) and Logstor Broad (b). The spatial relationship between stations is indicated with a dashed line between the nearest stations in the long-term study areas, and with a solid line in the short-term study areas. design (Bet'ore-After-Control-lmpact) that also tests for temporal and spatial variability in a study area. It is important for the results of experimental impact studies of trawling and dredging that im- pacted areas are large (Thrush et al. 1995). A meta-analysis of the impact of demersal fishing gear on benthos (Collie et al. 2000) demonstrated that the recovery time is shorter in small impacted patches because of larger edge effects. The results of experimental studies may then be strongly dependent on the experimental de- sign. This study investigated the impact of an unplanned mussel fishery, in which the scale of the study area is very large compared with the experimental studies. This ensures that the results are not affected by the experimental design. Contrary, the character of the study implies that a causal relation between the observed varia- tions in species and substrate compositions and fishery impact not can be established, although the study represents strong indices of the impact of dredging. Fishery impacts may significantly affect ecosystem function. Seme of the species that were partly or fully eliminated from fished areas were sessile filter feeders (poriferans. anthozoans, molluscs, and ascidians). In shallow-water bodies such as Limf- jorden. these filter feeders exert an important control mechanism over the pelagic primary producers (Cloem 1982, 1991, Petersen (a) fished control ^ 3000 ^ 4 ^ 4 ^ station (b) 1500 n fished control 1000 E 2 SI 55 500 - 0 -^= station Figure 4. The weights of shell debris (solid linel and gravel (dashed line) at two fished stations and two stations in a closed area in Logstar Broad (a) and the Agero area (b). The weights are given as mean ± SE. & Riisgiird 1992, Dolmer 2000) and reduction of a benthic filtra- tion capacity may weaken bentho-pelagic coupling. Apart from the filtration capacity, the coupling between the pelagia and benthos depends on the mixing rates of the water column and the transport rates of material and energy from the water column to the benthos. Seabed roughness is a determinant of near-bed mix- ing (Herman et al. 1999), so smoothing of the seabed by remov- ing mussel beds and larger particles reduces the transport of par- ticles to the seabed and reduces the population filtration rates. Consequently, the fishery interferes with benthic filtration both by removing filter feeders and by changing near-bed hydrodynamics. The distribution of blue mussels was patchy, and in L0gst0r Broad the density of mussels in the fished area was slightly higher than in the permanently closed area. This suggests that the density 1.5 0.5 o o oo % HUH Fn , , 1 o 1 1 Mussel Dredging 2 535 1000 2000 3000 4000 shell debris (g m'^) 5000 6000 Figure 5. Relationship between the amount of shell debris and Shan- non-Wiener indices for each sample at stations 7-10 in Logstor Broad. (D) indicates samples from the area open to mussel dredging and ( I indicates closed areas. Data were analyzed with linear regression. The dotted line shows the regression on data from the fished area (R' = fl..^85; P < 0.01 ), and the solid line that on data for the closed area {R- = 0.017; P > 0.05). I 1 0.5 O D IB O ° D ° 0 mBBD — Q — B- 10 15 20 25 Mytilus edulis {indviduals m ) Figure 6. Relationship between density of blue mussels and Shannon- Wiener indices for each sample at stations 7-10 in Logstor Broad. (D) indicates samples from the area open to mussel dredging and (O) indicates closed areas. Data were analyzed with linear regression. The dotted line shows the regression on data from the fished area {«' = 0.470; P < 0.01), and the solid line that on data for the closed area (R- = 0.152; P = 0.09). of mussels was much higher m the fished area than in the closed area before the fishery was initiated. As the mussels fonn a bio- genic substratum important for associated fauna, mussel density may influence the distribution of other species. A higher initial mussel density in the area open to mussel dredging, as in the short-term study area in L0gst0r Broad, would result in an under- estimate of the impact of mussel dredging, as a more diverse associated fauna probably characterized the fished area before the fishery was initiated. In L0gst0r Broad, no long-term impact of mussel dredging was apparent. A previous study also failed to show long-term effects (Hoffmann & Dolmer 2000). In the present study, the multivariate analyses of long-term impacts was conducted on average ranked data resulting from differences among stations within each treat- ment area. The power of this analysis is much lower than an analysis of the complete data set. which can be used when no difference is observed among stations. To detect an impact ot mussel dredging fishery impact must be separated from the noise of other factors affecting the ecosystem (Jennings & Kaiser 1998). Limfjorden is almost bi-annually perturbed by extensive oxygen depletion lasting for several weeks resulting in mass mortality of mussels and other benthic invertebrates. In 1994 and 1997 oxygen depletion caused mortalities of 25-50% of the mussel populations. The scale of these events has to be considered when evaluating the impact of the mussel fishery. Seabed heterogeneity and complexity is an important feature when discussing the character of benthic habitats. This study has shown that mussel dredging removes larger sessile animals, shell debris, and gravel from fished areas. Furthermore, dredging re- moves mussels forming an important biogenic substratum for as- sociated fauna. The investigations measured the largest impact on sessile species, but the fishei^ also affected mobile species after 4 mo. Mussel and shell beds are reported to reduce predation by creation of spatial refuges (Thiel & Dernedde 1994. Lee & Kneib 1994) and Kraeuter and Castagna ( 1977) reported that Mercenaria mercenaria had a 75% better survival when reseeded on shell and gravel substrata than on sand. The impact on epibenthic species is 2-fold: a direct impact of mussel dredging by killing and injuring individuals by direct contact with the mussel dredge and an effect from the changed habitat heterogeneity and complexity. The re- covery of the fished habitats is a function of immigration of mobile species and recruitment of sessile and mobile species. Recruitment may also be affected by the changed seabed structure. In many invertebrates larval settlement depends on solid substrata (Young 1983. 1985. Witman & Suchanek 1984. Baker 1997. Lapointe & Bourget 1999) but also changed near-bed hydrodynamic forces may modify larval distribution (Butman 1987, Jonson et al. 1991 ). Changing the handling of benthic animals and materials in the fishery by-catch can accelerate the recovery process of ecosystems following mussel extraction. Today mussels below the legal mini- mum size (<4.5 cm shell length) and waste material (other animals and shell debris) are relayed in certain areas (Kristensen & Lassen 1997). Mussel shells from the cooking process must not be re- cycled to the seabed and are deposited or used on land. As a consequence of this procedure, important materials are transported away from the fishing grounds. These important materials should be brought back to fished areas. Habitat restoration by relaying small inussels and other invertebrates, shell, pebbles, and larger stones after mussel dredging would reduce the recovery-time both in respect of the form and the function of the ecosystem and would improve the sustainability of the mussel fishery. The conclusion of this study is that the mussel dredging exerts a significant short-term impact on the benthic fauna. No unequivo- cal long-term impact could be demonstrated. The fishery changes the seabed structure by removing solid particles and biogenic par- ticles. This impact may be long lasting or even irreversible and may significantly change the function of the ecosystem. ACKNOWLEDGMENTS The authors thank Prof J Collie and Dr. S. A Ragnarsson and three anonymous referees for thoughtful comments and linguistic corrections on the manuscript, and to A. Hansen for practical help in the field. 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Effects of physical trawling disturbance in a previously unfished sheltered Scottish sea loch. Mar. Ecol. Prog. Ser. 162:227-242. Witman. J. D. & T. H. Suchanek. 1984. Mussels in flow: drag and dis- lodgement by epizoans. Mar. Ecol. 16:259-268. Young. G. A. 1983. The effect of sediment type upon the position and depth at which byssal attachment occurs in Mytilus edulis. J. Mar. Biol. .Ass. U.K. 63:641-651. Young. G. A. 1985. Byssus-thread formation by the mussel Myiihis edulis: Effects of environmental factors. Mar. Ecol. Prog. Ser. 24:261-271. Jintrmil of Slwllfixh Ri:u'uivh. Vol. 21. No. 2. 539-547, 2002. FOULING IN SCALLOP CULTIVATION: HELP OR HINDRANCE? KATHERINE A. ROSS, JOHN P. THORPE, TREVOR A. NORTON, AND ANDREW R. BRAND* Port Erin Marine Laboratory, University of Liverpool, Port Erin. Isle of Man. British Isles. IM9 6JA AB.STR.ACT We examined the effects of fouling on physiocheniical and food conditions inside nets used for suspended great scallop iPecten imixitiitis) cultivation. Conditions in clean nets and sites with no nets were similar for the parameters studied and differed fiom those in fouled nets. Fouling of nets reduced water movement and. contrary to cominon assuinptions, was associated with high levels of plankton and detritus. Obvious negative effects of fouling (e.g., accumulation of inorganic matter or nitrate and ammonia) were absent. It was concluded that in some areas negative effects of fouling may be caused by foulers parasitising or mechanically interfering with scallops rather than creating an unfavorable environment. Our results have important consequences for scallop growers, research- ers modelling cultivation in an ecosystem context, and those investigating relationships between the growth of cultivated scallops and environmental conditions. KEY WORDS: scallop, aquaculture. biofouling. Pecien nui.xiimis. suspended culture, pearl nets, environmental conditions, particulate matter, Irish Sea INTRODUCTION Immersion in plankton-rich water generally enables scallops in suspended cultivation to grow faster than on the seabed, under natural conditions (MacDonald & Thompson 1985. Wallace & Reinsnes 1985. Hardy 1991 1. Unfortunately, the conditions that promote scallop growth also encourage fouling of cultivation nets and scallop shells. This is costly because it increases the weight and drag of cultivation equipment and is difficult to remove (Hardy 1991 ). Fouling can also affect scallop growth and appear- ance. Fouling of scallop shells and cultivation nets has been shown to reduce the growth of immature scallops (Claereboudt et al. 1994a, Lodeiros & Himmelman 1996. 2000). It is assumed that fouling organisms reduce scallop growth by competing for food and space or by reducing water flow through nets, and hence the supply of food and o.xygen and removal of waste products (Duggan 1973, Leighton 1979, Huguenin & Huguenin 1982. Cote et al. 1993. Enright 1993, Claereboudt et al. 1994b. Lodeiros & Himmelman 1996. Lu & Blake 1997), Fouling of scallop shells increases the weight of upper valves and can bind upper and lower valves to- gether. This increases mortality or reduces growth, probably be- cause scallop feeding and respiration is inhibited (Paul & Davies 1986. Minchin & Duggan 1989, Roman 1991, Lu & Blake 1997, Lodeiros c& Hiinmelman 2000). Other potentially negative effects of fouling include irritation of the scallop mantle (Getchell 1991, Mortensen et al. 2000) and parasitism by species, including poly- chaete worms and amphipods (Leibovitz et al. 1984. Mortensen et al. 2000). However, the influence of fouling varies. Some re- searchers have concluded that fouling does not affect bivalve growth (Wallace & Reinsnes 1985, Widman & Rhodes 1991. Lesser et al. 1992, Lodeiros et al. 1993, Lodeiros et al. 1999). whereas other work suggests that fouling can have beneficial ef- fects (Ross 2002). Potentially beneficial effects include a positive influence on plankton abundance (inveilebrate assemblages re- lease nutrients, promoting primary production. Dame & Dankers 1988, Asmus & Asmus 1991, Peterson & Heck 1999. Arzul et al. 2001, Mazouni et al. 2001) and. in areas of high flow, reduction of fast water currents that might otherwise inhibit scallop feeding (Cote et al. 1993. Devaraj & Parsons 1997). The present study aimed to determine how fouling on pearl nets alters the environment for great scallops [Pecien ma.ximiis (L.)). Divers collected water samples from clean and fouled nets (both containing scallops) and from sites with no nets (i.e., the water column nearby), enabling us to isolate the influence of fouling on physiochetnical and food conditions. This is the first investigation to examine environmental conditions inside bivalve cultivation nets and thus provides a unique description of how fouling could affect scallop growth. Fouling is typically countered by frequent net cleaning with high-pressure water hoses or regular net changes (Hardy 1991. Laing & Spencer 1997). Both methods are labor intensive, increase equipment requirements, and are thought to stress scallops, reducing their growth rates (Wildish & Kristman- son 1988, Parsons & Dadswell 1992. Enright 1993. McDonough 1998, Ross 2002). The results of this experiment should help growers to tackle fouling efficiently, thereby saving time and money. The study was conducted in an exposed Irish Sea location off the Isle of Man. However, the major foulers (hydroids and amphipods) are ubiquitous (see e.g., Hidu et al. 1981. Arakawa 1990. Enright 1993. Claereboudt et al. 1994a) and thus the results are likely to be relevant where scallop cultivation is perfonned in other areas with hich water flow. MATERIALS AND METHODS Field Work ♦Corresponding author. E-mail: arbrand@liv.ac.uk Two longline systems (subsequently referred to as the north and south systems) were positioned off the Southwest coast of the Isle of Man in approximately 23 m of water (Fig. 1). Longline head-ropes were approximately 10 m below the sea surface. The tidal range in this area is 6 m and peak flows are approximately 1 m/s. Experiments were performed in June 2000 during spring tides. Gross tidal flow runs parallel to the systems, but nearby rocky outcrops cause erratic local flow patterns that sometimes extended to the south system. Salinity is 34 ppt. and water tem- peratures reach a summer maximum of about 15°C and winter minimum of 6°C (T. Shammon. personal communication 2(;)01). To the north east of the longlines untreated sewage from Port Erin (population ca. 2.800) is discharged in the lower intertidal (Fig. 1 ). Pearl nets were hung in strings of three, with a 2-kg weight attached below the lowest net. The nets had a plastic covered square wire frame base with sides of 34 cm and black, monofila- tnent mesh with 16 mnr spacing. The tnesh is the same as that 539 540 Ross ET AL. Figure 1. Location of longline systems off the Isle of Man. Irish Sea. commonly used for lantern nets, and thus the results of these investigations are probably relevant for on-growth in both pearl and lantern nets. For logistical reasons only the top nets, hung about 0.15 m below the head-rope, were sampled. The nets used for nutrient samples contained ten 2-3 yr old P. nui.xinnis with a shell length of 65-85 mm. For water flow experiments, nets con- tained 10 flat pebbles whose combined weight equalled that of 10 scallops. Pebbles were used instead of scallops because scallop movement may have abraded the plaster balls, leading to inaccu- rate estimates of water motion. Scallop-sized pebbles were chosen so that water flow and net movements matched those of nets con- taining scallops. Nets for flow measurements had loops in the central, supporting rope, and a door so that plaster balls could be inserted and fixed centrally. Door fastenings and support were on net seams so that they did not alter water flow. Ammonia, nitrate, particulate matter, plankton, and water mo- tion were measured in clean nets, fouled nets, and open water sites. Fouled nets had been immersed for 16 weeks before the sampling whereas "clean" nets had been deployed for only 2 wk. Open- water sites were positions under the head-rope of the longline. at the same depth as experimental nets. Treatments were arranged randomly, at 1-min intervals, along the two systems. On each system, treatments were replicated five times for nutrient experi- ments and four times for water flow experiments. Nutrient and water motion experiments were perfoimed side by side on the longlines. Water motion was measured for 48 h. during which time water samples were collected. Diving was performed from the R,V. Sula. Syringes with 120- mm Teflon tubing tips (.3-mm diameter) were used to collect samples in preference to permanent sampling tubes (which would have become fouled) or electronic probes, which are difficult to use accurately in situ. Divers collected a complete set of 15 or 20 nutrient samples (for example, all of the ammonia samples from one longline) using labeled syringes. To prevent disturbance, nets were not touched or moved either before or during sampling. To remove any trapped debris from the syringe tip, 5 niL of water was taken up outside the nets and expelled once the tip was in position. Water samples were then collected slowly to minimize disturbance and to avoid sampling water from outside the net. Dives lasted a maximum of 20 min, after which samples were returned to the boat. Samples were collected from nets by two pairs of divers deployed at lO-min intervals. Sampling was alternated between longlines so that no more than two samples, totalling 160 mL of v\ater ( 1 % of the net volume), were taken from a net in 4 h. Before sampling, all bottles and syringes were washed in dilute acid and rinsed in distilled water. I'hysiochemical Conditions Ammonia and nitrate were measured in lOO-niL water samples, which were kept in the dark on ice during the short boat journey back to the laboratory. In the laboratory ammonia and nitrate samples were filtered, through GF/F papers, into bottles and fro- zen. An Alpkem autoanalyser (RFA 2) was later used to determine nutrient concentrations. Plaster of Paris spheres can be used to accurately measure time integrated water motion (Thompson et al. 1994); in these experi- ments, they proved to be a reliable alternative to expensive micro- flow meters. The spheres were made by combining 100 g of Plaster o\ Paris (CaSOj) with 90 mL of distilled water. The plaster was mixed to a smooth paste, tapped to remove air bubbles, and then poured into moulds. Moulds were plastic spheres (70-mm diam- eter) with a central wire. Filled moulds were vibrated for 10 min to remove trapped air. Plaster spheres were removed from their moulds after approximately 12 h and placed in a well-ventilated area to dry. After at least 4 wk. spheres were dried at .30°C to a constant mass (accelerated drying at high temperatures can affect the crystalline structure of CaSOj (Muus 1968). Before immersion, plaster spheres were wrapped in soft cloths to prevent chipping and to minimize dissolution. Divers opened nets and fixed spheres centrally so that they were not abraded by contact with fouling organisms or nets. Al open water sites, wire was used to suspend spheres below the head-rope, at the same level as spheres inside nets. Once in position, the cloths were removed and the nets were resealed. After 48 h, divers retrieved spheres and wrapped them in soft cloths before returning to the boat. In the laboratory, the spheres were dried to a constant mass and their final surface area was calculated from volume measurements obtained by fluid displacement. Plaster dissolution rates (V,) provide an indication of relative water motion; K/ = (W| -wo Where VV, and W, are the weight of the sphere at the beginning and end of the experiment respectively, A is the mean surface areas of the sphere, calculated from start and end values, and T is the time over which spheres were immersed (Thompson et al. 1994). Food Conditions Water samples ( 100 niL) for particulate matter analysis were kept in the dark on ice during the boat journey back to the labo- ratory. Particulate matter was filtered onto preashed papers imme- diately on return to the laboratory. The papers were rinsed with isotonic ammonium formate and then dried at 40°C to a constant weight and ashed overnight at 450°C. The GF/F filter paper used to collect particulate matter had a pore size of 0.7 p-m. Particulate organic matter (POM), particulate inorganic matter (PIM), and total particulate matter (TPM) were calculated as follows: POM= dry weight of filter paper and sample - ashed weight of filter paper and sample Fouling in Scallop Cultivation 541 TPM = dry weight of filter paper and sample - filter paper ashed weight PIM = ashed weight of filter paper and sample - filter paper ashed weight As soon as the water samples were taken up to the boat. 60-mL samples were transferred to bottles with 1 .2 niL of neutral LugoFs iodine. The bottles were stored in the dark until plankton were counted, measured, and categorized using inverted microscopy and the computer programme SCION image analysis for Windows. Samples were settled in a counting chamber following the methods of Utermohl (Hasle 1978). Dense samples were diluted with fil- tered seawater so that all of the plankton in the chamber could be counted. To ensure that the precision of plankton counts was greater than 20% of the total count, the volume of sample enu- merated always contained more than 130 individuals of the most abundant organisms (Postel et al. 2000). Organisms were recorded according to type (small plankton, centric diatoms, pennate dia- toms, diatom chains, solitary chain-forming diatoms, pelagic cili- ates, benthic ciliates, dinotlagellates, flagellates, crustaceans, nem- atodes, invertebrate larvae, and eggs and spores) and maximum .^ o W X tion was used to slightly reduce the contributions to similarity of the most abundant species). The similarity matrices were ordinated and clustered using non-metric multi-dimensional scaling (MDS) and hierarchical agglomerative clustering (on group-average link- age), respectively (Clarke & Warwick 1994). The two-dimensional MDS plot had a low stress value and hence the dendrogram from CLUSTER analysis is not presented here. Instead, levels of simi- larity from cluster analysis are indicated on the MDS plot (Fig. 4). A priori tests of the differences between locations and treatments were performed using a two-way, crossed ANOSIM (analysis of similarity), and the plankton groups contributing most to any dif- ferences found between the groups were determined using length (5-10, 11-20, 21-50, 51-100, and >100 |jim). Small plank- ton were all organisms of 50-10 pirn; generally these were flagel- lates and diatoms. Statistical Analyses The experimental design was balanced; location was a random factor with two levels, north and south systems, and treatment was a fixed factor with three levels, open-water sites, clean nets, and fouled nets (Underwood 1997). Physiochemical conditions were measured in five replicates per treatment for each system, but time constraints meant that plankton data were obtained only for three replicates per treatment-longline combination. Concentrations of plankton and nutrients and rates of plaster erosion for each treat- ment were examined using two-way analysis of variance (ANOVA). Heterogeneity of variance was tested for using Co- chran's test (Winer 1971) and where necessary data were trans- formed. Some data were heterogeneous even after transformation, but ANOVA was still applied because the experimental design was balanced and large (Underwood 1997). However, such analyses increase the probability of a type I error, and therefore significant results should be interpreted with caution. When ANOVA showed that the probability of a treatment effect exceeded 0.05 and there was no interaction between location and treatment [P > 0.25). data for the two systems were pooled, thus increasing the power of ANOVA to detect treatment effects (Underwood 1997). When ANOVA indicated significant factors or interactions between fac- tors, post-hoc Student-Newman-Keuls tests were performed to determine which means differed. All analyses were performed using GMAV5 (Underwood et al. 1998). Ammonia measurements were analyzed by ANOVA and concentrations below the limit of detection were included as 5 (Jig/L, the highest undetectable value. This conservative approach increased the probability of type II error. As described above, plankton were classified as one of 45 groups according to their size and type. The data set was then analyzed using nonparametric, multivariate techniques included in the PRIMER (Plymouth Routines in Multivariate Research) soft- ware package (Clarke & Warwick 1994). Bray-Curtis similarity indices (Bray & Curtis 1957) were calculated between all pairs of samples to produce a data matrix (after a square-root transforma- E o bj c o o CO CO zi. 0 CO _i CD 'c o E E < 0.4 0.3 0.2 0.1 0.0 12 10 8 6 4 2 0 20 15 10 - ■ r- — 1 T p E 1 open- water clean fouleid North system open- water clean fouled South system Figure 2. Physiochemical conditions. A, Water motion; B. nitrate con- centrations: C. ammonia concentrations (mean ± SE) in clean and fouled pearl nets and open-water sites. 542 Ross ET AL. SIMPER (similarity percentages analysis. Clarke & Warwick 1994). RESULTS Macrofouling Communities Fouled nets were almost entirely covered (>909f) in the hy- droids Tubidaria indivisa L. and T. kuynx Ellis & Solander: the amphipod Jassa falcata (Montagu) and its silt tubes were common as were the nudibranchs Dendnmotus frondosiis ( Ascanius). Cory- phelki lineata (Loven). and Facelina bostnniensis (Couthouy). Small hydroids (e.g.. Obelia sp. and Clyiia hemispherica (L.)) were present but occupied little space compared with the large TubuUuia spp. "Clean" nets were sparsely colonized (<59(') by small hydroids. Physiochemical Cimditiuns The plaster spheres used to measure water motion remained spherical throughout their deployment. Water motion was similar on both longline systems (Fig. 2) and was only significantly re- duced by fouled nets (Table 1 ). Nitrate and ammonia concentra- tions were not affected by treatment (Fig. 2). However, only four ammonia measurements exceeded the minimum detection level of the autoanalyser (5 |jig/L): these were all on the north system, which therefore had significantly higher rates of dissolution than the south system (Table 1 ). Food Conditions Total particulate matter was most abundant on the south system and here there was significantly more in fouled nets compared with clean nets and open-water sites (Table 2). Although this trend was apparent on the north system (Fig. 3), differences were smaller and not significant. The ratio of PIM to POM was lowest in fouled nets, for both noilh and south sy.stems (Fig. 3. Table 2). Micros- copy revealed that particulate matter included detritus and inver- tebrate faeces, in addition to plankton. Plankton larger than 21 |xm were rare and thus numbers in the three largest size classes were pooled for univariate analysis. Total plankton and plankton in size classes 5-10 |xm and 11-20 |i.ni were most abundant in fouled nets (Table 2). Multivariate analysis also distinguished fouled nets from clean nets and open water sites (which clustered together on the MDS. Fig. 4). Interestingly fouled nets from the two systems were also distinct. ANOSIM revealed significant differences between both locations and treatments (/? = 0.42, P < 0.01 and R = 0.57, P < 0.01, respectively). Pairwise comparisons found significant differences between fouled nets, open-water sites [R = 0.78. P = 0.01 ). and clean nets (R = 0.89, P = 0.01), but, as indicated by the MDS plot, open- water sites, and clean nets, contained similar communities {R = 0.278. P = 0.08). SIMPER analysis showed that high abundances of centric diatoms, flagellates, pennate diatoins (all 5-10 )a.m). and small plankton primarily distinguished fouled nets from clean nets and open water sites. High abundances of these organisms also distin- guished samples from north and south systems. Although less common, eggs and spores, nematodes, pennate diatoms, and in- vertebrate larvae also appeared inost frequently or uniquely in fouled nets. Consistently low plankton abundance explains the atypical position of one sample from fouled nets on the MDS plot (Fig. 4): despite its low content, this replicate contained most of the plankton that characterized other samples from fouled nets. DISCUSSION Fouling of cultivation nets created a unique environment for the scallops inside: physiochemical and food conditions differed from those both in clean nets with scallops and those in the water TABLE 1. Two-way ANOVA and Student-Newman-Keuls multiple comparisons testing for the effect of location and treatment (clean nets, fouled nets, and open-water sites) on pliysiocliemical conditions. Source of Variation df MS F P F Ratio \ ersus Water motion C = 0.4799. P > 0.05 Location 1 0.0012 3.18 0.091 Residual Treatment 2 0.0425 196 0.005 Location x treatment Location x treatment 2 0.0002 0.57 0.575 Residual Residual 18 0.0004 Total 23 SNK multiple comparison of treatment results; North system: open-uater = c ean > fouled Nitrate C = 0..^539. P > 0.05 Location 1 0.1242 2.22 0.149 Residual Treatment 2 0.1141 2.91 0.256 Location x treatment Location x treatment 2 0.0392 0,70 0.506 Residual Residual 24 0.0558 Total 29 Ammonia C = 5574. P < 0.05 Location 1 160.5453 4,41 0.046 Residual Treatment 2 2 1 .8963 1.00 0.500 Location x treatment Location x treatment 2 21.8963 0.60 0.556 Residual Residual 24 36.3827 Total 29 Cochran's test results are given. (Bold type mdicates a significant result. P < 0.05). Fouling in Scallop Cultivation 543 TABLE 2. Two-way ANOVA and Student-Newnian-Keuls multiple comparisons effect of location and treatment (clean nets, fouled nets, and open-water sitesi on particulate matter and plankton. Source of Variation df MS F P F Ratio Versus TPM C = 0.3013. P>0.05 Location 1 1040 7.08 0,014 Residual Treatment 2 2378 4.35 0.187 Location X treatment Location x treatment 2 547 3.73 0.039 Residual Residual 24 147 Total 29 SNK multiple comparibon of interaction: Open- water: North system = South system North system: open-water = clean = fouled Clean: North system = South system South system: open-water = clean < fouled Fouled: North system < South system PIM:POM Transformation = Ln (X + 1 ). C = 0.4331, P > 0.05 Location I 0.0003 0.00 {).444 Pooled data Treatment 2 0,3369 5.61 0,001 Pooled data Location x treatment 2 0.0293 0.49 0,620 Pooled data Residual 24 0.0627 Total 29 Pooled data 26 0.0601 SNK multiple comparison of treatment results: Open- water = clean > fouled Plankton 5-10 |xm Transformation = Ln (Xl, C = 0.7020. P < 0,05 Location 1 0.15 0.17 0,686 Residual Treatment 2 11.44 22..56 0,042 Location x treatment Location x treatment 2 0.51 0,58 0,576 Residual Residual 12 0.88 Total 17 SNK multiple comparison of treatment results: Open- water = clean < fouled Plankton 1 1-20 |xm Transformation = Ln (X + 1), C = 0.7042, P < 0.05 Location 1 0.15 0,18 0,678 Pooled data Treatment 2 11. .34 13,88 0,001 Pooled data Location x treatment 2 0.50 0,61 0,559 Pooled data Residual 12 0.87 Total 17 Pooled data 14 0.82 SNK multiple comparison of treatment results: Open- water = clean < fouled Plankton 21-H jjim Transformation = Ln (X + 1), C = 0.5244. P > 0.05 Location 1 0.97 7,26 0,020 Residual Treatment 2 1.85 5,24 0,160 Location x treatment Location x treatment 2 0.35 2,66 0,111 Residual Residual 12 0.13 Total 17 Total plankton (>5 ixm) Transformation = Ln (X + 1). C = 0.4092, P > 0.05 Location 1 279000 1,25 0,283 Pooled data Treatment -) 16300000 7.29 0,007 Pooled data Location x treatment 2 2890000 1.29 0.305 Pooled data Residual 12 2130000 Total 17 SNK multiple comparison of treatment results: Open water = clean < fouled The results of Cochran's test are given, (Bold type indicates a significant result. P < 0,05,) nearby. Other work has indicated that sometimes scallops in fouled nets grow faster than scallops in clean nets (Ross 2002). High water flow can inhibit scallop feeding (Wildish & Saulnier 1993, Claereboudt et al, 1994b. Skjaeggeslad 1997) and it could be that, in a high current area, heavy fouling aids scallop feeding by roughly halving water movement. Similarly. Skjaeggeslad (1997) found that water-motion inside plastic cages was reduced by up to 68% by fouling. In addition, fouling might prevent seston deple- tion around scallops by creating turbulent flow and actively mixing the water (Frechette et al. 1989. Larsen & Riisgard 1997), Ammonia and nitrate concentrations were similar for all treat- ments, indicating that neither clean nor fouled nets caused a build 544 Ross ET AL. O) E O Q. E c CD cn O c o c ro Q. 70 60 50 40 30 20 10 0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7000 6000 5000 4000 3000 2000 1000 0 open- water clean Z^ '^rfifA fouled North system open- water clean fouled South system Fijjurt 3. Food conditions. A, Concentrations of total particulate mat- ter (TI'M): B. ratios of inorganic to organic particles (PIM:POM); C, concentrations of 5-1(1 pni (hatched areasi, 11-20 (im (white areas), and >21 jim (grey areasi plankton (mean ± SF.), in clean and fouled pearl nets and open-water sites. up of decay or excretory products. A few apparently high ammonia measurements may have been caused by the capture of detritus in water samples (kelp detritus was abundant and the northern longline was close to a sewage outfall pipe). Inoiganic nitrogen is absorbed by phytoplankton and it is possible that high numbers of phytoplankton reduced levels of nitrate and ammonia in fouled nets. In finfish cages, fouling can reduce water exchange, leading to oxygen depletion, fish mortality, or reduced growth (Cronin et al. 1999). Similarly, reduced oxygen levels have been invoked to explain reductions in scallop growth when fouling is heavy (Hu- guenin & Huguenin 1982, Enright 1993. Lu & Blake 1997). How- ,.--" "•sV \ .SF •spy .sc~\_ .,--' /' .SC ""^ \ SC ' • JJC 1 NO. so *'"'; NO, *S0 SO"NC •■. NC '',Nf . ,' Stress = 0.04 Figure 4, MDS ordination of Bray-Curtis similarity matrix for square-root transformed plankton-abundance-data. Samples are la- belled with their location: north system (N), south system (S), and treatment: clean nets (C), fouled nets (F), and open-water sites (O). Samples are grouped at a 70% level of similarity from CLUSTER analysis. ever, oxygen concentrations have been found to track the abun- dance of autotrophic plankton, peaking in fouled nets (Ross 2002). Thus, it seems unlikely that oxygen depletion is a consequence of fouling in scallop net culture in temperate locations with high water flow. Contrary to previous suggestions (Duggan 1973. Leighton 1979. Huguenin and Huguenin 1982, Cote et al. 1993. Enright 1993. Claereboudt et al. 1994. Lodeiros and Himmelman 1996, Lu and Blake 1997). fouling did not reduce the (quantity or quality of food particles available for scallops. Instead, net fouling was as- sociated with abundant plankton and detritus and a favorable PIM/ POM ratio. Trends in plankton abundance were strong enough to be identified, despite the low number of replicates and the noto- riously patchy distribution of plankton (Hasle 1978). Proximity to the shore and to a sewage outfall may explain why the study area was characterized by high seston loadings (ca. 20 mg/L); similar loadings were found by Cranford et al. (1998) and Lodeiros et al. (1998) in the sea off Canada and Venezuela, re- spectively. High levels of organic matter in fouled nets suggest that fouling may trap, produce, and perhaps support the production of organic matter — a potential energy source for scallops. Even at high seston concentrations ( \5 mg/L) scallop scope for growth can be reduced by dilution of POM by PIM (MacDonald et al. 1998). When seston concentrations are not limiting a low PIM/POM ratio (critical values are between 3.5 and 6: the exact number varies between authors) is required by scallops to maintain a positive energy balance and maximum scope for growth (Vahl 1980. Wal- lace & Reinsnes 1985, Cranford 1995, MacDonald et al. 1998). In this study, open-water sites had PIM/POM ratios of around three, close to the critical value, and significantly higher than in fouled nets. Enhanced le\els of POM in fouled nets could thus prevent high ambient PIM concentrations from depressing scallop growth rates. Plankton communities were dominated by autotrophs of 5-20 |xm. reflecting Graziano's conclusion that 65% of primary produc- tion in the northeast Irish Sea is from phytoplankton of 5-20 |xm (Gra/iano 1988). Fouling may have encouraged primary produc- tion by releasing nutrients, by retaining plankton in a favorable light environment or by providing a substrate for benthic auto- Fouling in Scallop Cultivation 345 trophs. Increased primary production seems, in turn, to have sup- ported lieterotrophic and mixotrophic organisms such as di- notlagellates and cihates. Although this finding contradicts com- mon assumptions of those interested in shellfish cultivation, ecolo- gists recognize that beds of suspension feeders have the potential to induce the growth of more phytoplankton than they consume (e.g., Asmus & Asinus 1991). This is because suspension feeders increase local inorganic and organic phosphate and nitrate concen- trations directly through excretion and indirectly via bacterial de- cay of their faeces (e.g.. Dame & Dankers 1988, Asmus & Asmus 1991, Peterson & Heck 1999. Arzul et al. 2001, Mazouni et al. 2001). A possible mechanism by which suspension-feeding foulers and scallops could produce and retain nutrients is described in Figure 5. This is important because in most marine systems, in- cluding the Irish Sea. phytoplankton are likely to be nitrate limited at certain times of year (Allen et al. 1998. Kennington et al. 1999). Mazouni et al. (2001), for example, suggest that during summer months nutrient recycling by oyster culture units may drive pri- mary production in a French lagoon. Benthic plankton are often suspended by coastal turbulence and generally survive well in the water column (Newell & Newell 1979); thus, the prevalence of benthic ciliates and pennate diatoms in our open-water samples. However, inany benthic species were most common in fouled nets where they may have proliferated because of conditions described above or because of the presence of a solid surface onto which they could attach or settle. Benthic organisms might have been suspended by the passage of the sam- pling syringe, but movement of scallops and water currents are also likely to make them readily available as food for scallops. Increased plankton abundances in fouled nets could promote scal- lop growth because ambient levels (<600 cells/niL in this study) are unlikely ever to exceed maximum concentrations for scallop uptake or assimilation (ca. 15.000 cells/mL. Cahalan et al. 1989, Skjaeggestad 1997). Proliferation of phytoplankton in fouled nets indicates that light levels were not reduced below their compensation point, even by thick Tubulaha fouling. Perhaps strong sunlight in June penetrated the translucent stalks of this hydroid. Fouled nets may also have encouraged plankton growth and reproduction by preventing cells from sinking below the euphotic zone. Future studies might try to measure light attenuation by different fouling communities. Inver- tebrate larvae (including decapods and echinoderms) and nema- todes were only found in fouled nets; although relatively rare, these potential predators and parasites could have deleterious ef- fects on scallop growth and survival (O'Connor et al. 1999. Freites et al. 2000). Net fouling could also be problematic if it promoted the growth of plankton responsible for shellfish poisoning. This is the first description of how fouling influences the en- vironment inside nets used for shellfish cultivation. The data con- Phytoplankton Nutnents via excretion Heterotrophs (Scallops, zooplankton. foulers) Nutnents via bactenal activity Detritus, faeces and pseudofaeces Figure 5. Mechanism by which nutrients might be retained and re- cycled in fouled pearl nets. tradict the common assumption that fouling reduces food levels. Instead, fouling can be associated with increased food availability and does not necessarily encourage a build up of decay products or inorganic matter, even when scallop densities are high. These find- ings may help to explain why in high current areas scallops inside pearl nets grow faster than scallops outside (Claereboudt et al. 1994b). They also support anecdotal evidence that a degree of fouling proinotes the growth of cultivated oysters (Arakawa 1990, Mazouni et al. 2001). It might be inferred that in some areas negative effects of fouling on scallop growth are caused by fouling organisms mechanically interfering with scallops (e.g.. binding them in unfavorable positions, inhibiting shell opening or disrupt- ing feeding behavior), or by harboring predators and parasites, rather than altering the environment. Growers of scallops in high current or oligotrophic offshore locations should perhaps strive to reduce the mechanical inteiference of foulers rather than trying to prevent fouling altogether. Strategies might include biological con- trol, which can keep bivalves clean and free to move, but does not completely remove biofouling (Hidu et al. 1981, Enright et al. 1983, Cigarria et al. 1998. Ross 2002). This approach could benefit scallop growth by reducing mechanical interference whilst retain- ing the potential food enhancing properties of fouling. That the en\ ironment inside scallop cultivation nets can differ significantly from the water-column also has important conse- quences for two areas of research. First, studies often relate pat- terns of scallop growth in suspended culture to environmental conditions (e.g., Wallace & Reinsnes 1985, Cote et al. 1993, Claereboudt et al. 1994a, Emerson et al. 1994. Lodeiros & Him- melman 1994. Thorarinsdottir 1994, Velez et al. 1995, Kleinman et al. 1996. Lodeiros et al. 1998, Lodeiros & Himmelman, 2000). This relationship may be better understood if future studies con- sider the influence of net fouling, or measure conditions inside nets. On a wider scale, water column data have recently been used by researchers assessing the affects of bivalve cultivation on nu- trient and seston dynamics of bays, or to predict the capacities of areas for shellfish cultivation (e.g., Penney et al. 2001, Pilditch et al. 2001). Because of its potential to uncouple scallop processes from water-column seston conditions, the influence of net fouling should also be included in such models. Here we have provided a snapshot examination of how fouling can alter environmental conditions. There is evidence that effects vary with season and the age or composition of the fouling com- munity (Ross 2002). Though difficult, simultaneous assessment of fouling communities, the environment inside nets and scallop growth would enable the influence of fouling to be better under- stood. We used wide-mesh peari nets containing intermediate sized scallops in a fast current area. Future work might examine the effects of fouling in low current areas or with the fine mesh nets used for growing spat. Studies to determine how common fouling assemblages (e.g., hydroid, tunicate and bivalve dominated com- munities) affect the net environment could help growers to choose cultivation sites, depths or methods of fouling control. ACKNOWLEDGMENTS We are very grateful to divers and boat crew for help with sampling in cold Manx seas. Thanks to T. Shammon, I. Allen, 1. Laing and J. Berges for advice on nutrient and plankton sampling and J. Ludgate for help with maps. 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ANNUAL FEEDING CYCLE OF THE PATAGONIAN SCALLOP ZYGOCHLAMYS PATAGONICA (KING AND BRODERIP, 1832) IN RECLUTAS BED (3rS-55°W), ARGENTINE SEA LAURA SCHEJTER,' - CLAUDIA S. BREMEC,' " * RUT AKSELMAN," DANIEL HERNANDEZ,- AND EDUARDO D. SPIVAK' ^Comejo Nacioiuil tie lnvestlt;aci<>nes Cientificas y Tecnicas (CONICET). Buenos Aires, Repi'iblica Argentina: -Institiita Nacional de Investigacion y Desarrollo Pesquero. Paseo Victoria Ocampo 1, (7600) Mar del Plata. Repiildica Argentina: and ^Universidad Nacional de Mar del Plata (UNMdP), Mar del Plata. Repiihlica Argentina ABSTRACT This article deals with the diet ol the Patagonian scallop. Zygochluiiiy.s p,ikis;i>niiu. during a yearly period at Recliitas bed (39°S-55"W). Argentine Sea, and relate.s the results with the oceanographic conditions and the growth pattern known tor this species. Scallops (n = 180) from six samples were dissected and the gut contents were identified and counted. Results showed a predominance of diatoms in the diet and maximum food ingestion in spring. Maximum somatic (muscle) growth for this species at the same study site was also found during spring. It is suggested that after the spring phytoplanktonic bloom, oceanographic conditions (thermocline in development) allow the sedimentation of food particles to the bottom; thts input of energy could produce the somatic growth documented for this filter-feeding species. KEY WORDS: diet, annual cycle. Patagonian scallop gut contents, Zxgochlamxs patagniiica. Argentine Sea INTRODUCTION Scallops are suspension-feeding organisms, feeding on detritus and phytoplankton (Bricelj & Shumway 1991). Particles aie mainly captured by cilia and mucus on the dorsal infrabranchial surface of the gill and the gill arch thus provides the major capture site (Beninger & Le Pennec 1991 ). Ciocco ( 1995) documented the importance of the labial palps in food particle transport to the mouth and also their contribution in preventing reflux and favoring agglutination and particle selection in Aequipeclen tehiiekhus (d'Orbigny). It is known that particle selection may occur at the labial palps and/or gills (Jorgensen 1990; Ward et al. 1997) and also that selection is not only based on particle size but on other important characteristics (Shumway 1985. Shumway et al. 1997). The effective lower limit of particle retention in studied pectinids ranges between 5 and 7 |jim. and therefore bacterioplankton (typi- cally between 0.3 and 1 jxm; Bricelj & Shumway 1991) and pi- coplankton are not available as food. Some gut content studies in pectinids showed the importance of benthic and/or tychopelagic algae in the diet of scallops (Vemet de Hall 1977. Shumway et al. 1987. Bricelj & Shumway 1991 ). Seasonal growth in bivalves, including scallops, is influenced by environmental changes, especially food supply (Bricelj & Shumway 1991). It is known that food availability is coirelated with growth in scallops (Griffiths & Griffiths 1987. Barber & Blake 1991 ), and that it is possible to find abnormalities in growth during diatom blooms (Lorrain et al. 2000). In addition. Ciocco (1992) concluded that differences in growth between populations of Aequipeclen lelniehluis were related with environmental fac- tors, such as temperature, depth, and food availability, and not with genefic factors. Valero et al. (2000) studied growth in Zy- gochlamys patagonica (King and Broderip) using satnples taken from the Reclutas bed of the Argentine Sea (Fig. 1 ). They found that maximum growth in shell, muscle and gonad occurred during different months of the year. The Patagonian scallop, Zygochlamys patagonica, is distrib- uted in the Magellanic Biogeographic Province, in the Atlantic ^Corresponding author. E-mail: cbremecCs'inidep.edu.ar Ocean, from 35°S to Tierra del Fuego, and in the Pacific Ocean, up to 42°S, between 40 and 200 m depth (Waloszek & Waloszek 1986, Ciocco et al. 1998). Zygochlanns pataganica and Aequipeclen lelmelchiis are the two commercial pectinid species in the Argentine Sea. There are nine Patagonian scallop beds in the Argentine Sea; Two are located in the intermediate shelf between 60 and 70 m depth and seven in the shelf break front area along the 100 m depth isobath (Lasta & Bremec 1995, 1998, 1999: Fig. 1), which is characterized by high productivity (Brandhorst & Cas- tello 1971, Carreto et al. 1981, 1995, Podesta & Esaias 1988). The Reclutas bed is located in the northern zone of this area, where oceanographic seasonal changes occur. During summer, the water column is stratified, and the surface and the bottom layers are separated by a pronounced pycno and thermocline between 30 and 40 m depth. During autuinn and winter, the water column is ver- tically mixed by convective circulation patterns, which result in breakdown of stratification (Guerrero & Piola 1997). Baldoni and Guerrero (2000) provided a more detailed study of the evolution of the temperature vertical structure of the water column in Reclutas bed area during the year. The objectives of this work were to study the diet of the Pat- agonian scallop. Zygochlamys patagonica, from samples taken at Reclutas bed during an annual period, to detect possible seasonal changes in diet, and to relate these results with the oceanographic conditions in the study area and with the growth pattern of the species, MATERIALS AND METHODS Gut contents of 180 scallops (35-50 mm shell height) were analyzed from samples (;? = 30) preserved in formaldehyde (10%). taken in June, August, October, and November 1996 and February and March 1997 in Reclutas bed (39°24'S-55°56'W, lOO-m depth). The protocol used involved the dissection of gut + digestive gland complex in all sampled scallops. The gut was cut open and the contents were washed in 2 mL of fresh water. A 1 -niL subsample of the gut content washed suspension was analyzed in a Sedgwick-Rafter counting chamber that was divided into 7 rows and 17 columns. All panicles within six columns were counted and idemified under the microscope ( 100-250x). This quantity repre- 549 550 SCHEJTER ET AL. -3500 ■^0 00 -5500 ARGENTINA -5500 -70.00 -65,00 -6C00 LONGITUDE Figure 1. Zygochlamys patagonica beds in the Argentine Sea. sents approximately 1/6 of the total gut content. The number of samples and columns studied were statistically tested and the es- timated error of the procedure was <16% (Schejter 2000). Multivariate MANOVA (Morrison 1976), univariate ANOVA, and the Tukey test (Sokal & Rohlf 1979) were used to establish the significance in the observed differences in food groups or species found in gut contents during the study. The significance level was P = 0.05 in all cases, but a Bonferroni correction (Morrison 1976) was used when necessary. Data were normalized using the square root transformation; the variance was found to be heterogenous. However, as MANOVA and ANOVA are robust tests when there is a balanced design (Ito & .Schull 1964, Ito 1988). it seemed correct to use them in this case in which sample sizes were the same in all months. The IR index (an index of relative importance and abundance; Bucher & Herrera 1981 ) was calculated for the main groups and species in gut contents. This index [IR = (Ni/Nt) x (Mi/Mt) x 100] considers not only the total abundance of an item (or group) per month in all guts (Ni/Nt), but also whether the item (group) was present in all guts or just in a few (Mi/Mt). RESULTS Twelve species and resting stages of diatoms, five species of dinotlagellates and cysts, two species of silicotlagellates, prasino- phytes, and a number of other organisms and remains grouped as miscellanea (tintinnids, foraminifers, nematodes, etc.) were found in gut contents of the Patagonian scallop (Table 1 ). Particle sizes varied between 19 and 170 (xm (mainly 25 to 60 jim), although some crustacean remains (-250 \x.m) and some small diatoms (<19 (jLm) were found (Table 2). The majority of items found in gut contents had a planktonic origin. This is the case of dinotlagellates. silicoflagellates, prasinophytes and tintinnids, and most diatom species. Nevertheless, some of them, namely Paralia sulcata, are tychopelagic. Dinoflagellate cysts and resting diatom stages are typically benthic. TABLE \. Food items found in Zygnchlamys patagonica gut contents during the stud> period at Reclutas bed, Argentine .Sea. Diatoms Pill alia siilciiki (Ehrenberg) Cleve Nitzschia ctuinuira Grunow Nilzschia sp. Thalassiosira spp. Pleurosigma nonnanii Ralfs in Pritchard Tluilassionema nilzscliioides (Grunow) Meereschkowsky Siepliaiiopyxis nirris (Amott in Greville) Ralfs in Pritchard Biililiilpliia sp. Aclinoptychus sp. Pennate 1 Centric 1 Centric 2 Resting stages Dinotlagellates Dinophysii inawsoni (Wood) Balech Dinopliysis rolumiala (Claparede et Lachmann) Dinophysis conlracia (Kofoid et Skogsherg) Balech Dinophysis acuminata (Claparede et Lachmann) Protoperidinium metananwn (Balech) Balech Gonyaiilax digitalis (Pouchet) Kofoid (cysts) Resting cysts Silicoflagellates Dictyocha speculum Ehrenberg Dictyocha fibula Ehrenberg Prasinophytes Ph'rospfrma spp. Tintinnids Dicryocysta elegans var. lepida (Ehrenberg) Kofoid el Campbell Dictyocysta elegans var. speciosa Jorgensen Acanlhostomella sp. Others Miscellanea Foraminifera Rotifers Nematodes Nauplii larval stages Invertebrate eggs Copepod spermatophores Radiolarian remains Unidentified remains Polychaete chaetae Sponge spicules Detritus Pollen The total number of ingested particles varied between 300 and 8,000 per gut and their mean number differed significantly among months (F,,; ,-,4] = 55.859: P < 0.001). Maximum food ingestion was registered in November, and minimum food ingestion periods were October and March (Fig. 2). Total abundance of diatoms, dinotlagellates, silicotlagellates, prasinophytes, foraminifera, and tintinnids differed significantly between months (MANOVA; F|3o.67si =-^6.86597; P < 0.001; Fig. 3). Diatoms were always the most abundant food item (1R= 45- 90%, with mean values between 320 and 1450 diatoms per gut) and were present in all guts during the entire sampled period (Fig. 4). The most abundant species was Paralia sulcata (maximum values in November: IR = 66%; Fig. 5). This species was present throughout all months and in all guts and was the dominant spe- Feeding of the Patagonian scallop 551 TABLE 2. Size ranges of main food ittms found in Zyguchlamys palagoiiica gut contents from Reclutas bed. Food Item Size ((im) Paralia sukalci Thalassiosini spp. Nitzschia spp. Pleurosignm nonnanii Pennate 1 Dlnophysis spp. Dictyocha fibula Dictyocha speculum Plerospenna spp. Dictyocysui elegans Acanthostomella sp. Foraminifera Rotifers Invertebrate eggs Sponge spicules Pollen 19-180-' 30-55 34-64 100-121 30-42 36-62 50-55 38-50 19-75 64-70 31-37 30-170 59-66 146-162 39-190 19-23 ' Minimum individual frustule diameter and maximum chain length, respectively. cies, except in February. In this month, the typical planktonic diatom genus Thalassiosira was almost the only food item found in gut contents (IR = 80%). In March, this species was still an important component of the diet (IR = 46%) but decreased in importance during the remaining months (IR = 7-16%; Fig. 6). Nitzschia spp. and Pleurosigina nonnanii made a minor contribu- tion to total gut content (IR < 19% and IR < 4.5%, respectively; mean = up to 140 items per gut and up to 23 items per gut, respectively). Dinoflagellates followed diatoms in importance (up to 1,000 items per gut, maximum IR = 25%, in November) in four of the six sampled months (Fig. 4). Dinophysis rotundata was present most of the time. Dinophysis mawsonii was present only in the spring, Gonyaulax digitalis cysts were found by the end of summer (February and March) and only a few in June. Unidentified di- noflagellates cysts were found in February, March, June, and Au- gust. Silicoflagellates (two species of Dictyocha) were always ob- served, and they were the second most abundant item in August ^ MeantSO CH MeaniSE a Mean Figure 2. Mean abundance of particles per gut of Zygachlamys patag- onica in the Reclutas bed during the study period. Different letters (a, b, c) indicate significant differences iP < 0.05 1. (IR = 0.02-12%; mean between 1 and 156 items per gut). Prasi- nophytes were relatively important in gut contents in June and March (IR = 9.6 and 5%. respectively, mean = 1 10 and 32 items per gut, respectively). Foraminifers were present in all months and in most guts (except in March) but with low IR and abundance values (IR = 0.7-6.2, mean between 8 and 79 items per gut). Tintinnids were frequent in spring (October and November) but even in these months had low IR and abundance values (IR < 1.7; mean <20 items per gut). Invertebrate eggs and copepod spennato- phores were abundant in February and March. DISCUSSION The diet of Zygachlamys patagonica is mainly composed of diatoms. These results agree with similar studies on bivalve gut contents, which also showed a predominance of diatoms (Vernet de Hall 1977. Pollovero 1984, Newell et al. 1989, Leonard! et al. 1996). In addition, it is remarkable that most of the food items found had a planktonic origin. Paralia sulcata, one of the more important diatoms recorded in the diet of Zygachlamys patagonica and known as a tychopelagic species, is frequent in the shelf and the shelf break area during most of the year(Lange 1985). Reclutas bed, approximately 1 10 nautical miles offshore and 100-m deep, is undoubtedly located in the pelagic zone. It must be pointed out that among the food items found in Zygochalnivs patagonica gut contents, there were two potentially harmful dinoflagellate species present, Dinophysis acuminata and Dinophysis rotundata. which could produce diairhetic shellfish toxins (Lee et al. 1989). The maximum food contents were recorded in November (up to 8,000 food items per gut; mean = 2,800 items per gut), being Paralia sulcata the main food item. Paralia sulcata was also dom- inant in gut contents during the year, except in February when Thalassiosira spp. predominated and represented about 80% of total gut content. It should be mentii>ned that samples were pre- served in formaldehyde, some food particles could have been de- stroyed because of the preservation, and also that highly digestible particles or naked cells were not recorded by this method. The vertical movement of particles in the seawater column is very important for those animals that live far away from the photic zone (Valiela 1995). Algal cells settling during spring and fall blooms are one of the main inputs of particulate organic matter from the pelagic to the benthic system. Downward mixing of plankton during certain times of the year undoubtedly plays a large role in making food organisms available to deep-water scallops (Smetacek 1982, Shumway et al. 1987). The northern Argentine shelf shows an annual phytoplankton growth cycle with two peaks of which the most important one occurs in spring (Carreto et al. 1995, Akselman 1998). This peak was reported to occur by the end of September-October at the boundary of the coastal system/ intermediate shelf (Akselman 1998), and maximum recorded chlo- rophyll a levels increase from the intermediate shelf to the shelf break (Can-eto et al. 1981, 1995, Bertolotti et al. 1996). Ther- mocline formation begins in spring at the intermediate shelf and extends all along the shelf (Carreto et al. 1995). In October and November, the thermocline is already in development in the Re- clutas bed area (Baldoni & Guerrero 2000), and oceanographic data for October and November 1996 were similar to mean esti- mated values for the study area by Baldoni and Guerrero (2000, Baldoni, personal communication). Smetacek (1982) indicated that nutrients accumulated in a 552 SCHEJTER ET AL. DIATOMS DINOFLAGELLATES c - c b 1 0 \" c m S 1 S 600 [ 3 ■B (00 _ _ < a a 4 4 JUN AUG OCT NOV FEB PRASINOPHYTES SILICOFLAGELLATES 200 a 5 a 1 160 o 5. 1 ™ [ I I 80 b b f b i 40 < - L r b b c 1 a 1 c + m JUN AUG OCT NOV FEB MAR FORAMINIFERS TINTINNIDS AUG OCT OCT NOV I Mean ± SD; Mean ± SE; I Mean Figure 3. Mean abundance of principal food items per gut of Aygochlamys piitiifidiiica in the Reciutas bed during the stud\ period. Different letters (a, b, c, d, e( indicate siyniflcanl differences (/' < 0.05). Feeding of the Patagonian scallop 553 ■ DIATOMS El PRASINOPHYTES Figure 4. Percent composition of different food items groups in gut contents of ZygocUlamys patagonica during the study period in the Reclutas bed. The group Miscellanea (see text) also includes tintinnids. 17-m water column at Kiel Bight over the winter were depleted within two weeks after phytoplankton bloom initiation. Sedimen- tation of phytoplankton took about one week and attained maxi- mum values three or four days after bloom initiation. Sedimenta- tion rates of diatoms and other phytoplankton components are related to their physiology, cell weight, and volume (Denman & Gargett 1983). As the organic matter sedimented was formed by a large number of living cells (and hence, was high in nutritional quality), benthic metabolic response to this input was rapid. For other deeper benthic systems a similar pattern is expected (Smeta- cek 1982). It is thus possible that, given the oceanographic conditions described during spring at the Reclutas bed (thermocline in devel- opinent). sedimentation processes or phytoplankton sinking down- wards to the bottom have facilitated food availability to benthic organisms after the phytoplankton bloom started at the surface. This would explain the high abundance of cells observed in gut contents from scallops collected in November. It is probable that at the beginning of October (when samples were taken) increased primary production started in surface waters, as previously docu- mented (Carreto et al. 198 1 ). but availability of food at the bottom remained low because of the time requirement for sedimentation processes in a lOO-m water column. During summer, the stratifi- cation of the water column is so pronounced that transport of phytoplankton cells to the bottom would probably be limited; un- der these circumstances we cannot explain the relatively higher abundance of Thalassiosira spp. during February. It is known that species of this genus are capable of producing blooms in other Figure 5. Mean abundance of Paralia sulcata per gut of Zygochlaiiiys patagonica in the Reclutas bed during the study period. Different let- ters (a, b. c) indicate significant differences (/' < ().(I5|. too c 200 000 d b 800 - 600 400 b T b 200 a rm b a i i nZ Mean t SD CH Mean i SE □ Mean JUN AUG OCT NOV FEB MftR Figure 6. Mean abundance of Thalassiosira spp. per gut of Zy- gochlamys patagonica in the Reclutas bed during the study period. Different letters (a. b, c) indicate significant differences iP < 0.05). 554 SCHEJTER ET AL. ureas of the Argentine Sea (Carreto et al. 1981). but oceanographic conditions at the Reclutas bed resulting in a strongly stratified water column would prevent cell sinking. However, occasional climatic events are able to disturb water column stratification, increasing sedimentation rates (Nielsen & Kiorbe 1991). On the other hand. Bode et al. ( 1998) conducted studies on the export of organic matter to the bottom, and found that most of it was pelagic in origin. Moreover, they determined that phytoplankton species found in sedimentation traps were not the same as those found at the surface, indicating that sedimentation traps were providing records of past production events. In summaiy, simultaneous sea- sonal information about gut contents and phytoplankton species composition in the study area is needed to establish the degree of particle selectivity in the feeding process, and whether the occur- rence and abundance of food items are due to higher availability of potential food after phytoplankton growth. The results of this study agree closely with recent findings of studies on biologic aspects of this species. Valero et al. (2000) studied the grow th pattern of Zyf><>clilamys patagDiika at Reclutas bed. As was already found for several species of scallops from shelf and coastal areas (Barber & Blake 1991. Ciocco et al. un- publ.). growth of different body components of the Patagonian scallop are not simultaneous: ma.ximum shell growth occurred in July, maximum gonadal growth was obtained in June, and maxi- mum somatic (muscle) growth occurred in November. In view of this pattern, it is possible to link the period of maximum muscle growth with that in which scallops showed higher cell abundance in gut contents, both of which occur in November. Consequently, we hypothesize that somatic (muscle) growth of Zyiiochlumys pu- uiiionica located in the Reclutas bed occurs during the period of higher food availability in the bottom, which follows the period of spring phytoplankton growth in the upper layer of the water col- umn and its subsequent sinking to the bottom before the develop- ment of the seasonal thermocline. ACKNOWLEDGMENTS The authors thank to Lie. R. Pinero, Lie. S. Incorvaia, Dr. H. Mianzan, and Dr, N. Ciocco for useful suggestions and bibliogra- phy. We are particularly grateful to Dr. S. Shumway and Lie. M. Lasta for their encouragement during our investigation. We also would like to thank the suggestions of the anonymous reviewers. LITERATURE CITED Akselman. R. 199^. 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Biometn'a. Principios y Metodos Estadi's- ticos en la Investigacion Biologica. Madrid. Espaiia: H. Blume Edi- ciones. 223 pp. Valero. J. L.. M. Lasta & D. Armstrong. 2000. Temporal and spatial varia- tion in growth of the Patagonian scallop (Zygochlamys patagonica) in the Argentinian continental shelf. Republica Argentina: Resumen. IV Jomadas Nacionales de Ciencias del Mar, Puerto Madryn. p. 122. Valiela. I. 1995. Manne ecological processes. 2nd. edition. New York; Springer-Verlag Inc.. 686 pp. Vemet de Hall. M. 1977. Alimentacion de la vieyra tehuelche [Chlamys tehuelchus). Comision Nacional de Estudios Geo-Heliofisicos. Centre Nacional Patagonico. Puerto Madryn. Argentina. Unpublished Report. 26 pp. Waloszek. D. & G. Waloszek. 1986. Ergebnmisse der Forschungsreisen des FFS "Walther Herwig" nach Sudamerika. LXV. Vorkommen. Re- produktion. Wachstum und mogliche Nutzbarkeit von Chlamys patag- onica (King and Brodenp. 1832) (Bivalvia. Pectinidae) auf dem Schelf von Argentinien. Arch. Fish Wiss. 37:69-99. Ward. J. E.. J. S. Levinton, S. E. Shumway & T. Cucci. 1997. Site of particle selection in a bivalve mollusc. Nature 360:131-132. Journal of Shellfish Resecinh. Vol. 21, No. 2, 557-561. 2002. COMPARISON OF THE PARASITES AND PATHOGENS PRESENT IN A CULTIVATED AND IN A WILD POPULATION OF SCALLOPS (ARGOPECTEN PURPURATUS LAMARCK, 1819) IN TONGOY BAY, CHILE KARIN B. LOHRMANN,'* ANDREW R. BRAND," AND STEPHEN W. FEIST' 'Universidad Catolica del Norte. Facidtad de Ciencias del Mar. Cas. 117. Coquimbo. Chile; 'Port Erin Marine Lahoratoiy. University of Liverpool. Port Erin. Isle of Man. IM9 6JA. United Kiniidoni: 'CEFAS Weymouth Laboratoiy. Barrack Road. The Nothe. Weymouth, Dorset DT4 SUB. United Kingdom ABSTRACT Cultivation of the "ostion del norte". ArgopecWn purpuratus. is an important economic activity in tlie 3rd and 4th Regions of Chile. Studies of disease v\'ere undenaken on wild scallops to gather baseline mformation on healthy populations. However, as cultivated scallops are kept at higher densities than the wild populations, the occurrence and prevalence of parasites and tissue pathology differed as indicated in this study that compares the types of parasites and their prevalence in wild and cultivated scallops from Tongoy Bay, a major center for scallop cultivation. In January 1999, 151 cultivated and 154 wild scallops were e.\amined histologically and via scanning electron microscopy (SEM). The same parasite taxa. as well as granulomas, were found in both populations, but their prevalence differed. The granulomas were small, with no apparent etiology, but the cultivated population harbored significantly more lesions than the wild population. The only parasites found were a prokaryote in the dige,stive gland and the ciliate protozoan Tricliodina sp. on the gills of the scallops. Trichodina did not seem to elicit a host response and its prevalence was significantly higher in the cultivated scallops. The prokaryote. a rickettsiales-like organism (RLO) was observed as basophilic inclusions in digestive gland tubule epithelial cells. The intensity of infection was low. but significantly higher in the wild stock. No host reaction was seen, and the prevalence showed no significant difference between the two populations. The infection with RLOs was therefore independent of the source of the scallops, unlike the Trichodina or the granulomas, which were significantly more prevalent in the cultivated population. KEY WORDS: scallop, parasite, long-line culture, nckettsiales. Trichodina INTRODUCTION Argopecten piirpurariis. the ""ostion del norte"" {northern scal- lop) is distributed on the Eastern Pacific coast from Sechura. Peril (6°S), to Tongoy Bay, Chile (31°S) (von Brand et al. 2002), and is harvested and cultivated in both countries. In Chile, the most abun- dant beds are in the 4th Region, in the bays of Tongoy, Guanaque- ros, and Coquimbo. These populations almost disappeared through overfishing, prompting a total harvesting ban for this species in 1986 and this ban is still in force for wild populations. In the early 1980s, A. purpuratus culture became established (DiSalvo et al. 1984, lUanes-Biicher 1987) and currently the 3rd and 4th Regions produce 97% of the cultivated scallop production of Chile ( Aqua- noticias 2000). Tongoy Bay is the single most important bay for production of cultivated scallops. A serious disease outbreak here could have significant consequences for this industry, so a survey was under- taken to gather baseline information on parasites and tissue pa- thology present in healthy members of the cultivated population. There is no information available on any diseases of A. purpuratus from Tongoy Bay, but some metazoan parasites have been re- ported for this species in other bays in Chile and Peru. Mateo et al. (1975) described germ sacs and cercaria of a hemiuroidean in the gonad of A. purpuratus in Perii, causing castration of heavily in- fected scallops. In Chile. Oliva et al. (1986) described two larval cestodes located in the gonads of A. purpuratus in Antofagasta. One of the cestodes belonged to the family Phyllobothriidae. and the other possibly belonged to the family Oncobothriidae. In Co- quimbo. Lohrmann et al. (1991) described a metacercaria of a fellodistomid trematode in the labial palps of A. purpuratus and an unidentified larval cestode in the intestine of a few animals of this species (Lohrmann & .Smith 1993). 'Corresponding author. E-mail; klohrmanCaucn.cl As there is still a small natural bed of A. purpuratus at the southern tip of Tongoy Bay. both wild and cultivated scallops are available for examination in this bay. The wild scallops from this natural bed live on the sea floor in depths of up to 15 m. at a density of 1 to 2 scallops m"- (Stotz & Gonzalez 1997). Cultivated scallops are kept in pearl nets at densities of about 166 individuals m"- for scallops at a size below 50 mm, and 19 individuals m"" in lantern nets at sizes above 50 mm (lUanes 1986). Since the culti- vated scallops are kept at greater densities than the natural popu- lations, it might be assumed that the occurrence and prevalence of parasites and other pathogens may differ. This investigation was carried out on wild and cultivated scallops, to test for differences in the prevalence of parasites and tissue pathology between the two populations of A. purpuratus. MATERIALS AND METHODS In January 1999, 151 cultivated, and 154 wild scallops were obtained from Tongoy Bay (Fig. 1). The 12-month-old cultivated scallops had been hatchery-produced, and transferred to the sea at age one month. They had been kept at a density of 19 scallops m^- in lantern nets hanging from long lines at a depth of about 10 m from the suil'ace. The natural scallops were obtained from a small bed located near the fishing community of Puerto Aldea, which is located at the southern tip of Tongoy Bay (Stotz & Gonzalez 1997). These scallops were adults, but their ages were not known. They were collected by "hooka" diving (this consists in delivering air to the diver through a hose from a compressor in the boat), from a depth of 8 to 10 m. Each sample of scallops was transported to the central aquaculture laboratory of the Universidad Catolica del Norte in Coquimbo. where they were placed in tanks with running seawater at ambient temperature. They were processed m batches of 50 scallops daily, starting on the day after their arrival. The soft tissues of the scallops were taken out of the shells, and 557 558 LOHRMANN ET AL. CHILE Pacific Ocean i <■" Coquimbo ,i j 25"S 30° S 4 Guanaqueros Bay ..^^ 4 Tongoy Bay !71O20-W 45°S I /\ Tongoy Bay ^ lengua: DE VACA POINT Puerto^ Aldea *■..-. i^M Scallop farms 0 1 2 / Km \ Si « / V Antarctica Figure I. Map of Tongoy Bay showing the area dedicated to scallop farming (F) and the small natural bed (N) near Puerto Aldea. were fixed for histology in Davidson's fluid (Sliaw & Battle 1957). including the gills, digestive gland, gonad, kidney, mantle, and the adductor muscle. They were prepared for histology using standard methods and stained with haematoxylin and eosin fH & E). Giemsa and Ziehl-Neelson methods were used as appropriate to detect parasites and ceroid-like pigment respectively. Slides were analyzed and photographed using a Nikon E600 Eclipse photomi- croscope. For assessing the intensity of infection of rickettsiales- like organisms (RLOs) and trichodinids. the most heavily infected area of the histologic section was selected, and the number of parasites present was counted at a magnification of 400 times, with a field diameter of 1.240 |xm. Three categories of intensity were defined: grade I (very light) one parasite present, grade II (light), two to four parasites present, grade III (moderate), five or more parasites present. For scanning electron microscopy (SEM), sections from selected wax blocks were cut at 12 p.m, and mounted on cover- slips. Sections were de-waxed in three changes of xylene, passed through three changes of H)09f ethanol (modified from Toner et al. 1992). and critical point dried using CO,. Samples were mounted on double-sided adhesive tape and ion sputtered with gold. The sections were viewed and photographed using a JEOL TS 300 microscope. Measurements of individual rickettsi- ales-like organisms and Trichodina were taken from SEM photo- graphs. For analyzing the prevalence of each parasite, a 2 x 2 contin- gency table was constructed with the frequency counts of each parasite in the wild and the cultivated scallops, and a x" test for goodness of fit was applied. The intensities of infection were coin- pared using the Mann Whitney rank sum test (Zar 1999). RESULTS From the total of .305 scallops analyzed, surprisingly few patho- gens were detected. These included rickettsiales-like organisms (RLOs) and ciliutes [Trichodina sp.). Small, granuloma-like tissue lesions were also detected. These are described in more detail later. Rickettsiales-Like Organisms (RLOs) Rickettsiales-like organisms (RLOs) were detected in digestive gland tubule epithelial cells in the form of spherical basophilic inclusions, with a diameter ranging between 7 and 14 fjLm. Occa- sionally, the inclusions were also seen in the lumina of the diges- tive tubules (Fig. 2A). In the interior of each inclusion there were darker staining bodies, which at the scanning electron microscope (SEM) level could be discerned as rod-shaped, and of fairly uni- form size (Figs. 2B & C), ranging from 0.8 to 1.3 |xm in length, and 0.38 to 0.46 ixm in width. The inclusions seemed to be en- closed by a thin membrane, separating the RLOs from the cyto- plasm of the host cell (Fig. 2B). The prevalence of these RLOs was 41'7f in hatchery-reared scallops and 37% for wild scallops (Table I ) but this difference was not statistically significant (P = 0.6247). The intensity of infection in both populations is shown in Table 1 . A significant difference [P = 0.001 ) was detected in RLO intensity between populations, the natural population showing a higher proportion of scallops with degree II and III of infection. Trichodina sp. Trichodina sp.. a ciliate protozoan, was found associated with the gills of the .scallops (Fig. 3A). It was dome-shaped, with a horseshoe-shaped macronucleus (Figs. 3 A & B). It measured 19 to 23 jxm in height, and the basal disc was 14 to 18 p.m in diameter. The basal disc was surrounded by a ciliary girdle (Figs. 3A & B). This trichodinid was always closely associated with the gill fila- ments, but no pathologic changes to the gill were detected. The prevalence was 56% for fanned scallops, but only 5.1% for wild scallops (Table 2). and this difference was highly significant (P < 0.001). The intensity of infection is shown in Table 2 for both populations of scallops. The difference in intensity between the cultivated and the wild population was not statistically different. Craiiiilomas In the base of the gills, small tissue lesions were found embed- ded in the connective tissue. These consisted of a central focus of pigmented material resembling ceroid that appeared to be con- tained within host cells and was surrounded by a thin capsule of fibroblast-like cells (Fig. 4). These lesions are hereafter referred to as granulomas. No evidence of infectious agents was detected in association with these granulomas. The prevalence was 12% in cultivated scallops, and 2.6% in the natural stock (Table 3) and this difference was highly significant (P = 0.0034). DISCUSSION In scallops. RLOs have been described as basophilic inclusion bodies in the gills of Placopeclen magellanicus (Gulka et al. 1983). Argopecten irradians (Leibovitz et al. 1984. Elston 1986, Karlsson 1991 ), and Pcclen maxinuis (Le Gall et al. 1988. Le Gall et al. 1991). They have also been found in the kidney of Ar- gopecten irradians (Morrison & Shum 1983. Karlsson 1991, McGladdery et al. 1993). as well as in the digestive gland (McGladdery et al. 1993). The basophilic inclusions oi A. pitrpii- Parasites from Wild and Cultivated Scallops 559 TABLE 1. Prevalence and intensity of infection with a rickettsiales-like organism in scallops from Toiihov Bay. No. +/No. Examined Prevalence Intensity of Infection {%) Scallop Group Grade Grade II Grade III Cullivated Natural 61/151 57/154 41 37 78 22 20 32 46 was found that indicates they are most likely rickettsiales and not chlamydiales-like organisms. Using SEM. the morphology and surface characteristics were discerned. However, as no other SEM images of RLOs were found in the literature, a comparison with other RLOs was not possible. Since the prevalence of this organism showed no significant difference between the cultivated and the wild populations, host density does not seem to be an important factor in its transmission. Figure 2. Rickettsiales-like organisms (RLOs) in the digestive gland of A. purpuratiis. A: Light micrograph showing several basophilic inclu- sions (arrows) with RLOs in the cells of one tubule. One inclusion can be observed in the lumen (L) of another tubule (short arrow). Stain: H & E. Bar: 5(1 (im. B: Scanning electron microscopy (SEM) image of a histologic section of one basophilic inclusion. IK': digestive tubule cell. RLOs (*). Arrow: membrane separating the inclusion from the diges- tive cell. Bar: 5 \im. C: RLOs at higher magnification. One RLO (*). Bar: 1 (im. rains were similar in size and structure to the rickettsial inclusions described by Morrison and Shum (1983) in the kidney of A. irm- dians. or in digestive gland tubules of clams by Elston and Peacock (1984). For the RLOs from A. piirpunitus only one life cycle stage Figure .^. Tricbodina sp on the gills of A. piirpuralus. A: Light micro- graph of a few individuals in different orientations. The horseshoe- shaped nucleus (short arrow) can be clearly seen, as well as the ciliary girdle (arrow). Stain: H & E. Bar: 5(1 pm. B: SEM image of a histologic section showing one complete, and part of another Trichodina sp. The whole individual appears longitudinally sectioned, showing the horse- shoe-shaped nucleus (n), and the ciliary girdle (eg). The incomplete individual shows the basal disc (arrow) surrounded by the ciliary girdle (eg). Bar: It) pm. 560 LOHRMANN ET AL. TABLE 2. Prevalence and intensity of infection with Trichodinu in scallops from I onuoy Bay. Intensity of Infection (%( TABLE 3. Prevalence of granulomas in scallops from Tongoy Bay. Scallop No. +/No. Prevalence Grade Grade Grade Group Examined i'7i\ I II III Cultivated 84/151 Natural 8/154 56.0 5.1 64.0 37.0 29.0 37.0 7.0 26.0 The RLOs observed in A. purpuniuts do not .seem to cause any harm, because there is no host response. Infections with RLOs are common in bivalves, where they usually cause only mild effects, if any (Lauckner 1983. Comps & Tige 1999). However, RLOs were reported to have caused a few serious diseases but the relationship between the presence of RLOs and the mortalities was not experi- mentally demonstrated. In 1983. a mass mortality of Placopecten magellaniciis occurred in Rhode Island, USA and a rickettsia-like organism was found in the gills and other tissues of these scallops (Gulka et al. 1983). Mass mortality due to branchial RLOs was also reported for Pecten inaxiimis in Brittany. France by Le Gall et al. (1988), for the giant clam Hippupiis hippopus (Norton et al. 1993). and for the clam Vi-iicnipis rlunnhoides from Spain by Villalba et al. ( 1999). With such limited knowledge it is important to undertake further studies on RLOs transmission and their effect on scallops of different ages and culture conditions such as density, temperature and depth. Tiicluidina sp. ciliates are very common in bivalves (Lauckner 1983. Bower et al. 1994). They have been described from the following scallop species: Mizuhopeclen yessoensis (Stein 1974, in Lauckner 1983), Chlamys farreri (Kuidong et al. 1995) and Pla- copecten magellaniciis (McGladdery et al. 1993). In invertebrates, trichodinids are considered to be harmless commensals, feeding on bacteria (Lauckner 1983). However, they are present in large num- bers in weakened animals (Bower et al. 1994), and also in organ- isms from areas polluted with chemicals and bacteria (Boussaid et al. 1999). Boussaid et al. (1999) found that Ciassostrea gigas heavily infected with Trichodina exhibited an intlamniatory re- sponse of the gill, and numerous desquamated epithelial cells, haemocytes, and tissue debris of host origin was observed sur- rounding the parasites. They also stated that an excessive mucus production covered the gill lamellae. This could interfere with the respiratory function of the gill, and may result in the death of the Figure 4. light micrograph of one granuloma located in the base of the gills. Some degrading material and ceroid (c) can be observed, encircled by flbroblast-like cells (arrows) encapsulating it. Stain: H & E. Bar: 50 fim. Scallop Group No. +/No. Examined Prevalence ( % ) Cultivated Natural 18/151 4/1.54 12 2.6 oyster (Boussaid et al. 1999). A significant difference in preva- lence of Trichodina was found between farmed (56%) and wild A. piirpiiialiis (5.2%). This may be a consequence of the crowding of the scallops, their proximity facilitating the transfer of this com- mensal. The granuloma-like tissue lesions found at the base of the gills are similar to lesions (that they called ""swirl" encapsulation) ob- served by McGladdery et al. (1991) in bay scallops Argnpecten inadians infected by what was thought to be a Perkinsus species. Goggin et al. (1996) made an assessment of these lesions, and concluded that they were not produced by a Perkinsus species, but were a general response to a foreign agent. In this study, the very low prevalence and intensity of the lesions precluded ultrastruc- tural investigations that are needed to determine the etiology of the granulomas. However, Gonzalez et al. ( 1999) identified apparently identical lesions in A. piirpuratiis from Valparaiso, which were heavily infected with a protistan. Macroscopically, infected ani- mals presented small dark brown pustules in the mantle, which was also retracted. Based on one electron microscopy image of the protistan they suggested that it could be an apicomplexan. How- ever, details were indistinct and this finding needs to be confirmed. Since pathogen involvement cannot be ruled out as a cause for these granulomas and there was a significantly higher prevalence in cultivated stocks, further investigations are needed to identify their cause. From the results of this study it can be concluded that both cultivated and natural A. piirpitratus from Tongoy Bay harbored very few putative pathogens, and those that were present, were the same for the two scallop groups. The main difference between the two groups of scallops was the density at which they lived: I to 2 scallops m'- for natural scallops in Puerto Aldea. and 19 scallops per m"~ for the cultivated scallops. They also differed in the lo- cation, with natural scallops living on the seabed and cultivated scallops in cages suspended in mid-water. The increased density can favor transmission of pathogens in two ways, either providing hosts that are in close vicinity, and/or increasing stress. Over- crowding can reduce food availability or increase the levels of toxic waste products, all of which contribute to stress (Newell & Barber 1988). Stressed organisms have less energy available to defend themselves from disease and this is recognized as an im- portant factor that can trigger disease in otherwise healthy animals (Lauckner 1983. Newell & Barber 1988. Sindermann 1990). It is surprising how few potential pathogens were harbored by these two populations of scallops, since some metazoan parasites have been previously found in A. piirpitratus in other northern Chilean bays. Although the scallops examined during this study were from healthy populations, the threat of disease is always present. Any pathogens that are new to A. purpiiratus could have a devastating effect on both cultivated and natural stocks. Patho- gens may be involuntarily carried by fouling organisms on boats, in the ballast water of big ships, or by transpoiling scallops or other bivalves from other regions to Tongoy Bay. Newly introduced Parasites from Wild and Cultivated Scallops 561 parasites can cause epidemics if the host's innate defense mecha- nisms are not able to destroy it. or if the host is not able to defend against a novel parasite strategy (Figueras & Fisher 1988). It is therefore recommended that A. purpuratHS stocl 0.2). However, individual spat showed differences in the proportion of times they were observed to be attached or to have moved over the seven days (P < 0.05). DISCUSSION Our investigation demonstrated that A. balloti consistently se- creted a transient byssus, contrary to, and clarifying previous re- ports (Rose et al. 1988, Sumpton et al. 1990, Cropp 1992, Robins- Troeger & Dredge 1993). However, unlike most scallops (Sastry 1965. Bourne et al. 1989, Benigner & Le Pennec 1991 ). there is no evidence of the byssal attachment in A. balloti before and during early metamorphosis. The first appearance of byssal threads occurs after the spat begins to synthesize the dissoconch shell. Spat of shell height less than I mm do attach to the substratum, although it is difficult to detect byssus even under microscopic inspection because it is very thin and transparent (future electron microscopy may resolve this issue). The thickness and visibility of threads increases as the animal grows. We do. however, find similarities between A. balloti and the reported behavior of other pectinids during byssal secretion (Be- nigner & Le Pennec 1991), specifically the crawling, and explo- ration phases prior to secretion, and the process of secretion itself. Amusium balloti spat of 2-3 mm shell height secreted only 2 or 3 transient byssal filaments under our experimental conditions. Compared with other scallops A. balloti appears to have a much reduced bvssus attachment. Gruffydd (1978) estimated the number "^ Figure 1. The foot, cornel, and byssus of Amusium balloti spat (shell height 2 mml (Al Foot extended. (Bl Foot retracted, b, byssal: f. fool; c. cornet. Scale bar = 1(1 (ini. 566 Wang et al. c 0.05, ANOVA) Byssal Attachment in Amusium balloti 567 Time (days) •distaDce (mean) ram ■survival rate (%) A percentage of moved animals )( percentage of attachment Figure 4. Amusium balloti spat (shell height 2 (±0.06 SE) mnil. mean movement distances with standard deviation, daily proportions of moved and attached animals, and daily survival rates over seven days. Values sharing same superscript letter do not differ significantly (P > 11.05, ANOVA) attachment rates over time for A. balloti young spat are similar to those reported tor Clilatnys opercularirs (L) (Paul 1980) and Plu- copecten magellanicus (Caddy 1972). The tinie required for most A. balloti spat to reattach is also similar to that reported for these species of scallop, with approximately 80% to 90% reattached after 30 and 50 min respectively (Caddy 1972, Paul 1980). Scallops appear to be susceptible to the effects of siltation, particularly in relation to gills (Naidu & Scalpen 1976. Tettelbach et al. 1988). It has been suggested that byssal attachment to el- evated substrates, by both juvenile and adult scallops, may help avoid burial by fine-grained sediments in areas of high near- bottom turbidity, as well as a means of avoiding predators (Bricelj & Shumway 1991, Pohle et al. 1991). Amusium balloti lives on medium to coarse sandy-mud seabed (Cropp 1994) where there is very fine sediment, but also no vertical substrate for juvenile at- tachment. Consequently A. balloti spat probably settle directly on the bottom with byssal attachment to coarse sand grains. As small spat, only a few filaments are required for them to attach onto the substrate and avoid being moved by currents, or perhaps concen- trated and buried in areas of fine silt. The small number of fila- ments, in addition to minimizing the energy requirements of byssal production, would also enable more rapid detachment if the need arose, perhaps in response to predators. However, with the growth of young spat, sand grains are no longer effective anchors, even if the spat could secrete more and stronger byssal threads. Interest- ingly, after 4-5 mm, A. balloti spat rapidly develop a strong swim- ming capability, which is also likely to be an effective means of avoiding siltation and predation. 60 50 40 ^ 30 Q 20 10 0 T J -i- ^ -| -r J T J -r T- T < . 1 1 1 1 1 L J lJ Mil Hill ill ^uDanmiTinuiui 16 21 26 31 Individual spat Figure 5. Mean daily movement of individual Amusium balloti spat (;i = 32), (shell height 2 (±0.06 SE) mm), over 7 days, mean distance (mm) with standard deviation. Data arranged In ascending order. 568 Wang et al. Methods for artificial and controllable detachment of spat have important practical applications in hatchery production. Growing spat need to be frequently graded and transferred onto screens of successively larger surface area and mesh aperture sizes to reduce screen fouling and to offset increasing biomass (Heasman et al. 1994). In Pecten fuimitus. mechanical methods, such as seawater jet and scrapers, can cause injury and subsequent high mortality, particularly in spat less than 5 mm shell height (Heasman et al. 1994) due to their more robust attachment. However, the delicate byssus oi A. balloti is readily broken with a gentle stream of water does not cause mortality to the spat. A hypersaline (45%() bath was found to be the most effective and safe means for inducing spat detachment in both P. fumatus (>95%) (Heasman et al. 1994) and Pinctada maxima, (>90%) (Taylor et al. 1997). This method was also effective for A. halloti with 40%o salinity inducing 98% de- tachment within 2 h. compared with 15'^/i in normal seawater (and after gentle rinsing). A prerequisite for any aquaculture operation is the reliable, plentiful, and inexpensive supply of seed (juveniles). In most bi- valve culture operations throughout the world, .seed is obtained from the wild, although hatchery-produced spat is an alternative (Bourne 2()()()). Both approaches have been used successfully to produce scallop spat and in all documented cases, procurement of scallop relies on the attachment of spat via byssal threads to a solid substratum (Bourne et al. 1989. Ito 1991, Wang et al. 1993, Neima 1997, Bourne 20(J0). The results of this study demonstrate that A. halloti secrete byssus. consisting of a few fragile filaments. In addition, although spat are usually attached to a substratum by the byssus, they do move frequently. These characteristics explain why wild collection is unsuccessful, with water movement even- tually removing most spat from collector bags, and supports the conclusion of Robins-Troeger and Dredge (1993), that fishery enhancement (and aquaculture) will be reliant on hatchery- reared spat. The results of our present study also suggest that to culture A. Inilliili spat in the hatchery, we cannot use standard methodologies reliant on a robust byssal connection. However, this problem can be overcome by using screen chamber settlement systems. Down- weller and upweller screen chamber settlement systems have been used successfully to rear many species of bivalve, such as oyster, clam, (Utting & Spencer 1991 ) and scallop (Heasman et al. 1998). Our work provides the first foundations for the development of a more reliable and commercial-scale hatchery production method for A. halloti. which has not previously been demonstrated. The potential for subsequent aquaculture or stock enhancement through large-scale hatchery production of spat may then be realized. ACKNOWLEDGMENTS The authors thank Ms Jan Rose for supply of microalgae. Mr. Mike Dredge and various fishermen for the supply of broodstock and to Mr. David Mayer for advice on statistical analysis. We also thank staff at the Bribie Island Aquaculture Research Centre and Dr. David Hewitt for their generous help in setting up the scallop hatchery. The authors thank Dr. Michael Heasman. Dr. Wayne O'Connor, Dr. Zhaoping Wang and an anonymous referee for helpful comments and advice. Benigner. P. G. & M. Le Pennec. 1991. Functional Anatomy of Scallop. In: S. E. Shumway. editor. Scallop: Biology. Ecology and Aquaculture. Developments in Aquaculture and Fisheries Science. Amsterdam: El.sevier Science Publishing, pp. 133-223. Bourne, N., Hodgson. C. A. & J. N. C. Whyte. 1989. A manual for scallop culture in British Columhia. Can. Tech. Rep. Fish. Aqual. Sci.. no. 1694. 230 pp. Bourne. N. F. 2000. The potential lor scallop culture - the next millenium. Acjiiaciilliire huernational 8:1 13-122. Brand. A. R. 1991. Scallop Ecology: Distributions and Bchuxiour. In: S. E. Shumway, editor. Scallop: Biology, Ecology and Aquaculture, Devel- opments in Aquaculture and Fisheries Science. Amsterdam: Elsevier Science Publishing, pp. 517-569. Bricelj. V. M. & S. E. Shumway. 1991. Physiology: Energy acquisition and utilisation. In: S. E. Shumway. editor. Scallop: Biology. Ecology and Aquaculture, Developments in Aquaculture and Fisheries Science. Am- sterdam: Elsevier Science Publishing, pp. 305-,M6. Caddy, J. 1972. Progressive loss of hyssus attachment with size in the sea scallop. Pliicopecten nicificllaiiiciis (Gmelin). J. Exp. Men: Biol. Ecol. 9:179-190. Cropp, D. A. 1992. Aquaculture of the saucer scallop .Aiiiii.siiim halloti. Final Report to FIRDC 89/58 WSFIZ. 35 pp. Cropp. D. A. 1994. Hatchery production of western Australian scallops. Mem. Queensl. Mus. 36:269-275. Gruffydd. L. D. 1978. The byssus and byssus glands in ChUimys isUmdica and other scallops (Lamellibranchia). Zool. Sci: 7:277-285. Gwyther, D., D. A. Cropp. L. M. Joll & M. C. L. Dredge. 1991. Fisheries and Aquaculture: Australia. In: S. E. Shumway. editor. Scallop: Biol- ogy, Ecology and Aquaculture, Developments in Aquaculture and Fish- eries Science. Amsterdam: Elsevier Science Publishing, pp. 835-851. LITERATURE CITED Heasman. M. P.. W. A. O'Connor & A. W. Frazer. 1994. Detachment of commercial scallop Pecten fiiituitus. spat from settlement substrates. Aqiuutiltiire 123:40l-+()7. Heasman. M. P., O'Connor, W. A.. O'Connor, S. J. & S. Walker. 1998. Enhancement and farming of scallops in NSW using hatchery produced seedstock. Final Report to FRDC. NSW Fisheries. CronuUa. NSW. Australia. 146 pp. Ito. H. 1991. Fisheries and Aquaculture: Japan. In: S. E. Shumway. editor. Scallop: Biology. Ecology and Aquaculture. Developments in Aqua- culture and Fisheries Science. Amsterdam: Elsevier Science Publish- ing, pp. 1017-1055. Joll. L. M. 1989. Swimming behaviour of the saucer scallop .Amii.siKiii halloti (Mollusca: Pectinidae). Mar. Biol. 102:299-305. McCuUagh. P. & J. A. Nelder. 1989. Generalized Linear Models. 2nd ed. London: Chapman and Hall. 551 pp. Naidu. K. S. & R. Scalpen. 1976. Settlement and survival of giant scallop. Placopecten magellanicus. larvae on enclosed polyethylene film col- lectors. In: T V. R. Pillay & W. A. Dill, editors. Adances in aquacul- ture. Famam. UK: Fishing News Books Ltd. pp. 379-381. Neima. P. G. 1997. Report on commercial scallop hatchery design. Can. Tech. Rep. Fish. Aquat. Sci./rapp. Tech. Can. Sci. Halieut. Aquat. 62 PP Paul. J. D. 1980. Upper temperature tolerance and the effects of tempera- ture on byssus attachment in queen scallop. Chlainys operciilaris (L). J. Exp. Mar. Biol. Ecol 46:41-50. Payne. R. W.. D. B. Baird. A. R. Gilmour, S. A. Harding. P. W. Lane, D. A. Murray, D. M. Soutar. R. Thompson. A. D. Todd. G. Tunnicliffe Wilson. R. Webster & S. J. Welham. 2000. GenStat Release 4.2 Ref- erence Manual. Oxford: VSN International. 782 pp. Pohic, D. G.. V. M. Bricelj & Z. Garcia-Esquivel. 1991. The eelgrass canopy: An above-bottom refuge from henthic predators for juvenile bay scallops Argopeclen irradians. Mar. Ecol. Prog. Ser. 74:47-59. Byssal Attachment in Amusium balloti 569 Robins-Troeger, J. B. & M. C. L. Dredge. 1993. Seasonal and depth characteristics of scallop spatfall in an Australian subtropical einhay- ment. J. Shellfish Res. 12:285-290. Rose, R. A., G. R. Campbell & S. G. Sanders. 1988. Larval development of the saucer scallop Amusium halloti (Bernardi) iMollusca: Pec- tinidae). Aust. J. Mar. Freshwat. Res. 39:153-160. Sastry, A. N. 1965. The development and external morphology of pelagic larvae and post-larval stages of the bay scallop. Argopecleii irradians concentricus Say, reared in the laboratory. Bull. Mar. Sci. 15:417^35. Sumpton, W. D.. I. W. Brown & M. C. L. Dredge. 1990. Settlement of bivalve spat on artificial collectors in a subtropical embayment in Queensland. Australia. J. Shellfish Res. 9:227-231. Taylor, J. J., R. A. Rose & P. C. Southgate. 1997. Inducing detachment of silver-lip pearl oyster [Pinciada maxima. Jameson) spat from collec- tors. Aquaculiure 159:1 1-17. Utting. S. D. & B. E. Spencer. 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Laboratory leaflet. Directorate of Fish- eries Research (Great Britain) No. 68. 31 pp. Wang. R., Z. Wang & J. Zhang. 1993. Marine Mollusc Culture. Qingdao, China: Ocean University of Qingdao Publisher. 397 pp. Joiinwl ofSlu'llfish Research. Vol. 21. No. 2, 571-576, 2U02. INTRASPECIFIC VARIATION IN THE REPRODUCTIVE CYCLE OF THE TEHUELCHE SCALLOP AEQUIPECTEN TEHUELCHUS (PELECYPODA, PECTINIDAE), IN SAN MATIAS GULF, PATAGONIA, ARGENTINA MATTE NARVARTE* AND MARINA KROECK Institute) de Biologia Marina y Pesqiiera "Almirante Storni, ' Negro. Argentina CC 104. (8520) San Antonio Oeste. Rio ABSTRACT The reproductive cycle of the tehuelche scallop. Aaiiiiivclen leluiclclui.s. from San Matias Gulf was studied for a two year period. Scallops were collected at monthly intervals from August 1997 to July 1999. Muscular and gonosomatic indexes were estimated and histologic sections of the gonad were prepared and used to determine gametogenic stages and to estimate the mean oocyte diameter. Gonosomatic index (GSI) data revealed a bimodal pattern, but the spring-summer spawn was larger and more synchronized than the autumn spawn. GSI and histologic observations had similar patterns. Massive spawning began in December. Gametogenesis lasted from June through late winter (September). The tehuelche scallop is a partial spawner. The spawning schedule showed between-year tlucluations. starting at any time during September through October, depending on the year. Partial spawnings and gonadal recovery alternated along^the summer, until March. Spawning was followed by the reab.sorption of unspawned gametes. A resting stage in the reproductive cycle occurred from late summer (March) to mid-autumn (May). In I of the two years studied, an early sexuatmatwation matched low temperatures. This suggests that in San Matias Gulf the reproductive cycles are not clearly synchronized with seasonal cycles of superficial seawater temperature and that other factors, presumably the amount of food supply, may be responsible for an anticipated gonadal maturation. KEY WORDS: reproductive cycle, intraspecific variation. Aequipecten tehuelchus. san Matias Gulf INTRODUCTION The tehuelche scallop Aequipecten lehuelcluis (D'Orb., 1846) is one of the most impoitant commercially fished bivalves in east- ern Patagonia (San Matias and San Jose gulfs). Landings have fluctuated from 4.700 to 100 tons during the last 30 years. Fishery measures include minitnum commercial size, rotation of fishing areas, and selection of catch on board (Orensanz et al. 1991 ). The fishery operates during winter months to obtain maximum yield and to prevent capture during reproductive periods. The tehuelche scallop is hermaphroditic (Christiansen & Olivier 1971). As in other pectinid species, the male gonad is white-yellow and the female gonad is orange ("corar'). Gamete development is easily viewed within the acini, and light varies with the gametogenic stage (Barber & Blake 1991). Christiansen et al. (1974) delineated the reproductive cycle of a San Mati'as Gulf population based on irregular samplings in the region. Fecundity ranges from 2 to 17 million eggs for scallop sizes of 35-90 mm (Orensanz et al. 1991). The reproductive cycle of the populations from San Jose Gulf (43°S) was studied by Lasta and Calvo ( 1978). Partial spawnings begin in late spring and pre- and post-spawning stages coexist as far as the end of the summer. Despite the high value of this fishery in San Matias Gulf (41°S). the reproductive cycle was not studied in detail until this work. There is extensive literature on the gametogenic cycle and the timing of spawning in many bivalve species (Giesse & Pierce 1974, Sastry 1979. Newell et al. 1982. Barber & Blake 1991). For several pectinid species in the northern hemisphere, spawning oc- curs at higher temperatures and later in the year in southern popu- lations (Barber & Blake, 1991) and is often more synchronized at higher latitudes (Bricelj et al. 1987). The objective of this study is to describe the reproductive cycle of the tehuelche scallop of San Matias Gulf and to compare the reproductive patterns between years for the same population. MATERIALS AND METHODS Specimens of tehuelche scallop were collected from the north- ern bank of the San Matias Gulf (Orengo, 40°32'S; 64"32'W) (Fig. 1), at a depth of 18-24 m, between August 1997 and July 1999. Monthly samples of 20 specimens, with a mean shell height of 67.05 mm (SD: 10.1), were used to determine the gonosomatic index (GSI) and muscular index (MI) before fixation. Scallops were dissected; the shells removed; and the total weight (TW). soft parts wet weight (SPW), muscular weight, and gonad wet weight (GW) were recorded. The GSI was calculated using the equation (Giesse & Pierce 1974, Jaramillo et al. 1993): GSI = GW X 100 SPW Mean GSI was estimated for each monthly sample. To analyze multiyear trends in reproduction, data collected in 1997 and 1999 were coupled to a GSI data set already available for the Orengo area from 1989 and 1991, and both data sets were related to sea water temperature. Minimal average values following high average Gonosomatic index (GSI) 40 32 24 16 8 ♦Corresponding author. E-mail: maitenarvarte@canaldig.com.ar Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun 1997 1998 1999 Figure I. Temporal changes in the GSI iif tehuelche scallop deployed in Playa Orengo, San Matias Gulf, from August 1997 to July 1999. Data points are mean values ± standard deviations; n = 20 scallops per sampling date. 571 572 Narvarte and Krofxk values were considered as indicators of spawning (Jaraniillo et al. 1993. Avendano & Le Pennec 1997). The mean muscular index (MI = wt. of adductor inuscle/TW X 100) was estimated monlliK in a similar \\a\ than GSI and was compared with GSI data. The gonads from those 20 specimens were then processed for histologic analysis. The whole gonad was fi,\ed in Davidson's solution, stored in cthanol i71)'f). and then dehydrated using a series of increasing ethanol solutions (Howard & Smith 1983). Then, the samples were embedded in paraffin and sections 5- to 7-|jLm thick were cut and stained with Harris hematoxylin and eosin. Gonad tissue was quantitatively examined using the schemes of Lasta and Calvo (1978). Dihacco et al. (1995). and Pazos et al. (1996) to assess developmental stages. The percentage of each gonadal stage was analyzed. The definition of each stage (early- mid mature, ripe, partial spawn, spawning and recovery, and spent) is provided in Table 1 . Photomicrographs were taken using a stan- dard microscope at magnifications of 100 and 400x. Oocytes were measured and analyzed according to Dihacco et al. ( 1995). Mean oocyte diameter was estimated by measuring the diameter of 250 oocytes of each individual. This process was repeated for five individuals from the total monthly sample. Only those oocytes with clear nucleolus were considered for the analysis. The GSI and adductor MI indexes were compared by means of a simple ANOVA using data that had been subjected to an arcsine transformation [x'(p)l (Sokal & Rohlf 1979). The maximum values of the oocyte diameter were compared between years using a simple ANOVA, RESULTS Gonosomatic Indices During this two year study, major peaks in GSI were observed in January and December 1998. Peaks were followed by decreases in GSI. representative of spawning events (Fig. 1 ). Maximum GSI was significantly different (F, 3y = 88.88, P < 0.001) between these two reproductive periods. Average values of GSI ranged between 27,58 (SD: 9,93) and 4.83 (SD: 0.85) from January to July 1998, and from 15,30 (SD: 5,17) to 4,01 (SD: 1,11) from Decem- ber 1998 to April 1999, GSI increased between July and October, indicative of early stages of gametogenesis, GSI then reached maximal values from November to January and subsequently dropped over a short pe- riod, which indicated a massive spawn. Spawning continued to April, when the last spawning events were detected and after which the GSI was annually the lowest (May through June), A resting period of 1-2 mo was observed after the autumn spawn. The magnitude of spring and autumn spawning periods, esti- mated by observ ing the extent of drops and peaks of the GSI. varied interannually. particularly for the spring spawn. Histologic Analyses Sperm and oocytes were observed together in the gonoduct (Fig, 2a), The male portion spawns o\er a longer period than the female gonad (Fig, 2b) and. consequently, the female gonad de- velopment is what determines the reproductive viability. For this reason gonadal cycle was described only for the female portion of the gonad. Oocyte Characteristics Absence of vitello was typical of the deselopmcnt oocytes, as well as a defined nuclei (Fin, 2c), Mature oocytes had a diameter TABLE \. Main histological characteristics of gonadal maturity stages of the tehuelche scallop, (ionadal stages Here determined h\ histological analjsis and scored using a modification of Lasta and Cal>o (lyVX). Stages Description Early maturation Follicles are well-defined. The lumen may occupy up to 50% of the follicle, The intertollicular space is highly visible, Male: few layers of cells (germ cells and spermatogonia) along the follicle wall. Female: presence of germ cells, oogonia. and primary oocytes with diameters varing between 10 and 25 |xm. Mid-maturation The lollicles occupy up to 75"(: of the gonad. Follicular lumen is much reduced. Interfollicular space is still present but limited. Male: follicles are full of spermatocytes I and II and spermatids. Female: stalked oocytes I. 25 to 40 (jLm, protrude into the follicular lumen but still attached to the wall (peduncled oocytes). Ripe The follicles are completely full of ripe gametes, and there is no interfollicular space. Mate: spermatozoa occupy almost 100'7r of the follicles. They are in order and oriented with the flagella toward the centre of the follicle, Fumatc: follicles are full of free oocytes up to 50 |xm diameter. They have polygonal shapes, being pressed against each other. Spawn and Ripe gametes are being released through the recovery gonoducts. Presence of a second series of germ cells attached to the follicular wall. Male: presence of spermatocytes I and II, and spermatozoa are disordered in the follicular lumen. Female: presence of oocyte I attached to the wall and polygonal oocytes free and disordered in the follicular lumen. Partial spawn Follicles remain lull of mature gametes, but some of them look lax because some gametes were released. Some phagocytes are present in the interfollicle space. Male: spermatozoa are disordered and separated in the follicular lumen. Female: remained oocytes still have polygonal shapes and they are free or attached at one side of the follicle. Spent Most follicles are empty. Interfolliclar spaces become very noticeable. Phagocytic cells are very abundant. Residual gametes may or not be present, but w ith no sians of cametosienesis. around 50-60 |jim, with a high quantity of vitello around a con- spicuous nuclei (Fig, 2d and 2e), Reabsorbed oocytes (both mature and non-mature) remained attached to the follicle wall or free inside it. They were easily distinguished by the loss of the round- polycdric shape, typical of mature oocytes, and by the diffuse contour of the cell membrane (Fig. 2f). The central portion of the gonad, or the transitional zone between female and male portions, had a high frequency of atresic oocytes inainly in the ripe stage during August-October 1497 (Fig, 2h), Gonadal Phases Gonadal phases, considered as the percentage of individuals of each gonadal stage over the monthly sample, differed between 1997 and 1998 and 1998 and 1999 (Table 1, Fi;;, 3), Reproductive Cycle Variation of the Tehuelche Scallop 573 4^ i> ■= > ^r.^- \ .1*^^' ^1 :ijM± . .♦: M- ^■ «" ■^^<^:. : ■ i ■> V . :. J -. ^ 1.3°C) were recorded from September to December for the period 1997-1998 and from Sep- tember to January in 1998-1999. Figure 6b represents the variations of GSI during 1989-1991 and 1997-1999 seasons. Inter-year variations were similar for the first period. Maximum mean values oscillated between 16.15 (SD: 4.28) and 17.56 (SD: 6.02). During the 1997 and 1999 periods, differences between years were greater, ranging from 27.58 (SD: 9.93) to 15.30 (SD: 5.17). Muscular Energy Resen'es Ml reached its ma.\inium mean value during winter-spring months, before the start of the reproductive season of 1997 to 1998 and 1998 to 1999 (Fig. 7). No differences in the MI were found between 1997 and 1998, for August, October, and November (P > 0.05). MI increased between January and July before gonadal de- velopment (Fig. 7). DISCUSSION The GSI of A. relnielchiis from the San Mati'as Gulf, studied over 2 consecutive years, indicated that this species undergoes its maximum reproductive activity during the spring-summer months. A small spawning occurred in autumn. Even when this pattern was repeated during the study period, seasonality and du- ration of spawnings were not identical between successive years. In both the 1997 to 1998 and 1998 to 1999 seasons, a massive spawning occurred between December and January. A second, lower-magnitude spawning occurred in March through April 1999. Even though a fall spawning was not supported by GSI in 1998. Water temperature (°C) a) Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun Gonosomatic index b) 30 24 _ 18 (0 a 12 6 A Aug Oct Dec Feb Apr Jun Aug Oct Dec Feb Apr Jun Figure 6. Inter-year variations of (a) the seawater temperature IT, C) and (b) GSI during two study periods, 1989 to 1991 and 1997 to 1999. »**v^ ^^^ 0 Jun-97 Sep-97 Ene-98 Abr-98 Jul-98 Nov-98 F6b-99 May-99 Ago-99 -GSI -Ml Figure 7. Variations of MI and GSI indexes from August 1997 to June 1999. evidence of spawning was cleariy observed in the oocyte fre- quency distribution and mean oocyte sizes. Temporal patterns of the reproductive condition of the tehu- elche scallop in this study revealed some other differences between histological analyses and GSI monitoring. The main spawning in 1997 to 1998 season was detected from January to February by GSI and from December to January by histologic studies. In ad- dition, the microscopy study on the female gonad of A. telmelchus revealed the coexistence of different components of the oocyte series in those successive years. Therefore, the tehuelche scallop has asynchronous maturation and continuous spawnings within the same reproductive season. The existence of an eariy mature stage in August was con- firmed by the oocyte diameter frequency distribution. Neverthe- less, the great percentage of atresic oocytes in August-October suggests that spawning did not occur. Greater than 50% atresia has also been observed in other pectinids (Lubet et al. 1987a, Lubet et al. 1987b, Avendatio & Le Pennec 1997, Roman et al., unpub- lished data). Dibacco et al. (1995) suggested that during unfavor- able environmental conditions, bivalves may have a mechanism to reabsorb and use the high energy content of mature oocytes. As in other pectinids (Motavkine & Varaksine 1983. Beninger 1987, Dorange & Le Pennec 1989. Avendatio & Le Pennec 1997). the highest levels of atresia in A. telniekluis were observed just before spawning. These authors have suggested that atresia should be related with an energy deficiency, caused by low food availability. This could have occurred during the winter months in 1997 in San Matias Gulf, when lower water temperature was recorded. Histologic observations of the female gonad during the fall spawning of both reproductive seasons showed a high relative proportion of oocytes in eariy stages, whereas larger oocytes were observed undergoing lysis. Metabolites produced by lysis of ma- ture oocytes by phagocytic cells, present in large quantities at the end of the reproductive period, would implicate an important en- ergy contribution for the recovery after the reproductive season. This phenomenon was previously observed for a variety of pec- tmids (Dibacco et al. 1995, Avendaiio & Le Pennec 1997). Ac- cordingly, we could infer that the second spawning of each season was smaller than the first, and constituted a minor contribution to that year class. The utilization of the adductor muscle energy reserves for re- production was cited for other pectinids (Barber & Blake 1991). Variations of MI observed along two annual cycles in this study showed a similar pattern, in which the energy stored in the adduc- tor muscle is then diverted to the reproductive system, to satisfy 576 Narvarte and Kroeck energetic demands for the synthesis of gonad components (Gould et al. 1988). When the MI was compared between years, no clear variations were observed between 1997-1998 and 1998-1999. Therefore, energy demands from the muscle seemed to be similar. However, the extraordinary GSI values in 1997-1998 suggest a higher energy availability for the gonad development. Because muscular reserves are limited by muscular degradation, other ex- ogenous mechanisms could help to sustain the energy budget for this development. A higher food availability during the spring 1997 was probably responsible for this extraordinary gonadal de- velopment. Thompson and MacDonald (1991) have indicated that temperate species such as Pkicopecteii iinini'lliiniciis are exposed to phytoplanktonic "blooms" in early spring and therefore have a short period of energy reserve accumulation. This early investment for reproduction would depend of the environmental conditions following the bloom (Dibacco et al. 1995). de Vido de Mattio 1 1984) and Orensanz ( 1986) indicated that energy for gonadal development for A. teliiielcliii.\ in the San Jose Gulf seems to come more from food than from the energy stored in the muscle. In the San Mati'as Gulf, a decrease of MI before spawning indicates that this population uses both energy sources, food and muscle. As was observed in other pectinids (Langton et al. 1987, Barber & Blake 1991, Dibacco et al. 1995. Rheault & Rice 1996. Avendano & Le Pennec 1997), exogenous factors, such as temperature and food availability, may produce great variations in the gonadal development and the spawning time of A. lehiiel- chiis. AKNOWLEDGMENTS This work was supported by funds from the Europe Union- Argentina Agreement ( 1996-1999). The authors thank the person- nel of the IBMP"Alte. Storni" for their cooperation and assistance. We specially thank Sandro Acosta. Tony Brochado, and Elda Cerecera for assisting with field and laboratory activities, and Mar- cela Pascual, Jose M. Orensanz, and an anonymous reviewer for their constructive criticism of the manuscript. LITERATURE CITED Avendano, M. & M. Le Pennec. 1997. Intraspecific variation in gameto- genesis in two populations of the Chilean molluscan bivalve, Ar- gupeclen purpuratus (Lamarck). Aquaculliire Res. 28:175-182. Barber, B.J. & N.J. Blake. 1991. Reproductive physiology. In; S. E. Shumway, editor. Scallops: biology, ecology and aquaculture. Amster- dam: Elsevier, pp. .V^7-128. Beninger, P. G. 1987. A qualitative and quantitative study of the repro- ductive cycle on the giant scallop, Placopecten miigellanicus. in the Bay of Fundy (New Brunswick, Canada). Can. J. Zool. 65:495^98. Bricelj, V. M., J. Epp & R. E. Malouf. 1987. Intraspecific variation in reproductive and somatic growth cycles of bay scallops Argopecten irradians. Mar. Ecol. Prog. Ser 36:123-137. Christiansen, H. E. & S. R. Olivier. 1971. Sobre el hermafroditismo de CbUtmys tehuelcba d"Orb 1846 (Pelecypoda. Filibranchia. Pectinidae). An. Sue. Cienti'f. Argentina 191:115-127. Christiansen H. E., M. E. Cabrera & S. R. Brodsky. 1974. Ecologia de las poblaciones de vieiras {Clilaniy.s tehuelcha d'Orb. 1846) en el Golfo San Matias (Rio Negro, Argentina). I. Estudio histologico del cicio reproductive. Ins. Biol. Mar. Mar del Plata. Contr. 225:17. De Vido de Mattio, N. 1984. Variacion estacional de la composicion bio- quimica de la vieira Chlainys tehuelcha (d"Orbigny) en el Golfo San Jose. Centra Nac. Patagonico. Contr. 92:22. Dibacco, C, G. Robert & J. Grant. 1995. Reproductive cycle of the sea scallop. PUuopeclen magellaniciis (Gmelin. 1791). on northeastern Georges Bank. J. Shellfish Res. 14:59-69. Dorange. G. & M. Le Pennec. 1989. Ultrasctructural study of oogenesis and oocyte degeneration in Pecteii ma.xinms from the Bay of St. Brieiic. Mar. Biol. 103:339-348. Giesse. A. D. & J. S. Pierce. 1974. Pelecypoda (excluding Ostreidae). In: Reproduction of marine invertebrates. New York: Academic Press, pp. I1,V265. Gould. E.. D. Rusanowsky & D. Luedke. 1988. Note on muscle glycogen as an indicator of spawning potential in the sea scallop, I'hicopecten magellaniciis. Fish. Bull. 86:597-601. Howard, D. W. & C. S. Smith. 1983. Histological Techniques for Marine Bivalve Mollusks. NOAA Technical Memorandum NMFS-F/NEC-25. Jaramillo, R.. J. Winter. J. Valencia & A. Rivera. 1993. Gametogenic cycle of the Chiloe scallop (Chlamys amaiidi). J. Shellfish Res. 12:59-64. Langton, R. W., W. E. Robinson & D. Schick. 1987. Fecundity and re- productive effort of sea scallops Placopecten magelUmicus from the Gulf of Maine. Mar. Ecol. Prog. Ser 37:19-25. Lasta, M. L. & J. Calvo. 1978. Ciclo reproductivo de la vieira [Chlamys tehuelcha) del golfo San Jose. Com. Soc. Malacol. Uruguay 5:1—42. Lubet, P., J. Y. Besnard, R. Faveris & I. Robbins. 1987a. Physiologic de la reproduction de la coquille Saint Jacques {Pecten maxiinus L. ). Oceanis 13:26.5-290. Lubet. P.. J. Y. Besnard, R. Faveris & I. Robbins. 1987b. Competition energetique entre tissus musculaire et gonadique chez la coquille Saint Jacques (Pecten maximus L.). Haliotis 16:17.3-180. Motavkine. P. A. & A. A. Varaksine. 1983. Histophysiologie du systeme nerveux et regulation de la reproduction chez les mollusques bivalves. Paris: Editions IFREMER. 208 pp. Newell. R. I. E.. T. J. Hilbish, R. K. Kohen & C. J. Newell. 1982. Tempo- ral variation in the reproductive cycle of Mytilus edulis L. (Bivalvia: Mytilidae) from localities on the east of the United States. Biol. Bull. Mar Biol. Lab. Woods Hole 162:299-310. Orensanz. J. M. 1986. Size, environment and density; the regulation of a scallop slock and its management implications. Can. Spec. Piihl. Fish. Aquat. Sci. 92:195-227. Orensanz. J. M.. M. Pascual & M. Fernandez. 1991. Scallops in Argentina. In: S. Shumway, editor. Scallops: Biology, ecology and aquaculture. Amsterdam, Elsevier, pp. 981-999. Pazos. A. J.. G. Roman. C. P. Acosta. M. Abad & J. L. Sanchez. 1996. Stereological studies on the gametogenesis cycle of the scallop. Pecten maximus. in suspended culture in Ria de Arousa (Galicia. NW Spain). Aquaculture 142:119-135. Rheault. R. B. & M. A. Rice. 1996. Food-limited growth and condition index in the eastern oyster. Crassostrea virginicu (Gmelin 1791 1, and the bay scallop. Argopecten irradians irradians (Lamarck 1819). J. Shellfish Res 15:271-283. Sastry. A. N. 1979. Pelecypoda (excluding Ostreidae). In: A. C. Giese & J. S. Pierce, editors. Reproduction of marine invertebrates. New York: Academic Press, pp. 1 13-292. Sokal, R. R. & F. J. Rohlf 1979. Biometri'a. In; H. Blume. editor. Princip- ios y metodos estadi'sticos en la investigacion bioliigica. Madrid, 817 pp. Thompson. R. J. & B. A. MacDonald. 1991. Physiological integrations and energy partitioning. In: S. Shumway. editor. Scallops: biology, ecology and aquaculture. Amsterdam: Elsevier, pp. 347-372. Journal of Shellfish Reseciirh. Vol. 21. No. 2. 577-584, 2UU2. REPRODUCTIVE AND RESERVE STORAGE CYCLES IN AEQUIPECTEN OPERCULARIS (L., 1758) IN GALICIA, NW SPAIN G. ROMAN,'* M. J. CAMPOS,' J. CANO,' C. P. ACOSTA," PILAR IGLESIAS,' AND OSCAR GARCIA' 'inslituto Espanol de Oceunografi'a. Mmisterio de Cienciu y Tecnologiu. Ceiiiro Ocecmogrdfico de A Contna. P.O.Box 130. 15080 A Coniiia. Spain: -CMA Coron. P.O.Box 208, 36600 Vilagarci'a de Aroiixa. Pontevedni. Spcdn: ^Departameuto de Bioqiiimica y Biologi'a Molecular. Facultad dc Fannacia. Universidad de Santiago de Compostela. Campus Sur. A Contna. Spain ABSTRACT A study was conducted on the reproductive and reserve storage cycles in Aequipecten opercularis (L.. 1758) from deep waters (50-60 m) in the Ri'a de Arousa (Galicia, NW Spain). The reproductive cycle was studied by visual inspection of the gonad, the use of the gonad condition index, the gonad dry weight of a standard queen scallop 50-mni height, the average oocyte diameter and the percentage of the gonad occupied by oocytes. The same results were obtained with each of these methods. Following a restmg period in autumn, which varied in length depending on the year, there was a period of recovery in winter, followed by a spawning period lasting until the end of summer. There were several partial spawnings, followed by recovery periods until the gonad was totally spent. Accumulated reserves in the adductor mu.scle (glycogen and proteins) and digestive gland (lipids) were used up during gametogenesis. The weight and condition indices of both organs and their reserve substrate contents showed parallel changes throughout the two years of the study. KEY WORDS: Aequipecten openidari.s. Ria de Arousa, breeding season, reproduction, energy reserves INTRODUCTION Aequipecten operciiluris (L., H.'SS) is distributed along the North Atlantic coast, ranging between WH and 70°N, and also extends to the Mediterranean and the Adriatic Sea (Brand 1991). There is one commercial fishery of this species in Galicia, which in the 1960s caught up to 700 tonnes year"', although nowadays the catch rarely exceeds 100 tonnes year"'. Despite the commercial interest in A. opercidaris in Galicia, investigation of the reproductive biology of natural populations in this region has not been performed. Only the reserve storage cycle and breeding season have been previously been described, by Ro- man et al. (1996), for cultured queen scallop in the Ri'a de Arosa. Mason (1983) and Barber and Blake (1991) reviewed the pe- riods of gametogenesis in queen scallop populations in the North Atlantic. However, the information provided is scant and the re- productive behavior of geographically separated populations of the same species may vary (MacDonald & Thompson 1988, Bricelj et al. 1987, Devauchelle & Mingant 1991). As gametogenesis, the storage and use of energy reserves in scallops is also cyclical and underlies the |-eproductive cycle. The importance of stored reserves in the reproductive cycle of pectinids in temperate waters is well documented (see Barber & Blake 1991 for a review). In general, protein and/or glycogen are .stored in the adductor muscle and lipids in the digestive gland. Barber and Blake (1981) reported that in Argopecten irradiuns. the relative importance of different parts of the body as energy storage sites is highly adaptive, and results from genetic divergence or non genetic adaptation to different environments within the geographical range of the species. This study is part of an investigation of the feasibility of cul- tivating A.opercularis in Galicia from spat obtained from natural environments using collectors. Studies of the reproductive behav- ior of this species are required to optimize spat collection for commercial purposes. The main objectives of this study are to describe gametogenesis, the breeding season, seasonal changes in weight of reserve storage organs and their biochemical composi- tion in relation to the reproductive cycle, and the influence of environmental factors on gametogenesis and spawning. MATERIALS AND METHODS Sliidv Area *Corresponding author. E-mail: guillermo.roman@co.ieo.es The largest population of queen scallops in the Ri'a de Arosa are found in "The Channel" (Fig. I ), at a depth of between 50 and 60 m. The study was therefore centered on this area, although dis- perse, smaller banks are found in shallower waters. Environment From January 1996 until December 1997 weekly recording was made of temperature, salinity and chlorophyll a at a depth of 45 m using a CTD. From March to September 1996 fortnightly deter- minations were made of the paniculate organic matter, by filtering a sample of 2 L of seawater, collected from a depth of 30 ni, using pre weighed Whatman GFC/C filters (Whatman International Ltd., Maidstone. England). After drying at 60°C for 24 h. the filters were weighed then reweighed after combustion at 450°C for 12 h. The organic matter content was calculated as the difference be- tween the two weights. Animals Adult specimens of A. opercidaris were collected between January 26,1996 and November 18, 1997. Captures were made at fortnightly intervals in "The Channel"" from a commercial fishing boat, by trawling. The animals measured between 35 and 65 mm in height, which corresponds to age year classes I and 2. The gonads of 10 scallops were placed in Bouin"s fixative immediately following capture. The fixative was removed by frequent washing with 70* ethanol saturated with lithium carbonate and then stored in 70% ethanol until being processed. Classic histologic techniques were used; gonad was embedded in paraffin and serial sections of ovary (5 (xm) stained using modified Wheatley"s stain. Histologic study was only made of ovary tissue, using stereological methods (Paulet & Boucher 1991, Pazos et al. 1996) with a Visilog 3.1.1 image analyzer. The average diameter of the oocytes (Dm) and the 577 578 Roman et al. 43'«)' MbftG/vRCIA 43 "JS' 43°3D' I SAVORA 43*25' 1D°5' 9° 9°55' 9°50' Figure 1. The Channel (shadowed area) in the Ria de Arosa. 9P46' percentage volume occupied by oocytes (OVF = % of ovary occupied by non atretic oocytes) were calculated. Another 30 animals were transported to the laboratory in a coolbox and then maintained under running water for 18 h to allow sand to be expelled. The specimens were then opened and the soft tissue separated into 4 components: gonad, adductor muscle, di- gestive gland, and remaining tissues. After draining for 10 min on filter paper, the wet weight (WW) was detennined; an aliquot of each organ was used to determine the dry weight (DW lOO'C. 24 h). We recorded the appearance of the gonad, noting if transparent and colorless or if there were two clearly differentiated areas of color (white or cream in the testicle, orange or red in the ovary). The height of the shell was measured to the nearest 1 mm. using calipers. The shells were dried (lOO'C. 24 h) and weighed. Con- dition indices were calculated for gonad (Gci). digestive gland (DGci). muscle (Mci) and remaining tissues (RTci) using the fol- lowing equation: CI organ = 1985). 100 X DWorsan/DWshell (Lucas & Beninger, Reproduction of Queen Scallop 579 Regression equations were used to describe the relationships be- tween shell height and the dry weight of each component, and the weight of each component for a standard animal having a shell height of 50 mm was calculated according to Taylor and Venn (1979). Aliquots of muscle, digestive gland and gonad were lyophilized before biochemical analyses were carried out (glycogen and pro- tein content of muscle and total lipid content of digestive gland and gonad) following previously described techniques for lipids and glycogen (Roman et al. 1996). Protein contents were determined from the N content, measured using an elemental analyzer Carlo Erba 1 108. The amount of N was multiplied by 6.25 to estimate the amount of proteins (Ansell 1974. Beninger 1982). Grams of lipids in gonads and digestive gland and glycogen and protein in muscle were estimated by multiplying the percentage of each reserve sub- strate by the previously calculated standard dry weight of each component. Statistical Methods One-way ANOVA was used to test for significant differences among the average values of the condition indices. Dm and OVF. The normality of the distribution of the variables was tested using the Kolmogorov-Smirnof test and the homogeneity of the vari- ables using Bartlett's test. Where necessary, arcsine transforma- tions (.v' = arcsin^i.v) were carried out. Correlation coefficients were calculated for the relationships among gonad indices. Dm and OVF, and among the condition indices of the reserve storage or- gans, the standard dry weight and biochemical composition. RESULTS Environment The temperature varied little throughout 1996, ranging between 12.7°C and 14.4°C. In 1997 very high temperatures of 17 to I8"C were recorded in May and June. High values of 16.5 to 17°C were also recorded from the end of October until the middle of Novem- ber. The chlorophyll a content was low in both years, ranging between 0.6 and 1.3 |Jig L"'; the lowest values were registered between October 1996 and May 1997 and did not rise above 0.8 jjLg chlorophyll a L~'. Particulate organic matter ranged between 0.3 and 2.7 mg L"'. Salinity remained practically constant at 35.7- 35.8%c throughout most of the sampling period, with slight de- creases occurring in winter to below 35.59{(. The minimum value. 34.2%t, was recorded in November 1997 (Fig. 2). Gonad During winter and spring of 1996 the gonads were well differ- entiated, with testicle and ovary clearly visible in all specimens. From July 23, 1996 onwards there was an increasing percentage (reaching a maximum value of 90% on September 10) of queen scallops with spent gonads, which were tlat. colorless and watery, histologic analysis revealed empty follicles in the undeveloped gonads. In these queen scallops that were in a period of sexual repose, the average diameter of the oocytes and the percentage volume occupied by oocytes was considered to be zero (Fig. 3). Following the period with the maximum percentage of sexually reposing animals, there was a period of gonad recovery, which involved only part of the population, peaking (at 60%) on October 26, then a new minimum (30%) was observed on November 19. From December 1 996 onwards there was a new period of gonad D) 1.2 CD 1,0 r 0,8 Q- O O 0.6 O 04 E O Q. ,#•" .s^-- ,^^'~ ,.^^ ,.^^ Figure 2. Environmental conditions in The Channel. ^K-^ recovery, which this time involved the whole population, reaching full development on January 28, 1997. Following a decrease ob- served on February 12, which affected 40% of the population, all animals had developed gonads. This situation persisted until the end of summer, then from August 12 until October 28, 1997 all of the population was in sexual repose and displayed spent gonads. In 1997, gonad recovery took place between October 28 and Novem- ber 19. The value of the gonad condition index (Gci) ranged between 0.1 and 2.0 (Fig. 3). Maximum values corresponded to the period between March 13 and 26 1996 followed by a significantly lower value on April 10. There were then a series of recoveries followed by decreases to significantly lower values on June 4. July 2. and August 20; minimum values were recorded coinciding with gonads being totally spent. There was a new peak (significantly higher) on October 20, the gonads then became totally spent before undergo- ing recovery from November 19 onwards. The peak recorded on January 28, 1997 was followed by a significant decrease on Feb- ruary 12 and then again, as in 1996 there were a series of recov- eries and decreases, with significant decreases observed on March 25, May 17, July 1, and August 12, which coincided with the 580 Roman et al. ■o (0 c o O) c 'l nj n w o o X 0) ■D C c o c o o ■D TO c o O) E E Q Figure 3. From top to bottom, a) percentage of scallops bearing gonad, bl gonad condition index, c) oocyte mean diameter and percentage volume of the ovary occupied by oocytes, and d) gonad dry weight of a standard 50 mm height scallop and lipid content of the gonad. gonads being totally spent; recovery did not take place until No- vember 19. The decreases were assumed to be associated with spawning. Changes in the average diameter of the oocytes and of the percentage volume of the ovary occupied by oocytes (OVF) were similar (Fig. 3). Maximum values occurred in winter (February to March 1996) then a sharp decrease was observed on April 10. 1996. The values had significantly increased by April 24 and there was then a gradual decrease until reaching minimum values be- tween September 10, and October 8. 1996, with two recoveries observed on June 19 and August 6. There were significant in- creases in both Dm and OVF between December 16 and January 14, coinciding with gonad recovery. In 1997 there were 4 peaks observed on January 28, March 10. June 3. and July 29 followed by decreases, which only on the latter date was not followed by gonad recovery. The changes in all of the parameters studied followed the same pattern and each parameter was significantly correlated with the others: Gci vs. Dm (/■„ = 0.588. P < 0.0001 ); OVF vs. Dm (/; = 0.772. P < 0.0001). and Gci vs. OVF {i\ = 0.716. P < 0.0(J01). The gonad dry weight of a standard queen scallop ranged be- tween 0.006 and 0.204 g. The seasonal variations are shown in Figure 3. Although the mean percentage value of the total weight was low {9.09c) the range of values varied greatly, between 0.7% and 23.0% of the dry weight of the animal. There was a large variation in weight, which followed the pattern of variation of Gci. Biochemical Compositian of the Gonad The percentage values of lipid contents are shov\n in Table 1. The total lipid content of the gonad of a standard queen scallop varied in a similar way to the Gci and to the standard DW. with maximum and minimum values coinciding with the maximum and minimum values of the condition index and the standard dry weight (Fig. 3). Somatic Tissues Seasonal changes in the standard dry weight of the muscle and the digestive gland, as well as in their respective condition indices and in their reserve substrate contents (lipids in digestive gland, glycogen and proteins in adductor muscle) are shown in Figure 4. Somatic growth began in early summer and was demonstrated by increases in dry weight, condition index and specific reserve sub- strate content, which reached maximum values between autumn and winter. There was then a decrease in the values of all of these parameters. Adductor muscle accounted for most of the w eight of the queen scallop, on average 54.5%; the mean dry weight of the muscle varied between 35.6% and 68.1%. representing the greatest varia- tion in the weight of the animal. The dry weight of the muscle of a standard animal varied between 0.273 and 0.993 g. (i.e.. the mean dry weight varied by up to 3 times). The condition index of the adductor muscle (Mci) varied in a similar way. ranging be- tween 3.3 and 8.5. Digestive gland accounted for a small proportion of the total weight of the animal (mean. 10.3%. range 6.9%- 14.2% ). Although the weight varied more than that of the muscle, ranging between 0.042 and 0.198 g, (i.e., varying by almost 5 times) the condition index (DGci) ranged between 0.5 and 1.7 (i.e.. it varied by more than three times). The dry weight of the remaining tissues accounted for. on average, 26.0% of the weight of the animal, ranging between 17.3% and 39. 1 % of the somatic components of the standard queen scallop. The dry weight of the remaining tissues of the standard specimen ranged between 0.144 and 0.310 g (i.e.. it varied by approximately two times). The percentage amounts of glycogen and proteins in the mus- cle and of lipids in the digestive gland in the gonad are shown in Table 1. In January 1996. high values were recorded of standard dry weight of muscle. Mci. percentage, glycogen, and total protein and Reproduction of Queen Scallop 581 TABLE 1. Seasonal changes in the composition of A. opercularis. Adductor Muscle Digestive Gland Gonad Date % Lipids % Lipids % Glycogen % Protein Jan 25 57,52 28.07 14.74 71.68 Feb 28 42,70 25.27 7.28 80.19 Mar 13 32,04 25.88 5.19 80.22 Mar 26 34.96 26.63 5.32 77.34 Apl 10 27.40 27,04 4.27 7 1 .95 Apr 24 28,72 26,89 4,41 77.61 May 07 22.08 24,20 3,34 80.17 May 23 35.00 24,67 3,47 72.19 Jun05 33.75 23,30 4,49 69.38 Jun 19 44,26 27.89 4,39 76.00 Jul 02 42,77 29.14 6,11 72.5 1 Jul 23 50,55 29.41 6,84 70,64 Aug 06 54,86 25.87 7,85 69,77 Aug 20 61,02 27,05 9,21 73.42 Sep 10 48,04 29,57 16,68 67.23 Oct 08 54,81 26,71 18,02 64.44 Oct 26 49,50 22,64 18,92 69.33 Nov 19 44,34 18.86 14,03 72..30 Dec 10 41,61 21.80 12,56 70.01 Jun 14 47,20 20.61 13,03 74.83 Feb 12 62,45 23,54 8,55 74.89 Feb 25 41,8! 22.12 5,63 75.66 Mar 10 34,63 17.32 8,20 74.52 Mar 25 28,66 18.21 6,25 73.07 Abr22 25,76 18,54 2,65 77,87 May 06 31,80 22,03 6,61 77.02 May 17 25.38 23,73 2,93 76.26 Jun 03 36,65 23,73 2,76 74.26 Jun 17 39,01 18,95 5,76 78.27 Jul 01 39,31 22,31 6,36 73,45 Jul 15 44,40 22,11 8,44 — Jul 29 49,11 23,70 8,48 — Aug 12 52,66 23,87 11.98 — Aug 26 57,81 30,00 13.91 72.10 Sep 09 59,36 25,67 14.74 71.58 Sep 29 57,64 23.31 14,79 72.76 Oct 28 60,75 21,28 18,68 66.25 Nov 18 62,99 19,91 14.90 68.80 glycogen content; these then decreased rapidly and minimum val- ues were registered at the beginning of spring. During summer and autumn there was rapid somatic growth, with maximum weight values in November. Values of all parameters then began to de- crease and the cycle was repeated, with minimum values occurring at the beginning of spring 1997, before beginning to increase once again. The percentage amount of proteins in the muscle showed a more irregular pattern of variation, with frequent oscillations be- tween 707f and 80% between March and September. Following a minimum of 65% in November the values began to increase show- ing a similar pattern to that of the previous year. The changes in standard dry weight of the digestive gland, DGci, percentage lipids, and total lipid content showed parallel variation, with the pattern being very similar to that observed for the studied muscle parameters. The greatest differences were that in both years growth of the digestive gland began later than that of the adductor muscle and also that in May of both years there was a sharp increase in growth, only found in the muscle in 1997. There was no clear pattern of variation for the remaining tis- sues, or any correlation with the other somatic components. There were highly significant positive correlations among con- dition index, standard dry weight and lipid content of the digestive gland and among condition index, standard dry weight and glyco- gen and protein contents of the muscle (simple regression. P < 0.01). Likewise, there were statistically significant correlations between the muscle and digestive gland condition indices (P < 0.01 ). between the "standard"" dry weight of the muscle and of the digestive gland (P < 0.01) and between the lipid content of the digestive gland and glycogen and protein contents of the muscle iP < 0.01). Only the percentage amount of proteins in muscle corre- lated negatively with the other parameters. Spearman's correlation analysis did not reveal any significant correlation between environmental variables and the parameters used to study reproduction and the cycle of reserve storage. DISCUSSION In this study we describe the spawning cycle and the cycles of storage and use of reserves in a population of A.opeixidahs from relatively deep waters (50-60 m) in the Ria de Arousa. 1/05/96 1/09/96 1/01/97 1/05/97 1/09/97 1/01/98 - Digestive gland lipids (g) Muscle glycogen (g) - Muscle protein (g) 0,6 ■ 05 0,2 S ..v<*^ .^^ Figure 4. From top to bottom, a) muscle and digestive gland conditi(m indices, b) dr> weight of the somatic components of a standard scallop 50 mm height, and cl biochemical composition of the specific reserve substrates of the organs of energy storage. 582 Roman et al. Spawning Cycle The spawning cycle of A. opercidaris was sludied by visual observation (presence or absence of developed gonad) as well as by quantitative (Gci) and qualitative methods (Dm and OVF) (Bar- ber & Blake 1991). Despite the simplicity of the first method, its use provided a general idea of when spawning took place — from winter until the middle of summer — there being a gradual decrease in the number of specimens with gonad as the summer progressed, culminating in the gonads being totally spent. There then followed a period of gonad recovery in autumn. Taylor and Venn ( 1979) also found the maximum number of spent scallops in summer in the Clyde Sea Area, although both the time of appearance of animals without gonad and the recovery period were earlier than found in the present study. It is possible that the rough estimate provided by this method would only be applicable in species or populations with very marked seasonal variations and in which gonads become totally spent. The consistency of the results obtained b) the different meth- ods of establishing the different processes involved in the repro- ductive cycle (Gci, OVF, Dm) may be because Gci decreases due to release of mature, larger oocytes. As a result, there is an obvious decrease in the diameter of the remaining oocytes and of the per- centage space occupied by them. Furthermore, as seen in the graphs outlining the changes in Dm and OVF (Fig. 2), increases in Gci coincided with cytoplasmic growth and vitellogenesis, (there- fore larger oocyte diameter and greater percentage space occu- pied). Spawning was indicated by the decrease in the Gci, the de- crease in the mean diameter and the percentage space occupied as well as by the increase in the percentage number of spent gonads and the decrease in "standard" dry weight and lipid content. Optimization of spat collection in natural environments de- pends on knowledge of the reproductive cycle, the changes in which must be monitored over time. The consistency of results independent of the method used allows monitoring of the repro- ductive cycle of this species without the need for use of sophisti- cated, expensive techniques by, simply recording the changes in Gci. Following spawning, we recorded a period of gonad recovery, characterized by undifferentiated gonads, without gametes, as pre- viously described by Allarakh ( 1979), who observed a very short recovery period of less than a month. In this study, this period lasted for I month in 1996 (September 10 to October S) and for almost 2 months in 1997 (September 9 to October 28). In 1996 the period of sexual repose was shorter because there was partial maturation in autumn (from October 8), which was not observed in 1997. In both years gonad recovery, which involved the whole population, started at the end of autumn or beginning of winter and was rapid, as the first spawning look place in January; there were then a series of successive periods of maturation and spawning that lasted until the middle of summer. As the summer progressed, the number of individuals participating in reproduction gradually de- creased (1996), or all suddenly displayed spent gonads (1997). We conclude that the reproductive strategy of this species con- sists of a very protracted spawning period with frequent partial release of mature oocytes, followed by recovery due to the rapid maturation of oocytes present in the follicles, by vitellogenesis. These ovocytes are in turn spawned while a new cohort of oocytes matures. The successive recovery and spawning periods involve gradually less oocytes, until the gonads are totally spent; the final spawning is therefore not the most important. The whole popula- tion participates in reproduction during the period of greatest ga- metic activity, from February to July, but between August and January, only part of the population is involved. Descriptions of the reproductive cycle of A. opercularis in northern areas, revised by Mason (1983) and Barber and Blake ( 1991 ), are mainly based on visual observation of gonads, although Taylor and Venn ( 1979) used the dry weight of gonad to describe the spawning cycle in Clyde Sea area; only Allarakh (1979) used histologic methods to study the reproductive cycle in scallops from Rade de Brest. In general these studies describe partial spawning taking place at different times, and the main spawning in autumn. Allarakh ( 1979) for example, described three natural spawnings in Rade de Brest, the first two in January to February and June to July and the third in September, ending with gonads being totally spent. Soemodihardjo ( 1974) and Paul ( 1978) reported various spawning periods in the Isle of Man. two partial spawnings in winter (Janu- ary to February) and spring or the beginning of summer (May to July) and the main spawning in autumn (August to October). Amirthalingam (1928) observed spawning between January and July in Plymouth but did not record any spawning activity in autumn. Our results are generally consistent with those of the authors cited, although the more frequent sampling and the inclusion of histologic analyses allowed confirmation that partial spawning takes place more often than previously described in other popula- tions and that the final spawning that resulted in the gonads being totally spent, is not the most important, but merely the last before gonad recovery. In summary, partial spawning forms part of the sexual strategy consisting of staggered maturation of 4 to 3 cohorts of oocytes that are shed when mature, throughout a long breeding season that may span from January to July. The generally small number of oocytes obtained by artificial stimulation of these animals is possibly due to this behavior (Le Pennec. 1982). Conversion of the measurements obtained to those of a stan- dard 50-mm animal allowed us to compare our results with those of Taylor and Venn (1979). In one of the two years of their study, these authors recorded a lower standard dry weight of gonad than in this study (range between 0.087-0.105 g), whereas in the other year the values were similar (0.170-0.198 g). In this study, the maximum values ranged between 0.168-0.204 g. This may be partly due to the lower frequency of sampling by Taylor and Venn (1979), who may have recorded dry weight values as they were rising or falling but not while at their maximum values, as we have observed considerable increases and decreases in weight within 15 days. A similar explanation may be given for the greater number of spawnings that were observed. The lipid contents of scallops from the Ria de Arousa (maxi- mum levels of 0.043 and 0.045 g in 1996 and 1997. respectively) were considerably higher than in those from the Clyde Sea Area (0.()LM).026 g). Spawning took place at approximately the same times in both years, despite the large differences in tentperature observed. Tem- perature therefore probably does not affect spawning, as previ- ously thought by other authors; Broom (1976) investigated the possible relationship between spawning and changes in tempera- ture, but with inconclusive results. Reproduction of Queen Scallop 583 Cycles of Storage and Use of Reserves By autumn somatic growth was completed, the muscle and digestive gland had reached their maximum weights and had maxi- mum levels of reserves, the breeding season was over and gonads were spent. A new period of gonad growth began in November and from this time onwards in both years, the accumulated reserves began to diminish. This decrease was associated with gametogenesis and the main spawning that affected the whole population during most of the breeding season. The final, partial, spawning that affects only part of the population, is apparently carried out using external energy enough to maintain gonad growth, without having to use the re- serve energy stored in organs. In both years accumulation of re- serves began (at the end of spring for muscle and at the beginning of July for digestive gland) even though spawning was still taking place. The variation in the weights and indices of digestive gland and muscle indicate their status as reserve energy storage organs; the variation in the lipid content of the digestive gland and of glycogen and protein in the muscle indicate that these are the substrates used as energy reserves. Although the percentage amount of protein in the muscle al- ways remained high (6-1— 80'7f ), a large amount of protein was used (ranging between 0.189 and 0.657 g). The glycogen con- tent varied greatly (3-18%), but the total amount used was relatively small (ranging between 0.010 and 0.185 g). As a con- sequence of fluctuations occurring during the cycle of storage and use of reserves, the weight of the muscle varied between 0.273 and 0.617 g and between 0.301 and 0.993 g in the first and second years, respectively. Observations were consistent with those of Taylor and Venn (1979) who describe a marked seasonal variation in the weight of the adductor muscle, with minimum values at the end of winter, gradual increases during spring and summer and maximum values in September to October, the weight sometimes doubling between March and October. Ac- cording to these authors, and in agreement with our results, the seasonal variation in weight is due to changes in protein and car- bohydrate contents. Taylor and Venn ( 1979) attributed the variations in lipids of the remaining tissues to the digestive gland, although they did not carry out specific analyses of this organ. Observed in the Ri'a de Arousa were large variations in the lipid content of the digestive gland (minimum values of 0.012 and 0.016 g and maximum values of 0.133 and 0.198 g in 1996 and 1997. respectively), thereby confirming their speculations. In contrast with the findings of Barber and Blake ( 1981 ). who observed that Argopecten irradians concentricus uses dif- ferent substrates (glycogen, protein or lipids) of different origin (muscle or digestive gland) depending on the phase of gameto- genesis, A. opercularis apparently uses all of its reserves si- multaneously (muscle glycogen and proteins, as well as diges- tive gland lipids). However, these authors indicate that which are the important substrates, where they are stored and how their use is related to reproduction varies among species and among different populations of the same species. It should also be taken into account that A. irradians concentricus is se- melparous. and has a straightforward reproductive cycle, with one spawning after which the animal dies, whereas A. opercularis is iteroparous, and spawns several times in one year for several years. ACKNOWLEDGMENTS This study was financed by CYTMAR, project MAR95-1893- CO3-01. The authors thank Carmen Presas, Carmen Vazquez, and Eamon Loewe and the crew of the Chipre II. Jose Luis and Manuel Charlin. LITERATURE CITED Allarakh. C. 1979. Histological and experimental studies on sex differen- tiation and on the reproductive cycle of ChUimvi opercularis L. (Mol- lusque lamellibranche). These 3" cycle, Caen. 148 pp. Ansell, A. D. 1974. Seasonal changes in biochemical composition of llie bivalve Chlamys seplemradiata from the Clyde Sea Area. Mar. Biol. 26:85-99. Amirthalingam. C. 1928. On lunur periodicity in reproduction of Peaen opercularis near Plymouth m 1927 to 1928. / Mar. Biol. Ass. U.K. 15:605-641. Barber. B. J. & N. J. Blake. 1981. Energy storage and ulilizalion in relation to gametogenesis in Argopecien irradians concentricus (Say). / E.vp. Mar. Biol. Ecol. 52:121-134. Barber. B. J. & N. J. Blake. 1991. ReproducUve physiology. In: S. E. Shumway, editor. Scallops: Biology, Ecology and Aquaculture. Am- sterdam: Elsevier, pp. 377-428. Beninger. P. G. 1982. Comparative biochemical study of two populations of the clams Ruditapes decussatus (Linne) and Ruditapes phiiippi- narum (Adams and Reeve). These de Doctoral. Universite de Bretagne Occidentale. 193 pp. Brand. A. R. 1991. Scallop Ecology: Distributions and Behaviour. In: S. E. Shumway. editor. Scallops: Biology. Ecology and Aquaculture. Am- sterdam: Elsevier, pp. 517-584. Bricelj. M. v., J. Epp & R. Malouf. 1987. Comparative physiology of young and old cohons of bay scallop Argopecten irradians irradians (Lamarck): mortality, growth and oxygen consumption. J. Exp. Mar. Biol. Ecol. 112:73-91. Broom, M.J. 1976. Synopsis of biological data on scallops Chlamys (Aequipecten) opercularis (Linnaeus), Argopecten irradians (Lamarck), Argopecten gibbus (Linnaeus). FAO Fish. Synops., No 1 14. 44 pp. Devauchelle, N. & C. Mingant. 1991. Review of reproductive physiology of scallop Pecten maximus. applicable to intensive aquaculture. Aquat. Living Resour. 4:41-51. Le Pennec. M. 1982. L'elevage experimental de Chlamys opercularis (L.) (Bivalvia. Pectinidae). Vie Marine 4:29-36. Lucas. A. & P. G. Beninger. 1985. The use of physiological con- dition indices in marine bivalve aquaculture. Aquaculture 44: 1 87-200. MacDonald, B. A. & R. J. Thompson. 1988. Intraspecific variation in growth and reproduction in latitudinally differenciated populations of the giant scallop Placopecten magellanicus (Gmelin). Biol. Bull. 175: 361-371. Mason. J. 1983. Scallop and Queen Fisheries in the Bntish Isles. Fishing News Books Ltd. Surrey: Farnham. 1 14 pp. Paul. J.D. 1978. The biology of the queen scallop. Chlamys opercularis (L.), in relation to its prospective cultivation. PhD thesis. University of Liverpool. 127 pp. 584 Roman et al. Paulet. Y.-M. & J. Boucher. 1991. Is reproduction mainly regulated by temperature or photoperiod in Pecten maximiis'.' Inv. Reprinl. Dev. 19:61-70. Pazos, A. J.. G. Roman. C. P. Acosta, M. Abad & J. L. Sanchez. 1996. Stereological studies on the gametogenic cycle of the scallop Pecten maximus in suspended culture in Ria de Arousa (Galicia, NW Spain). Aqiuuiitliire 142:119-135. Roman. G., M. J. Campos & C. P. Acosta. 1996. Relationships among environment, spawning and settlement of Queen scallop in the Ri'a de Arosa (Galicia. NW Spain). Aquacul. Inl. 4:225-236. Soeinodihardjo, S. 1974. Aspects of the biology of Chlamys opercularis (L.) (Bivalvia) with comparative notes of four allied species. PhD thesis, University of Liverpool. 1 10 pp. Taylor. A. C. & T. J. Venn. 1979. Seasonal variation in weight and bio- chemical composition of the tissues of the queen scallop. Chlamys opercularis, from the Clyde Sea Area. ./. Mai: Bini Ass. U.K. 59:605-621. Jcnimal of Shellfish Resvanh. Vol. 21. No. 2. 585-590. 2002. KARYOTYPE ANALYSIS AND CHROMOSOME BANDING OF THE CHILEAN-PERUVIAN SCALLOP ARGOPECTEN PURPURATUS (LAMARCK. 1819) GONZALO GAJARDO.* MARGARITA PARRAGUEZ, AND NELSON COLIHUEQUE Laboratory of Genetics & Acjuacultiire. UniversiclacI tie Los Lagos. P. O. Box 933. Osorno. Chile ABSTRACT The Chilean-Peruvian scallop. Argopecten purpitrahis. is the southernmost representative of the genus and has become an aquaculture commodity in Chile as a result of 20 y of culture practice promotion after natural beds were almost exhausted by overexploitation. Chromosome preparations of A. purpuratus D-larvae from three samples, two from commercial hatcheries, and one from the few remaining wild populations were subjected to karyotype analysis by the usi al measurements of chromosome pairs, Ag-NOR staining, as well as by the Hoechst 3325S/Actinomycin D counterstain and restriction endonuclease banding techniques. The species has a modal diploid number of 2/) = 32, and the karyotype consists of 1 1 pairs of telocentric and t~ive pairs of subtelocentric chromosomes. NF = 32. The banding techniques, attempted for the first time in the species, served as a good complement for identifying a number of homologous chromosomes. The NOR was located terminally on the short arm and in the pericentromeric region in three chromosome pairs. The Hoechst 33258/Actinomycin D counterstain revealed tiny fluorescent heterochromatic areas in the centromeric region of chromosome pairs 1, 2, 4, 5. 6. 7, 8. 9. 11, 15, and 16, whereas a quenching zone was observed in chromosome pair 6. Endonuclease restriction banding revealed pericentromeric and telomeric bands in one chromosome pair {Alu\) and interstitial bands in other two chromosome pairs (WacIII). The karyotype and banding pattern provided in this work are likely to help an industry seeking for improved stocks. Such information will he also valuable for further investigating both intra- and interspecific genomic relationships in the Chilean Pectinids. where the number of species karyologically screened is still very limited. KEY WORDS: Arf>opecleiu karyotype. Chilean scallop, genetics, banding INTRODUCTION Classic techniques tor chromosome analysis generally have permitted accurate assessment of chromosome numbers and mor- phology in a wide variety of aquatic invertebrate species (Thiriot- Quievreux 1994). Yet the number of species investigated from the cytogenetic point of view is rather scarce, i.e.. no more than 200 of approximately 15.000 species in the Bivalvia class (Gonzalez- Ti/()n et al. 2000). Recent molecular and banding techniques have contributed to a better identification of whole or chromosome parts, and hence to a more detailed characterization of the genome organization in different organisms. Analysis of the distribution and composition of heterochromatin and identification of the nucleolar organizer regions iNORs) are among these advances (Insua et al. 1998; Gonzalez-Tizon et al. 2000). In commercially exploited species, these techniques have enabled the obtaining of better karyotypes, although not fully standardized, allowing more reliable intra- or interspecific comparisons of genetic resources either for basic studies (evolutionary inferences) or for practical purposes (taxonomy, chromosomal manipulations). The Chilean-Peruvian scallop, Argopecten purpuratus. is a functional hermaphrodite inhabiting sedimentary substrates in sheltered bays along the Pacific Ocean from Paracas Bay. Peru (13° South) to the Gulf of Arauco. Chile (37' South). The species used to be a commercially important member of the subtidal macro-invertebrate fauna along the northern part of the Chilean coast until overfishing almost exhausted natural beds. Since 1982, A. purpuratus has been farmed successfully and currently repre- sents an important aquaculture product (Gajardo & Nunez 1992). Because of its economic importance, the species has been trans- planted to southern Chile, where it does not occur naturally. Von Brand et al. ( 1990) ascribed it to the genus Argopecten instead of Clilaiins as previously described based on the fact that both dip- loid number (2n = 32) and chromosome types (4 m+ 14 m-Sm-t- 6St-i- 8t) were shared with other Pectinids. particularly Argopecten irradians (Wada 1978). Instead, most Clilamys species studied so far exhibit a diploid number of 2/; = 38 and few bi-armed chro- mosomes (Komaru and Wada 1985). A. purpuratus is replaced by other Chlamys species in the southern Chilean coast, such as Chlamys patagonica. Chlamys anumdi. and Chlamys vitrea. As part of a broader effort aimed at the genetic characterization of Argopecten purpuratus. this study seeks to provide a more reliable karyotype description and chro- mosome pairing based on the chromosome index and banding analysis of this important aquaculture commodity. The information piovided is likely to help in future cytogenetic studies of intra- and interspecific karyotype comparisons of Chilean scallops. MATERIAL AND METHODS Chromosome Preparations Argopecten purpuratus D-larvae were obtained by artificial hatching of ripe animals from three different sources. Two samples were from commercial hatcheries. Cultivos Marines Internaciona- les S.A (Caldera). northern Chile (24 South), and Germont S.A in southern Chile (Calbuco; 51" South), whereas the Quintay sample corresponds to animals collected in Laguna Verde (33° South). This latter location is one of the few natural beds remaining in the Central coast of Chile, close to Valparaiso. TABLE 1. Diploid chromosome number determination in three populations of .4. purpuratus. No. of Cells Chromosome Number Locality Observed 25 26 27 28 29 3(1 31 32 Modal 2n *Corresponding author. E-mail: ggajardo@ulagos.cl Caldera 48 0 ] 1 3 4 3 7 5 23 32 Calbuco 49 1 1 5 8 11 23 32 Quintay 28 1 1 4 4 5 12 32 585 586 Gajarado et al. TABLE 2. Chromosome measurements in three samples of A. purpuratus. mean relative length, 9S'^e confidence interval (X ± ci). and standard deviation ISD) of the short arm (SA) and long arm (I. A I. Chron\os(mie type according to the centromeric index iC'll. Chromosome No. of Pair SA LA Length ± SD (fim) T> Locality X ± ci (SD) X ± ci (SD) CI ± SD pe Caldera (« = 9) 1 1.64 ±04 7 (0.23) 7.75 ±0.41 (0.56 I7.47± 1.93 4.28 ±0.81 s 2 1.24 + 0.10(0.13)-' 6.37 ± 0.36 (0.48 16.26 ± 1.54 3.57 ± 0.83 s 3 1.15±0.08 (0.11) 5.60 + 0.28 (0.37 17.02 ± 1.37 3.10 ±0.63 s 4 1.10 ±0.08 (0.1 If 5.08 ±0.39 (0.52 17.87+1.74 2.88 ± 0.53 s 5 1.30 + 0.19(0.26) 4.66 ± 0.49 (0.65) 21.78 ±3.20 2.78 ±0.44 s 6 0.61 ±0.28 (0.37f 8.42 ± 0.30 (0.41 6.61 ±3.93 4.14 ±0.83 t 7 0.63 ± 0.20 (0.27)'- 7.24 ± 0.48 (0.65 8.02 ± 3.50 3.65 ±0.81 t 8 0.55 ±0.1 8 (0.24)" 5.70 ±0.35 (0.48 8.86 ±3.91 2.97 ±0.78 t 9 0.52 ± 0.22 (0.30)" 5.14 ±0.23 (0.31 9.11 ±5.20 2.64 ± 0.56 t 10 0,48 + 0.21 (0.28)" 4.71 +0.37 (0.50 9.45 ±5.44 2.40 ± 0.46 t 11 0.03 ±0.06 (0.08) 5.89 + 0.32 (0.43 0.45 ±1.34 2.80 ±0.76 I 12 0.05 + 0.10(0.14) 5.40 ±0.38 (0.51 0.87 ±2.61 2.57 ± 0.70 I 13 0.04 ±0.08 (0.11) 4.98 ±0.19 (0.26 0.69 ± 2.06 2.33 + 0.53 t 14 0.00 ±0.00 (0.00) 4.81 ±0.23(0.31 0.00 ± 0.00 2.26 ±0.57 t 15 0.00 ± 0.00 (0.00) 4.55 ± 0.29 (0.40 0.00 ± 0.00 2.15 + 0.57 t 16 0.04 + 0.10(0.13) 4.33 ±0.29 (0.39 1.02 ±3.07 2.07 ± 0.55 t Calbuco {II = 10) 1 1.47 ±0.28 (0.39) 8.24 ±0.74 (1.05 15.08 ±3.11 5.01 ±1.84 s 2 1.28 + 0.27 (0.38)° 6.57 ±0.64 (0.91 16.23 ±4.09 4.02 ±1.. ^6 s 3 1.26 ±0.24 (0.34) 5.73 + 0.39 (0.56 17.96 + 4.37 3.64 ±1.36 s 4 1.13 ±0.22 (0.32)=' 5. 18 ±0.29 (0.42 17.89 ±4.88 3. 30 ±1.23 s 5 1.00 + 0.31 (0.44) 4.75±0.83 (1.17 17.10±7.42 2.98 ±1.06 s 6 0.12 ±0.15 (0.22)" 8.07 + 0.68(0.97 1.46 + 2.67 4.34 ±1.85 t 7 0.10 ±0.15 (0.22)" 6.98 ± 0.62 (0.88 1.47 ±3.14 3.74 ±1.62 t 8 0.10 ±0.15 (0.21)" 6.30 ±0.51 (0.72 "■" 1.64 ±3.50 3.40 ±1.48 t 9 0.06 ±0.1 4 (0.1 9)" 6.07 ± 0.46 (0.65 " 1.08 + 3.41 3.27 ±1.41 t 10 0.06 ±0.1 2 (0.17)" 5.80 ± 0.30 (0.43 " 1.05 ±2.88 3.15+1.37 t 11 0.07 ±0.13 (0.19) 5.40 ±0..30 (0.43 1.33 ±3.71 2.92 ± 1 .23 t i: 0.01 ±0.02 (0.03) 5.31 +0.24(0.34 0.19 ±0.59 2.86 ±1.25 t 13 0.00 ± 0.00 (0.00) 5.07 ±0.26 (0.37 0.00 ± 0.00 2,71 ±1,17 t 14 0.00 + 0.00 (0.00) 4.85 ± 0.26 (0.37 0.00 ± 0.00 2.58 ± 1 .07 t 15 0.00 ± 0.00 (0.00) 4.68 + 0.27(0.38 0.00 ± 0.00 2.50 ±1.06 t 16 0.01 ±0.02(0.03) 4.32 ± 0.24 (0.34 0.19 ±0.60 2.31 ±0.98 t Quiiilay (/i = 5) 1 1.23+ 1.13 (1.13) 7.06 ±1.59 (1.60 14.69 ±13.99 1.99 ±0.42 s t 2 1.81 ±0.20 (0.20)" 6.30 ±0.61 (0.61 22.47 ± 3.22 1.94 ±0.16 s t 3 1.46 ±0.24 (0.24) 5.53 ±0,35 (0.35 20.84 ±2.91 1.68 + 0.15 s t 4 1.61 +0.18 (0.18)" 4.85 ± 0.63 (0.64 25.08 ± 3.97 1 .55 ± 0. 1 2 s t 5 1.28 ±0.28 (0.28) 4.66 ±0.72 (0.73 21.63 ±4.45 1.42 + 0.14 s t 6 0.00 + 0.00(0.00)" 7.78 ± 1.09(1.09 0.00 ± 0.00 1.87 ±0.31 t 7 0.00 ± 0.00 (0.00)" 6.90 ±0.23 (0.23 0.00 ± 0.00 1.66±0.16 t 8 0.00 + 0.00 (0.00)" 6.57 ±0.10 (0.10 " 0.00 + 0.00 1.58 ±0.12 t 9 0.00 ± 0.00 (0.00)" 6.19±0.18(0.18 " 0.00 ± 0.00 1.49±0.13 t 10 0.00 ± 0.00 (0.00)" 5.95 ±0.18 (0.18 " 0.00 + 0.00 1.43 ±0.14 t 11 0.00 + 0.00 (0.00) 5.71 ±0.11 (0.11 0.00 ± 0.00 1.37 ±0.10 t 12 0.07 ±0.16 (0.16) 5.51 ±0.22 (0.22 1.17 + 2.61 1 .34 ± 0. 1 1 I 13 0.18 ±0.40 (0.40) 5.30 ±0.22 (0.23 2.82 ± 6.30 1.31 ±0.15 t 14 0.00 ± 0.00 (0.00) 5.18 ±0.22 (0.22 0.00 ± 0.00 1.24 ±0.09 t 15 0.00 + 0.00 (0.00) 4.69 ±0.1 5 (0.15 0.(K) ± 0.00 1.13±0.10 1 16 0.00 ± 0.00 (0.00) 4.16±0..34(0.35 0.00 ± 0.00 1 .00 + 0. 1 2 t Values with a different letter are significantly different (Tukey, P < 0.05). The splash method was developed in our laboratory for D- larvae (24— 1-8 h after hatch) according to the following protocol: initial colchicine (0.05%) treatment of a pool of D-larvae from each locality in sea water for 2 h. then mechanical disruption of cells, including centrifugation at 1000 rpm/10 min, hypotonic treatment in distilled/ seawater (1:1), washing to eliminate hypo- tonic solution (centrifuge at 800 rpm/10 min). fixation in metha- nol-acetic acid (3:1 ) for 20 min. and washing (centrifuging twice at 800 rpm/ 10 min). The pellet was re-suspended in 0.5 mL of cold fixative methanol/acetic acid (3:1) and a drop splashed onto clean slides and stained in 4% Giemsa. For diploid number deter- minations, between 28 and 49 of the best metaphase plates (with- out overlapping) were studied. These were then photographed on a Nikon Labophot microscope (with epitluorescence ) for chromo- Karyotype and Banding Pattern of A. PURPURATUS 587 St 2n = NF = 32 32 ^'^ 1 /A 3 6 7 8 1-1 ie 13 Afi ■♦..\ /.'I O' ^* some counting. In all populations, between 5 and 10 suitable cells were used for the estimation of the centromeric index (CI) follow- ing the protocol of Levan et al. (1964). Chromosome measure- ments were obtained and mean and standard deviation for CI were calculated for each chromosome. Total chromosome relative length (expressed as percentage) and the relative length of short (SA) and long arms (LA) over the total length of haploid comple- ment were also obtained. Chromosome differences within and be- tween karyotypes were evaluated from karyo-ideograms elabo- rated on SA and LA relative lengths (Spotomo 1985). Confidence intervals were established for SA and LA for each chromosome pair to assess the significance between comparisons. For chromo- some arm number (NF) determinations, subtelocentric and acro- centric chromosomes were considered uni-armed according to Matthey ( 1945). This karyotype descriptor (NF). which only con- siders the main arm of a chromosome, is quite reliable as very often the size of the short arm of a subtelocentric chromosome is variable due to technical artefacts during spread preparations. Figure \. Karytitypv »( Argopeiteii piirpiiratiis. 2ii represents 10 microns. : 32; NF = 32. Bar Banding Methods Howell and Black's (1980) silver-NOR staining method was used to identify NOR-bearing chromosomes. The counterstain- o -I Figure 2. Karyo-idiogram for three populations of A. purpuratus. Diagonal lines separate chromosome morphologies in metacentrics (m), submetacentrics (sm), subtelocentrics (st), and telocentrics (t). Dotted lines show those chromosome pairs (identified by numbers) of the studied population that are easily distinguished from the rest. FZach point in the karyo-idiogram represents a mean value (in percent! of the relative length of the short and the long arm of 16 homologous pairs. Bars represent 95 '7f confidence intervals of the mean for short and long arms. 588 Gajarado et al. enhanced chromosome banding technique (Hoechst 33258/ Actinomycin D; Schweizer 1981 ) was used to identify heterochro- matic regions. Metaphase plates were photographed using a Nikon epifluorescence microscope (filter block UV-2) with a Kodak T- Max 100 ASA film. For restriction endonuclease (RE) banding, RE enzymes sus- pended in the appropriate buffer were added to one drop of an air-dried cell suspension and covered with a coverslip. Enzyme concentration and digestion time depended on each endonuclease type, as follows: for EcoRl. 0.5 U/[jlL for 5 h: for B((//)HI. 0.5 U/jiL for 8 h; for Akil. 0.3 U/fJiL for 7 h: and for «(«'III. 1 .7 U/|jiL for 5 h. Slides were incubated in a moisture chamber al 37°C, washed in distilled water, and stained in 4% Giemsa solution for 10 min. RESULTS The modal diploid number of 32 was established for all three A. purpiimtiis populations screened (Table 1 ). The CI revealed that all karyotypes had similar chromosomal constitutions (5 pairs of subtelocentrics and 1 I pairs of telocentrics (Table 2 and Fig. 1 ). The karyo- ideogram showed that various chromosome pairs could be easily identified because of size differences (pairs 1 to 7). Interpopulational chromosome differences were evident, particu- larly for the short arm (ANOVA. P < 0.05) for chromosome pairs 2, 4, 6. 7, 8, 9, and 10 (Table 2 and Fig. 2). The Ag-NOR staining revealed three pairs of NOR-bearing chromosomes (Fig. 3) with NOR location varying, i.e.. it was pericentric in the long arm of one pair and telomeric in the short arm of two other pairs (see Fig. 3). The number of NOR-bearing chromosomes was in agreement with the number of nucleoli ob- served in the interphase nucleus (3 to 6. Fig. 3b). The Hoechst/Actinomicin D counterstain (Fig. 4) revealed a tiny positive fluorescent heterochromatic block in the centromeric Ag-NOR Metapl Metap2 Metap4 r/ldtapS dmjk. IVIetapl & — » • • » Poirl PolrJ PoW ^A V.-O ".'1 •- l^ \- l- y 1- U r ((f If <- a area of chromosome pairs 1,2, 4 to 9, II, 15, and 16 (Fig. 4). This stain produced a quenching zone in the middle of the long arm of pair 6. which corresponded to an A-T low-content chromatin se- quence, according to the pair specificity of the Hoechst 33258. Figure 5 shows the pattern after restriction endonuclease digestion. Only two REs, Alid and HcieUl. produced a banding pattern in such a way that pericentric and telomeric bands were evident in one chromosome pair (A/id) and interstitial bands in two pairs DISCUSSION This work confirms the chromosome number of 2/; = 32 re- ported for A. purpwatus (Von Brand et al. 1990; Canello et al. 1992: Alvarez-Sarret & Lozada 1992: Winkler et al. 1993). How- ever, the chromosome formulae obtained (5 st -I- 1 1 t chromosome pairs) differs from that reported by Von Brand et al. ( 1990), who indicated the presence of 2 m pairs, 7 m-sm pairs, 3 st pairs, and 4 t pairs in the karyotype. Such a difference is likely to be pro- duced by the fact that in the latter study the karyotype was tenta- tively arranged using the size criteria without the chromosome measurements required to determine the centromeric position. Our results consistently show, in all three samples that were analyzed, a chromosome complement of sub-telocentric chromosome types (see Fig. 2). A. piirpiiratus. along with other related species, such as A. irradians inadians (Wada 1978) and C. nohilis (Komaru and Wada 1985), are among the few Pectinids sharing a diploid num- ber of 32. Instead, the majority of species studied (i.e., genus Clilamys. Pecten. Patiuopeclen. and Placopeclen) exhibit In = 38 (Thiriot-Quievreux 1994: Insua et al. 1998), with Aequipecten opcirularis [2ii = 26) being the exception (Beaumont & Gruffydd 1974). Molluscs and crustaceans are among the most primitive groups considering those karyologically compared by Thiriot-Quievreux ( 1994). Nevertheless, a typical diploid number does not emerge in most groups, Ostreidae being a special case in which a diploid number of 2;; = 20 is common and very likely the ancestral one. Some marine species frequently exhibit greater numbers of chro- mosome than those of freshwater species, although in some cases, for example, within the bivalvia, both an increase and decrease are evident. The diploid number in most animal species ranges widely, between 12 and 40 (White 1978). Whereas evolution to higher chromosome numbers in certain aquatic organisms is often paral- leled by an increase of subtelocentric and telocentric chromo- somes, probably by fission of chromosomes (see review by - -1 $§ «* ^^ ^ 2 3 4 i *^*t^ ^f) |< h0 t« n u Figure .^. .Xg-NOR staining, (a) Metaphase plate showing six NOR- bearing chromosomes. Below. NOR-bearing chromosomes obtained from different metaphase plates, (b) Resting nuclei showing 3 to 5 nucleolus. Figure 4. Hoescht .V'258/ Actinomycin I) counterstain. Intense lluo- rescent bands are indicated by small arrows. Large arrows depict a less lluorescent area in chromosome pair 6. Karyotype and Banding Pattern of A. purpuratus 589 b EcoRI ? .v^r Metap.2 Metap.5 ■i ?>^ Metap.2 Metap.3 Metap4 ll 8- Metap.7 MetafxS Melapd — Figure 4. RE digestion: lal BaiiiHl (no digestion): (bl EcoRl (no di- gestion); (c) Aliil (+ digestion), photo depicts a pericentronieric band in one pair of subtelocentric chromosome (arrows): (d) HaelU (+ di- gestion) depicts an interstitial band in two chromosomes. Below, banded chromosomes obtained from difTerenI metaphase plates. Thiriot-Quievreux. 1994: 379). Such phenomenon is hkely to be related with speciuli/ution or adaptation to a certain type of marine environment. As mentioned previously, the diploid number in Pectinids varies between 26 and 38; hence, the chromosome formulae observed for A. purpuratus could be considered a de- rivative state within the group. Although it is debatable whether such a trend could yet be inferred in a group with few species exhibiting standardized, or comparable, karyotypes, it is worth mentioning that A. purpuratus is one of the southernmost Ar- gopecten representatives. Indeed, this group originated in the tropi- cal/subtropical region of the Caribbean and Atlantic (Von Brand et al. 1990). A. purpuratus along with A. opercularis (Beaumont & Gruffydd 1974) are among the few Pectinids of 14 species karyo- typed so far with 2/i = 32 and 2n = 26. respectively (see Insua et al. 1998). Our results (2n and NF) and similar information from other species within the group (reviewed by Mendez et al. 2000). sug- gest that NF (Matthey 1945) is a valuable de.scriptor for species comparisons and to evaluating factors affecting chromosome shape variations such as chromosome rearrangements (White 1978). Because of technical procedures, the size of the short arm can vary widely and this can be deduced froir) the deviation ob- served between the average values of the centromeric index (CI ) in Table 2 as well as from the presence or absence of short arms in the telocentric chromosomes. The combination of classic and banding techniques used in this work allowed a better description of the karyotype and a more reliable pairing of certain homologous chromosomes. Reproduc- ible banding patterns are still uncommon in aquatic invertebrates; hence, chromosome markers described in this work, for the first time in A. purpuratus, are considered valuable for an improved and reliable characterization of the species karyotype and its variations. Three NOR-bearing chromosome pairs were preliminary identified in the karyotype with the NOR regions, varying its location in the chromosomes (Fig. 3). Further analysis of these chromosome re- gions (using chromomycin A3 and a rDNA probe) will be required to clarify a probable polymorphism for both number, position, or NOR size in A. purpuratus. The three NOR-bearing chromosome pairs observed in this work are within the range reported for Pte- riomorphia subclass (1—4 pairs; Martinez-Lage et al. 1997; Insua et al. 2000). The Ag-NOR number reported for A. purpuratus is higher in comparison with A. opercularis. which shows only one pair (Insua et al. 1998). The combined use of fluorescent dyes and RE digestion al- lowed the detection of different kinds of chromatin in A. purpu- ratus. suggesting specific DNA composition of certain chromo- some sectors. The Hoechst 32258/Actinomycin D counterstain highlights heterochromatic areas in humans (Schweizer 1981) and lower vertebrates (Colihueque et al. 2001 ). Although we observed highly fluorescent areas in the A. purpuratus karyotype, these were restricted to the cent)omeric regions of few chromosomes, indi- cating that heterochromatin is not common in this species (see Fig. 4). However, the differential response to digestion with re- striction enzymes is also evidence of chromatin differences in A. purpuratus chromosomes. For example, the positive detection (no bands), at least preliminarily, of sites for Alu I (AGACT) and HaeWl (GCiCC; see Fig. 5) indicates that these target sequences should be highly repeated for the digestion to be evident (Sanchez et al. 1991). It should be stressed, however, that heterochromatic areas are few and mainly restricted to the centromeric areas as revealed by the fluorescent banding. Digestion with RE of fixed metaphase plates is not so common in invertebrates, with the exception of Drosopliila (Mezzanotte 1986). Therefore, the results of this work confirm the finding of Martinez-Lage et al. (1994), who indicated that the digestion of bivalve chromatin produces specific chromosome bands (Fig. 5). However, the pattern of di- gestion is simple and restricted to few chromosomes similar to what is observed in fishes (Sanchez et al. 1991) and mammals (Burkholder 1989). The fanning of the Chilean scallop was greatly promoted by the fact that natural beds were almost depleted. Nowadays, a bloom- ing, fast-growing industry is still far from realizing its potential because of the limited, or unreliable, production of quality seeds (Gajardo & Niiinez 1992). This is in part related with proper brood- stock management. By providing a better karyological description (quantitative measurements and banding pattern of chromosomes), this work offers a reliable and comparable way to evaluate the chromosome formulae of A. purpuratus and hence, a proper way for comparing probable differences among the few wild popula- tions remaining, or hatchery stocks, if any can be identified yet. This is expected to assist to the management, improvement and conservation of this economically valuable resource. At the same time, it will help in the understanding of the processes affecting chromosome evolution in this species as well as in the bivalvia class. 590 Gajarado et al. ACKNOWLEDGMENTS Support from the European Union, INCO-DC project (contract no. IC18-CT97-0188) is greatly appreciated. We are indebted to Dr. Josefina Mendez, University Da Coruiia, Spain, for sugges- tions and valuable literature. Two anonymous reviewers made con- structive comments to improve the original manuscript. LITERATURE CITED Alvarez-Sarret. S. & L. Lozada. 1992. Spontaneous triploidy and chromo- somic study in Chilean scaWop Argopecteii purpuranis (Lamarck 1819) (Bivalvia, Pectinidae). Invest. Pesqueras 37:119-126. Beaumont. A. R. & L. L. L. Gruffydd. 1974. Studies on the chromosomes of the scallop Peclen maximii.s (L.l and related species. J. Mar. Biol. A.«. U. K. 54:713-718. Burkholder. G. D. 1989. Morphological and biochemical effects of endo- nucleases on isolated mammalian chromosomes in vitro. Cliromosontii 97:347-355. Canello. F., L. Paredes & J. Toro. 1992. Induccion a la triploidfa en el oslion del norte Argopecten purpuratus. por medio de shock termico de calor. Invest. Pesqueras. 37:5-1 1. Colihueque. N., P. Iturra. F. Estay & N. Diaz. 2001. Diploid chromosome number variations and sex chromosome polymorphism in five cultured strains of rainbow trout i^Oncorhynchus inykiss). Aqiiaciiltiire 198:63- 77. Gajardo, G. & J. Nufiez. 1992. Seed production, the critical factor for mollusc farming in Chile. World Aquaculture 24:72-77. Gonzalez-Tizon, A. M.. A. Marti'nez-Lage. I. Rego. J. Ausio & J. Mendez. 20fJ0. DNA content, karyotypes, and chromosomal location of 18S- 5.8S-28S ribosomal loci in some species of bivalve molluscs from the Pacific Canadian coast. Genome 43:1065-1072. Howell, W. M. & D. A. Black. 1980. Controlled silver staining of nucleo- lus organizer regions with a protective colloidal developer: a I step method. E.xperientia 36:1014-1015. Insua. A.. M. J. Lopez-Pifion & J. Mendez. 1998. Characterization of Aeqttipeclen opercularis (Bivalvia: Pectinidae) chromosomes by dif- ferent staining techniques and fluorescent in situ hybridization. Genes Genet. Sy.st. 73:193-200. Insua, A., R. Freire & J. Rios. 2000. Localizacion cromosiimica y patrones de variacidn del DNA ribosomico en moluscos bivalvos. In: J. Mendez, editor. Los moluscos bivalvos; aspectos citogeneticos, moleculares y aplicados. Spain: Universidade Da Corufia, pp. 77-99. Komaru, A. & K. T. Wada. 1985. Karyotypes of four species in the Pec- tinidae (Bivalvia: Pteriomorphia). Venus 44:249-259. Levan. A. K., K. Fredga & A. A. Sandberg. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201-220. Martinez-Lage. A.. A. Gonzalez-Tizon & J. Mendez. 1994. Characteriza- tion of different chromatin types in Mytilus galloprovincialis L. after C-banding. tTuorochromc and restriction endonuclease treatment. He- redity 72:242-249. Marti'nez-Lage. A.. A. Gonzalez-Tizon, J. Ausio & J. Mendez. 1997. Karyotype and Ag-NORs of the mussels Mytilus californiunus and M. trossiilus from the Pacific Canadian coast. Aquaculture 153:239-249. Matthey. R. 1945. L' evolution de la forniule chromosomiale chez les vertebres. Experientia l:5(.)-56. Mendez. J.. A. Insua & J. Lopez. 2000. Caracterizacion citogenetica en moluscos bivalvos. In: J. Mendez. editor. Los muluscos bivalvos: as- pectos citogeneticos, moleculares y aplicados. Spain: Universidade Da Coruiia, pp. 15—48. Mezzanotte, R. 1986. The selective digestion of polytene and mitotic en- donuclease. Chroinosoma 93:249-255. Sanchez, L.. P. Marti'nez. C. Baeza & A. Vinas. 1991. Chromosomal het- erochromatin differentiation in Salino iriittci with restriction en/ymes. Heredity 6&.24\-249. Schweizer. D. 1981. Counlerstain-enhanced chromosome banding. Hum. Genet. 57:1-14. Spotorno. A. E. 1985. Conceptos y metodos en cariologia descripliva y comparada. In: R. Femandez_Donoso. editor. El nucleo. los cromo- somas y la evolucion. Santiago de Chile: UNESCO, pp. 135-165 Thiriot-Quievreux. C. 1994. Advances in cytogenetics of aquatic organ- isms. In: A. R. Beaumont, editor. Genetics and evolution of aquatic organisms. London: Chapman & Hall, pp. 369-388. Von Brand. E.. G. Bellolio & K. Lohrmann. 1990. Chromosome number of the Chilean scallop Argopecten puipuratns. Tohoku J. Agr. Res. 40: 91-95. Wada. K. 1978. Chromosome karyotypes of three bivalves: the oysters, Isogitomon alatus and Pinctada imbricata. and the bay scallop. Ar- gopecten irradians irradians. Biol. Bull. 155:235-245. Winkler. F., B. Ladron de Guevara, B. Estevez & L. Jollan. 1993. Induc- cion a la triploidia en Argopecten purpuratus Lamarck. 1819, mediante Cilocalasina-B. Rev. Biologia Marina 28:239-246. White. M. J. D. 1978. Modes of speciation. San Francisco: W. H. Freeman. 455 pp. Jounuil ot Shellfish Research. Vol. 21. No. 2. 591-5y6. 2002. MOLECULAR CLONING AND CHARACTERIZATION OF A FRUCTOSE- 1,6-BIPHOSPH ATE ALDOLASE cDNA FROM THE DEEP-SEA SCALLOP PLACOPECTEN MAGELLANICUS MOHSIN U. PATWARY,'* AKELIA WAUCHOPE,' TIMOTHY W. SHORT,^ AND EDWARD J. CATAPANE' ^Departmeui of Biology Medgar Evers College of the City University of New York 1 150 Carroll Street Brookhn. New York 11225: "Queens College and the Graduate Center of the City University of New York. 65-30 Kissena Boulevard. Flushing. New York 1 1367 ABSTRACT The deep-sea scallop Phicopeclen magellanicus is an important member of commercial fisheries along the coast of Northeastern United States and Atlantic Canada. A cDNA encoding a glycolytic pathway enzyme fructose- 1 ,6-biphosphate aldolase was isolated from a sea scallop adductor muscle-specific cDNA library and sequenced from both directions. The full-length cDNA is a 1627 base-pair (bp) long sequence that has a 62 bp 5' untranslated region, a 1092 bp open-reading frame, and a 47.^ bp 3' untranslated region including a 24 bp polyA tail. The open-reading frame encodes a .^9.,^ kDa protein with 36.3 amino acids. The protein has 183 nonpolar, 94 polar uncharged, and 86 polar-charged amino acids. Several amino acids show bias for codons with G/C at their third position. The cDNA has 28 unique restriction sites, including common restriction enzymes such as Sod, B<;mHl, 7se-1.6-hiplios|)liate aldolase cDNA and the deduced amino acid seifiience of the enzyme. The nucleotide residues are numbered from the 5' end. The amino acid residues are numbered from llrst in-frame methionine (M). The potential polyadenylation signal is bolded. Underlines indicate primers for amplification of 3'LITR. The star indicates the stop codon. Note that the 57 bp obtained by RACE system is added to the 5' end of the clone PniC 82. 594 Patwary et al TABLE 1. Codon usage in fructose-l,6-biphosphate aldolase gene. uuu phe F 5 UCU ser S 1 UALI tyr \ — UGU cys C 4 uuc phe F K UCC ser s 6 UAC tyr Y 9 UGC cys c 2 UUA leu L — UCA ser s 3 UAA OCH Z — UGA OPA z — UUG leu L T UCG ser s 1 UAG AMB Z 1 UGG trp w 3 cuu leu L 1 ecu pro p 3 CAU his H 1 CGU arg R 1 cue leu L 12 ccc pro p 7 CAC his H 4 CGC arg R 4 CUA leu L 3 CCA pro p 6 CAA gin Q 1 CGA arg R T cue leu L 13 CCG pro p -> CAG gin Q 16 CGG arg R 1 AUU ile I 3 ACU Ihr T 1 AAU asn N 4 AGU ser S 6 AUC ile I 13 ACC Ihr T 9 AAC asn N 14 AGC ser S 2 AUA ile I 1 ACA Ihr T 5 AAA lys K S AGA arg R 1 AUG met M 7 ACG Ihr T 9 AAG lys K 16 AGO arg R 6 GUU val V ~> GCU ala A 7 GAU asp D 4 GGT gly G 9 GUC val V 7 GCC ala A 21 GAC asp D 13 GGC gly G 13 GUA val V 4 GCA ala A 7 GAA glu E 10 GGA gly G 1 GUG val V 10 GCG ala A 10 GAG glu E 13 GGG gly G 3 In each block, the first, second, third, and fourth columns are codons. three-letter abbreviations for amino acids, one-letter abbreviations for amino acids, and number of amino acid residues, respectively. other codons for this amino acid. Likewise, one (CAG) of the two codons for glutamine code for 16 of the 1 8 residues of this amino acid. This conspicuous nonrandom usage of synonymous codons in the FBP aldolase gene in sea scallops is consistent with the bias found in highly expressed genes of several other organisms (Shaip & Li 1986, Sharp et al. 1988). This strong codon-usage bias may be the result of selection for translation efficiency and accuracy in highly expressed genes like FBP aldolase. The number of FBP-aldolase amino acid residues in different organisms varies from 358 in Oriza sativa to 366 in Caenorhab- ditis etegans. However, a wide range of organisms such as Droso- phila. rat. and Xenopits have 363 residues as in sea scallops. Based on the high amino acid sequence homology, we conclude that this is a subunit of class I aldolase. Because of its essential role in glucose metabolism, the primary structure of this enzyme has re- mained highly conserved across kingdoms. The rate of evolution of aldola.se was estimated to be about Wc amino acid residue changes every 100 million years ( Sawyer et al. 1988). The muscle- specific aldolase evolved at an even slower rate, with only about 2% amino acid residue changes per 100 x 10'" years (Freemont et al. 1988). The BlastP search (Altschul et al. 1997) results show that the sea scallop aldolase is approximately 669^ identical with that of Schistosoma mansoni, 65% with that of Drosophita melaiwgaster, 64% Sahno salar, 63% with Galhis galliis and Xenopits leavis, 62%' with human muscle aldolase A. 62% with caenoihahdilis elegans, 5T7c with Oiyza saliva, and 55% Plasmodium falciparum. The approximate number of genes encoding FBP-aldolase in sea scallop was established by Southern blot analysis. Hybridiza- tion of cDNA coding region probe with a blot containing DNA samples frotn a single sea scallop but digested with several restric- tion en/.ymes produced signals of varying intensity in all lanes (Fig. 2). From the number of signals we estimate that sea scallops have a family of 4 to 10 FBP-aldolase genes. Aldolase constitutes a medium-sized gene family in comparison to the estimated family size of 12-15 for the actin gene (Patwary et al. 1996) and 1 to 3 for the tropomyosin gene (Patwary et al. 19991 in sea scallops. The organization of FBP aldolase as a small multigene family was also observed in Euglena (Plaumann et al. 1997). kb 21.2- 5.2- 3.5- 2.0 Figure 2. Detection of fruclose-l,6-biphosphte aldolase gene in the sea scallops. In this Southern hyhridi/.alion. digoxigenin-labcled aldolase coding region probe was hybridized to genomic DNA from a single animal. Kach lane has U\ pg of DNA digested with /uiiRI (lane a), /?<7)RV (lane b). Himl\\\ (lane ci. Sal\ (lane d). and \ha (lane el. M is digoxigenin-labeled DNA molecular weight marker (Boehringer Mannheim I. FBP Aldolase cDNA in Sea Scallops 595 kb Mabcdefg 4.40- - 2.37- - 1.35- - **" kb Mabcdefghijklmno 21.2-1 Figure 3. Northern blot analysis. A 3' untranslated cDNA region was labeled with "P and hybridized to blots each lane with 12 fig of pooled total RNA obtained from adductor muscles (lanes a and b), gonads (lane cl, hearts (lane d), livers (lane e), mantles (lane fl, and gills (lane g). M is RNA molecular marker lane. Figure 3 shows that the size of sea scallop FBP aldolase mes- senger RNAs in the Northern blot corresponds closely to the length of the sea scallop FBP aldolase cDNA (Fig. 1 ). This result suggests that the clone PmC-82 represents a FBP aldolase cDNA with a very small truncation at its 5' untranslated region. We have used the 3' non-translated region of cDNA as a probe in Northern hybridization to determine the tissue specificity of aldolase expres- sion, and obtained signals in the adductor muscle lanes only (Fig. 3). In absence of any indication of RNA degradation in the gel or in the blot, we conclude that the aldolase gene in question is expressed tissue-specifically in the adductor muscle of sea scallop. This finding suggests that, like vertebrates (Penhoet et al. 1966. Penhoet et al. 19691, sea scallops may have different class I aldo- lase genes expressed in different tissues. The extent of structural differentiation of these genes and the pattern of their distribution among different sea scallops tissues remain to be studied. We examined the utility of FBP-aldolase cDNA as a probe to reveal polymorphisms. The probe revealed RFLPs in three loci in an /AftT-digested blot (Fig. 4) and in two loci in a Drffl-digested blot (data not shown), Pogson and Zouros (1994) and Pogson (1994) obtained three types of RFLPs in sea scallops when they used unidentified sea scallop cDNAs as probes. These were re- striction site polymorphisms, polymorphisms caused by variable numbers of tandem repeats (VNTR) and complex fingerprinting patterns (Pogson 1994). In this study the FBP aldolase cDNA probe revealed restriction site polymorphisms at the approximately 15 kb and 3 kb loci and a VNTR type polymorphism at the 5 kb locus in the Accl digested genomic blot (Fig. 4). Each of these loci has one or two bands, suggesting that the genomic DNA was ^ ••» «« Figure 4. RFLPs in the region surrounding the aldolase genes in sea scallops. Each lane has 10 Mg of genomic DNA from a different animal obtained from live different sea scallop beds (a-c from Yarmouth, d-f from Sable Island, g-i from Georges Bank, J-l from Brow ns Bank, and m-o from Newfoundland) digested with restriction enzyme .icc\ and hybridized with the complete aldolase cDNA coding region. M is digoxigenin-labeled DNA molecular weight marker (Boehringer Mannheim). completely digested and that the gene lacks restriction sites within it for the enzyme concerned. This finding of polymorphisms at multiple loci supports the presence of multiple copies of the aldo- lase gene probably distantly placed in the genome. Some of these genes possibly carry one or more variable sized introns and or VNTRs within their introns or in their fianking regions, producing multiple alleles at a particular locus. These results demonstrate that FBP cDNA is a useful addition to our collection of cDNAs that can be used as a probe to reveal RFLP markers for varieties of genetic studies in deep-.sea scallops and possibly in other related bivalves. The isolation of the first bivalve aldolase cDNA may also assist in understanding the mechanism of aldolase function in this unique group of organisms. ACKNOWLEDGMENTS M. U. P. received support for the construction of the cDNA library from the Department of Fisheries and Oceans. Canada through a contract to the NRC Institute for Marine Biosciences, Halifax, Canada. The authors thank Dr. Ellen Kenchington, De- partment of Fisheries and Oceans, Bedford Institute of Oceanog- raphy, Dartmouth, Canada for initiating this contract with NRC- IMB. We acknowledge the support received from American So- ciety for Cell Biology through a Visiting Professorship awarded to M. U. P. and from the Office of Academic Affairs, and Health Science Research Assistance Center, School of Science, Health and Technology, Medgar Evers College of the City University of New York. LITERATURE CITED Altscliul. S. F.. T. L. Madden. A. A. Schaffer. J. Zhang. Z. Zhang. W. Miller & D. J. Lipman. 1997. Gapped Blast and PSI-Blast: a new generation of protein databa.se search programs. Nucleic Acids Res. 25:3389-3402. 596 Patwary et al Alefounder. P. R.. S. A. Baldwin. R. N. Perham & N. J. 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Allozyme and physiological variation in the scallop Placopecten magellanicus and the general model for the effects of heterozygosity on fitness in marine mollusks. Mar. Biol. 103:51-61. Journal of Shellfish Research. Vol. 21. No. 2, 597-603. 2002. COMPARATIVE ANALYSIS OF OOCYTE TYPE FREQUENCIES IN DIPLOID AND TRIPLOID CATARINA SCALLOP {ARGOPECTEN VENTRICOSUS) AS INDICATORS OF MEIOTIC FAILURE ROSALIO MALDONADO-AMPARO AND ANA M. IBARRA* Centra de Investigaciones Biologicas del Noroeste. S.C. Aqiiaciilninil Genetics Labonitoiy. A. P. 128, La Pa: B.C.S. 23000. Mexico ABSTHACT Differences between diploid and triploid female mollusks in their gametogenic process had previously centered on comparing the general gametogenic cycle, not the oocyte types in each ploidy class, which by their presence or absence might be used as indicators of completion of specific meiotic events. In this study we compared oocyte type frequencies at three different stages of the gametogenic cycle of tnploid and diploid catarina scallops. Regardless of age of sampling (81 d, 1 18 d, 205 d of grow out), the most abundant oocyte type in diploids was always the vitellogenic. and in triploids the previtellogenic. The frequency of occurrence of vitellogenic oocytes in tnploids was only 4.7-19'7f of that in diploids, and that for postvitellogenic oocytes was only from 0-8% of that in diploids. This indicates that the oocyte stage in triploids at w hich gametogenesis was largely arrested was the previtellogenic oocyte, at meiosis I. The principal cause for the meiotic arrest might be associated with problems during zygotene at the time of synapsis between the three homologous chromosomes in triploids as previously suggested by other authors. KEY WORDS: triploid, oocyte-frequency, scallop, Argopecten rentricosiis INTRODUCTION Previous research comparing diploid and triploid female mol- lusk gonad development have centered on comparisons of the ga- metogenesis cycle as described for diploids. Those studies have led to the general conclusion that triploid gametogenesis is retarded when compared with diploids (Allen & Downing 1986, Allen & Downing 1990. Allen 1987, Komani & Wada 1989. Co.x et al. 1996). Whereas some studies have based the comparative analysis of diploid and triploid gametogenic cycle in the qualitative occur- rence of some particular type of oocyte at an age (Cox et al. 1996), a different approach to study the abnormal gonad development in triploid females is to estimate oocyte type frequencies in each ploidy class at different stages of the gametogenic cycle as it has been done for some tlsh species (Carrasco et al. 1998, Felip et al. 2001 ). The most common method of comparing diploids and trip- loids (by gametogenesis cycle) results in that it is not clear if the delayed gametogenesis in triploids is caused by a lower develop- mental rate of most oogenic stages, or by an inhibitory effect resulting in delay or an arrest of a particular stage so further development is impaired, or by both. What is clear is that a final consequence of the triploid condition at the end of the gametogenic cycle is the occurrence of partial or total sterility observed for most moUusk species, measured as the qualitative observation of low numbers of either spermatozoa or mature oocytes (Komaru & Wada 1989, Allen & Downing 1990, Guo & Allen 1994a. Guo & Allen 1994b, Cox et al. 1996, Eversole et al. 1996). As with other mollusk species, in the functional hermaphrodite catarina icsWo^. Argopecten ventricosus. gametogenesis of triploid scallops is apparently delayed when compared with diploids, re- sulting in partial sterility of triploids. The partial sterility results in fecundity being largely reduced in triploid females, with the male gonad generally not developing further than the spermatocyte stage, and becoming replaced by female germinal cells later in the life cycle of tripkiids (Ruiz-Verdugo et al. 2000, Ruiz-Verdugo et al. 2001, Maldonado-Amparo & Ibarra 2002). However, as is the case with most other mollusk species, an oocyte stage at which *Corresponding author: aibarrarow out. Light microscopy (x20). In diploids at 81 d lAl (here were ahundant vitelloRenic oocytes (Vit), and few previtelloyenic (Prevl; by 118 d (C) postvitellogenic oocytes (Postv) were abundant; by 2(15 d (K) some mature oocytes (Mat) were also present. In triploids prcvitellogenic oocytes were the most common type at all ages although a few vitellogenic (IS) were already present at 81 d, increasing in numbers by 118 d (0): some postvitellogenic oocytes were evident by 205 d (F). also indicated that there is a difference between ploidy groups in the type of oocytes most frequently occurring, with the most com- mon one in triploids being the prcvitellogenic oocyte. This points toward the first oocyte stage at which the triploid condition results in an inhibitory effect of further development being the previlel- logenic oocyte, such that more advanced oocyte stages occur at much lower frequencies. In scallops it is known that only those previtellogenic oocytes that complete the pachytene .stage and en- ter the diplotene stage of meiosis I will begin the process of vi- tellogenesis and become mature (Beninger & Le Pennec 1991, Dorange & Le Pennec 1989). Our I'esults clearly point to the fact that oogenesis in triploid catarina scallop [Araopcctcn Yenliicosiis) was arrested at the previtellogenic oocyte, and the reduced num- bers of ooycte stages other than the previtellogenic type in triploids point to the halt of oocyte development at prophase of meiosis I, before the diplotene stage. In the male gonad part of this hermaph- 602 Maldonado-Amparo and Ibarra 81 days o Oogonia ♦ Previtellogentc ■ Vilellogenic o o o o o o o o o ^ o o o -, , 1 T T I f * T ¥ 123456789 123466789 Scallop No 118 days Oogonia Prevltellogenic Vitellogenic ' ■ - ■ 9 f » 9 — >>*>?» -^ — $ — 4> — $ — ^ — ^ — * — * — * — 9 — 123466789 10 123466789 10 Scallop No Oogonia Previlellogenic Vitellogenic 205 days t f 2 3 -^ — tf — .ji — 0.058; Fig. 4A; Table 1 ). except at the first observation period when cold-stressed scallops stayed shut more than twice as long as the control scallops [P < 0.0001 ). At virtually all observation periods, control scallops recuperated more of their initial response (i.e.. in tenn of percent- age) than cold-stressed scallops (Fig. 4B. 4C. 4D). This was par- ticularly clear for the total number of claps (Fig. 4B). Cold Shock with Air Exposure Experiment (Control + Air vs. Cold-Stressed + Air) In this experiment, all scallops were initially exposed to air (18°C) for 4 h to simulate conditions during transfer from culti- vation sites to seeding grounds. Control scallops were then re- turned to warm water ( 18°C) whereas cold-stressed scallops were transferred to 8°C. Scallops from these treatments made an average of 50 claps in the first escape test (Fig. 5A). At this time, the 1.5 min clapping time of control scallops was significantly shorter than the 2.5 min time for cold-stressed scallops (P = 0.0002) (Fig. Cold Stress Slows Escape Responses of Juvenile Scallops 609 TABLE 1. Statistical analysis of the effects of treatment and time on escape performance of juvenile sea scallops from Cold Shock Experiment. The scallops were transferred from pearl nets in the lagoon (18"C) to seawater at either 18 C (control! or 8 C (cold-stressed) in the laboratory to simulate the cold stress accompanying seeding operations. p:ach scallop (;i = 24 per treatment) was measured during each sampling time (12. 24, 36, 48. 6(1, 84, 120 and 156 h). Initial Response df Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time 1 7 7 1 7 7 I 7 7 1 7 7 1 7 7 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 2.63 4.73 1 .08 33.67 7.33 5.78 23.25 2.44 L71 48.79 3.26 0.67 3.68 4.39 3.09 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 0.11 <0.0001 0.37 <().()0()1 <0.0(M1 <0.0001 <0.()()(11 0.019 0.11 <0.0001 0.0023 0.70 0.061 <0.()0(11 0.0036 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. Response After 15 min of Recuperation df Number of claps Clapping time Clapping rate Ma.Kimum number of claps in a .series Time spent closed % initial number of clapst initial clapping ratet % initial maximum number of claps in a seriesS 1 45.44 <0.0001 7 2.31 0.026 7 1.22 0.29 1 13.33 0.0007 7 1.54 0.15 7 1.83 0.081 1 63.12 <0.0001 7 0.80 0.59 7 1.30 0.25 1 104.38 <0.0001 7 0.44 0.88 7 1.16 0.32 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 1 49.86 <0.0001 7 2.47 0.018 7 0.49 0.84 1 15.54 0.0003 7 1.11 0.35 7 0.12 0.99 1 26.39 <0.0001 7 1.33 0.24 7 1.53 0.16 t 1. t5. or §7 observations were removed from the analysis as recuperation exceeded 200%. n.a. = not applicable. ?C). Thus, initial clapping rate was higher tor control scallops (38.7 vs. 23.6 claps • min"'). Control and cold-stressed scallops had. respectively, average maxima of 10 and 7 claps in a row (Fig. 5E. 5G). One day (24 h) after re-immersion, the escape performance of cold-stressed scallops resembled that of control scallops. The ini- tial number of claps of cold-stressed scallops remained constant throughout the experimental period while it gradually decreased for control scallops (Fig. 5A; Table 2).). The initial clapping time was only greater for cold-stressed scallops at the H4th (P = 0.032) and the 120th h (P = 0.0002) (Fig. 5C). Initial clapping rales remained similar between 24 and 156 h (Fig. 5E; Table 3). In spite of the slightly greater mean values for control scallops, the 2 treatments did not differ in the initial maximum number of claps in a series, except at the 4Sth h (P = 0.038) (Fig. 5G: Table 3). After the 15 min recuperation, control and cold-stressed scal- lops differed in terms of total number of claps for the first six sampling periods (P < 0.046) (Fig. 5B). This difference was al- leviated at 120 and 156 h after transfer, when both groups made 22-24 claps. After recuperation, the clapping time of the two groups never differed (Fig. 5D; Table 3), even though the clapping time of cold-stressed scallops remained steady, whereas that of control scallops decreased slightly (Table 2). After recuperation, clapping rate and maximum claps remained stable for control and cold-stressed scallops (Table 2). Clapping rate tended to be higher for control scallops (Fig. 5F). Control scallops consistently made a maximum of 7-8 claps in a series in contrast to 4-5 claps for cold-stressed scallops (Fig. 5H; Table 3). Overall, cold-stressed scallops improved their performance relative to that of control scallops with time after transfer, either sustaining their perfor- mance as control scallops decreased (Fig. 5 A, 5B; Table 2) or improving their performance while control scallops remained stable (Fig. 5E. 5G; Table 2). During the first test 12 h after re-immersion, the time spent closed after exhaustion differed between control and cold-stressed scallops (3.8 vs. 6.7 min. respectively P = 0.0019). Subsequently. 610 LaFrance et al. TABLE 2. Temporal stability of escape performance by juvenile sea scallops either maintained at 18°C (control: C), transferred from 18°C to 8°C (thermally-stressed: TS), air-exposed for 4 h (control + air: C'A) or air-exposed for 4 h during a transfer from 15.5 C to 8°C (thermally-stressed + air: TSA). Tests of effect slices analogous to the ANCOVAs shown in Tables I and 3. Kach scallop (h = 24 per treatment! was measured at each sampling time (12, 24. 36, 48, 60, 84, 12(1, and 156 h). Initial Response df Time X Treadiienl C 7 3.30 ().()(i:i Time X Treatment TS 7 2.51 Odlh Time x Treatment CA 7 3.03 ().()()42 Time X Treatment TSA 7 1.10 0..^6 Time X Treatment C 7 1.73 0.10 Time X Treatment TS 7 11.38 <().0001 Time X Treatment CA 7 4.24 0.0002 Time x Treatment TSA 7 6.50 <0.0001 Time X Treatment C 7 l.(.)4 (1.41 Time X Treatment TS 7 3.12 0.00,34 Time x Treatment CA 7 1 .93 0.065 Time X Treatment TSA 7 6.72 <0.(X)01 Time X Treatment C 7 3.13 0.0033 Time x Treatment TS 7 0.80 0.59 Time x Treatment CA 7 1 .04 0.40 Time X Treatment TSA 7 3.06 0.0040 Time x Treatment C 7 1.32 0.24 Time X Treatment TS 7 6.16 <0.()001 Time X Treatment CA 7 1.72 (1.1(1 Time x Treatment TSA 7 4.54 <0.0(1(11 Time X Treatment C n,a. n.a. n.a. Time x Treatinent TS n.a. n.a. n.a. Time x Treatment CA n.a. n.a. n.a. Time x Treatment TSA n.a. n.a. n.a. Time x Treatment C n.a. n.a. n.a. Time x Treatment TS n.a, n.a. n.a. Time x Treatment CA n.a. n.a. n.a. Time x Treatment TSA n.a. n.a. n.a. Time x Treatment C n.a. n.a. n.a. Time x Treatment TS n.a. n.a. n.a. Time x Treatment CA n.a. n.a. n.a. Time x Treatment TSA n.a. n.a. n.a. n.a. = not applicable. Response .After 15 min of Recuperation df Number of claps Clapping time Clapping rale Maximum number of claps in a series Time spent closed % initial number of claps % initial clapping rate % initial maximum number of claps in a series 7 2.94 0.0053 7 0.59 0.77 7 2.93 0.0055 7 1.04 0.40 7 1.92 0.065 7 1.44 0.19 7 2.28 0.028 7 0.45 0.87 7 0.62 0.74 7 1.48 0.17 7 0.84 0.56 7 0.97 0.45 7 0.54 0.81 7 1.06 0.39 7 0.74 0.64 7 0.76 0.62 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 7 1.63 0.13 7 1.33 0.24 7 0.84 0.56 7 0.79 0.60 7 0.77 0.61 7 0.46 0.86 7 1.80 0.087 7 2.41 0.021 7 1.65 0.12 7 1.19 0.31 7 0.63 0.73 7 1.22 0.29 both control and cold-stressed scallops mainlained their valves closed 4-7 min (Fig. 6A; Table .3). Towards the end of the obser- vations, control scallops stayed closed longer than cold-stressed scallops with this trend becoming significant at 156 h (f = 0.044). As noted during the first experiment, control scallops recovered more of their initial responses than cold-stressed scallops (Fig. 6B. 6C. 6D; Table 3). This difference was again particularly marked for the percent initial number of claps (Fig. 6B). The overall re- covery of control scallops was between 65-75'7r compared with 42-52% for cold-stressed scallops. Evaluation of the Impact of Air Exposure Statistical comparison of the escape performance (number of claps, clapping time, clapping rate, etc.) of the control groups from the two experiments did not reveal any differences. As the values for each escape parameter were similar, with P-values ranging from 0.12 to 0.94, 4 h of air exposure did not seem to affect the escape response capacity of the scallops. Sixty to ninety percent of the air exposed scallops responded to the starfish within 5 sec of the initial contact whether they were Cold Stress Slows Escape Responses of Juvenile Scallops 611 TABLE 3. Statistical analysis of the effects of treatment and time on escape performance of juvenile sea scallops from Cold Shock with Air Exposure Experiment. A 4 h air exposure during the transfer of scallops from pear! nets in the lagoon (15.5°C) to seawater at either 18°C (control) or 8°C (cold-stressed) in the laboratory was used to simulate the cold stress with air exposure accompanying seeding operations. Each scallop (II = 24 per treatment) was measured during each sampling time (12, 24, 36, 48, 60, 84, 120, and 156 h). Initial Response df Response After 15 min of Recuperation df Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time Treatment Time Treatment x Time 1 7 7 1 7 7 1 7 7 1 7 7 1 7 7 n.a. n.a. n.a, n.a. n.a. n.a. n.a. n.a. n.a. 2.85 2.93 1.20 7.78 6.60 4.13 1.89 3.86 4.80 3.20 2.03 2.06 0.11 2.98 3.28 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a 0.010 0.0055 0.30 0.0077 <0.0001 0.0002 0.18 0.0005 <0.0001 0.080 0.051 0.047 0.74 0.0049 0.0022 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. Numtier of claps Clapping time Clapping rate Maximum number of claps in a series Time spent closed initial number of claps % initial clapping ratef initial maximum number of claps in a seriest 1 20.40 <0.0001 7 1.93 0.064 7 2.04 0.050 1 1.94 0.17 7 1.96 0.060 7 0.77 0.61 1 7.35 0.0094 7 1.03 0.41 7 0.78 0.61 1 15.94 0.0002 7 0.76 0.62 7 0.73 0.64 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 1 57.24 <0.0001 7 0.77 0.61 7 0.85 0.54 1 9.32 0.0038 7 3.24 0.0025 7 0.98 0.45 1 15.65 0.0003 7 0.75 0.63 7 1.10 0.36 n.a. = not applicable. t observations were removed from the analysis as recuperation exceeded 200%. cold-Stressed or not (Fig. 7C, 7D). Interestingly, scallops from the Cold Shock Experiment (without air exposure) took more time (5-15 sec) than those given 4 h air exposure before initiating an escape response (Fig. 7A, 7B). Very few individuals (1 to 3 out of 24) required more than 15 sec following the initial contact with the starfish. In both experiments, control scallops swam in virtually all tests (Fig. 8), while a greater proportion of cold-stressed scallops re- TABLE 4. Carbohydrate and protein contents in the adductor muscle, and muscle index of juvenile sea scallops that were used in repeated escape tests or that remained inactive during the Cold Shock with Air Exposure Experiment. Mean (SE. ii). In a given line, values for treatments that do not share the same letter differed (P < 0.05) according to Scheffe's multiple comparisons. Control (I8°C) + Air Thermally-stressed (8°C) + Air Variable Repeated Escapes Rest Repeated Escapes Rest Muscle indext Carbohydrate concentration (mg • g"' wet tissue) Protem concentration (mg • g"' wet tissue) 65.7(1.2.24)' 68.7(2.5,8)-' 10.6(0.8.24)-' 17.5(1.9,8)'' 145.4 (2.0, 24)" 138.7 (3.8, 8)-' 66.4(1.6,24)-' 59.7(2.9,8)-' 11.1(1.1.24)-' 17.5(1.6,8)" 138.9(2.5.24)-' 139.6(5.3,8)° t (M„„,„^(M, digestive gland ■M, oiher soft lissues )) X 100. 612 LaFrance et al. Initial response Response after 15 min recuperation 0 Clapping time (min) D 4 fi I I I 4 -I- ^ 50 E 40 -1 I 30 20 lo- ll II Clapping rate (no. of claps ■ min' ) 40 F tjH t } 30 20 10 0 { I 16 iG Maximum no. of claps in a series 12 nH 0 12 24 36 48 60 84 120 156 12 24 36 48 60 84 120 156 Time (h) after transfer Figure 3. Effect of a transfer from 18°C to 8"C (thermal stress) vs. maintenance at 18 C (control) in seawater on the escape behaviors of juvenile sea scallops, I'lacopecten magellaniciis. measured before and after 15 min of recuperation from exhaustive exercise. Means are shown for all parameters. Vertical bars indicate 95% confidence limits and ;i = 24 for each treatment. sponded with jumps. In the experiment with air exposure, jumps were again more frequent for cold-stressed individuals, particu- larly within the first 60 h. Biometrics and Muscle Energetics At the end of the Cold Shock with Air Exposure Experiment, we compared shell characteristics and soft tissues of cold-stressed and control scallops. Furthermore, we compared resting scallops with those that had taken part in escape tests to assess the impact of the repeated escape measurements on tissue masses and muscle reserve levels. Shell mass was marginally smaller in resting scal- lops (P = 0.040). However, we found no differences between the adductor muscle mass {P = 0.67), digestive gland (P = 0.13) and the mass of other soft tissues (wet and dry; P > 0.26) of scallops that were induced to swim and those left undisturbed. Nonetheless, the muscle index of resting scallops exposed to air prior to the cold stress was slightly lower than that of control scallops {P = 0.05 1 ) (Table 4). Muscle carbohydrates were decreased in scallops used for es- cape tests (Table 4). The phasic muscle of resting scallops con- tained about 18 mg of carbohydrates ■ g"' wet tissue in comparison Cold Stress Slows Escape Responses of Juvenile Scallops 613 10 8 6 4 ] 2 0 Time spent closed (min) Thermally-Stressed (8°C) I Control (18°C) -1 1 r % initial number of claps % initial clapping rate 00 ^ C . 90 - 80 - 70 - 60 " 1 1 1 < 1 1 . I t I ! ! ' 50 - ■' '- . 1- ■ - '- n 1 \ 1 1 ] 1 1 1 1 1 r 12 24 36 48 60 84 120 156 Time (h) after transfer Figure 4. Effect of a transfer from 18 C to 8 C (thermal stress) vs. maintenance at 18 C (control) in seawater on (A) the time spent closed after initial exhaustive stimulation and (B. C, D) performance after 15 min of recuperation relative to initial response for the main escape behaviors of juvenile sea scallops. Placopecten magellanicus. Means are shown for all parameters. Vertical bars indicate 95% confidence limits and n = 24 for each treatment. to 11 mg ■ g"' wet tissue for exercise(J scallops. Muscle carbohy- drate concentrations did not vary with cold stress. The mean muscle protein content was about 140 mg ■ g~' wet tissue (Table 4); it did not differ between control and cold-stressed scallops or between the scallops that were forced to swim and those left un- disturbed. DISCUSSION During seeding operations, juvenile scallops are often subjected to a cold stress. Although sea scallops can live at low temperatures, a rapid decrease in temperature will reduce the capacity to escape predators or colonize suitable habitats. Our results indicate that scallops cannot acclimate their clapping rate to such a cold stress within 6 days and thus may he more vulnerable to starfish pieda- tion. Indeed, the transfer of juvenile scallops from I8"C to 8°C markedly slowed their rate of clapping and the maximum number of claps in a series, but did not significantly alter the total number of claps carried out during an escape response. Nonetheless, after 6 days at 8°C, scallops reduced the time spent closed after ex- hausting escape responses and became similar to control scallops. Over the 6 days of study, cold-stressed scallops reduced their clapping time during an initial stiirulation and increased their ini- tial clapping rate whereas control scallops remained quite constant. Nevertheless, as the cold-stressed scallops did not attain the rates typical of control scallops, only partial thermal compensation oc- curred. Effectively, rate processes usually take considerable time to acclimate when an organism is moved into a colder environment (Bennett 1990). Dickie (1958) stated that the loss of the acclima- tion to warm temperatures may require up to 3 mo in nature and that sea scallops are extremely slow to recover their normal be- havior when exposed to a sudden decrease in temperature. A marked impact of cold stress was apparent in the recupera- tion from escape responses. Cold-stressed scallops were less able to recover their total claps and clapping rate than control scallops. Fifteen minutes after exhaustion, scallops transferred to cold water only recuperated 40-50% of their total claps relative to 60-70% for the control scallops. In the Iles-de-la-Madeleine. predators ag- gregate within three days after seeding of juvenile scallops (Cliche et al. 1994). Since seed scallops are unlikely to have recuperated from cold stress in such a short time, this may place them at a disadvantage when faced with predators that elicit active escape responses. Temperature is likely to be a major determinant of predation rate since it involves many behaviors (location, attack, capture, and ingestion of prey) that are sensitive to temperature. Up to a certain point, predator activity is known to increase with temperature. For example, predation by starfish on 5-9 mm juvenile scallops shows a (2,0 of 6.9 (between 4-15 °C) (Barbeau & Scheibling 1994). On the other hand, scallop escape reactions also benefit from higher temperatures (Barbeau & Scheibling 1994). Valve contraction rate increases with temperature (Dadswell & Weihs 1990, Manuel & Dadswell 1991, Olson & Marsh 1993). Sea scallops (5-35 mm in shell height) increase their clapping rate between 9 and 14.5°C (2,0 of 1.9) (Manuel & Dadswell 1991). The clapping rates of our 35^5 mm sea scallops gave Q,„ values of 2.4 (8-1 8°C) and 1.6 (8-15. 5°C) in our first and second experiments, respectively. The thermal sensitivity of clapping rate may reside in any of its components: latency period, time to peak force and time to relax- ation (Olson & Marsh 1993). Thus, a greater clapping rate may come from more rapid adductions, more rapid opening of the 614 LaFrance et al. Initial response Response after 15 min recuperation Number of claps before exhaustion 45 iB 40 35 30 25 'M\\\ Control(18X) + Air Thermally - Stressed(8°C) + Air 15 ^ Clapping time (min) 3 D 2 ^ 0 0 55 ^E 45 35 ' 25 - 15 4 13 11 9 7 5 Clapping rate (no. of claps ■ min' ) 50 F G T r . 1 0 H p- 1 K5 H 1— 1-0 — m 1 n 1 r :: t • -< 1 , Maximum no. of claps in a series H 9 T ■ ■ 1 |.f-^r:; 1 1, ^^^ ^: ' {{ill 3 4—. : : — r- I 70 mm scallops (Dadswell & Weihs 1990). Further research should assess the re- sistance of different size classes to tluctuating temperatures (Dickie & Medcof 1963). Cold Stress Slows Escape Responses of Juvenile Scallops 615 Time spent closed (min) 10 8 6 ^ 4 2 -\ 0 Control(18°C) + Air Thermally-Stressed(8°C) + Air ~i 1 1 1 I I I i r % initial number of claps % initial clapping rate % initial maximum number of claps in a series 110 100 90 80 70 - 60 - 50 40 30 — 1 — I — I — I — I — I — I — I — I — I — I — I — I I 12 24 36 48 60 84 120 156 Time (h) after transfer Figure 6. Escape response behaviors of juvenile sea scallops, Pla- copecten magellanicus, after 4 h air exposure and transfer to seawater at 8 C (thermal stress + air) or 18°C (control + airl. (A) Time spent closed after initial exhaustive stimulation and (B, C, D) performance after 15 min of recuperation relative to initial response for the main escape behaviors. Means are shown for all parameters. Vertical bars indicate 95% confidence limits and » = 24 for each treatment. Starfish from two thennal regimes were used to provoke escape responses. They were put in tanks at 8°C or 18°C the day before the first escape test. Although we manipulated the predator arms to stimulate the scallops, the amount of chemical cues emitted by cold-stressed and control starfish may have modified escape re- sponses. However, no differences were detected in the response times of scallops at 8°C and 18"C. In the Cold Shock with Air Exposure Experiment, the differ- ences between control and cold-stressed scallops were smaller than those in the Cold Shock Experiment. The drop of water tempera- ture in the lagoon from 1 8°C to 15.5°C (September 23-26; see Fig. 2) may have initiated acclimatization to cold water in the scallops remaining in pearl nets. Thus, a transfer to 8°C would have been less of a stress during the second than the first experiment. This suggests that seeding juvenile scallops later in fall when thermal shock is reduced could reduce losses from predation. The 4 h air exposure seemingly had no effects on the escape parameters of the two groups of control scallops. This is consistent with previous studies of Pecten niaximus showing no significant differences between 0 and 3^ h of air emersion on various as- sessments of scallop's quality (Maguire et al. 1999b. Christo- phersen 2000, Minchin et al. 2000). Also, our preliminary inves- tigations showed that the righting time of juvenile sea scallops was not significantly prolonged by 4 h emersion (unpublished data). The 4 h emersion period of juvenile scallops during their transfer from the lagoon to open sea seems not to be an issue in terms of vulnerability to predation. Air exposure for 4 h led the scallops to react more quickly to contact with the starfish. The occurrence of a stress such as air exposure, even if it may not be injurious, may increase the general excitability (Wilkens 1981). Scallops held in laboratory conditions react more rapidly to any perturbation during the first days of their captivity (unpublished data). As contact of a starfish near the re- gion of the dorsal ears often triggers a swimming response (Wilk- ens 1981), we consistently stimulated in this region, making it unlikely that, the greater occurrence of jumps in cold-stressed scallops was due to differences in our method of stimulation. Total tissue mass of the control scallops was similar to that of cold-stressed scallops in the air exposure experiment. This variable was not affected by thermal regimen when juvenile sea scallops were subjected to constant ( 10 'C) or Ouctuating temperatures (6- I5°C. 8 d temperature cycle) during 48 d (Pilditch & Grant 1999). The muscle carbohydrate content of resting scallops from the Cold Shock with Air Exposure Experiment did not differ from that obtained for cultured scallops of the same size, same origin and under similar grow-out conditions (Lafrance et al., submitted). Thus, a 4-h air exposure did not influence muscle carbohydrate levels. The muscle of scallops that performed escape responses contained carbohydrate levels 40% lower than that of resting scal- lops. Thus, repeated exhausting escapes markedly reduce the scal- lop's energy reserves in an environment with no food. Extrapolat- ing results from laboratory to field is risky since laboratory con- ditions can lead to abnormally high levels of stress (Pilditch & Grant 1999). Nonetheless, repeated attacks by predators on the sea bottom, even if not successful, could make scallops more vulner- able to other stresses (sustained food shortage, diseases, etc.). Hence, predator elimination (Ventilla 1982) on seeding grounds should be considered to increase their chance of survival. The metabolism of Placopecten mageUaiiicits is tightly coupled to thermal fluctuations, as shown by Pilditch and Grant (1999) who observed that metabolic rates did not acclimate to thermal oscil- 616 o A Control ( I ,S°C) LaFrance et al. B Thennally-Stressed(8°C) 1(1(1 - 12 24 36 4ti 60 84 120 156 12 24 36 48 60 S4 120 156 D + 30 sec 15-30. sec C Control(18°C) + Air DThcrmaliy-Stressed(8°C) + Mr Q 5-15. sec 100 0-5 sec 36 4S 60 84 120 156 12 24 36 48 60 84 120 156 Time (h) after transfer Figure 7. Effect of a thermal stress (transfer from -18 C to 8 C") and a 4 h air exposure on ttie delay before initiation of escape reaction of juvenile sea scallops stimulated by contact with a starfish. One-hundred percent of scallops corresponds to ;; = 24 for each treatment from (A, B) Cold Shock Experiment and (C'.Dl Cold Shock with Air Exposure Experiment. latioiis between 6'C and 13 C during 6 x 8 d cycles. On the other hand in our study, temperature only decreased at the start of our observations. During long term exposure to low temperatures, scallops are likely to acclimate and at least partially compensate for the decrease in temperature, as shown by the changes in mem- brane fluidity during thermal acclimation of P. iiiagellcmiciis (Na- politano et al. 1992). In a study of the time course of changes in membrane fluidity during transfer of f. mas^eUaniciis from 13°C to 5°C. compensation of membrane fluidity through increases in the proportion of polyunsaturated fatty acids required 1.3 lo 21 days (Hall 1999). This study provides information that should help planning of a crucial step in bottom culture of sea scallops. A 4-h air exposure does not enhance the impact of cold shock on escape responses of Pkicopeclen inagellanicus. While it has been argued thai seeding of juvenile scallops should be performed at cold temperatures to decrease predation (Barbeau & Scheibling 1994), cold tempera- tures also reduce the scallop's viability in terms of escape behav- ior. As the scallop's escape capacities require more than 6 days before complete themial acclimation, seeded juveniles may benefit from operations minimizing thermal shock or from the use of an acclimation period with no predators at intermediate seawater tem- peratures prior to seeding operations. This need for an acclimation period agrees with the recommendation of Barbeau et al. 1 1994). However, since crab attacks overwhelm the anti-predator defenses of iuvenile scallops at virtualh' all temperatures (Barbeau & Cold Stress Slows Escape Responses of Juvenile Scallops 617 ex a u u Oh c o o Q. O ■ Control (18°C) n Thermally-Stressed (8°C) D Control ( 1 8°C) + Air ■ Thermally-Stressed (8°C) + Air 12 24 36 48 60 84 120 156 Time (h) after transfer Figure 8. Influence of a thermal stress (transfer from -18°C to 8"C) and a 4 h air exposure on the proportion ( % ) of juvenile sea scallops using swimming behavior as a means of escape from a starfish. A "swimmer" was defined as a scallop that made at least 3 claps during most series of claps during the stimulation (evaluated on the response after 15 niin recuperation), n = 24 for each treatment from Cold Shock Experiment and Cold Shock with Air Exposure Experiment. Scheihling 1944. Barbeau et al. 19%). such considerations would not be relevant in an environment dominated by crabs. ACKNOWLEDGMENTS This study was supported by funds from MAPAQ (Ministere de TAcriculture des Pecheries et de TAlimentation du Quebec) to M. Lafrance and H. Guderley and by an operating grant from the NSERC to H. Guderley. The authors thank the staff of the Station Technologique Maricole des iles-de-la-Madeleine of MAPAQ for assistance. Special thanks are extended to C. Cyr for technical help, to H. Paradis and G. Daigle for their statistical support, and to J. H. Himmelman and B. Myrand for their insightful comments on the manuscript. LITERATURE CITED Barbeuu. M. A.. B. G. Hatcher. R. E. Scheihling. A. W. Hennigar. L. H. Taylor & A. C. Risk. 1996. Dynamics of juvenile sea scallop (P/k- copecten magelUmiciis) and their predators in bottom seeding trials in Lunenburg Bay, Nova Scotia. Can. J. Fish. Aquat. Sci. 53:2494-2.'il2. Barbeau. M. A. & R. E. Scheihling. 1994. Temperature effects on predation of juvenile sea scallops (Placopecten magellunicus (Gmelin)) by sea stars (Asterias vulgari.s Verrill) and crabs {Ciiini'r inciniliis Say). ./. E.xp. Mar. Biol. Ecol. 182:27-17. Barbeau. M. A.. R. E. Scheihling & B. G. Hatcher. 1994. 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Biol. 20:309-382. Wilkens, LA. 1981, Neurobiology of the scallop {Pecten ziczac): I. Star- fish-mediated escape behaviors. Proc. R. Soc. Land. B Biol. Sci. 211: ,W 1-372. Young-Lai, W, W. & D. E. Aiken. 1986. Biology and culture of the giant scallop, Placopecten magellanicus: a review. Can. Tech. Rep. Fish. Aqual. Sci. 1478:iv + 21 pp. Joiirmil of Shellfish Research. Vol. 21, No. 2. 619-625. 2002. SELECTIVE INGESTION OF PELAGIC VERSUS BENTHIC ALGAE BY THE COCKLE CERASTODERMA EDULE (LINNE, 1758) JOSE L. RUEDA'* and AAD C. SMAAL' ' Marine and Estuaiine Ecology Unit. Department of Zoology and Entomology. The Univenfity of Queensland. Brisbane Qld 4072, Australia: ^Netherlands Institute for Fisheries Research (RIVO), P. O. Box 77. 4400 AB Yerseke. The Netherlands ABSTRACT The pre-ingestive selection of microphytobenthic algae by the cockle Cerastoderma ediile was studied in comparison with diets containing the pelagic diatom Phaeodacryliim tricornutum. Treatments with the different diets covered a range of seston concentrations and organic content similar to field conditions. Rejection rates of C. edule exposed to the different treatments were significantly correlated with the concentration of total particulate matter. No significant differences in total rejection rates were found between pelagic and benthic diets. Organic rejection rate was significantly correlated with particulate organic matter of the treatments and no significant differences were found between both diets. Selection efficiency was significantly correlated with particulate organic matter concentration In both diets and no significant differences were found between the diets. Analysis of the pseudofeces composition by fiow cytometry from cockles exposed to a mixed diet of microphytobenthic algae and P. iriconuilum. showed a preferential ingestion of the pelagic diatom. Benthic species, such as small pennates and Navwula sp., were preferentially Ingested In comparison to larger microphytobenthic species. The largest microphytobenthic species, Cylindrotlxeca sp., was significantly rejected. In general, C. edule Is an opportunistic filter feeder that takes advantage of both pelagic and benthic algal cells. KEY WORDS: Benthic diatoms, cockles, fiow cytometry, rejection, selection efficiency, Cerastoderma edule INTRODUCTION Filter feeding bivalves are able to sort particles with their gills and labial palps (Kiorboe & M0hlenberg 1981, Newell & Jordan 1983, Newell et al. 1989, Ward et al. 1993) resulting in preferential ingestion of organic material and rejection of inorganic particles in pseudofeces. This preingestive selection has also been observed within phytoplankton with preferential ingestion of some groups of algal cells, both benthic and pelagic (Shumway et al. 1983, Bou- grier et al. 1997, Baker et al. 1998). Preingestive selection of particles may be influenced by Iheir chemical composition or by their size and shape. Some studies about preingestive selection in bivalves have been carried out by using microscopic techniques to compare wa- ter and biodeposits or stomach contents in different species of deposit or filter feeder bivalves (Hummel 1985, Kamermans 1994, Shumway et al. 1987). Flow cytometry offers opportunities for the analysis of algal cells in experiments on the feeding behavior of bivalves (Shumway et al. 1983, Bougrier et al. 1997. Baker et al. 1998). Differential selection of various algal species has been dem- onstrated, using flow cytometric techniques by Peirson ( 1983) in the scallop Argopecten irradians (Lamarck 1819) and by Shum- way et al. (1985) in Ensis direetus (Conrad 1843). Ostrea edulis Linne, 1758, Plaeopecten magellanicus (Gmelin, 1791) and Arc- tica islandica (Linne, 1767). Shumway et al. (1985) showed that the flat oyster Ostrea edulis fed with a mixture of three algae of similar size (Phaeodacryliim tricornutum. Prorocentrum minimum. and Chroomonas salina) preferentially ingested the dinotlagellate Prorocentrum minimum. In contrast, Cucci et al. (1985) have shown that individuals of MytUus edulis Linne, 1738 fed with a mixed diet of the diatom Phaeodactylum triconuitum. the di- noflagellate Prorocentrum, and the cryptomonad flagellate Chroomonas .udina. showed no differential ingestion of cells from suspension. Bougrier et al. (1997) found differences in the pre- ingestive selection of different algal cells (mainly pelagic) by *Correspondlng author. E-mail; J.rueda@zen.uq.edu.au Mytilus edulis and Crassostrea virginica (Gmelin, 1791) and re- lated to differences in the shape of the algal cells. Recently Loret et al. (2000) have shown preferential ingestion of nanoflagellates and cryptophytes by the bivalve Pinctada margaritifeni (Linne, 1758), by applying in situ HPLC techniques. There is some controversy about the feeding behavior of bi- valves, especially C. edule, exposed to benthic algae compared with pelagic algae, or the importance of microphytobenthos as a food source. Kamermans ( 1994) found microphytobenthic algae in the stomachs of the filter feeding bivalves Mya arenaria (Linne, 1738), C. edule and M. edulis, although the percentage of their abundance compared with pelagic algae, was higher in the deposit feeders Scrobicularia plana (Da Costa, 1778) and Macoma hal- thica (Linne. 1738). Recently two studies have been done measur- ing ratios of C and N isotopes in different primary producers and benthic invertebrates (Kang et al. 1999, Riera et al, 1999). These studies concluded that C. edide may prefer to ingest microphyto- benthos and phytoplankton compared with detritus from fragments of benthic macro-algae and seagrasses or detritus from other ori- gins. However, Herman et al. (2000), using also stable isotopes ratios, suggested that suspension feeders, such as C. edule and M. arenaria, depend almost exclusively on pelagic rather than micro- phytobenthic algae. The aim of this study is to quantify the pre-ingestive selection of microphytobenthic algae in comparison with a pelagic species by the cockle Cerastoderma edule. A mixture of benthic and pe- lagic micro-algae occurs in the tidal flat environment of this filter feeder bivalve. The response of cockles exposed to the different diets was studied separately and also when both diets were mixed in the water column. In the latter case analysis were done by flow cytometry to analyze differential algal cell acceptance or rejection. MATERIALS AND METHODS Animals In total 187 cockles (Cerastoderma edule) were used in the different experiments. Cockles had a shell length of 29,78 mm ± 619 620 RUEDA AND SMAAL 1.27 (mean ± standard ernir) in experiments with microphyto- benthos and 24.75 mm ± 0.46 in e.\periments with Phaeodactylwn trkdnuttiimy. ash free dry weight was 255.75 mg ± 30.15 in ex- periments with microphytobenthos and 259.87 mg ± 20.55 in ex- periments with P. irkonwtum. No significant differences were found in shell length or ash free dry weight of the animals used in both experimental treatments. All individuals were collected from an intertidal mudtlat situated in the Oosterschelde estuary (SW Netherlands). Experiments were performed in spring 1997 and summer 1999, in a field laboratory at Jacobahaven (Oosterschelde estuary — SW Netherlands). Shell length, dry tissue weight (DW) and ash-free dry tissue weight for each animal were determined. After measuring the shell length, animals were immersed in boiling water until the shell was opened. Soft tissues were excised, dried at 70°C for minimum 48 h in a drying oven and weighed (= dry weight). Ash-free dry weight is determined as the weight loss after ignition in a furnace at 520"C for 3 h. Diets and Treatments Two different diets were used in the different experiments: microphytobenthic algae (Benthic) and cells of Phaeodacr^htin triconuiliiiii (Pelagic). Microphytobenthic algae were collected in the Oosterschelde estuary. The top layer of sediment (2-3 mm) was sampled and after sampling, coarse sediment and microphy- tobenthos were separated by mixing them in filtered seawater. After sedimentation of the large particles the water containing mainly benthic algae (based on observations with microscopy) was used for the experimental treatment. The microphytobenthic spe- cies were benthic diatoms, including species from the genus Nitzs- chia (length 61 ± 1 1 jxm; width 7 ± 1 ixm), Naviciila (length 46 ± 1 1 p.m: width 22 ± 3 \xm). Cytiiutiothecu (length 81 ± 5 (xm: width 23 ± 2 |xm), and small pennate cells (length 30 + 2 jjim; width 6 ± 1 fj.m). The pelagic diatom P. iricunuitiiiii was cultured outdoors in 100-1 reservoir tanks. Pelagic and Benthic algae were added separately to filtered seawater in different concentrations (treatments) for both diets (Table 1 l. Each treatment was completed by adding silt particles collected in the same location as cockles and microphytobenthos. This silt was dried and burned at 520"C during 4 h for removing the organic content. A coulter counter was used to determine par- ticle concentration, which was monitored during the experiment for differences of the particle concentration from the experimental treatments. Total particulate matter in the different experiments ranged from 40.2 mg.r' to 161.2 mg.l"' and the organic content ranged from 9.08^-58.91% (see Table 1). Analysis of Particulate Matter Water samples of the different treatments were collected from the control chamber (without animal) at the start of the experiment and before collection of the biodeposits. Total particulate matter (TPM; mg.r') was determined by filtering one liter sample onto pre-weighed and pre-ashed Whatman GF/C filters, rinsing with ammonium formate and drying at 70°C (minimum 24 h). Particu- late inorganic matter (PIM: mg.l"') was estimated from the same filters as the weight after ignition during 3 h at 450'C. Particulate organic matter (POM) resulted from the difference of TPM and PIM. In all weight measurements an electronic balance was used which determined the nearest 0.01 mg. Dietary composition was TABLE 1. Characteristics of the different experiments f(ir each diet and treatment. Concentrations of total particulate matter (TPM: my • I 'l. particulate ornanit matter (POM: mg • I"'), particulate Inorganic matter (PIM: mg • !') and percentage of organic matter from the seston ('> OMi. Treatment TPM POM PIM OM code (mg-!"') (mg-l"') (mgl"') (%) Diet with Ph, [U'iitliutyhtni inc >niuUini IP 4S.y(l 4.44 44.46 9.08 ^P 55.32 29.56 25.76 53.43 3p 58.82 8.18 50.64 13.91 4p 83.64 24.88 58.76 29.75 5P 92.40 17.00 75.40 18.40 6p 95.28 32.32 62.96 33.92 7p 125.84 15.04 110.80 11.95 Xp \5234 17.66 134.68 11.59 Diet with Microphytobenthos Im 48.68 28.68 20.00 58.92 :m 53.92 10.10 43.82 18.73 3m 72.52 13.76 58.76 18.97 4m 76.44 30.72 45.72 40,14 .sm 80.96 28.04 52.92 MM dm 87.48 34.24 53.24 ,^9.14 7ni 98.40 20.98 77.42 21.32 Hm 161.20 37.92 123.28 23.52 Mixed diet Imx 5 1 .56 20.80 30.76 40.34 2mx 59.52 17.42 42.10 29.27 .Imx 28.05 5.87 22.18 20.92 characterized in terms of organic content by weight (9cOM = POM X 100/TPM). POM and TPM. Experiments with Separate Diets A flow-through system was used in all experiments. The water containing the different treatments was pumped to the experimen- tal chambers from a diet-reservoir (300 L) in which a mixer and an air pump promoted the resuspension of particles. Peristaltic pumps (Watson Marlow) were used for pumping the water to the indi- vidual experimental chambers. The animals were placed individu- ally in chambers of 300-ml volume and one chamber was used as a control for sedimentation of particles. The flow in all chambers during the experiments was between 3 and 4 1/h. and the concen- tration of particles in the outflow of the chambers containing ani- mals was always more than 70% compared w ith the control cham- ber (without animals). Collection of pseudofeces started after an acclimation period of 3 h to each treatment. Pseudofeces rejected during 3-h periods were collected 2 to 3 times separately for each individual. Pseu- dofeces samples were filtered on preweighed Whatman GF/C fil- ters and measured according to the same procedure as described for samples of the diets. Rejection rates (mg.h"') of total (RRl, oi-ganic (ORR) and inorganic (IRR) particulate matter were calcu- lated. Rates were standardized to an equivalent 500 mg ash free dry tissue cockle by calculating the expression Ys = Ye (0.5AVe)''. where Ys: rate of standard-sized cockle: Ye: uncortected physi- ologic rate; We: measured ash free dry weight of experimental animal: b: allometric coefficient for clearance rate of cockles (b = 0.57; Umjtia 1997, Smaal et al. 1997). Selective Feeding in Cerastoderma edule 621 Selection efficiency (SE) was calculated using the values for organic fraction of seston (f = POM/TPM) and pseudofeces (p = ORR/RR) from the different experiments. Values were calculated as: SE = (l-(p/f)) x 100. Experiments with a Mixed Diet In these series of experiments, cockles were exposed to a mixed diet and the same flow through system described in the previous paragraph was used. The diet contained a mixture of microphyto- benthic algae and the diatom P. triconuttum (si/e 17 ± 2 ixm). The different size of this pelagic species, and its fluorescence charac- teristics make it clearly distinguishable from the microphyto- benthic species used (sizes between 30 ± 2 p.m and 81 ± 1 1 iJim). Individuals were acclimated 3 h to the mixed diet and pseudofeces produced by each animal were collected during periods of 1 h. Sample analysis by flow cytometry was done directly after the experiment. Another series of samples of pseudofeces was col- lected in the experiment for selection efflciency measurements. Algal composition of diet and pseudofeces was analyzed using a flow cytometer (EurOPA: European Optical Plankton Analyser). Standard beads (1.07 |jim, Duke Scientific, USA) were used for calibration and optical adjustments of the EurOPA instrument. A 529 nm and 633 nm laser were used for excitation. Laser light is scattered when a particle traverses the laser beam and is measured in forward and perpendicular (PLS) direction. Laser light irradiat- ing autotrophic phytoplankton is partly emitted as fluorescence. Fluorescence emission excited by the green laser is measured in the red (FOR) and orange (FGO) bandwidth. The number of par- ticles processed in the flow cytometric analysis was 5.000 and 20,000. Only data derived by FGR-triggering (i.e.. fluorescent particles) were used to distinguish between groups of particles with different optical characteristics. The grouping or clustering of data was calculated by using the software program Matlab ver- sion 1.0. The algal species studied are easy to distinguish in the scatter plots of the graph representing FOR and PLS data. The star-shaped form of Phaeodactytwn tricomutum occupied a large area with values of PLS. between 1.500 and 2.200. and values of fluores- cence FOR in general below 2.200 and above 1.900. Microphyto- benthos were composed with mainly four subgroups (related to the most abundant species). The benthic diatom species with lower FOR and lower PLS was Navicula sp.. together with a group of small pennate cells, which could not be identified with values of PLS (1.800-2.300) and FGR (1.900-2.200). Nilzdmi species had a higher PLS value (2,400-2,500) and higher FGR (2,500-2.700). Cylindrotheca species displayed the highest PLS (almost 2.500) and the highest FGR (almost 3.000). To examine the degree of acceptance or rejection of particle types (pelagic or benthic algae), we calculated an electivity index (EI) (Jacobs et al. 1974). modified by Baker et al. (1998), as follows: EI = -[(P - S) / ((P -H S) - (2 * P * S))] where P is the particle ratio in the pseudofeces and S is the particle ratio in the suspension. Electivity index can range from -1.0 to 1.0. A positive El for a given particle type indicates preferential inges- tion (depletion of the particle type in the pseudofeces compared with the suspension), and a negative EI indicates rejection (enrich- ment of a particle type in the pseudofeces compared with the suspension). Subsaniples of the diet and pseudofeces were also fixed in LugoPs and the algal composition was determined using micro- scopic techniques for determination in addition to the flow cytom- eter results. Statistics All statistical tests were performed using the program SYSTAT for PC version 9.0. Multiple stepwise regression analyses were used to test for significant relationships between physiologic rates and parameters of the treatments. Analysis of covariance was used for comparing the rejection rates and selecdon efficiencies of cockles with the different diets. Electivity indices were compared with zero using a one-sample, two-tailed, nonparametric Wilcoxon signed-rank test. These analyses test the null hypothesis that se- lectivity of a particular type is equal to zero (no sorting). A t- student test was used to compare concentrations of the different algal species in samples of the diet and pseudofeces. RESULTS Response to Diets Data about quality and quantity of the experimental treatments are shown in Table I . All treatments were above pseudofeces threshold and the obtained physiologic values with the different diets and treatments have been listed in Table 2. Multiple stepwise regression analyses indicated a significant positive relationship between rejection rate (RR) and total particulate matter (TPM) from the treatments of both diets (Fig. 1; Table 3). The model rejected parameters POM and %0M. Analysis of covariance in- TABLE 2. Physiological response of cockles with the different diets and treatments: Selection efficiency (SE: %), rejection rate of pseudofeces (RR: mg • h"'), organic rejection rate (ORR: nig • h ') and inorganic rejection rate (IRR: nig • h"'). Mean value ± standard error. Number of measurements (n). Treatment SE RR ORR IRR code (%) (mg- h~') (mg-h') (mg- h"') n Diet with Phueodactyhim tricornumin IP 4.9 ± 6..S 29.3 ± 1.7 2.6 ± 0.3 26.8 ±1.4 6 2p 46.1 ±4.3 38.3 ± 7.2 10.8 ±2.0 27.5 ±5.5 10 3p 13.9 ±9.4 22.3 ± 6.2 2.4 + 0.7 19.9 ±5.5 6 4p 36.8 ± 9.3 24.3 ±5.0 5.9+ 1.9 I8.4±3.1 12 ^P 9.4 ±2.5 54.3 ± 5.5 8.9 ±0.7 45.3 ± 4.9 16 6p 4I.9±2.2 45.8 ± 2.7 9.1 ±0.6 36.7 ± 2.3 12 7p 26.1 ±3.4 96.4 ±8.7 8.4 ± 0.6 88.0 ±8.1 6 8p 14.5 + 2.6 80.3 + 5.5 8.0 ±0.7 72.3 ±4.9 10 Diet with Mi icrophytobenthos Im 31.9 + 4.1 34.7 + 2.9 13.8 ± 1.1 20.9 ± 2.2 12 :ni 18.7 ±y.4 17.2 ±3.1 2.6 ± 0.3 14.6 ±2.9 6 3ni 13.1 ±9.6 27.4 ±4.9 4.3 ± 0.6 23.2 ±4.4 6 4m 34.9 ± 2.4 42.7 ± 1.6 11.1 ±0.4 31.6± 1.5 15 ."^m 34.3 ± 2.5 70.9 ± 4.5 16.2 + I.I 54.7 ± 3.7 12 6m 43.1 +3.5 47.9 + 6.4 10.1 ±0.9 37.7 ± 5.7 10 7 111 22.5+ 1.9 50.8 ± 6.8 8.2 ± 1.0 42.7 + 5.8 16 8ni 37.5 ± 7,7 41.0 ± 13.2 1 3.6 ±0.2 77.4 ± 13.0 6 Mixed diet Imx 25.1 ±4.4 - -; - 10 2mx 15.6 ±3.5 - - - 6 3mx 6.6 ± 1.6 - - - 8 622 RUEDA AND SmAAL 100 o 40 20 50 100 150 Total particulate matter (mg. 1'^) Figure 1. Rejection rate (mg ■ h ') of pseudofeces as a function of tfie seston concentration in Ijoth diets. Pelagic: Results for treatments with Phaeodaciyliiiii IrkornMiim (line). Bentliie: Results for treatments with microphytohenthos (dotted line). Vertical bars indicate standard er- ror. Sec Table 2 for number of measurements. obtained with the benthic diet, analysis of covariance indicated that there were no significant differences between diets (/t/ = 0,791; P >0.05). Response to a Mixed Diet Selection efficiency values as a function of POM concentration (see Fig. 3) were generally lower for the mixed diet of pelagic and benthic cells. Higher values of SE were registered at higher values of particulate organic matter. Flow cytometer results showed significantly lower cell concen- trations (expressed as '/r-age) in pseudofeces in comparison to the diet composition (Fig. 4). and therefore significant positive dec- livity indices (EI) (acceptance). For both diets, differences of algal concentration in pseudofeces and diet were significant using a t-student test (Pelagic: DF = 12, t = 14.09, P < 0.001; Benthic: DF = 12. t = 4.86. P < 0.01 ). However, the EI of cockles was significantly higher for pelagic (EIp^.,^,g,^. = 0.31 ± 0.01, mean ± standard error) than for benthic cells (ElB^,„,h,c = 0.20 ± 0.03, mean ± standard error), indicating a preferential acceptance of the Pelagic rather than the Benthic component of the mixed diet, A lower EI for the benthic species was due to differences in the acceptance or rejection of the different benthic species within the Benthic group (Fig, 5). The smallest benthic species, such as Na- viciila sp. and the group of small pennates, were significantly ingested, displaying higher EI values than larger species such as Nilzchia sp. However, Cylindrotheca sp.. the largest species, was significantly rejected, resulting in a negative EI value. dicaled that there were no significant differences between the diets (N = 0.2.';6; P>0.05). Organic rejection rates (ORR) were only significantly corre- lated to the particulate organic matter (POM) (Fig. 2, see Table 3), with TPM and %OM as nonsignificant parameters related to ORR. According to the regressions obtained in each case no significant differences were found between both diets (/t/ = 1 . 1 36; P > 0.05 ). Multiple stepwise regression analysis indicated a significant positive relationship between selection efficiency (SE) and par- ticulate organic matter (POM) (after log transformation) in both diets (Fig. 3 see Table 3). Although lower values of SE were TABLE 3. Stepwise multiple regression analyses of physiological parameters of Cerastoderma cdiile with total particulate matter (TPM: mg • T ). particulate organic matter (POM: mg • I'), particulate inorganic matter (PIM: mg ■ r') and percentage of organic matter from the different diets (% OM). se: standard error. DISCUSSION Production of pseudofeces of cockles was significantly related to the seston concentration in both diets. This relationship between Pelagic diet Benthic diet Coefricient Coefficient Rejection rate (RR) Terms retained Constant 7,97 TPM 0.64 Terms rejected POM % OM TPM X POM Organic rejection rate (ORR) Terms retained Constant 2.45 POM 0.24 Terms rejected TPM % OM TPM X POM Selection efficiency (SE) Terms retained Constant -28.85 Log POM 19,92 Terms rejected Log TPM Log <7r OM 0.73 0.16 <0,01 <0.01 -1.67 <0.0I 0..S8 0.72 0.14 <0.01 <0.01 <0.0] 0.63 0.(J8 <().().5 <0.01 -0.27 <0.05 0.41 0.68 0. 1 1 <0.()l <().01 <0.01 0.67 5.46 <0.0I <0.01 -33.49 <0.01 19.94 0.82 3.85 <().()0.S <0,()(1.S <0.005 Selective Feeding in Cerastoderma edule 623 20 T U Pelagic I 115 £ n c 5 10 O Benthic o "S.J^. r o S 5 ^^^^"'"'^ 1 1 o - i° 0 10 20 30 40 Particulate organic matter (mg. 1'^) Figure 2. Organic rejection rate of pscudofeces (nig • h ') as a function of the organic seston concentration in botii diets. Pelagic: Results for treatments with Pliaeodactyltiin tricornutum (line). Benthic: Results for treatments with microphytobenthos (dotted line). Vertical bars indi- cate standard error. See Table 2 for number of measurements. 60 . QDiet 40 D fteudofaeces Percentage O T 1 T i Pelagic Algal cell Benthic Figure 4. Percentages of the different algal cells, from the total num- ber of particles, both in the diet and the pseudofeces. Pelagic: Pliaeo- dactylum trkontiitum. Benthic: Microphytobenthic algae Vertical bars indicate standard error. In all measurements n = 14. RR and the seston concentration has been observed in several studies and rates were comparable with our outcomes (Iglesias et al. 1992. Navarro & Widdows 1997, Urrutia 1997). Similarly, the rejection rate of organic matter increased with organic content of seston. At a constant clearance rate, filtration rate increases at increasing seston concentrations and the digestive system reaches maximum capacity, hence pscudofeces production increases. Together with the production of pseudofeces, selection of or- ganic material occurs prior to ingestion. Selection efficiency (SE) values found in our experiments were very similar to values docu- mented for C. edule under comparable conditions of food quality (Iglesias et al. 1992, Urrutia 1997). The response by the cockle to the pelagic diet did not differ significantly from the benthic diet. The SE maximizes in C. edule as a response of an increment in the organic content in the diet (Iglesias et al. 1992. Urrutia 1997) reaching a maximum value in our study of 409}-. This maximum value of gross SE (measurements based on the organic content of pseudofeces) for the cockle is similar to those obtained in previous 60 ■ Pelagic r efficiency (%) O Benthic [ o T .^ Selection 1 ' 1 10 20 30 40 -10 J Particulate organic matter (mg. ■') Figure 3. Mean values of selection efficiency ( % ) as a function of the organic seston concentration in each dietary condition. Pelagic: Re- sults for treatments with Phaeodaclyliim tricormilum (line). Benthic: Results for treatments with microphytobenthos (dotted line). Mix: Re- sults for treatments with a mixed diet. Vertical bars indicate standard error. See Table 2 for number of measurements. studies using pelagic algae as a food source (Iglesias et al. 1992. UiTutia 1997). The percentage of organic matter in the different treatments covered a range between W7c-6Q%. Several authors (Iglesias et al. 1992, Urrutia 1997) have described a decrease of the SE in C. edule exposed to treatments above 50% organic content. The decrease of SE values obtained in treatments containing Benthic algae could be related to a lower acceptance or the pref- erential rejection of some benthic species as it has been found with Nitzchia sp. and Cylindrotheca sp. In this study, we have documented the selection and acceptance of microphytobenthos as a benthic food source by the cockle. However, pelagic algae were accepted preferentially when mixed with microphytobenthic species. Moreover, only some microphy- tobenthic species were preferentially accepted by C. edule. Micro- X CD ■D _C >^ -^ o LU O ' a ro Q. o o < c o o 0) Figure 5. Electivity indices (El) for the different microphytobenthic species, as separate species or as a group, relative to the total concen- tration of particles. The studied species are small pennate cells (SmP), Navicula sp. (Nav), Nitzchia sp. (Nit), Cylindrotheca sp. (Cyl) and the benthic group (All), which includes all microphytobenthic species. Vertical bars indicate standard error. * Indices significantly different than zero (f < 0.05). In all measurements n = 14. fi24 RUEDA AND SMAAL phytobenthos seems Ui represent a food source in several species of bivalves and its importance for filter feeding bivalves has been addressed previously. Kamemians ( 1994) found relutii>ns between algal composition of the water column (30% were resuspended benthic diatoms) and the stomach contents of the bivalves C. ed- ulf, Mya arenaria and Mytihts edulis in the western Wadden Sea. She concluded that selection of algae by the bivalves was absent based on comparison of water and stomach samples. Recently two studies measured ratios of C and N isotopes in different primary producers and benthic invertebrates (Kang et al. 1999. Riera et al. 1999). These studies concluded that C. edute preferentially in- gested microphytobenthos and phytoplankton compared with de- tritus from benthic macro-algae or sea grasses fragments. How- ever. Herman et al. (2000) indicated that C. edule depends basi- cally on pelagic algae as a food source and benthic algae hardly contribute to the metabolism. Our experiment showed a differen- tial pre-ingestive selection by C edule of the different benthic species. This may explain the actual controversy about microphy- tobenthos as a food source for suspension feeders. More detailed information about acceptance and/or rejection of single microphy- tobenthic algal species is needed to clarify the actual controversy. Results obtained in this study, using flow cytometry tech- niques, showed a preferential ingestion of Pelagic over Benthic species and a differential selection of the benthic species. Within the Benthic group, some species such as Cylindrolheca sp. was rejected in significantly higher concentrations than small pennates or Navicida sp. The mi.xed composition of the benthic diet is a feature that resembles the natural conditions where C. edule lives and a preferential selection of some species may occur also in the field. Studies on sorting of food in bivalves using flow cytometry to determine the preferences within mixed samples are scarce (Cucci et al. 1985. Shumway et al. I9«3. Chretiennot-Dinet et al. 1991. Bougrieret al. 1997. Baker et al. 1998). Cucci et al. (1985) did not observe preingestive selection in mussels fed with a diet of a diatom, a dinoflagellate and a cryptomonad. However. Bougrier et al. ( 1997) observed in the oyster Crassostrea gigas (Thunberg, 1793) that a preferential ingestion of flagellates species occuned compared with diatom species. Moreover, Baker et al. ( 1998) ex- amined the ability of zebra mussels Dreissena polyiiiorphci (Pallas. 1771 ) to preferentially ingest or reject various phytoplankton spe- cies in the Hudson River (New York). In their study, zebra mussels selected more efficiently small algal cells, such as cyanobacteria. explaining the changes in the Hudson River phytoplankton com- munity in the last decade. Accepted benthic species by C. edule had smaller and similar sizes (Navicula sp.: measured size 46 ± 1 1 iJim; small pennates: measured size 30 ± 2 p.m) than the significantly rejected species such as Cylindnnheca sp. (measured size 81 ± 5 p,ni). Therefore. a possible relation may be found with a preferential ingestion or rejection of those algae and their size. Nevertheless, preingestive selection of algae in bivalves could also be related to chemical characteristics (e.g.. taste of the particles) or to morphologic char- acteristics (e.g., shape of the particles). In this context. Shumway et al. (1990) discussed that not only size is an important factor in the preingestive selection of particles but also mucoid trapping and chemoreceptors. Chretiennot-Dinet et al. (1991 ) reported that the relative ingestion or rejection was not dependent on the size of the algae in C. gigas and M. edulis. Bougrier et al. (1997) reported similar results on those bivalves and no relation was found be- tween preingestive selections of algal species with their size. Mac- Donald and Ward ( 1994) showed that rejection is not dependent on particle size in the scallop Placopecleii inagellauicus. The cockle. C. edule. preferentially accepted small benthic dia- toms such as small pennates and Navicula sp.. both also with an elliptical smooth shape, and rejected others such as Cylindrniheca sp.. which has a larger size and large lateral spicules. The actual mechanisms whereby particles are selected on the gills and the labial palps have been studied using endoscopy (Ward et al. 1991. Ward et al. 1993) but not much information is known about their relation with morphologic characteristics of the particles. Bougrier et al. (1997) indicated that a differential rejection or ingestion of diatom and flagellate species might depend on their shape and flexibility, with a preferential rejection of species with inflexible spicules or the presence of a non-flexible membrane (i.e., silicate frustule). which may hamper the pass through the gill filter. Some authors (Shumway et al. 1985. Bougrier et al. 1997) have also detected a preferential ingestion of flagellates in comparison to diatoms and have speculated that the presence of the silicate frus- tules from the diatoms may appear as mineral particles to sensory receptors of the labial palps. According to this hypothesis the preferential selection of the pelagic diatom P. thcornutum could be influenced because benthic species are. generally, attached to silt particles and it may be possible that some species will have more chance to be rejected as inorganic particles by C. edule. In this study, similar values of selection efficiency were regis- tered when C. edule was exposed to Pelagic and Benthic treat- ments, and lower values obtained in mixed treatments. Riera and Richard (1995) have reported in C. gigas a preferential ingestion of benthic diatoms and detritus compared with phytoplankton in shal- low estuarine waters. But they also reported that C. gigas had a preferential ingestion of phytoplankton. when compared with benthic diatoms and detritus, in deeper waters from the same bay. Shumway et al. (1987) found a preferential ingestion of benthic algae by P. magellanicus collected from deep-water populations, however individuals from shallow water populatiiins seemed to ingest more pelagic than benthic algae. Gut contents of P. magel- lanicus generally reflected available organisms in the immediate habitat. The preingestive selection of algae in some filter feeding bi\ alves. such as C. edule. could be adapted to the composition and the percentages of the different species available in the water col- umn. The preferential ingestion of benthic species in comparison with pelagic species has been observed in the mussel M. edulis in conditions with low concentrations of pelagic algae in the water column (Prou et al. 1994). In our experiments, microphytobenthos is selected at the same efficiency as P. tricornutum in nonmixed treatments. In a mixture of pelagic and benthic species, the rejec- tion of microphytobenthos is higher and this could be related to the dominance of the pelagic algae in the diet. Preingestive selection of benthic compared with pelagic species has been studied previ- ously in the oyster C. gigas by using the flow cytometry technique (Bougrier et al. 1997). In those experiments oysters preferentially ingested pelagic species than benthic species, in a dominant pe- lagic species treatment. Baker et al. (1998) observed that the se- lectivity of different phytoplankton species by the zebra mussel Dreissena polymorplia differed depending on the complexity of the offered suspension. Summarizing, the pre-ingestive selection of benthic algae by C. edule is similar and nonsignificantly lower than the pre-ingestive selection of pelagic species, when benthic and pelagic species are not mixed. This situation of maximum presence of benthic diatoms in the seston could be similar to environmental characteristics of Selective Feeding in Cerastoderma edule 625 tidal flat areas with high resuspension rates of benthic diatoms, or seasons with low pelagic productivity (e.g.. autumn and winter). However, acceptance of benthic algae is lower, compared with pelagic, and not equal for the different components of the benthic group when mixed in a pelagic dominant treatment. These condi- tions may be present during warm periods of the year (e.g., spring and summer) when phytoplankton blooms occur in their natural environment. C. edule may be considered as an opportunistic filter feeder that may take advantage of certain algal species, both pe- lagic and benthic, in relation to their availability in the field. ACKNOWLEDGMENTS This work is a contribution to the ECOFLAT (Eco-Metabolism of a Tidal Flat) project carried out under contract ENV4-ct96- 0216, jointly sponsored by the ENVIRONMENT and MAST pro- grams of the EU. The first author has also been supported by a Marie-Curie Training Research Grant from the European Commis- sion within the SIMCERE project (Fair GT 97-4325). The Na- tional Institute for Coastal and Marine Management (RIKZ). Mid- delburg (The Netherlands) offered us the possibility to use part of their equipment and technology. Many thanks go to researchers from RIKZ such as Thomas Rutten and Ben Sandee for their help in the analysis of the samples with the flow cytometer. Louis Peperzak for his help in the identification of the algal species and Fred Twisk and Richard Eertman for considerable support at dif- ferent stages of this research. We also thank Dr. Pauline Kamer- mans and Dr. Sandra E. Shumway for their comments in earlier versions of the manuscript. 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No. 2. 627-634. 20U2. REPRODUCTIVE CYCLE OF THE BIVALVE CLAMS SEMELE SOLIDA (GRAY. 1828) (SEMELIDAE) AND GARI SOLIDA (GRAY, 1828) (PSAMMOBIIDAE) FROM CHILE DONALD BROWN,'* BERNARDITA CAMPOS," AND H.-JORG URBAN' ^ Departiuneiito de Biologi'a. Iiistlluto de Ciencias Bioldgicas y Qiiimicas, Faciiltad de Ciencias, Universidad de Valparaiso, casilla 5030, Valparaiso, Chile; 'Facultad de Ciencias del Mar. Universidad de Valparaiso, casilla 13-D. Vina del Mar, Chile: ^Alfred-Wegener-Instititte for Polar and Marine Research, Section of Comparative Ecosystem Research, Columbusstr. 27568 Bremerhaven, Germany ABSTRACT Coinmercial clam landings reached an average of almost 91.000 tons annually in Chile over the last decade. In spite of the high value of this resource, few efforts have been made to understand the basic biology of the exploited species, data that might in the future be needed to aid in their protection or even artificial culture. This study is a contribution to the knowledge of the reproductive cycles of two valuable species. Semele solida (Gray ) and Gari xolida (Gray). Representative samples of these species were collected at two widely separated localities in Chile and examined histologically to determine their seasonal reproductive cycles. It was found that the species were of separate sexes, and had annual gonadal cycles. In S. solida from northern Chile, the reproductive period extended from June 1991 to February 1992. In C. solida from southern-central Chile, the reproductive period was relatively short, from October 1991 to February 1992. In both species, most specimens have empty gonads by March. The data obtained are useful in developing management plans related to their reproductive periods. Relevant to culture strategies. S. solida has the comparative advantage of a lengthy reproductive period, wherein mature individuals may be frequently encountered in nature for spawning inductions. G. solida. with its shorter annual reproductive cycle may have the advantage of being induced to mature in artificial conditioning systems over relatively short periods of time. KEY WORDS: clam reproduction, reproductive cycle, bivalves. Semele solida, Gari solida. Chile INTRODUCTION Chilean coastal waters host very productive and diverse clam fisheries due to the rich coastal upwelling and fasorable water temperatures. The largest clam populations occur in the protected bays and fjords of southern Chile, Over a number of years official fisheries records in Chile (SERNAPESCA 1990-1999) considered all clam species as one generic group ("clams") among which were included the venerids Protoihaca tluica (Molina). Venus antiqita (King and Broderip). Eiirhomalea exalbida (Chemnitz), E. lenticu- laris (Sowerby). E. rufa (Lamarck), and the mactrid Midinia edit- lis. Semele solida (Gray) and Gari solida (Gray) belonging to the Semehdae and the Psammobiidae respectively, and objects of this study are also included in this group. They are primarily exploited in artisanal fisheries, and commercialized mostly in canned form. G. solida is highly valued from the culinary standpoint. In 1994. the first year of its listing as an individual species. 461.^ tons of S. solida were harvested, declining to 2071 tons by 1999; G. solida. recorded separately beginning in 1990, was registered at 31.37.^ tons, which declined to 9931 tons by 1999 (SERNAPESCA 1990- 1999). The only regulation for the fishery of these clams is a minimum size limit of 53 mm for 5. solida and 60 mm for C. solida (Subsecretari'a de Pesca 1996). Despite their great economic value, not much research has been done on reproduction in Chilean clams, particularly in S. solida and G. solida. One recent report (Jerez et al. 1999) suggested that G. .wlida in southern Chile had a continuous reproductive cycle throughout the year, a pattern apparently common among the heav- ily commercialized clams such as V. antiqua (Lozada & Bustos 1984) and P. thaca (Henri'quez et al. 1981 ). This also was true for E. lenticidaris (Campos & Brown 1997. Campos et al. 1999) and M. edulis (Jaramillo et al. 1998). * Corresponding author. E-mail: donald.brownCgiuv.cl Semele .solida (Fig. lA). locally termed "tumbao", occurs par- tially buried in sand and gravel bottoms froin the intertidal (Osorio et al. 1979) to the subtidal zone (Urban 1994). Its geographic distribution ranges from 12°S to 47°S. (Viviani 1979). Gari solida (Fig. IB), locally termed "culengue", occurs completely buried in bottom sands and gravels, usually at greater depths than S. solida from the intertidal to 15-m depth (Urban 1994). Its range of dis- tribution along the Pacific Coast, as given by Viviani (1979) and later by Guzman et al. (1998). was between 12°S and 47°S. Biologic data for species of economic importance is fundamen- tal for proposing regulatory recommendations for sustainable har- vest of these resources over time. The obvious declines in harvest over the years enhances the need for more information on the reproduction and survival of these species to support efforts di- rected towards their artificial culture, repopulation. or management as a renewable resource in over-exploited beds. In this study, we describe the reproductive cycles of G. solida and S. solida by means of histologic observations of gametogen- esis during different seasons of the year. Patterns in reproductive cycles, including gametogenesis and resting gonadal periods were studied in a population of S. solida from northern Chile and in a G. solida population from central-southern Chile, representing the first study of this nature for these two clam species in their re- spective regions. MATERIALS AND METHODS Adults of each species were obtained by diving at monthly intervals from June 1991 to July 1992. S. solida was collected in La Herradura Bay (29°58'S) and G. solida from Coliumo Bay (36 '32'S) (Fig. 2). Maximum anterior-posterior length of the shell was measured on each specimen, to the nearest 0.1 mm. Matura- tional status of the gonad was determined monthly on around 30 animals of each species. Tissue samples 5 mm in thick- 627 628 Brown et al. FiKurc 1. Species under study. A: Semele sulida. 78 mm total length (maximum anterior-posterior shell dimension); B: Gari solida, 89 mm total length. ness were obtained and fixed 24 h in Bouin's fluid and prepared by routine histologic procedures as follows: dehydration with graded series of ethanol. clearing in xylol and embedding in Paraplast. Five micrometers histologic sections obtained from three levels of each gonad separated 300 p.m, were stained with hematoxylin and yellowish eosin, dehydrated in graded series of ethanol. cleared in xylol and permanently mounted with Canadian balsam (Gabe 19681. The gametogenic cycles of the two clams were followed by describing the histologic appearance of the gonadal sections and classifying them into different stages of maturity using a modifi- cation of the scale proposed by Lucas (1965). Each individual was assigned to one of the following stages based on the degree of morphologic development of its germ cells: (dl) = initial devel- opment or maturation; (d2) = advanced development or matura- tion; (d.^) = complete development or maturation; (rl) = initial regression or evacuation; and (r2) = total regression or evacua- tion. The results were expressed as percentage frequency histo- grams of: (1) males in each gonadal stage; (2) females in each gonadal stage; and (3) males plus females in each stage, separately for S. solida and G. solida during the sampling period from June 1991 through July 1992. Figure 2. Location of collection sites for A: S. solida and B: G. Solida on the Chilean coast. RESULTS Semele solida specimens measured from 38.9 to 86.0 mm and Gari solida from 41 .4 to 88.0 mm. The two clam species were of separate sexes, with no hermaphroditism and no sexual dimor- phism evident. Histologic analysis of the gonad in both species showed a multilobulate organization of the acini connected to evacuation tubes covered by simple ciliated epithelium similar to that observed in other bivalves (Sastry 1979). The acini consisted of a basal lamina of variable thickness depending on the stage of gonadal maturity. Its relative thickness was greatest in specimens initiating gametogenesis. and in those that had spawned. In these specimens an intra-acinar reticulum consisting of vesicular so- matic cells and an intra-acinar space containing groups of ame- bocytes may be found (Figs. 3A and 4F). The cells of the male germinal line that characterize spermato- genesis may be restricted to two zones of the gonadal acinus; ( I ) a basal region representing the early germinal line that includes spermatogonia and spermatocytes that form a band of circular voluminous nuclei, and recently formed round spermatids that also form a band of small circular nuclei (Figs. 3A, 3B and 4A. 48). that is evident in G. solida: and (2) a lumen region, representing an advanced germinal line with spermatids undergoing cytodifferen- tialion with heavy stained elongated nuclei, gathered by their heads in double columns, giving a ■■feathered" appearance (Figs. 38. 3C and48. 4C). Reproductive Cycle of Chilean Clams 629 Fiyurt 3. A-K: Linlil plKPlomiiroyraphs ol diltiTi'iil histiilii}iic:il slanis ol male and K-,J: li'inali' ;im the majority ol the specimens, a phenomenon more pro- chrony of the reproductive cycles. nounced in S. solida. There was a predominance ot advanced Every monthly sampling during the reproductive period stages of gonadal maturity, and also those with complete regres- showed a few individuals having gonadal conditions differing as an evidence that spawning had occurred. These observa- Reproductive Cycle of Chilean Clams 633 100%-i 80% a 60%' 40%' 20%- 0%' B Gari solida. Male cycle H 5 11 11 11 14 14 13 14 14 12 7 2 7 12 I I ■ J mm m I J JASONDJ FMAMJ J 1991 1992 Months 100%-| 80% 60% 40% 20%4 Gari solida. Female cycle N 23 15 15 18 14 15 16 11 16 18 12 10 19 13 0% 11 iii i i pn I Lmi il I JJASONDJFMAMJJ 1991 1992 Months Gari solida. Reproductive cycle □ MR2 niMRI nMD3 BMD2 0MD1 □ FR2 QFRI DFDS SFD2 HFDI DR2 DRI aD3 SD2 BD1 Months Figure 6. Distribution of different gonadal stages in A. males; B. Females and C. males + females of G. solida collected in Coliumo Bay from June 1991 to July 1992, with sea surface temperature added. The length of each area represents the percentage frequency of specimens in each histological stage of the gonadal acini. N = number of specimens examined. tions suggest intrapopulation asynchrony of gametogenic activity. firmed this asynchrony and strengthened the hypothesis of con- with partial evacuations of gametes over a longer period. The fact tinuous gametogenesis with various cycles of gametogenic activity that the specimens showing signs of having spawned showed a and spawning by each individual during the reproductive period, new wave of maturation in the germinal layer of their acini, con- A difference was observed in the degree of maturity of the 634 Brown et al. piipulation samples of 5. solida between June and July 1991 where advanced and complete maturity were well represented; in the same period of 1992, on the contrary, maturity was just beginning. This difference may be explained by normal adaptation to envi- ronmental conditions such as temperature and fcH)d availability, which may vary within a limited range from year to year. Although Urban and Campos (1994) suggested that the repro- ductive cycles of S. solUla and C. soluhi were influenced by tem- perature, Jerez et al. (1999) working on a G. solida population from the south of Chile found the annual reproductive cycle to be continuous without a marked resting period. Further studies are required to evaluate seasonal variations in gonadal cycles of these species with latitude, as they are distributed over a broad latitudi- nal range from Callao, Peru ( 12°S) to Chile's Chonos Archipelago (44°S). The hypothesis here is that the reproductive cycles of these clams become shorter in populations the farther south they occur on their distributional gradient. Some data available on other clam species with extensive distributions support this hypothesis. Popu- lations of Protothaca thaca (Henn'quez et al. 1981), Tagelus dombeii ( Acufia et al. 1994) and Eiirhomalea lenlicularis (Campos & Brown 1997) from central and north-central Chile exhibit con- tinuous gonadal activity with various important spawning peaks throughout the year. Nevertheless, T. dombeii from south-central Chile showed a period of gonadal regression in the fall (Jaramillo et al. 1998). This phenomenon is not clear across other clam spe- cies inhabiting the south-central zone of Chile, such as Venus antiqiia, Tawera gayi. Midinia ediilis and £/;.v/,s macha that show continuous reproductive cycles without resting periods (Lozada & Bustos 1984, Jerez et al. 1999). From the practical point of view, regulation of harvesting these clams should be based on considerations of their reproduc- tive cycles by limiting their harvest during the major spawning season. Consideration of the reproductive cycles is also important in obtaining broodstock for aquaculture. Experimental studies should prove this a feasible alternative for production or protect- ion of the resource. In studying resource management of these clams, S. solida shows a comparative advantage in having a more extensive reproductive period, as mature individuals may be encountered over an extended period. This implies that mature broodstock would be available in nature for artificially induced spawning (e.g.. in aquaculture experimentation) over compara- tively lung periods. Although G. solida. in contrast, has a more restricted reproductive period, it may be a species amenable to artificial conditioning in aquaculture systems, given that its gonads almost always contain high numbers of immature gametogenic cells. ACKNOWLEDGMENTS The authors thank Ms. T. Jeri for sampling G. solida at Coliumo Bay. and Ms. G. Bellolio (U. Catolica del Norte at the time) for providing S. solida from Herradura Bay. We also thank Dr. R. Guerra for her supervision of the histologic processing at the U. de Valparaiso, and Mr. C. Olivares for assistance in histo- logic analyses. Corrections and comments by anonymous review- ers helped to improve the manuscript. The work was financed by FONDECYT Grant 91-.S02 to B. Campos. Acufia, E., Ch. Guisado & M. Bem'os. 1994. Cicio reproductive de Tagelus (/o/Hfcfii (Bivalvia: Helerodonta: Solecunidae). provenienles de la bahi'a La Herradura de Guayacan. IV Region. XIV Jornadas de Ciencias del Mar. Chile. 131 pp. Campos, B. & D. Brown. 1997. Aspectos reproductivos de la almeja Eii- rhomalea lenlicularis (Sowerby) proveniente de la rada El Algarrobo (V Region). Informe Final Proyecto DIPUV 20-95. Universidad de Valparaiso, Chile. Campos, B., D. Brown, L. Duran. C. Melo & J. Urban. 1999. Estudio de edad y reproduccion del recurso almeja en la IV y V Regiones. Informe Final Proyecto FIP 97-32, Subsecretaria de Pesca, Chile. Gabe, M. 1968. Techniques hi.stologiques. Paris: Masson et Cie. 1113 pp. Guzman, N., S. Saa & L. Ortlieh. 1998. Catalogo descriptivo de los mo- luscos litorales (Gastropoda y Pelecypoda) de la /,ona de Antofagasta. 23 S (Chile). Esnid. Oceanol 1 7; 1 7-86. Henriquez. R.. P. Barboza, R. Ramos. E. Tapia & C. Toro. 1981. Variacion anual de la gonada de la almeja Protothaca thaca (Molina 1782): analisis histologico. I Jornadas de Ciencias del Mar. Chile. Resiimen. 34 pp. Jaramillo, E., E. Clasing, M. Avellanal, P. Quijon, H. Contreras. P. Rubilar & G. Jerez. 1998. Estudio biologico pesquero de los recursos almeja. navajuela y huepo en la VIII y X Regiones. Informe Final Proyecto FIP 96-46, Subsecretaria de Pesca. Chile. Jerez, G.. N. Barahona. H. Miranda. V. Ojeda, D. Brown. C. Osorio, A. Olguin & J. Orensanz. 1999. Estudio biologico pesquero de los recur- LITERATURE CITED SOS tawera [Tawera gayi) y culengue (Gari solida) en la X Region. Infomie Final Proyecto FIP 97-29, Subsecretaria de Pesca, Chile. Lozada. E. & E. Bustos. 1984. Madurez sexual y fecundidad de Venus antiqua antiqua King & Broderip. 1835 en la bahi'a de Ancud (Mol- lusca: Bivalvia: Veneridae). Rew Biol. Mar. Valparaiso 20:91-112. Lucas, A. 1965. Recherche sur le sexualite des mollusques bivalves. The- ses Doclorat. Fac. Science. Universite de Rennes. France. Osorio, C. J. Atria & S. Mann. 1979. Moluscos marinos de importancia economica en Chile. Biol. Pesq. Chile 1 1:3— 47. Saslry, A. 1979. Pelecypoda (excluding Ostreidae). In: A. Giese & J. Pearse, editors. Reproduction of Marine Invertebrates. Vol. V. New York: Academic Press, pp. 113-292. SERNAPESCA. 1990-1999. Anuario estadistico de pesca 1990 a 1999. Servicio Nacional de Pesca. Chile. Subsecretaria de Pesca. 1996. Medidas de Administracion Pesquera. Min- isterio de Economi'a, Desarrollo y Reconstrucci6n (Chile). 32 pp. Urban, H.-J. 1994. Adaptations of six infaunal bivalve species of Chile: coexistence resuhing from differences in morphology, burrowing depth and substrate preference. Arch. Fish. Mar. Res. 42:183-193. Urban. H.-J. & B. Campos. 1994. Population dynamics of the bivalves Gari solida. Seinele solida and Protothaca thaca from a small bay in Chile at 36 °S. Mar. Ecol. Prog. Sen 1 15:93-102. Viviani. C. 1979. Ecogeografi'a del literal chileno. Stud. Neotrop. Fauna & Environ. 14:6.5-123. Joiinuil of Shellfish Research. Vol. 21, No. 2, 635-642. 2002. GROWTH OF NORTHERN [MERCENARIA MERCENARIA (L.)] AND SOUTHERN [M. CAMPECHIENSIS (GMELIN)] QUAHOGS: INFLUENCE OF SEAGRASSES AND LATITUDE K. L. HECK, JR.' - * L. D. COEN/ ' AND D. M. WILSON' ^ ^Dauphin Island Sea Lab. 101 Bienville Boiilevanl. Dauphin IslanJ. Alabama 36528; 'Department of Marine Science, University of South Alabama. Mobile Alabama 36688: ^Marine Resources Research Institute. SCDNR. P. O. Box 12559. Charleston. South Carolina 29422: and ^Marine Science Program. Department of Biological Sciences. University of Alabama. Dauphin Island, Alabcuna 36528 ABSTRACT To better understand how seagrasses influence the growth of northern and southern quahogs (Mercenaria mercenaria and Mercenaria campechiensis). we collected and compared the growth rates of clams from seagrasses and adjacent unvegetated locations along the Atlantic (Massachusetts and New Jersey) and Gulf of Mexico coasts (Alabama and Florida) using identical methods. In particular, we sought to test hypotheses that clam growth is inlluenced not only by the presence or absence of seagrass {Tlialassia testudinum. Halochile nrighlii. and Zosrera marina), but also by location within seagrass beds. Walford plots constructed using annual shell-growth band analyses showed that Mercenaria spp. growth was significantly affected by the presence of seagrass habitat, although the magnitude of this effect varied with clam age (size) and latitude. Specifically, first-year growth was significantly greater in unvegetated than in adjacent vegetated sites, whereas a measure of lifelong growth (Ford's growth coefficient) was not significantly different between adjacent vegetated and unvegetated sites. We hypothesize that these conflicting results may be due, in part, to differing patterns of energy resource allocation in Mercenaria at various life cycle stages. During the first 1.5 y or so, a clam's energy resources are allocated primarily to somatic growth, whereas in subsequent years energy is also allocated to reproduction. Therefore, factors affecting Mercenaria growth, such as the presence or absence of submerged vegetation, should be most easily detectable in analyses examining the first year's growth. Even though our data showed greater first-year growth at lower latitudes (Gulf of Mexico sites versus New Jersey and Massachusetts), overall lifelong growth rates were greatest at the Massachusetts sites. We attribute this pattern to the fact that higher first-year growth rates quickly become asymptotic in the warm waters of the Gulf of Mexico, whereas slower but more constant growth rates are typical of clams from colder water sites. We found little evidence for the effects of location (as measured as distance from bed edge) on growth of individuals collected from grassbeds, except where patterns of water movement showed consistent directionality (e.g.. Gulf of Mexico sites). Given the multitude of variables that can interact to influence clam growth rates and the unresolved variability in results from single-site studies performed in different locations, some complex, carefully controlled experiments are still needed to elucidate the dominant factors that regulate both short and long-term clam growth. KEY WORDS: hardclams, Mercenaria. seagrass, latitude, growth, quahog INTRODUCTION Growth and survival' of suspension-feeding animals may be strongly influenced by the presence of submerged aquatic vegeta- tion (SAV), presumably because macrophytes reduce flow and enhance the deposition of suspended materials (e.g.. Scoffin 1970, Orth 1977, Tegner & Dayton 1981, Fonseca et al. 1982, Harlin et al. 1982, Eckman 1983. Peterson, et al. 1984, Dayton 1985. Wild- ish & Kristmanson 1997), thereby altering available food supply (Peterson & Beal 1989, Iriandi & Peterson 1991, Irlandi 1996, but see Judge et al. 1992, Nakaoka 20(J0). However, studies of the suspension-feeding bivalve Mercenaria mercenaria (The Noilhem Quahog), have continued to produce conflicting results (e.g.. field- ing 1912, Kerswill 1941, 1949. Haskin 1952; Pratt & Campbell 1956; Ansell 1968, Greene 1979, Hadley & Manzi 1984, Peterson et al. 1984, Arnold et al. 1991, Coen & Heck 1991, Irlandi & Peterson 1991, Judge et al. 1992, Coen et al. in prep.) with regard to the effect of seagrasses. For example, early studies by Kerswill (1941, 1949) found decreasing M. mercenaria growth rates as eelgrass (Zostera marina) density increased, whereas a later study by Peterson et al. (1984) found just the opposite. More recent studies (Peterson & Beal 1989; Coen & Heck 1991; Slattery et al. 1991, Nakaoka 2000, Coen et al. in prep.) found that M. mercenaria in seagrass beds sometimes grew faster, slower, or at the same rate as M. mercenaria in unvegetated habi- tats. Peterson and Beal ( 1989) cited two possible explanations for "Corresponding author. the variability in their results. First, as water velocities decrease, the flux of suspended food particles passing by the clam's siphons will decrease. Alternatively, reductions in velocity could enhance the settlement of suspended materials and increase food concen- trations near the bottom. The net effect of decreasing suspended horizontal food flux while increasing the number of food particles settling from the water column will determine whether the impact of vegetation on Mercenaria growth is positive or negative (Peter- son & Beal 1989). Subsequently, Irlandi and Peterson ( 1991 ) have argued that measured variations in food concentration between vegetated and nearby unvegetated areas are insufficient to explain the observed between-habitat differences in clam growth. There must also be an upper limit to current velocity beyond which bottom instability inhibits feeding, either directly by smoth- ering or indirectly by requiring the clam to expend energy rebur- rowing to maintain a desired burial depth (Myers 1977, Orth 1977, Turner & Miller 1991, Iriandi &Mehlich 1996, Iriandi 1996) or by decreasing assimilation efficiency (Bricelj & Malouf 1984). Greater bottom stability in vegetated habitats produced by the baftling effect of seagrasses could also lead to greater growth of Mercenaria located within seagrass beds (cf. Peterson et al. 1984; Irlandi & Peterson 1991: Iriandi 1996, Coen, unpublished. 2000). Location within a landscape of habitats and the associated sys- tem states (i.e., food, flow, competitors, predators, see Micheli 1996) can also significantly affect bivalve populations. This can result from local food depletion from upstream-feeding individuals (e.g., Okamura 1986. Peterson & Black 1987, Frechette et al. 1989), within or among habitat conditions (e.g., Iriandi & Peterson 6,35 636 Heck et al. 1991. Kamermans 1993) or the physical activity of neighboring animals interfering with feeding or space acquisition (Vimstein 1977, Peterson 1979, Okamura 1986. Nakaoka 2000). These can manifest themselves under certain situations as density-dependent effects (Grizzle & Morin 1989, Grizzle & Lutz 1989. Grizzle et al. 1992, Kamermans et al. 1992). The growth of suspension-feeding bivalves may also be influ- enced by lethal or nonlethal (siphon nipping, reduced foraging) predation by numerous species, such as fishes, gastropods, and crabs (e.g., Coen & Heck 1991, Irlandi & Peterson 1991. Ka- mermans & Huitema 1994, Peterson & Skilleter 1994, Skilleter & Peterson 1994. Bonsdorff et al. 1995. Nakaoka 2000). Although the siphon tip is easily regenerated, this requires an expenditure ot energy (see Peterson and Fegley 1986) that could otherwise be used for shell and soft-tissue growth/repair and reproduction (e.g., Trevallion et al. 1970. Hodgson 1982, Peterson & Quammen 1982. Festa 1975. De Vlas 1985, Zwarts 1986, Coen & Heck 1991. Skilleter & Peterson 1994. but see Irlandi 1994. 1996. and Irlandi & Mehlich 1996 for counter examples). In addition, the mere presence of potential disturbers or predators in the vicinity of Mercenaria may cause individuals to cease feeding and thereby lead to reduced growth rates where predators are abundant (e.g.. Blundon & Kennedy 1982, Irlandi & Peterson 1991, Nakaoka 2000). This study was designed to investigate how the presence of vegetation and location within a bed can influence the growth ot selected Atlantic and Gulf of Mexico (U.S.) populations of the northern and southern quahogs, M. mercenaria and M. campechiensis. We were especially interested in determining whether 1 ) generali/'ations could be made that would apply to populations of both the northern and southern quahog species re- garding the effect of seagrasses on growth: and 2) whether the distance of individual clams from the bed's edge could be an indirect measure of habitat quality as a result of potential differ- ences in food supply, and therefore help explain some of the con- flicting evidence regarding the effects of seagrasses on quahog growth rates. MATERIALS AND METHODS Sd/rfv Locations M. mercenaria and M. campecliiensis populations were sampled at six different locations in four states (Massachusetts. New Jersey, Florida, Alabama) along a latitudinal gradient from about 42° to 30°N. The Massachusetts sampling area was located on Cape Cod in the Nauset Marsh system (Nauset Harbor and Salt Pond Bay; 42°N, 70°W), whereas M. mercenaria were collected from both vegetated (/; = 51 clams) and unvegetated (sand) sub- strates (/I = 52 clams) in May 1987. Vegetation consisted of pure stands of Z. marina, and water depth at low tide was less than 1.0 m. The New Jersey sampling site was in southern Bamegat Bay, near Marshelder Island (39°N, 72°W), and M. mercenaria were also collected from both vegetated in = 51 clams) and adjacent unvegetated (sand) substrates (/; = 84 clams) in May 1987. Veg- etation was dominated by eelgrass (Z. marina), with lesser amounts of widgeon grass {Ruppia maririma). Water depth (at low tide) was less than 1.0 m. Collections were also made in the Perdido Bay system (30°N. 87°W), with one site at the western end (see Fig. 1 in Coen & Heck 1991; Perdido Pass. Alabama), and one site at the eastern end of the Bay (in the Gulf Islands National Seashore, Big Lagoon, Florida, see Fig. 2 in Spitzer et al. 2000). Native M. campechiensis were collected at the Perdido Pass site in April. May, and June 1987 ()i = 32 clams) and at the Gulf Islands National Seashore site (30 N, 87 W). in June 1987 (« = 21 clams). All clams collected from these two sites were taken from vegetated habitats because there were no hard clams found in the unvegetated sand adjacent to the seagrass beds. Submerged vegetation at the two sites con- sisted of shoal grass. Halodule wriglitii (Alabama) and mixed stands of H. wrif^lilii and turtle grass. Tlialassia testttdinum (Florida). Water depth (at low tide) at both sites was less than 1.5 m. Additional details on vegetation composition and physio- chemical conditions at these study sites can be found in Heck et al. ( 1989. Massachusetts); Wilson etal. (1990. New Jersey); and Coen and Heck (1991 ), Judge et al. ( 1992. 1993). and Wilson (1991 ) for the Gulf of Mexico sites. Clams were collected by hand after being located by treading at each sampling location. Clam location within grassbeds relative to the edge of the bed was estimated as the minimum distance from collection point to the nearest grass bed edge along one of the four compass directions. All Mercenaria were frozen after collection and later were thawed to allow removal of the soft tissue without damaging the shell. Shells were then washed and allowed to air dry. Valves of each clam were separated, with one valve used for age and growth estimates and the other archived (see also Wilson 1991). We assumed that all specimens collected from Atlantic locations are Mercenaria mercenaria (L.). and all individuals col- lected from Gulf sites are Mercenaria campecliiensis (Gmelin). Analysis of Growlli Incremenls For age and growth determinations, one valve from each clam was embedded in epoxy resin before sectioning with a Buehler Isomet"^" low-speed saw. Sections were made through the umbo to the ventral margin, along the axis of maximum growth (Kennish et al. 1980). A second cut was made approximately 10 tj.ni from the first to produce a thin section. To facilitate examination of annual growth increments, each thin section was ground and polished on a Buehler Ecomet \\\^'^ grinder/polisher with appropriate polish- ing compounds. During the polishing procedure, visual inspection of the thin section under a microscope determined when the thin section was readable (see Wilson 1991 for more detail). When thin sections were viewed under the microscope, growth increments appeared as alternating translucent narrow dark and wide white (opaque) regions corresponding to periods of slow (narrow microgrowth increments) and rapid (wide microgrowth increments) growth, respectively (Barker 1964, Rhoads & Pan- nella 1970, Kennish 1980. Fritz & Haven 1983, Peterson et al. 1983. 1985. Grizzle & Lutz 1988. Arnold et al. 1991. 1996. Bert & Arnold 1995). One annual growth cycle, therefore, consists of two opaque regions (spring and fall) and two translucent regions (summer and winter). In older Mercenaria. there is often only one opaque region, with the fall band being faint or absent. Annual growth was marked at the end of each successive winter band for Atlantic sites (Pannella ct MacClintock 1968. Rhoads & Pannella 1970, Kennish & Olsson 1975, Fritz & Haven 1983), and at the end of each successive summer band for the Gulf of Mexico sites (Clark 1979, Fritz & Haven 1983). Thus, one year's growth was measured as the linear distance between consecutively marked annual growth bands. To obtain a quantitative measurement of annual growth, the distance between consecutive bands was traced on paper at 60x Influence of Seagrasses on Growth of Hardclams 637 with a camera lucida. Tracing followed the boundary between the outer, prismatic and middle, homogeneous shell layers with the end points located at the point where each successive annual band crossed this boundary (rather than the point where each successive annual band reached the outer edge of the shell), serving to delin- eate each year's growth. Distances were measured with a cali- brated ocular micrometer. Visual examination of the thin sections indicated that annual growth increments corresponding to ages >12 y could not be re- solved with certainty (Wilson 1991, Lowell Fritz, Rutgers Univer- sity, personal communication). In older Menenaria. annual growth increments become narrower and more difficult to distin- guish owing to decreased lateral shell growth (Hopkins 1941, Fritz & Haven 1983). This slowing of lateral growth may be accompa- nied by an increase in shell thickness, especially along the shell's margin. This pattern of shell growth was evident in most of the larger (i.e., older) Menenaria .specimens in this study. Visual ex- amination of the thin sections identified clear annual growth in- crements corresponding to ages <1 1 y. Therefore, only the first 10 annual growth increments from each individual specimen were used for growth rate determinations. Walford plots (see Ricker 1975) of yearly clam growth in length were constructed for individual M. mercenaria and M. campechiensis (see above assumption) for each site by plotting total length in a given year (designated G,,^, ) versus total length in the previous year (designated G,). Total length was calculated directly from the summation of annual measured growth incre- ments measured from thin sections. In all cases, the first data point represents length at year two versus length at year one. "Popula- tion" Walford plots for each site, constructed by plotting 0,^., versus G, values across all ages and clams from a given site, contained a minimum of 63 and a maximum of 756 data points (Wilson 1991). A linear regression of G,.^, versus G, yielded two values of interest: the slope of the regression line and its y-intercept. The y-intercept is the growth for the first year (McCuaig & Green 1983, hereafter referred to as the first year growth). The slope (also called Ford's growth coefficient or simply growth coefficient) is the fraction of total growth still to be attained after the first year (Walford 1946, McCuaig & Green 1983). The slope represents the decrease in growth increments over age classes (Ricker 1975) and has been used to characterize clam growth rates after the first year. The point at which the regression line for the Walford plot inter- sects the line of slope = I, indicates the point at which growth stops and maximum size is reached. Walford plots were constructed for each site to determine the site-specific population growth coefficient and first year growth. SAS (Version 5) regression procedure (Proc Reg) and its "Test" option (Freund & Littell 1986) were used then to simultaneously test for differences in growth coefficient (slope), first year growth (intercept), and coincidence of the regression lines among pairs of sites. Pairwise comparisons of M. inerceiuiria between vegetated and unvegetated habitats were only analyzed for sites in Massa- chusetts and New Jersey. Between-habitat (within site) compari- sons were not made at the two Gulf sites because no M. campechiensis were found in any of the suiTounding unvegetated habitats in either Alabama or Florida. Walford plots were also constructed for individual M. merce- naria and M. campechiensis to determine growth coefficients and first year growth for each clam. Ford's growth coefficient and first year growth for each clam were then tested for the significance ot correlations between these parameters and the linear distance to the grassbed edge (see above). Because proper construction of the Walford plots requires a minimum of three sets of data points (i.e., three G,^,, G, pairs), only those individuals having four or more annual growth bands were used to calculate the growth coefficient and first year growth for individual Mercenaria and sites (see Wilson 1991 for more details). RESULTS Year I and Overall Growth First year growth, calculated for each site (Fig. 1: Table 1) ranged from a high of 2.11 cm for the Gulf Islands National Seashore vegetated site in Florida (mixed H. wriglnii and T. restiidinum) to a low of 1.10 cm for the vegetated site in Massa- chusetts (Z. marina). First-year growth was significantly greater in sand ( = unvegetated) than in seagrass at both the Massachusetts and New Jersey sites (see Table 2). There was also significantly greater first year growth at the vegetated sites in New Jersey, Alabama, and Florida than at the » - A 4 - 2 - -•- Slope = 1 -V- NH -•- NHU -*- FL -1- HI -»- NS -e- Hiu A 0 8 ■ 6 - 4 n ^ ^ f Length (t) Figure L Walford plots for all six sites constructed by plotting length at year t,., versus length al year t for all ages of all clams combined. Only the regression lines fitted to each site sampled and a line of slope = 1 (solid circle) are displayed. The six lines displayed correspond to the following sites: (1) unshaded diamond line = vegetated Gulf Islands National Seashore, Florida site (mixed Thalassia tesludinum and some Halodule wrightii). (2) open circle line ' = unvegetated New .Jersey site (sand), (3) solid diamond line ♦ = vegetated Perdido Pass, Alabama site {Halodule). (4) large + line = vegetated New Jersey site {Zoslera marina and some Ruppia marilima), (5( large shaded triangle line A = unvegetated Massachusetts site (mud/sand), and (6) large unshaded triangle line 7 = vegetated Massachusetts site (Z. marina). 638 Heck et al. TABLE 1. Growth parameters for Walford plots calculated from clams collected at each study site. Study Sites Intercept (First-Year Growth I cm Slope (Ford's Growth Coefficient) Massachusetts, vegetated (Zoslera) Massachusetts, unvegetated New Jersey, vegetated iZostera) New Jersey, unvegetated Gulf Islands National Seashore, vegetated iTInilassUi) Perdido Pass, vegetated (Halodule) 51 1.10 0.91 0.96 84 1.28 0.88 0.96 51 1.64 0.82 0.95 52 1.88 0.80 0.94 21 2.11 0.82 0.94 32 1.73 0.81 0.87 Intercept represents the first year's growth and the slope represents Ford's growth coefficient. Massachusetts site (Table 2). and first-year growth was signifi- cantly greater in Florida [Thalassia/Hadodule) than New Jersey (Zostera) grassbeds. However, no significant difference in growth was observed between clams from New Jersey and Alabama (Per- dido Pass, Halodule) grassbeds. Growth rates of the 291 clams measured, while not presented individually here, varied over about an order of magnitude. For example, first-year growth ranged from a low of 0.67 cm for a clam at the vegetated site in Massachusetts (in Z marina) to a high of 6.31 cm for an individual at the Perdido Pass vegetated site (taken from H. wrightii). Ford's growth coefficients for individual clams ranged from a low of 0.14 for a clam at the vegetated site in Perdido Pass (W. wrightii) to a high of 0.97 for an individual at the vegetated site in Massachusetts (Z marina). Overall, site-specific Ford's growth coefficients ranged from a high of 0.91 for the vegetated site Massachusetts (Z marina) to a low of 0.80 for the unvegetated site in New Jersey (Table 1; Fig. 1). The only significant differences in the growth coefficients were between both the Massachusetts sites and all other sites in New Jersey. Alabama, and Florida. These significantly higher growth coefficients at the Massachusetts sites were unexpected because first-year growth was lowest at these sites. Growth and Location in the Grassbed There were two significant positive relationships between first- year growth and distance to the edge of the grassbed, which were found at the vegetated sites in Massachusetts (Z marina) and Perdido Pass, Alabama (W. wrightii: r = 0.315 and 0.432; P < 0.05 and 0.01. respectively), indicating greater growth in year I with increasing distance from the edge of the bed at these two sites (Table 3). The only significant correlation between Ford's growth coefficient for individual clams and distance from the grassbed edge was found at the vegetated site at Perdido Pass (//. wrightii: Table 3, /■ = -0.435; P < 0.05). a result that indicates greater lifelong growth near the edge of the bed. DISCUSSION The effects of habitat (seagrass versus unvegetated substrate) on clam growth did not vary consistently when examined by either clam age or geographical location. With respect to age, we found significantly greater growth during year one in clams collected from the unvegetated habitats at both Massachusetts and New- Jersey (Table 2). We attribute this to the fact that M. merceiwria growth is greatest during the first 1.5 y of life, with decreasing growth thereafter as sexual maturity is reached (Haskin 1952, Gustafson 1955, Walker 1985. Eversole et al. 1986). This suggests to us that habitat effects would be most evident in analyses of the rapid growth attained in the first year. However, this finding con- flicts with that of Iriandi and Peterson (1991), who found faster growth of juvenile clams (mean size of 2.6-2.87 cm in length) planted for up to 6 months in seagrass (vs. sand), and who con- cluded that "'. . . results should dispel any remaining doubts about TABLE 2. Pairwise comparisons of first-year growth and Ford's growth coefficient among study locations. First-Year Growth Ford's Growth Coefficient Pair-Wise Comparisons N Site 1/Site 2 F Value Significance Level {P <) F Value Significance Level (/»<) Massachusetts, vegetated vs. unvegetated 5I/S4 New Jersey, vegetated vs. unvegetated 5l/.'i2 Massachusetts, unvegetated vs. New Jersey, unvegetated 84/52 Massachusetts, vegetated vs. New Jersey, vegetated 51/51 Gulf Islands National Seashore vs. Perdido Pass 21/32 Massachusetts, vegetated vs. Gulf Islands National Seashore 51/21 Massachusetts, vegetated vs. Perdido Pass 51/32 New Jersey, vegetated vs. Gulf Islands National Seashore 51/21 New Jersey, vegetated vs. Perdido Pass 51/32 All four vegetated subsites (GINS. MA. NJ. and PP) 21/51/51/32 7.90 9.61 78.80 55.47 5.92 89.21 38.03 19.98 0.83 35.38 001 0.01 0.01 0.01 0.05 0.01 0.01 0.01 Not significant 0.01 3.10 3.72 50.71 37.28 0.29 22.66 21.10 0.00 0.62 13.00 Not significant Not significant 0.01 0.01 Not significant 0.01 0.01 Not significant Not significant 0.01 Comparisons were performed on the regression equations calculated for the Walford plots at each location using the test option in the regression procedure of SAS version 5. Influence of Seagrasses on Growth of Hardclams 639 TABLE 3. Pearson product moment correlations between first-year growth and Ford's growth coefficient and proximity of clams to the edge of the grassbeds at each study site. First-Year Growth Ford's Growth Coefficient Correlation Significance Level Correlation Significance Level Study Sites n Coefficient (^O Coefficient (P , Annual band deposition within shells of the hard clam. Mercenaria mercenaria: consistency across habitat near Cape Lookout, North Carolina. Fish. Bull. 83:671-677, Peterson. C. H.. P. B. Duncan, H. C. Summerson & G. W. Safrit Jr. 1983. A mark-recapture test of annual periodicity of internal growth band deposition in shells of hard clams. Mercenaria mercenaria. from a population along the southeastern United States. Fish. Bull. 81:765- 779. Pratt, D. M. & D. A. Campbell. 1956. Environmental factors affecting growth in Venus mercenaria. Limnol, Oceanogr. 1:2-17. Rhoads. D. C. & G. Pannella. r970. The use of molluscan shell growth patterns in ecology and paleoecology. Letluiia 3:143-161. Ricker, W. E. 1975. In: Computation and interpretation of biological sta- tistics of fish populations. Bulletin 191, Department of the Environ- ment. Fisheries and Marine Service. Ottawa. Canada. 382 pp. Robhins. B. D. & S. S. Bell. 20(10, Dynamics of a subtidal seagrass land- scape: seasonal and annual change in relation to water depth. Ecology 81:1193-1205. Scoftm, T. P. 1970. The trapping and binding of subtidal carbonate sedi- ments by manne vegetation m Bimini Lagoon, Bahamas. J. Sed. Parol. 40:249-273, Skilleter, G. A. & C. H. Peterson. 1994. Control of foraging behavior of individuals within an ecosystem context: the clam Macoma halthica and interactions between competition and siphon nipping. Oecolngia 100:268-278. Slattery. J. P.. R. C. Vrijenhoek & R. A. Lutz. 1991. Heterozygosity, growth and survival of the hard clam. Mercenaria mercenaria. in sea- grass vs. sand flat habitats. Mar Biol. 1 1 1:335-342. Spitzer. P. M.. K. L. Heck Jr. & J. Mattila. 2000. The effects of vegetation density on the relative growth rates of juvenile pinfish, Lxigodon rhom- hoides, in Big Lagoon. Florida. J. Exp. Mar. Biol. Ecol. 244:67-86. Tegner. M. J. & P. K. Dayton. 1981. Population structure, recruitment, and mortality of two sea urchins iStrogylucentralus franciscanus and 5. purpuralis) in a kelp forest near San Diego. California. Mar. Ecol. Prog. Ser. 5:255-268. Trevallion. A., R. R. C. Edwards & J. H. Steele. 1970. Dynamics of a benthic bivalve. In: J. H. Steele, editor. Marine food chains. Berkeley: University of California Press, pp. 285-295. Turner. E. J. & D. E. Miller. 1991. Behavior and growth of Mercenaria mercenaria during simulated storm events. Mar Biol. 111:55-64. Virnstein, R. W. 1977. The importance of predation by crabs and fishes on benthic infauna in Chesapeake Bay. Ecology 58:1199-1217. Walford. L. A. 1946. A new graphic method of describing the growth of animals. Biol. Bull. 90:141-147. Walker. R. L. 1985. Growth and optimum seeding time for the hard clam, Mercenaria mercenaria (L). in coastal Georgia. Nautilus 99:127-133. Wildish. D. & D. Knstmanson. 1997. Benthic suspension feeders and fiow. New York: Cambridge University Press. 423 pp. Wilson. D. M. 1991 . The effect of submerged vegetation on the growth and incidence of siphon nipping of the northern quahog {Mercenaria mer- cenaria) (Bivalvia). M.S. Thesis. University of Alabama. Tuscaloosa. AL. 56 pp. Wilson. K. A.. K. W. Able & K. L. Heck Jr. 1990. Predation rates on juvenile blue crabs in estuarine nursery habitats: Evidence for the im- portance of macroalgae (Ulva lactuca). Mar. Ecol. Prog. Ser. 58:243- 251. Zwarts. L. 1986. Burying depth of the benthic bivalve Scrohiciilaria plana (da Costa) in relation to siphon-cropping. J. Exp. Mar. Biol. Ecol. 101:25-39. Journal of Shellfish Research. Vol. 21. No. 2, 643-648. 2002. REPRODUCTION OF CALLISTA CHIONE L., 1758 (BIVALVIA: VENERIDAE) IN THE LITTORAL OF MALAGA (SOUTHERN SPAIN) C. TIRADO.' C. SALAS.'* AND J. I. LOPEZ" 'Deparlaniento Bi()loi>ia Animal. Faciihiul de Ciencias. Universidad de Malaga. E-29071- Malaga. Spain: 'Delegacion Provincial de Agricultura v Pesca. Avda, Aurora 47. E-29071- Malaga. Spain ABSTRACT The reproduclive cycle of Callisui chione Linnaeus. 1758 was studied using histology and changes in fiesh dry weight, in the littoral of Malaga (southern Spain), from June 1999 to May 2000. Histologic study of the gonads showed a long reproductive period, with spawning throughout the year. Three peaks of spawning have been observed through histology as well as flesh dry weight variation. The first one lasts from February to March and is accompanied by the highest decrease of weight; the second one is in spring, with the highest percentage of population in spawning but with a lower decrease of weight, and the third one is in summer. The latter peak represents a new activation of the gonads from postactive stages, without passing through a resting period. The absence of a resting period in the reproductive cycle of the studied population could be influenced by the mild seawater temperatures and high levels of chlorophyll a (attributable to the occurrence of upwellings) in the littoral of Malaga. The decrease of the average size in the population of Malaga from 1981 (70 mm) to 1999-2000 (54 mm length), points out an overexploitation of this resource. The above data, together with the removal of the close of season since 1990 in Malaga province, make it necessary to regulate a close of season in this area. We propose a close of season from February to March, months during which there was the most intense release of gametes in the population. A'£)' WORDS: Callisiu chione. histology, biomass, reproductive cycle, fishery INTRODUCTION Callista chione Linnaeus. 1758 is an Atlantic-Mediterranean species ranging in the Atlantic from the southwestern British Isles to Morocco, and also in the Canary, Madeira, and Azores islands (Tebble 1966, Poppe & Goto 1993). and in the Mediterranean. It lives in fine and clean sand, from low tide down to ISO m (Poppe & Goto 1993). Most of the studies on this species regard growth (Hall et al. 1974. Cano 1981, Forster 1981. Valli et al. 1983-1984, Strada & Zocco 1985, Valli et al, 1994). Because C. chione is a commercial species, some studies in Mediterranean waters were conducted in relation with the monitoring of infections by protozoans (Bravo et al, 1990, Canestri-Trotti et al. 1998. Canestri-Trotti et al. 2000) and heavy metals (Belmonte & Grasso 1986. Valli et al, 1994). Others were related with the influence of the dredge design on the size of the individuals captured and on the damage caused to the associated macrofauna (Gaspar et al. 1999). and with the fishery of this species in the Gulf of Trieste (Italy) (Del Piero 1994). Some few studies have addressed physiologic aspects. Charles et al. (1999) analyzed the selective utilization of bacteria and microalgae by C. chione. Cano ( 1983) analyzed different indexes of condition over a year in Malaga. Although C chione is a target-species of local fisheries in the whole Mediterranean area, there are few studies on reproduction. Valli et al, (1983-1984) presented preliminary data on the repro- duction of C. chione in the Gulf of Trieste (northern Adriatic) later completed in Valli et al. (1994). Nicotra and Zappata (1991) ana- lyzed the ultrastructure of the mature sperm and spermatogenesis from an Italian population. In southern Spain, C. chione. locally known as "concha fma," is a very popular shellfish with a considerable consumer market and an average of about 599 tons/year from 1985-1996. according to the data of regional authorities. Most of 90'7r of this amount was *Corresponding author: Tel,: 34-952-131857; Fax: 34-952-132000; E-mail: casanova@uma,es obtained and consumed in the littoral of Malaga, Moreover, there is an significant illegal market and a quantity of catches that could be of the same order of magnitude as the official catch, but is not taken into account by official statistics. The law in Andalucia (an autonomous region including eight southern provinces of Spain) rules that the fishing season for C. chione must be closed from 1 February to 30 September (order of Consejerfa de Agricultura y Pesca, November 12, 1984), However, the provincial delegations are authorized to change it within this interval; in the case of Malaga, the close of season has been removed since 1990. The latter decision has been justified by the absence of biologic studies on the reproductive cycle of the species in the area, together with the fact that Malaga is a tourist area, which implies greater demand for shellfish species during almost all year, pailicularly in summer. The absence of previous studies on the reproductive cycle of C. chione in southern Spain together with the overexploitation of this resource because of the absence of close of season, led the regional authorities of fishery (Consejerfa de Agricultura y Pesca) to pro- mote this research. This is part of a project on the reproductive cycles of the most important commercial bivalves of Andalucia supported by the Consejerfa de Agricultura y Pesca (Department of Fishery) and entrusted to D,A,P, enterprise (Tirado & de la Rua 2000), MATERIALS AND METHODS A total of 3,882 specimens of C. chione were examined and measured for shell length, ranging from 23-89 mm. The samples were collected from June 1999 to May 2000, with monthly fre- quency from October to February and with fortnightly frequency in the other months. The specimens were captured using a dredge with a toothed aperture, teeth length of 26 cm, and 6.7 cm of mesh, usual among the fishermen of the area. The samples were taken in Fuengirola (36°28'N, 4''43'W) (Fig. 1), at 20 m depth, in a sandy bottom. To evaluate the possible influence of environmental factors on the cycle, the temperature of the seawater at 20 m depth was measured. Samples of water (2 L) were taken from the bottom for 643 644 TiRADO ET AL. 36° 30' 4° 45" Figure 1. Saniplinj; area. determination of chlorophyll a. Pigment analyses were carried out by filtering the water through Whatman GF/C glass filters. The pigments of the retained cells were then extracted with acetone for twelve hours in cool, dark conditions, following the recommenda- tions of Lorenzen and Jeffrey (1980). Concentrations of chloro- phyll a were calculated using the trichromatic equations of Jeffrey and Humphrey (1973). A total of 3.371 specimens were used for the analysis of tlesh dry weight variation (about 200 specimens/sample). The length of every specimen was measured, and the soft parts were then pulled out of the shell, placed in the drying stove at 100°C for 24 h, and weighed to the nearest milligram. Two different indexes of con- dition were applied, the flesh dry weight/L' variation, and thai proposed by Crosby and Gale (1990) Condition Index (CI) flesh dry weight x 1.000/volume of the internal cavity of the shell. The regression of flesh dry weight on the length was calculated for each sample to estimate the variation in biomass of a standard individual, based on the logarithmic transformation of Ricker's function W = aL'' (Ricker 1975), where W is the weight. L is the length, a is the ordinate at origin, and b is the slope. The histologic study was performed on 51 1 specimens (usually 30 per sample), with shell length ranging between 23-85 mm. For the histologic processing, specimens were anesthetized with MgCK, fixed in 10% formaldehyde, embedded in paraffin, sec- tioned at 10 |jLm. and stained with hematoxylin of Carazzi and eosin, and a trichromic staining (V.O.F according to Gutierrez 1967) of hematoxylin of Carazzi. light green, orange G. and acid fuchsine. The stages of gonad development were scored according to the scale proposed by De Villiers (1975) for Donax serra Roding 1798 in South Africa: cytolized, preactive, active, spawn- ing, and postactive |the equivalent stages from Seed (1969) and Boyden ( 1971 ) are provided in Table 1 ). The test of Kolmogorov-Smimov and Kendal and Pearson's rank correlations included in the program SPSS 8.0. were used to check the distribution of the data. Cross correlation between both contlition indexes and percentage of spawning with seawater tem- perature and chlorophyll a levels were calculated to assess the influence of the environmental factors on the reproductive cycle. RESULTS Sex Ratio The sex of the specimens of C. cluone cannot be distinguished macroscopically by the color of the gonads. Therefore, sex deter- mination must be made microscopically. A total of 51 1 specimens were microscopically examined, but it was impossible to deter- mine the sex of some individuals in several months. These samples were not considered for the sex ratio estimation. From the remain- TABLE 1. Different scales of gonad condition. Authors De Villiers (197.';) Seed (1969) Boyden (1971) Cytolized Resting G. (0) Indeterminated G (I) Scales of Gonad (G) Condition Preactive Developing G ( 1.2.3) Developing G. Active Spawning Ripe G. (5) Ripe G (ill) Postactive Spawning G. (4.3) Spawning (IV) Spawning G (2.1) Resting G. (V) gonad. Reproduction of Calusta ch/one Linnaeus. 1758 645 ing 272 individuals. 125 (45.96%) were males and 147 (54.04 7p) females. The sex ratio for all them can be considered as 1:1 (x~ = 0.012. P>0.95) (Fig. 2). Sexual Cycle Biomass Analysis The variation of flesh dry weight/L' ratio during the annual cycle is shown in Figure 3. The mean values of both variables. flesh dry weight and size (L'), were considered. The standard deviations were between 10.59^8.32%. A broad size range (Fig. 4) can be observed in the samples, which explains, in part, these differences. Also, a broad weight range can be observed in most of the samples (Fig. 5), which is related, in part, with the presence of different stages of development of the gonads. From June to October. C. chione shows two decreases of the above ratio (Fig. 3). with a small increase in August. During the autumn months, there is another increase, followed by a decrease from January to March. During the spring, there is a continuous slight increase, before the first spring decrease. The other index (CI) shows less pronounced increases and de- creases (Fig. 6). Two decreases can be detected, one of them from late August to December and the other one from January to March. To minimize the bias introduced by the somatic growth of individuals during the cycle and by the variation in the size of the specimens between successive samples, the variation of flesh dry weight was estimated for a standard individual of 54-mni length, taking into account the regression lines for every sample (Table 2). This size is close to the mean size of the population. Figure 7 shows a major decrease in the flesh dry weight from January to March, although there are also drops from June to September. It is interesting to observe the difference in flesh dry weight between the first half of June 1999 and second half of May 2000. which could indicate that the sexual cycles are out of phase between contiguous years. In autumn, the values remain more or less stable. The two major increments of the flesh dry weight of the standard individual were registered between March and April and between the two samples of June. Ganietogenic Cycle Data from the histologic study are presented in Table 3. which shows the total number of specimens analyzed and their develop- ment stages, according to the scale of De Villiers (1975). The studied population of C. chione shows continuous spawning throughout the year, with values higher than 30% of the population in 12 of the 17 samples examined. The whole population was spawning in May, while the lowest percentage of spawning (30% of the sample) was registered in the first half of September (Fig. 8). If we considered the absence of any individual in cytolized or % 100 Ag Sp Oc Dc Fb Ap My Figure 3. Flesh dry weight (FDW)/Lenglh (I/) ratio throughout the year of study. Bars sho» standard deviation. postactive stage, the main period of sexual activity lasts from February to June. In February, the presence of active individuals points to the beginning of the active period (Table 3). There is not, however, a true resting period because of the existence of spawn- ing in the population at any time of the year. The regression of the gonads begins in June and July, with the occurrence of individuals in postactive stage, which were predominant from the second half of September to December, together with the presence of some individuals in cytolized stage. The ganietogenic cycle is asynchrononic in the population, which is evidenced by the presence of at least two developmental stages in nearly all the samples. An asynchrony is also detected in the individuals attributable to the coexistence of areas with differ- ent stages in the same gonad. Several cohorts of ovocytes can be detected throughout the year, together with the direct step from postactive to active stage, without a previous cytolized and preactive phases. F^nvironmental Factors The maximum temperature (Fig. 9) was registered in the sec- ond half of August (22'C) and the minimum (13.8°C) in January. The maximum of chlorophyll a (Fig. 9l levels occurs in the first half of August 2000 and the second peak in the first half of May 1999. Between these extremes, we observed several minor peaks, at the end of summer (first half of September), in autumn (Octo- ber), and at the end of winter (March). Coefficients of correlation of Pearson were estimated between temperature and FDW/L^ and temperature and CI, because the variables showed a normal distribution (according to the test of Kolmogorov-Smimov). However, between percentage of popula- tion in spawning and temperature, chlorophyll a levels, and the different condition indexes, the coefficients of correlation of Ken- Jn Jn Jl Fb Mr Ap Ap My My Figure 2. Relative frequency (% ) of sexes during the year of study. Ag Sp Ap My Figure 4. Monthly average length (L) throughout the year of study. Bars show standard de\iation. 646 TiRADO ET AL. M 7000 ^ 6000 a 5000 4000 TABLE 2. Linear regression calculated for each month. Figure 5. Monthly average flesh dry weight (FDVV) throughout the year of study. Bars show standard deviation. dal were estimated, because the percentage of spawning dataset showed a non-normal distribution. These coefficients of correlation have been calculated simulta- neously and with one and two months of delay. The temperature was directly correlated, with one month of delay, with CI (r = 0.629. P < 0.05). In the same way, the temperature was also directly correlated, with a delay of two months, with the FDW/L' of the population (r = 0.573. P < 0.05). A significant coefficient of Kendal's correlation has been obtained between the temperatuie and the tlesh dry weight of the standard individual, with a month of delay (tau = 0.394, P < 0.05). No other correlation was sig- nificant. DISCUSSION Sex Rath) Callista chione is a species that does not show sexual dimor- phism, and the sex ratio is 1:1. Our data regarding the sex ratio are similar to those of Valli et al. (1994) in the Gulf of Trieste, who obtained percentages of 46.25% males and 53.759r females. Also, we have not found any hermaphrodites in the studied population. Reproductive Cycle According to the histologic data. C. chione from the littoral of Malaga had individuals in spawning stage throughout the year (Fig. 8), while in the Gulf of Trieste (northern Adriactic) the spawning extends from February to September (Valli et al. 1983- 1984). or from March to September (Valli et al. 1994). This dif- ference is easily explained by the much lower winter seawater temperatures (in the order of 6°C) registered in the Northern Adri- atic. However, the high amount of phytoplankton and zooplankton of the Gulf of Trieste resulted in the absence of a resting period, because the individuals remain in a preactive stage during autumn and winter months (Valli et al. 1994J. In Malaga, most of the W (L = 54 Months Lni Regression ^ines R- R n mm) June 62.93 Y = 2.927 X -1.774 0.967 0.984 128 1983.59 June 66.20 V = 3.017 X -1.837 0.908 0.953 94 2445.84 July 59.23 V = 3.218 X -2.204 0.935 0.967 197 2346.59 July 50.40 V = 3.205 X -2.192 0.958 0.979 200 2286.74 Aug 51.10 V = 3.297 X -2.371 0.969 0.985 202 2194.50 Aug 52,94 V = 3.294 X -2.358 0.966 0.983 200 2229.42 Sept 51.57 y = 3.261 X -2.363 0.958 0.979 199 1934.74 Sept 49.49 V = 3.345 X -2.510 0.945 0.972 201 1928.17 Oct 48.90 Y = 3.533 X -2.837 0.952 0.976 200 1917.97 Nov 56.56 Y = 3.423 X -2.645 0.974 0.987 152 1930.79 Dec 51.30 Y = 3.524 X -2.820 0.979 0,990 200 1928.28 Jan 62.00 V = 3.246 X -2.309 0.900 0.949 203 2057.40 Feb 51.75 Y = 3.115 X -2.150 0.998 0.999 198 1 760.88 Mar 47.30 Y = 2.265 X -0.762 0.511 0.715 200 1451.32 Apr 55.04 Y = 3.449 X -2.675 0.938 0.968 199 1997.13 Apr 53.06 Y = 3.168 X -2.141 0.919 0.959 200 1982.24 Mav 56.38 V = 3.137 X -2.124 0.972 0.986 199 2045.41 May 53.30 y = 3.335 X -2.451 0.972 0.986 200 2123.80 LM = average length; R- = coefficient of determination; R = coetTicient of correlation; /I = number of observations; W (L = 54 mm) = weight of a standard individual of 54-mm long. individuals remained in postactive and spawning stages during the autumn and winter months (Fig. 8). Cano ( 1983) studied different condition index in C. chione from the littoral of Malaga, detecting only one strong decrease of dry weight (between January and February of 1981). Other species from southern Spain, such as D. truncuhis. D. veniistus. and D. semislriatus, show very long spawning periods, but in these species, there was at least one month without spawning (Tirado & Salas 1998. Tirado & Salas 1999). The mild seawater temperature (between 13.8°C-23°C) and the high concentration of phytoplankton (because of the presence of upwellings) in the littoral of Malaga (Fig. 9) would favor, prob- ably, a long reproductive cycle and the absence of a resting period. The temperature is the most influential factor, according to the coefficients of correlation. It is correlated with weight increments, a consequence of development of the gonads. According to the hypotheses of most authors, the gamete re- lease seems to be controlled by such physical environmental vari- ables as changes in temperature, salinity, or photoperiod (Sastry 1979 and references therein). An alternative hypothesis is that phytoplankton induces spawning (Ruiz et al. 1992). Starr et al. 220 200-1 180 160 140 120 100- 80- 60 40 Jn Jl Ag Sp Oc Dc Fb Ap My Figure 6. Index of condition of Crosby & Gale (CI I: tlesh dry weight X 1,(H)()/ volume of the internal cavity of the shell, throughout the year Figure 7. Variations in Hesh dry weight in a standard of study. Ap My Callista chione animal 54-mm long. Reproduction of Callista chione Linnaeus. 1758 647 TABLE 3. Developmental stages of the gonad over the year. Months Pr EA Ps June 1 11 20 32 July 1 6 24 30 July 2 I 4 9 1 13 1 29 Aug 1 6 3 9 1 10 30 Aug 2 9 1 II 9 30 Sept 1 4 1 14 11 30 Sept 2 4 4 1") 30 Oct 3 6 21 30 Nov 7 5 18 30 Dec I 9 20 30 Jan 1 14 15 30 Feb 4 1 19 30 Mar 2 3 4 19 2 30 Apr 1 1 29 30 Apr 2 3 27 30 May 1 30 30 May 2 1 29 30 C = eytolized; Pr = preactive; EA = early active; A = active; S = spawning; Ps = postactive; n = number of observations. (1990) showed that blooms of phytoplankton should be sufficient to induce spawning in tnussels as well as in urchins. The spawning of Crassostrea gigas in El Grove (Galicia, northwestern Spain), with water temperature below 18°C. was correlated with phy- toplankton bloom (Ruiz et al. 1992). Although the chlorophyll a levels in the littoral of Malaga during this study did not show significant correlation with any analyzed index or variable, the coincidence of some decreases in weight (Figs. 3. 6) with peaks of chlorophyll a (Fig. 9). seem to indicate some influence. The data of biomass showed three important decreases: be- tween January-March, second half of August-September, and June and July (Figs. 3. 7). The latter was not reflected in CI (Fig. 6). The first drop is coincident with the start of activation of the gonad and with the increase of percentage of population in spawn- ing (Fig. 8). At the first half of summer, there are high percentages of individuals in postactive stage, which begin a new gonadal activation in the second half of July. This direct step from post- active to active stages without a previous eytolized phase, has been found in Donax tnincidus (L. 1758) from the littoral of Malaga (Tirado & Salas 1998) and seem to be related with the presence of peaks of chlorophyll a in this area (Fig. 9). Ansell (1961) reports DC BPrilEASA HS SPs % 100 Figure 8. Relative frequency of different stages of development of the gonads in C. chione. Figure 9. Seawater temperatures and changes in concentration of chlorophyll a in seawater throughout the year of study. that in the population otChamclea striatiila from Kame Bay (Mill- port), the ovary passes directly from the spawning condition to an early stage of development, because of the development of young ovocytes in the ovary before the end of spawning. Also, according to the data for the standard individual (Fig. 7). the main decrease of flesh dry weight was registered in winter; whereas, those of summer seem to be less intense. Although the percentage of the population spawning in spring was the highest of the year (Fig. 8). the resulting decrease of biomass was less than in winter or summer ( Fig. 3 1. This is consequence of the fact that the emissions from winter were more intense, with implication of the entire gonad; whereas, in spring and summer, there were only partial spawnings. The latter, together with the coexistence of dif- ferent stages of development in the same gonad, could indicate that the spring/summer spawning is at least the second one for an individual during the cycle. In the field, it is difficult to know if a particular individual has more than two spawnings by reproductive cycle. In the laboratory, the venerid Chamelea striatula spawns repeatedly at intervals throughout the spawning season (Ansell 1961). In the littoral of Malaga, two spawning periods per indi- vidual were detected in D. mmculus: whereas, in other such spe- cies as Donax vemistus and Donax semistiiatus, only one spawn- ing per individual and cycle was observed (Tirado & Salas 1998. Tirado & Salas 1999). The asynchronic gametogenic cycle in the population is re- flected by the high standard deviations of the flesh dry weights (Fig. 5), the existence of several cohorts of ovocytes, and the coexistence of several stages of development in the same gonad. The coexistence of different stages has been found in many bi- valves from temperate areas, among them, Chamelea striaiiila (Ansell 1961), Donax serra (De Villiers 1975), Tapes rhomboides (Morvan & Ansell 1988), D. mmculus (Tirado & Salas 1998). D. venustus and D. semistriatus (Tirado & Salas 1999). Although the total of captures of C. chione in the littoral of Andalucia is high (about an average of 599 tons/year from 1985- 1996). in the last few years (from which statistical data have been published) this volume has decreased to values of about 188 tons in 1995. or about 259 tons in 1996. On the other hand, the average length of the Malaga population has decreased from about 70 mm in 1979-1981 (Cano 1981) to 54 mm in 2000 (present study). All these points indicate an overexploitation of this resource. More- over, the recorded data on growth indicate a slow growth of C. chione. Hall et al. ( 1974) registered a growth of 2 mm by year in the Gulf of Trieste; Forster ( 1981 ) suggested that a specimen of Plymouth reaches 9 cm in 40 y. The above data make it an urgent necessity to provide a close of season that, according to the data of this study, must be February-March period during which there was the most intense release of gametes in the studied population. 648 TiRADO ET AL. ACKNOWLEDGMENTS The aullidis thank David Lope/, Daniel Gome/, and M" Jose Garci'a-Patino for helping in the laboratory process. This study is part of a project supported by the Junta de Andalucia, (Department of Fishery). M" Dolores Atienza. general manager of the Department of Fishery is thanked for her trust in our work and the permission to publish. We are grateful to Manuel Castanon (Provincial Manager of Fishery) for his encourage- ment in this research. The project was entrusted to D.A.P. enterprise, which is thanked for the use of facilities given for realization of the work and publication of the results. We are grateful to Ildefonso Marquez and Manuel Aguilar for their management and the facilities given for the development of this research. Eva Garcia is thanked for her help with the refer- ences. LITERATURE CITED Ansell, A. D. I%1. Reproduction, growth, and niorlaiity of Vfiuis striuluhi (DaCosia) in Kame Bay. Millpon.7. Mar. Biol. .As.s. U.K. 41:191-21.s. Belmonte, G. & M. Grasso. 1986. Dati preliminari siilla presenza di metalli pesanti in alcuni bivalvi presenti a Porto Cesareo (Mar Jonio). Tluila.s- ,v/ii Salem. 16:59-63. Hoyden, C. R. 1971 . A comparative study of the reproductive cycles of the cockles Cerastoderma edule and C. ^Icnicum. J. Mar. Biol. Ass. U.K. 51:605-622. Bravo, I., B. Reguera, A. Martinez & S. Fraga. 1990. First report of Gymnoclinium calcnutiiin Graham, on the Spanish Mediterranean coast. In: E. Graneli, B. Sundstroem, L. Edler & D. M. Anderson, editors. To.xic marine phytoplankton . Elsevier Science, New York. pp. 449- 452. Canestri-Trolti, G.. E. M. Baccarani & F. Paesanti. 1998. Gregarines del genere Neniatopsis (Apicomplexa: Porosporidae ) in Molluschi bivalvi del mare Adriatico. Boll. Soc. Ital. Patol. Itlica IO(23):58-66. Canestri-Trotti, G.. E. M. Baccarini, F. Paesanti & E. Turolla. 2000. Moni- toring of infections by protozoa of the genus Nematopsis, Perkinsus and Porospora in the smooth venus clam Callista chione from the northwestern Adriatic Sea (Italy). Dis. Aqiiat. Org. 42(2):157-16l. Cano, J. 1981. Biologia y crecimiento de Callista chione IL. 1758). Iberus. 1:67-78. Cano. J. 198.V Indices de condicion, humedad. y cenizas en Callista chione (L. 1758), Veneriipis rhomhoides (Pennant 1777) y Cerastoderma tu- berciilatmn (L. 1758). Iberus. 3:29-39. Charles, F., J. M. Amouroux & A. Gremare. 1999. Comparative study of the utilization of bacteria and microalgae by the suspension-feeding bivalve Callista chione. J. Mar. Biol. Assoc. U.K. 79(4):577-584, Crosby, M. P. & L. Gale. 1990. A review and evaluation of bivalve con- dition index methodologies with a suggested standard method. J. Shell- fish Res. 9(l):233-237. De Villiers, G. 1975. Reproduction of the sand mussel Donax serra Roding. Invest. Report Sea Fish. Brch. Repub. S. Afr. 109:1-31. Del Piero. D. 1994. The clam fishery in the Gulf of Trieste. Coastal Zone '94. Cooperation in the Coastal Zone. Conference Proceedinii 4: 1645- 1660. Forster, G. R. 1981. The age and growth of Callista chione. J. Mar. Biol. Ass. U.K. 61:881-883. Gaspar, M., L. Chicharo, M. D. Diaz, P. Fonseca, A. Campos, M. N. Santos & A. Chicharo. 1999. The intluence of dredge design on the catch of Callista chione. J. ShellJ. Res. 18(2):717. Gutierrez, M. 1967. Coloracion histologica para el ovario de peces, crusta- ceos, y moluscos. Inv. Pescj. 31(2):265-271. Hall, C, A., Jr., W. A. Dollase & C. E. Corbato. 1974. Shell growth in Tivela stulturmn (Mawe, 1823) and Callista chione (L, 1758) (Bi- valvis): annual perioricity, latitudinal differences and diminution with age. Paleogr. Paleoclimatol. Palaeoecol. 15:33-61. Jeffrey, S. W. & G. T. Humphrey. 1975. New .spectrophotometric equation for determining chlorophylls a. b. c\ and c' in higher plants, algae, and natural phytoplankton. Biochein. Physiol. Pflanz. 167:191-194. Loien/en, C. J. & S. W. Jeffrey. 1980. Determination of chlorophyll in seawater, UNESCO. Tech. Pap. Mar. Sci 35:1-20. Morvan, C. & A. D. Ansell. 1988. Stereological methods applied to re- productive cycle of Tapes rhomhoides. Mar. Biol. 97:355-364. Nicotra, A. & S. Zappata. 1991. Ultrastructure of the mature sperm and spermiogenesis in Callista chione (Mollusca. Bivalvia), Invertehr. Re- prod. Dev. 20(3):213-218. Poppe, G. T. & Y. Goto. 1993. European seashells. vol. 2. Scaphopoda. Bivalvia, Cephalopoda. Wiesbaden: C. Hemmen. 221 pp. Ricker, W. 1975. Computation and interpretation of biological statistics of fish population. Bull. Fish. Res. Board Can. 191:1-382. Ruiz, C, M. Abad, F. Sedano, L. O. Garcia-Manin & J. L. Sanchez Lopez. 1992. Influence of seasonal environmental changes on the gamete pro- duction and biochemical composition of Crassostrea gigas (Thunberg) in suspended culture in El Grove, Galicia, Spain. J. E.xp. Mar. Biol. Ecol. 155:249-262. Sastry, A. N. 1979. Pelecypoda (excluding ostreidae). In: A. C. Giese & J. S, Pearse, editors. Reproduction of marine invertebrates, volume V. mollusc: pelecypods and lesser classes. New York: Academic Press, pp. 1L3-292. Seed, R. 1969. The ecology of Mytilus edulis L. (Laraellibranchiata) on exposed rocky shores. I. Breeding and settlement. Oecologia. 3:277- 316. Starr. M., J. H. Himmelman & J. C. Therriault. 1490. Direct coupling of marine invertebrate spawning with phytoplankton blooms. Science. 247:1071-1074. Strada, R. & M. Zocco. 1985. Dati preliminari suU'accrescimento di Cal- lista chnme in Adriatico settentrionale. Oebalia. 1 1(2):829-831. Tebble, N. 1966. British bivalve seashells. Trustees of the British Museum (Natural History). 212 pp. Tirado, C, & C. Salas. 1998. Reproduction and fecundity of Dona.x tnm- culus L. 1758 (Bivalvia: Donacidae) in the littoral of Malaga (southern Spain). J. Shellfish Res. 17( 1):169-176. Tirado, C. & C. Salas. 1999. Reproduction of Doita.x venu.ytns Poli 1795, Dona.x semistriatns Poli 1795. and intermediate morphotypes (Bi- valvia: Donacidae) in the littoral of Malaga (southern Spain). P. S.Z.N. Mar. Ecol.. 20(2): 1 1 1-130. Tirado. C. & A. Rodriguez de la Riia. 2000. Estudio del ciclo reproductor de los moluscos bivalvos y gasteropodos del liloral andaluz. Informe Tecnico. Conseierfa de Agricultura y Pesca, Junta de Andaluci'a. 357 PP- Valli. G., E. Bidoli & C. Marussi. 1983-1984. Osservazioni preliminari sulla riproduzione e sulla biometria di Callista chione (L.) (Mollusca, Bivalvia) del Golfo di Tneste. Nova Thalassia. 6:97-103. Valli, G., N. Marsich & M. G. Marsich. 1994. Riproduzione, biometria, e contenuto di metalli in Callista chione (L.) (Mollusca, Bivalvia) del Golfo di Trieste nel corso di un ciclo annuale. Boll. Soc. Adriat. Sci.. 75(2):441-I64. Joiinwl ofShellthh Reseanh. Vol. 21, No. 2, 649-658. 2002. INGESTION, DIGESTION, AND ASSIMILATION OF GELATIN-ACACIA MICROCAPSULES INCORPORATING DEUTERIUM-LABELED ARACHIDONIC ACID BY LARVAE OF THE CLAM VENERUPIS PULLASTRA S. NOVOA,' D. MARTINEZ,' J. OJEA,' P. SOUDANT," J.-F. SAMAIN,' J. MOAL,' AND J.-L. RODRIGUEZ^ * ^Centra de Ciiltivos Marinas de Rihcideo. CIMA. Conselleria de Pesca, Marisqueo y Acuicultiira. Miielle de Ponilldn s/n 27700 Rihcideo, Spcuii: -(Jniversite de Bretague Occidentcde. lUEM. UMR 6539 LEMAR, Teclmopole Brest-Iroise. Place Nicolas Copernic, 29280 Plouzane. France: DRV/A. Laboratoire LPI. IFREMER Centre de Brest. BP 70. 29280 Plouzane. France: and '^Departmento de Bioquinucu y Biol. MoL, Facultad de Veterinaria. campus univ. s/n, 27002 Lugo, Spain ABSTRACT Olive oil gelatin-acacia microcap.sules (GAMs) enriched with deuterium-labeled arachidonic acid (*AA) were prepared and fed to Veiienipis pullaslra larvae. In a first experiment, larvae were either starved or ted these microcapsules (*AA-GAM) for 12 h. The *AA-GAM-fed larvae incorporated *AA and oleic acid (OA) in their neutral and polar lipids. The incorporation yield was around 5% for *AA in neutral and polar lipids and 2% and \'7c for OA in neutral and polar lipids, respectively. In a second experiment, larvae were fed for 7 days with a mixed algal diet supplemented with or without *AA-GAM. The microcapsules were ingested and digested without any harmful effect on larvae. OA was incorporated preferentially in neutral lipids whereas *AA was equally distributed in neutral and polar lipids. The incorporation yield was higher in the second experiment probably in relation to the presence of algae. However, the incorporation rate of both tracer fatty acids decreased with time in both lipid classes. *AA dropped form 16-2% in both neutral and polar lipids and OA decreased more rapidly in polar (16-3%) than in neutral lipids (33-15%). These changes in the incorporation yield could correspond to the growth slowing down at the approach of the metamorphosis or to a saturation in the levels of fatty acids because the percentage of natural arachidonic acid stayed stable in neutral lipids or tended to decrease in polar lipids. The easy fabrication and use of GAM associated to the direct measurement of a deuterated fatty acid by gas chromatography is a promising tool for studying lipid metabolism in mollusks. KEY WORDS: clam, Venerupis piilliisiru, nutrition, microcapsules, larva, fatty acids INTRODUCTION The production of living microalgae (LMA) as food for the larvae and spat of bivalves in commercial hatcheries accounts for approximately 30% of operating costs (Coutteau & Sorgeloos 1992). Because of the high cost and unpredictability of the algal culture, the development of artificial diets for bivalve mollusks was attempted by several investigators (Jones et al, 1974). Micro- capsules of the cross-linked protein-walled type were used to ex- amine aspects of protein (Langdon & Siegfried 1984) and carbo- hydrate (Kreeger et al, 1996) metabolism of bivalves and gelatin- acacia microcapsules were investigated in lipid nutrition (Langdon & Waldock 1981, Chu et al. 1987, Numaguchi & Nell 1991, Knauer & Southgate 1997a, 1997b), Some potential alternatives, such as dried microalgae, microalgal pastes, lipid emulsions, and microcapsulated or yeast-based artificial diets (for revision, see Coutteau & Sorgeloos 1993; Robert & Trintignac 1997), have shown promising results. The total or partial substitution of mi- croalgae by artificial particles has been proposed, where, ideally, nutritional requirements of bivalves should be satisfied using diets whose composition could be precisely controlled. The gelatin- acacia microcapsules (GAMs) are not a suitable vehicle to deliver complete artificial diets to the larvae of bivalves because only water-insoluble nutrients can be encapsulated using this method. However, they may be a useful tool in studies addressing aspects of the lipid nutrition in addition to being a suppleinent for mixed microalgal diets poor in some essential fatty acids, GAM could be a useful supplement if sufficient quantities of LMA were unavail- *Corresponding author. E-mail address: jrodritsiugo. usees able to feed spat (Numaguchi & Nell 1991), GAMs are simple to produce, relatively easy to use. and can be prepared rapidly on demand. However, the potential of GAM as a substitute for LMA under large-scale culture conditions must still be assessed (Knauer & Southgate 1997a). A number of studies have been undertaken to prove GAM ingestion, digestion, and assimilation and have shown that GAM were readily digested by bivalves (Chu et al, 1982, Southgate 1988) and that the lipids supplied were assimilated with high efficiency (Knauer & Southgate 1997c). The data generated in such studies will facilitate the further development of suitable artificial diets for the larvae of marine bivalves. The knowledge of the nutiitional requirements of the larvae of cultured bivalves is necessary to improve the efficiency of algal diets used in hatch- eries and to design convenient artificial diets. Previous studies pointed out the energetic role of lipids during the larval develop- ment of mollusks (Holland 1978), as well as the changes in the composition of the polyunsaturated fatty acids (PUFAs) occurring in neutral and polar lipids during embryogenesis. This nutritional transition suggests that a metabolic control takes place in devel- oping larvae (Marty et al. 1992). It is probable that this control results from a selective incorporation of dietary fatty acids by acyltransferases, indicating a preferential incorporation of long- chain PUFA, especially the 22:6(n-3) in the case of the larval development of Pecten maximus (Marty et al. 1992). This study was proposed as an attempt to confirm the assimilation of the fatty acids incorporated in GAM and their bioconversion in endogenous fatty acids of neutral and polar lipids in the larvae of marine bivalves. The data generated could indicate whether GAM could serve as a tool in studies of lipid metabolism in marine bivalves as well as demonstrate their use as a nutritional supplement in mixed microalgal-microcapsulated diets. 649 650 NOVOA ET AL. MATERIALS AND METHODS Chemicals Lipid Standards and Reagents Deuterium-labeled arachidonic acid (FA-503 arachidonic acid- 5,6,8,9, 11, 12. 14. 15-ds)wa.s obtained from BIOMOL Research Laboratory. Inc. (Plymouth Meeting. PA). Identification and quan- tification were based on standard fatty acid methyl ester mixture (Sigma-Aldrich. Chemical Spain). Internal standard, Tricosanoic acid (C2.'':0). was purchased from Nu-Chek-Prep. Butylated hy- droxytoluene (BHT) and fluorescein isothiocyanate (FITC) were supplied by Sigma Chemical Co. (St. Louis. MO) and sodium hydrogen bicarbonate was from Merck (Damistadt. Germany). Solvents Hexane. chloroform, and methanol were high-performance liq- uid chromatography (HPLC) grade from Merck (Darmstadt. Ger- many). Boron trifluoride (10%. w/w) in inethanol (BF,) was ob- tained from Supelco (Bellefonte. PA). Fabrication of G A Ms Two different sets of GAM were fabricated following a modi- fied method described by Rodriguez et al. (1992). HTC-GAM GAMs were prepared with 500 (jlL of cod liver oil and by later addition of FITC. Briefly. 500 (xL of cod liver oil was homog- enized with antioxidant BHT (5%. w/v). Then. 500 mL of cod liver oil plus antioxidant was emulsified with a 1:1 mixture of a 2% (w/v) solution of gelatin and a 2% (w/v) solution of acacia, which had been made up separately in distilled water. The emulsion was stirred in a Virtis Tempest IQ" blade homogenator programmed at .30.000 rpm for 2 min The pH of the mixture was reduced until the coacervation pH (4.3). by the drop-by-drop addition of dilute HCl. The mixture was stirred for 40 min and pH was then raised to 9.3 by the addition of dilute NaOH. The resulting GAM suspension Figure L Larva of V. piillastra observed under fluorescence micros- copy. Accumulation of yellow lluorescence indicates ingestion of mi- crocapsules. (Author: .I.-L. Rodriguez) Figure 2. Digestion of microcapsules throughout a period of 20 min. Sequence A-C shows the disappearance of the yellow fluorescence inside a larva of V. piillaslra. (.Author: J.-L. Rodriguez) was poured into 500 mL of cold distilled water and kept in a refrigerator for 2 h. Previously a .solution of FITC 0.25% (w/v) in 100 mM sodium hydrogen bicarbonate (NaHCO,) was prepared. Two milliliters of stock microcapsules were incubated at 4"C for 12 h with 0.5 mL of FITC solution diluted with 2.5 inL of 100 mM NaHCO, solution. The mean diameter of FITC-GAM was 4.0 ± 1 .0 (Jim (n = 100). Two milliliters of FITC-GAM was used to feed a batch of 23-day-old Venenipis piillastra larvae and subsequently were observed under fluorescent microscopy. Ingestion, Digestion, and Assimilation of GAM 651 340 320 |300 =5 260 240 - 220 200 J 100 ^ 60 I d 20 - □ AB2 Feeding algal laivae -■- AB2 Feeding 'AA -GAM + algal larvae Q AB2 Feeding algal laivae ■ AB2 Feeding "AA -GAM + algal larvae. 9$°„ 96°; 16 20 22 23 25 Figure i. Shell length (fim) of the AB2 batch, fed with algal (open squares) and *AA-GAM + algal (solid squares). Survival rate (%) of the same batch fed with algal (open bars) and with *AA-GAM -h algal (solid bars). Results are expressed in (im (%); Mean ±. SD, n = 100. Deuterated Arachidonic Acid (*AA)-GAM GAMs were prepared with olive oil supplemented with deute- rium-labeled arachidonic acid. Briefly. 30 (xL of olive oil was homogenized with 5 mg of deuterium-labeled arachidonic acid and antioxidant BHT (5%. w/v). Deuterated *AA-GAM was obtained following the method previously described. The resulting GAM suspen.sion was poured into 250 mL of cold distilled water and held in a refrigerator for 2 h. The mean diameter of *AA-GAM was 3.5 + 0.9 ixm (;? = 100) and the stock suspension of GAM was kept at 4°C and shaken daily. Feeding Experiments A population of V. piillastra D-larvae termed AB2 was selected by its good growth and survival rates and distributed in 500-L tanks at a density of 5 larvae-mL"'. Larvae were fed daily with a microalgal mixture of Pavlova lutheri (Droop). Isochnsis aff. gal- bana Green (clone T-iso; Tahiti Isochiysis). Skeletoneina coslaliim (Greville), and Chaetoceros calcitrans (Tanako) at a density of 15/15/15/15 cells-M-L-'. Experiment 1 Larvae 14 days of age from the AB2 culture were collected and transferred at the same density into two 6-L flasks for a 24-h experiment. One batch was starved whereas the other was fed with 60 |xcap-|a.r' *AA-GAM. *AA-GAM-fed larvae had seawater renewed 12 h after feeding, and no food was supplied then. Twelve hours later, both fed and starved larvae were collected on a 45-(xni mesh. Experiment 2 Larvae 17 days of age from the same AB2 culture were dis- tributed in two 150-L tanks at a density of 4 larvaemL"' and fed daily with two different diets for 7 days, either a microalgal diet of P. lutheri. I. aff. galhana. S. costatum, C. calcitrans. and Tetra- selmis suecica (Butcher) at a density of 20/20/15/15/10 cellspiL"' or a diet consisting of the same microalgal mixture supplemented with 5 |jLcap-|jiL"' *AA-GAM. Samples for Fatty Acid Analyses Larvae from the 24-h experience were collected as indicated above. Larvae from the 7-day experience that were 18. 19. 21. and 24 days of age were collected 4 h after feeding on a 45-(j.m mesh ('( = 1). All larval samples were crushed and stored in a CHCl,-MeOH mixture (2:1. v/v) under nitrogen at -30°C until fatty acid analysis could be pert'ormed. Samples of microalgal mixture (n = 3) and microcapsules {ii = 3) were filtered on GF/F (Whatman) glass fiber filters and stored similar to larvae before analysis. Fatty Acid Analysis The separation of the polar and neutral lipids was performed by micro-column liquid chromatography as described by Marty et al. ( 1992). Total lipids were evaporated to dryness and dissolved three times using 500 |xL of chloroform/Tnethanol (98:2). Neutral and polar lipids were separated on a silica gel 6% (w/w) hydrated microcolumn (30 x 5 mm) using chloroform/methanol (98:2) and methanol successively as eluting solvents. The fractions were col- lected under nitrogen in screw-capped flasks containing a known amount of 23:0 as internal standard for quantitative determina- tions. Fatty acid composition and quantification of polar and neu- tral lipids were determined using gas chromatography (CG). after purification of fatty acid methyl esters (FAMEs) by HPLC. FAMES from the neutral and polar lipid fractions were transes- 652 NOVOA ET AL. terit'ied with \0% (w/w) BF, in melliunol (Metcalfe and Scliniit/ iy61 ) for 10 niin at lOO'C. After cooling. FAMEs were extracted with hexane. The organic phase was evaporated under nitrogen and dissolved in chlorofomi/methanol (98:2) for purification by HPLC (Hennion et al. 1983. modified). FAMEs present in each lipid fraction were injected in a CG System HP6890 series equipped with a split/splitless injector, a tlame ionization detector, and a DBWAX capillary column (30 m x 0.25 mm ID; 0.2-|xm film thickness). The carrier gas was H^, at an initial pressure of 80 kPa. The oven was programmed to stay at the initial temperature of 60°C for 2 min. rise from 60 to 160X at a rate of 5()°C/min. stay for 2 min and from 160 to 170°C at 1.5°C/min. then 170-185°C at 2°C/min. next 185-240^C at 3°C/min. and tmally remain at 240'=C for 10 min. Injector and detector temperatures were 230°C and 250°C. respectively. The flow rates of compressed air. hydrogen and make up gas (nitrogen) were 300. 30. and 20 niLiiiin"'. re- spectively. Fatty acids were identified by comparison of their re- tention times with those of standards. The C:X (n-Y) notation was adopted, where C was the number of carbons. X the number of double bonds, and n-Y the position of the first double bond from the terminal methyl group. TABI.K 1. Fatty acid composition of the deuterium-labeled microcapsules (*AA-GAM). Incoi-poration yield was calculated using the following equa- (AA-GAM„-algae„)ng FA x lar\'ae" *AA-GAM ( % ) Fatty Acid Mean SD Mean 14:U 0.061 ().()03 0.028 16:0 10.830 0.066 4.920 18:0 2.606 0.075 1.182 20:0 0.390 0024 0.176 22:0 0. 1 3 1 ().()t)7 0.059 24:0 0.073 0.027 0.032 16:1 (n-9) 0.171 0.018 0.077 16:1 (n-7) 0.872 0.036 0.395 18:1 (n-9) 65.691 0.310 29.863 18:1 (n-7l 2.481 0.493 1.145 20:1 (n-9) 0.326 0.019 0.147 18:2 (n-6) 10.056 0.333 4.564 18:2 (n-4) ().()()() 0.000 0.000 18:3 (n-6) 0.000 0.000 0.000 18:3 (n-3) 0.749 0.029 0.339 18:4 (n-3) 0.000 0.000 0.000 20:2 (n-6) 0.000 0.000 0.000 20:.^ (n-6) (),()()() 0.000 0.000 20:4 (n-6)-d« 5.363 0.238 2.428 20:4 (n-6) 0.000 0.000 0.000 20:4 (n-3) 0.000 0.000 0.000 20:5 (n-3) 0.000 0.000 0.000 22:4 (n-6) 0.000 0.000 0.000 22:5 (n-6) 0.000 0.000 0.000 22:5 (n-3) 0.000 0.000 0.000 22:6 (n-3) 0.076 0,012 0.034 TO.SAT. 14.215 0.208 6.453 TO.MONO 69.541 0.728 31.627 TO.POLY 16.244 0.520 7.366 (n-3)/(n-6) 0.053 0.001 0.002 ng/larvae 45.446 AA-GAM (ng/larvae) SD 0.002 0.583 0.118 0.011 0.004 0.009 0.002 0.033 3.804 0.37 1 0.010 0.497 0.000 0.000 0.028 0.000 0.000 0.000 0.192 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.711 4.210 0.711 0.000 5.613 X 100 incorporation %: (AA-GAM-algae) ng dietary FA x larvae where the numerator is equal to fatty acid difference between larvae fed the microcapsules + algae and larvae starved or fed the algae and the denominator is the fatty acid supplied by the micro- capsules. RESULTS Ingestion and Digestion of Microcapsules After feeding on FITC-GAM for 4 h. larvae were observed under fluorescent tDicroscopy. The ingestion of FITC-GAM was TABLE 2. Fatty acid composition of the diets supplied in experiment 2: algal diet (mixted niieroalgae only) and *A.4-GAM + algal diet (mixted microalgae supplemented with microcapsules). ,\lgal Diet (ng/larvae) *AA-GAM + Algal Diet (ng/larvae) Fatty Acids Mean SD Mean SD Values in hold are. respectively, the major fiitty (n-9)] and the deuterium-labeled ;inichid(inic ac Results are expressed in percentages (%) and (ng/larva) in experiment 1. Values are means icid pre.sent in olive oil |18:1 id added |20:4 (n-6)-d8|. in ng supplied to each larva + SD (« = 3). 14:0 16:0 18:0 20:0 22:0 24:0 16:1 (n-9) 16:1 (n-7) 18:1 (n-9) 18:1 (n-7) 20:1 (n-9) 16:2 (n-7) 16:2 (n-4) 16:3 (n-6) 16:3 (n-4) 18:2 (n-6) 18:2 (n-4) 18:3 (n-6) 18:3 (n-3) 18:4 (n-3) 18:5 (n-3) 20:2 (n-6) 20:3 (n-6) 20:4 (n-6)-d« 20:4 (n-6) 20:4 (n-3) 20:5 (n-3) 22:4 (n-6) 22:5 (n-6) 22:5 (n-3) 22:6 (n-3) TO.SAT. TO.MONO TO.POLY ne. Diet/larvae 15.220 14.1(.)5 0.694 0.189 0.109 0.122 0. 1 8 1 19.401 7.766 1.289 0.083 2.356 0.692 0.000 0.964 4.876 0.216 0.623 5.152 6.767 1.663 0.048 0.074 0.000 1.407 0.052 4.015 0.043 0.964 0.105 6.047 30.975 29.582 37.027 98.304 1 .520 1.670 0.290 0.245 0.014 0.046 0.029 1 .695 0.648 0.142 0.012 0.236 0.045 0.000 0.137 0.308 0.029 0.040 0.354 0.575 0.146 0.027 0.007 o.ooo 0.095 0.007 0.155 0.008 0.007 0.032 0.145 3.403 2.470 1.979 8.358 15.223 14.590 0.811 0.207 0.115 0. 1 25 0.000 0.188 19.440 10.710 1.401 0.097 0.000 2.356 0.692 0.000 0.964 5.326 0.216 0.623 5.185 6.767 1.663 0.048 0.074 0.239 1.407 0.052 4.015 0.043 0.964 0.105 6.050 31.611 32.701 37.753 102.785 1 .520 1.721 0.300 0.244 0.014 0.047 0.030 1.698 0.997 0.175 0.012 0.236 0.045 0.000 0.137 0.345 0.029 0.040 0.357 0.575 0.146 0.027 0.007 0.019 0.095 0.007 0.155 0.008 0.007 0.032 0.145 3.461 2.846 2.034 8.845 Values in bold are, respectively, the major fatty acid present in olive oil [18: 1 {n-9)) and the deutenumdabeled arachidonic acid added [20:4 (n-6)-d8]. Data are means ± SD (« = 3). Ingestion. Digestion, and Assimilation of GAM 653 V ' p/vl Liaxvae starved LP (b) p*. Lam U) *AJ V-GAM I ^P 18- 10- 14- 12- 10- *o 8- 1 so 0- <*- ' c ^ '-I \ 4 f \ R i , Js . i. i-V, 2- 2B (c) pA~| Larvae starved LN (d) pAl Larvae *AA-GAM LN JU ^ s o ^ ^ O OS JUiiM ae Figure 4. Partial chromatograms of FAMEs prepared from larvae of V. puUastra corresponding to retention time between 26 and 28 min. Figure shows the incorporation of 20:4(n-6)d8 in polar (b) and neutral (d) Upids in larvae fed with microcapsules with respect to starved larvae (a) and (c). proved by an accumulation of yellow fluorescence (the dye color) inside the larva (Fig. 1 ). A sequence of photographs taken through- out a period of 20 min showed the decrease of yellow fluorescence in the digestive gland of a larva fed with microcapsules, due to digestion (Fig. 2). Growth and Survival Throughout the 7-day experiment, larvae were sampled with the daily water renewal to determine growth (anterior-posterior shell length) and survival. Growth and survival were similar for the 654 NOVOA ET AL. batches fed either microalgae supplemented with microcapsules or the microalgal diet (Fig. 3). Fatty Acid Composition of*AA-GAM The mean fatty acid compositions of *AA-GAM are listed in Table 1 . The fatty acid profile of microcapsules fabricated with 50 fj,L of olive oil and 5 mg of deuterated arachidonic acid 20:4 (n-6)-dis showed a predominance of 18:l{n-9) (65.7 ± 0.31%), 16:0 (10.8 ±0.07%). 18:2(n-6)(10.1 ± 0.33%). and 20:4{n-6)-dH (5.4 ± 0.24%). A single microcapsule contained 3.8 pg total fatty acids. Dietary Fatty Acid Supply The diet consisting of *AA-GAM exclusively was used in the 24-h experiment and supplied 60 GAMjjiL"', which corresponded to 29.86 nglarva"' for the 18:l(n-9) and 2.43 nglarva"' for 20: 4(n-6)-ds (Table 1). Table 2 shows the fatty acid composition of the two different diets (algal diet and *AA-GAM-supplemented diet) used in the 7-day experiment. The first supplied 7.77 nglarva^' of 18:l(n-9) and the second supplied 10.71 ng-larva"' of 18:l(n-9) and 0.24 nglarva"' of 20:4(n-6)-ds. Assimilation of Microcapsules in the 24-h Experiment The FID-CG analysis allowed the separation and the quantifi- cation of deuterated *AA in polar and neutral lipis of larvae (Fig. 4). Larvae fed *AA-GAM exclusively showed incorporation of 20:4(n-6)-ds in polar and neutral lipid fractions (0.10 and 0.12 ng-larva"'. respectively). The amount of the major fatty acid in olive oil, 18:l(n-9), also increased in neutral and polar lipids when compared with that of starved larvae: 1.08 ng-larva"' versus 0.33 in neutral lipids and 0.66 ng-larva"' versus 0.28 in polar lipids (Fig. 5a). Incorporation yield percentages of 20:4(n-6)-dg were nearly equal in both lipid fractions (4.30% for polar lipids and 4.80% for neutral lipids), whereas for 18:l(n-9) they were 2.53% and 1.31% for neutral and polar lipids, respectively (Fig. 5b). Fatty Acid Composition of iMrvae Fed with Different Diets Larvae fed with -^AA-GAM-supplemented diet showed an in- crease in the incorporation of the two monitored fatty acids [20: 4(n-6)ds and 18:l(n-9)| in both lipid fractions when compared with those fed the microalgal diet (Fig. 6). With regard to 20:4(n-6)-ds. incorporation profiles were similar in both lipid fractions throughout the 7-day experiment (16.3% for neutral lipids and 16.8% for polar lipids between days of culture 18 and 19). Assimilation percentages were higher during the early days of the culture and decreased in the late days from 16.3 to 5.6% in neutral lipids and from 16.7 to 6.1% in polar lipids (Fig. 7). Incorporation of 18:l(n-9) was better in neutral than in polar lipids. Throughout the experience, the incorporation yield de- creased in both fractions, although the phenomenon was more pronounced in polar lipids (Fig. 7). The Hpid content, the percentage of neutral and polar fractions and the fatty acid composition of larvae were similar in both 7-day experiments (Table 3). Supplementation with microcapsules did not affect the incorporation of the essential PUFA provided by microalgae. However, larvae fed microcapsules appeared to have a higher FAME content if compared with larvae fed microalgae only. DISCUSSION Numerous artificial particles have been tested to supplement or partially replace the living algal diet for mollusks. These included yeast (Epifanio 1979). fiour (Albentosa et al. 1999). dried algae (a) 18:l(i>-9) 20:40.06 0.04 0.02 0 -r— algae -m— 'AA-GAM -f algae 17 19 21 23 Days of larval development 25 Figure 6. Dynamics of 18:l(n-9) and 20:4(n-6)d„ in neutral, polar, and total lipids of larvae fed microalgae and ""AA-GAM + microalgae during experiment 2. (Laing & Millican 1991). emulsions, and liposomes (Coutteau at al. 1996. Caers et al. 1999. 2000, Soudant at al. 2000). Davalop- mant of particles like gelatin-acacia spraybeads (Buchal & Lang- don 1998) was mostly devoted to identifying essential molecules and to understanding their metabolism. In this article, we report data on the incorporation efficiency of the essential PUFA AA using GAM. Because mollusks contain AA in their corporal fatty acid, we incorporated deuteratad AA (*AA), which can be easily discerned from the natural one by direct GC analysis, allowing a good estimation of its assimilation. This straightforward method- ology avoids the use of radioactivity and its additional measure- ments. Indeed, it may be applied to other essential fatty acids. Although deuteratad metabolites of AA were not detected in our study, their determination is possible with this methodology. Small-scale GAM fabrication is easy and suitable for experi- mental studies on metabolism, in particular for lipids, which are easily encapsulated. Olive oil was chosen as lipidic support be- cause its composition is very simple. Oleic acid (OA) represents 659!- of the total fatty acid present in olive oil. Moreover, olive oil lacks most very long-chain PUFA that are essential for marine mollusks (Trider & Castell 1980, Enright et al. 1986, Soudant et al. 1996). Therefore, the fatty acids from this oil would not interfere 656 (a) Polar Hpids NOVOA ET AL. (b) Nei80%l of gonadal neoplasia. Neoplasia reduces the life span of Indian River hard clams relative to their northern congeners and probably reduces fecundity of those clams that do survive. The second strategy involved seeding juvenile clams at very high densities (843-7165 m~' depending upon seed size), again with the intent of maximizing fertilization efficiency but with the additional goal of maximizing residual reproductive value. Various planting treatments were tested in an effort to minimize monality of seed clams, but losses were still high (generally >70%1, and the yield did not appear to justify the cost. The final strategy involved spawning large numbers of hard clams in the laboratory, fertilizing the resultant eggs, and immediately releasing the larvae at a predetermined location in the lagoon. Large numbers of larvae did appear to survive the 8-day lar\'al life span, but it remains to be seen whether those larvae will translate into harvestable hard clams. In any event, enhancement of hard clam populations appears to be feasible only when the density of naturally occurring clams is so low that reproducfive viability is compromised. Otherwise, natural reproductive potential will swamp any artificial efforts at population enhancement. KEY WORDS: Mercenaria. Indian River Lagoon, population enhancement, spawner transplant, seeding, larval release INTRODUCTION Hard clams of the genus Mercenaria support an economically and culturally important fishery in the Indian River Lagoon on the east central coast of Florida. This is a relatively new fishery that developed in the early 1980s (Fig. 1) and was originally centered near the town of Grant in southern Brevard County (Fig. 2). The clam population that supported the original fishery collapsed in the late 1980s, probably in response to decreased salinity caused by the release of St. Johns River floodwaters into the Indian River basin (Barile and Rathjen 1986). Another substantial set of hard clams was detected in the northern Indian River Lagoon, between Cocoa and Titusville. in the early 1990s. That population sup- ported a profitable and productive tlshery throughout the mid- 1990s (Fig. 1), but the population again collapsed and again the proximate cause appeared to be decreased salinity resulting from above-average rainfall in the watershed. As of the year 2001, the fishery for naturally occurring hard clams in the Indian River Lagoon has remained depressed, although a small number of fish- ermen continue to pursue the few clams that are available. The Indian River is a shallow, narrow, bar-built lagoonal sys- tem that stretches from Oak Hill to Stuart along the central Atlantic Present Address of Dan C. Marelli; Florida State University. 036 Mont- gomery. Tallahassee. Florida. 32306-2310. Present Address of Philip Hoffman: Pinellas County Department of Envi- ronmental Management. Environmental Resources Management Division. 300 South Garden Avenue. Clearwater. Florida. 33756. *Corresponding Author: E-mail bill.amoldta'fwc.state.fl.us coast of Florida. For our study, we considered only that area of the Indian River Lagoon (IRLl system that is located north of Sebas- tian Inlet (Fig. 2) and that is composed of the Indian River (IR). the Banana River (BR), and Newfound Harbor. Prehistorically. the IRL was a single continuous basin, and the IR and BR were con- nected to the south at Dragon Point and to the north through Banana Creek. In recent years, development and bridge construc- tion have resulted in the subdivision of the IRL into a series of basins that are defined by the causeways that span the lagoons. Water exchange between basins is restricted to the areas under the bridges that connect the causeway dikes (Evink & Morgan 1982). Additionally, the pattern of water exchange between the IR and BR has been altered in the last 40 years. Development of the Kennedy Space Center essentially severed the Banana Creek connection between the two lagoons (McCall et al. 1970). and construction of the Canaveral Barge Canal in the eariy 1960s (Yusof 1987) created a new connection south of Banana Creek. Reported clam landings from the Indian River Lagoon for the past 20 years suggest that at least under certain conditions, the capacity for production of hard clams in the lagoon is substantial. However, no single basin of the lagoon appears to be consistently productive. Instead, an individual basin may support a dense clam population for several years, after which the population collapses and remains depressed until another major set of Juvenile clams occurs either in that basin or in another area of the lagoon. Envi- ronmental conditions can vary substantially among basins, creating a potential mismatch between successful spawning events (Hes- selman et al. 1989) and the environmental conditions necessary to support the survival and development of that spawn. 659 660 Arnold et al. 2000 0'°" ^0!°' 0'° o'o s<>!°" ^^ v in the Indian River lagoon. Season Plant Date 2-wk Sample 3-mo Sample 6-nio Sample 9-mo Sample 12-mo Sample Fall 10/28/98(100) 11/9/98(127) 2/3/99(107) 4/28/99(114) 8/10/99(57) 10/26/99(17) Winter 1/27/99(100) 2/3/99 (.'59) 4/28/99(238) 8/10/99(53) 10/26/99(18) N/A (N/A) Spring 4/20/99(100) 4/28/99 080) 8/10/99(131) 10/26/99(38) N/A(N/A) N/A(N/A) Summer 8/3/99(121) 8/10/99(171) 10/26/99(6) N/A(N/A) N/A(N/A) N/A (N/A) Numbers in parentheses indicate sample si/e of hard clams collected on that date. Note that on each initial seasonal sampling date, clams were randomly sampled from all of those harvested, whereas on later dates sample size reflects the number of clams actually collected from each study plot. Clam Population Enhancement in Florida 663 TABLE 2. Planting and sampling dates for the liard clam (Mercenaria spp.) spawner transplant study in the Banana River lagoon. Season Plant Date 2-wk Sample 3-mo Sample 6-mo Sample 9-mo Sample 12-mo Sample Fall 10/28/98(100) 11/9/98(73) 2/3/99 (73) 4/28/99 (96) 8/11/99(97) 10/25/99(3) Winter 1/27/99 ( 100) 2/3/99(200) 4/28/99(144) 8/11/99(91) 10/25/99(16) 1/25/00 (2) Spring 4/20/99(100) 4/28/99(161) 8/11/99(87) 10/25/99(31) 1/25/00(10) 4/25/00 ( 1 ) Summer 8/3/99(121) 8/11/99(229) 10/25/99(20) 1/25/00(7) 4/25/00(9) 8/16/00(2) Numbers in parentheses indicate sample size of hard clams collected on that date. Note that on each initial seasonal samphng date, clams were randomly sampled from all of those harvested, whereas on later dates sample size reflects the number of clams actually collected from each study plot. of this enhancement strategy, it is necessary to be able to track and sample the animals during the planktonic phase of their life. Spawning The larval release strategy requires the production of large numbers of viable hard clam embryos that can be successfully transported to and released at the site targeted for enhancement. Adult hard clams were collected from various areas of the Indian River Lagoon on several dates during 1999. The clams were trans- ported to a holding area at Harbor Branch Oceanographic Institu- tion, where they were conditioned in preparation for spawning. Conditioning consisted of holding the clams for several months in a small lagoon on the Harbor Branch campus. On the day before spawning, the clams were transferred from the holding lagoon to a refrigerated storage area and held overnight. On the following day. the clams were removed from the storage area, placed in equally spaced rows on each of three spawning tables, and submerged in approximately 10-15 cm of 28 p.s.u. seawater. During the next six hours, the clams were exposed to cycles of cool and warm water and induced to spawn. As each individual clam initiated spawning, the sex of the animal was identified and the clam was removed from the table and isolated in containers with other clams in small groups of males or females. The clams continued to spawn within the containers, and the resultant eggs were pooled and concen- trated on a 35-jjim-mesh sieve and exposed to a sperm concentra- tion adequate to ensure fertilization of all eggs. Total egg produc- tion and fertilization success were determined microscopically, after which the developing embryos were transferred to 20 L TABLE 3. Gonad staging scheme for female hard clams {Mercenaria spp.) collected from the Indian River Lagoon, including female clams transplanted to study sites in the Indian River and Banana River lagoons and sampled during various times of the year, and from their undisturbed conspecifics. Reproductive Numerical Status Description of Gonadal Tissue Stage No Data Tissue unreadable 0 Inactive Gonad tissue undifferentiated 1 Developing Tissue differentiated, eggs present 2 Ripe Tissue full of eggs 3 Early spawning Eggs being shed, but follicles still full in appearance 3.5 Spawning Many eggs shed, follicles appear partially empty 4 Spent Follicles nearly empty 5 aquarium bags and transported to the study site for immediate release. Larval Tracking Hard clam embryos were transported from the spawning facili- ties at Harbor Branch to our study site in the Banana River lagoon and released at 20.30 EDT on May 16. 2001. at a site approxi- mately 1 .75 m deep. Water temperature at the site was 28°C and salinity was 22 p.s.u.. whereas the temperature of the water in which the larvae were transported was 25.8"C and the salinity was 27.7 p.s.u. Before larval release, five subsurface drifters (Davis 1985. Hitchcock & Arnold, unpublished data) were deployed in a box-and-one pattern (one drifter at each comer of a 10 m x 10 m box. with a single drifter in the center of the box), and their initial positions were recorded using a differential Global Positioning System. Then, at a depth of approximately 0.5 m. the clam larvae were gently poured from the bags into the center of the drifter array. Gradual mixing between the transport water and the la- goonal water was allowed in an attempt to minimize osmotic shock. During daylight hours on May 16, we collected thirteen 200 L water samples from the targeted release area to determine the prerelease concentration of hard clam larvae in the study basin. On May 17 and 23. 2000. post-release water samples were collected to determine the distribution and density of the larval mass. For the May 17 sampling, when the larval mass was predicted to be rela- tively concentrated, the subsurface drifters were visually located and the position of each drifter recorded. Sample collection loca- tions for hard clam larvae were then selected based upon the lo- cation and distribution of the subsurface drifters. On May 23. after diffusive processes were anticipated to have spread the larvae throughout the study basin, samples were collected at each of 23 grid nodes equally distributed throughout the basin. On each sampling date, samples of hard clam larvae were ob- tained by using a Jabsco Model 34600-0000 diaphragm pump to collect volumes of water that ranged in size from 100-400 L, depending upon the projected density of larvae. Water was pumped through a 150-jim-mesh sieve to remove large objects, and then captured in a 63-|xm-mesh plankton net. Each resultant sample was removed from the cod end of the plankton net and carefully distilled to a volume of approximately 30 mL, then trans- ferred to a 50-mL screw-cap centrifuge tube. labeled, and placed on ice until arrival at the laboratory, where it was frozen at ap- proximately -5°C. Within one month of the completion of the study, all of the frozen water samples were sent to the Skidaway Institute of Oceanography for determination of the presence and abundance of clam larvae. Samples were analyzed for the presence of hard clam larvae using a previously developed genetic probe 664 Arnold et al. that is both quantitative and Menenaria-speafic (e.g.. Frisctier et al. 20001. RESULTS Spawner Transplants At botli tlie Indian River and Banana River study sites, the mortahty of relayed hard clams was severe, particularly during the summer and early fall of 1999. Also at both sites, considerable loss of clams from the plots was associated with the initial transplant. At each site, on all dates, we transplanted an average of 25 clams m"-. and within 2 wk the densities for all plantings had decreased by more than 50% (Fig. 3). After the initial transplant event and the loss of clams associated with that event, clam densities stabi- lized throughout the winter, spring, and early summer at both sites (Fig. 3). During late summer and early fall of 1999, the clams experienced substantial mortality, possibly as a result of decreased salinity associated with Hurricane Irene (Fig. 4). As noted previ- ously, that storm destroyed our study plots in the Indian River. It also appears to have had a severe detrimental effect on the clams planted in the Banana River, as clam density decreased substan- " In JA ifl i\ CNJ E E O 15 10 5 • B) i ti C) ti i -^- 1D- 5 ■ D) -^ ^ ^^ /^ #" .^ .J' J" .^# Date Fidurt 3. Mean density of hard clams (Mercenaria spp.) on various sampling dates after transplantation during (A) fall IW8; (B) winter IVW; IC) spring 1999; and (I)) summer 1999 al the Indian River lagoon (filled bars) and Banana River lagoon (open barsi study sites. Sec Figure 2 for the location of each study site. Note that at the Indian River study site, all four study plots were destroyed l)y Hurricane Irene during October of 1999 and no further sampling was conducted after that date. F>ror l>ars represent one standard deviation. On the November 9, 1998, sampling date, samples were pooled and no stan- dard deviation was calculated. lially in all Banana River plots between the August I 1 and October 25, 1999. sample dates (Fig. 3). Hurricane Irene swept through our study area on October 16. 1999, and salinity near our Banana River study site decreased to a study-period minimum of less than 15 p.s.u. at the end of October 1999 (Fig. 4|. The high levels of mortality that we obsei-ved in our transplant plots may have been inlluenced by the inability of clams (espe- cially large clams) to reburrow following initial harvest. The In- dian River study site was characterized by a soft sand/mud sub- strate that appeared to provide little resistance to burrowing clams. Upon re-sampling that site two weeks after the fall transplant. 26% of all clams collected remained on the surface, and three months after the fall transplant 20% of all clams collected still remained on the surface. In contrast, the Banana River study site was charac- terized by a hard sand bottom that appeared to provide consider- able resistance to clams attempting to burrow. Two weeks after the Banana River fall transplant. 47% of all clams collected remamed on the surface, and three months after the fall transplant 34%^ of all clams collected remained on the surface. At both sites, failure to huiTow was related to clam size. A comparison of the mean SH ot buried versus unburied clams at each site two weeks after trans- plant during both fall and winter, indicated that the clams that failed to burrow were significantly larger than those that success- fully reburrowed (/-test, see Fig. 5A. B. E. and F for respective P values). We detected no significant difference in SH between bur- ied and unburied clams at either site three months after transplant (Fig. 5C and D). although only clams transplanted during the fall were compared. During the spring and subsequent sampling epi- sodes, we discovered few clams at either site that were both alive and unburied. After the initial episode of transplant mortality, overall mortal- ity of relayed clams did not appear to be size-related. At the Indian River study site, the size distribution of hard clams did not differ significantly among sampling dates (Simultaneous Test Procedure; Sokal & Rohlf 1995) except during the summer transplant study (Fig. 6). During the final sampling episode (October 26. 1999) of the summer transplant study, the size distribution of planted clams differed significantly from the size distribution recorded during the previous two sampling dates and appears to have shifted towards a preponderance of small clams (Fig. 6). For all four of the trans- plant episodes at the Banana River study site, a significant shift in clam size distribution was detected for the October 25. 1999. sample date, and for all sample dates subsequent to October 25. relative to all sample dates preceding October 25 (Fig. 7). The only exception to this pattern was from the spring transplant study, for which the size shift was not detected until the January 25 sampling episode (Fig. 7). During each seasonal harvesting event, a subsample of 15 clams from each plot was returned to the laboratory for analysis of reproductive condition and for a comparison with control samples collected from the natural population on the same date. However, the results from only the fall planting date at both study sites are included in the present analysis because that is the only planting date for which adequate sample numbers were available for all sample dates from both sites. The female clams in the control samples had a pattern of reproductive development typical of In- dian River hard clams (Hesselman et al. 1989). During fall and winter, when Hesselman et al. (1989) reported that spawning oc- curred in Indian River hard clams, most clams that we sampled (control and transplant) were either spawning or were spent (Fig. 8). During spring, the season of peak spawning in Indian River Clam Population Enhancement in Florida 665 3 (A d "E (0 (5* O ^- .^^- .^^- .^^ J^ O^ Q"-" ^^" ^^" s^" 4^ A\^ cb^

uun (closed triangles) and Banana River lagoon (closed circles) study sites during September 1998 through October 2(((MI. The dotted line provides a 20 p.s.u. reference. Data courtesy of the St. Johns River Water Management District Surface Water Quality Monitoring Program. hard clams (Hesselman et al. 1989). most of the clams from the control sample were ripe, and lesser proportions were either spawning or spent. In contrast, most animals collected from the Indian River and Banana River transplant plots during spring were in some stage of spawning. Finally, during summer the vast ma- jority of control and transplant clams that we sampled were in the spent condition, which agrees well with the observation of Hes- selman et al. (1989) that most Indian River hard clams are repro- ductively spent during summer. Gonadal neoplasia is extensive in Indian River hard clams (Hesselman et al. 1988, Bert et al. 1993), and this condition ap- pears to be related to hybridization between the two species of Mercenaria (M. mercenaria and M. cainpechiensis) that occupy the lagoon (Bert et al. 1993). We recorded neoplasia in 85% of the male clams and almost 92% of the female clams that we collected from the natural clam population of the lagoon. Male clams suf- fered 93% and 86% neoplasia when harvested after transplant to the Indian River and Banana River, respectively. Female clams suffered 96% and 93% neoplasia when harvested after transplant to the Indian River and Banana River, respectively. When all samples of clams collected for reproductive analysis during the course of the spawner transplant study were pooled, there were significantly more female than male clams (0.53 males: 1.00 females, x" = 42.13, df= 1, P< 0.0001 ). However, , sex ratio was dependent upon clam size-class. For all clams that were < 60 mm SH, we detected no significant difference in sex ratio (0.75 males: 1.00 females, x" = 1-43, df = \.P = 0.232). In contrast, for clams > 60 mm SH, the sex ratio was significantly skewed towards female clams (0.50 males: = I, P< 0.0001). 1.00 females, x" = 42.89, df Seeding Mortality of 2 mm hard clams was substantial within 2 wk of planting under all treatment conditions (Table 4). Only under mesh protection did the 2 mm size-class suffer less than 90% mortality, but even with mesh protection the small clains experienced a mean mortality of 85.6%. Survival of clams in the 8 mm size-class was not much better: those clams also experienced >90% mortality in the open plots and at least 50% mortality within 2 wk after being planted in the remaining plots. In contrast, clams in the 16 mm size-class experienced <10% mean mortality in the mesh plots and a mean mortality of 1 1.3% in the open plots. However, those clams suffered 30.4% mean mortality under the combined protective cover and >70% mean mortality in the shell plots. Hurricane Irene also severely impacted our seed clam plots. Nevertheless, on November 1 1, 1999, we attempted to reconstruct the experimental plots and we did conduct suction dredge sam- pling of the reconstructed plots. We found no live clams in the seed plot I y after planting. However, we cannot determine whether that lack of clams was due to the effects of Hurricane Irene or due to factors independent of the hurricane. iMrval Release Spawning On May 16. 2000. approximately 550 million hard clam eggs were spawned and collected. The eggs were then exposed to an 666 Arnold et al. 40 H 30 1 B) p = 0.0065 20 10 - 0 ■ , fl , ,n -P »U- lllll ,. , >< o c (U a- c 0) o a. C) , , , , . ,n . J,n l,n 1 p = 0.9551 In ,n , , . c\ p = 0.0043 . , , f--^--^ J1 ,n l)l 1,11 1,11 Ijl Ip Ip , , ^ F) p = 0.0001 ,- ,n f1 r i" T 'Jl l|" l| 11.., , , A ^ ■P >° >'' ? ^'^ a'' ^° ^'' =P ■4'= K# ^o^ Size-Class (mm) Figure 5. Percent frequency of occurrence of hard clams {Mercenaria spp.) at each of the Indian River lagoon and Banana River lagoon study sites during fall and winter sampling events. Filled bars repre- sent the percentage of clams that were recovered on the surface of the plots, and open bars represent the percentage of clams that were bur- ied upon recovery. (A) Indian River fall planting, 2-wk sampling: (Bl Banana River fall planting, 2-wk sampling: (C'l Indian River fall plant- ing. 3-mo sampling; (D) Banana River fall planting, 3-mo sampling: (E) Indian River winter planting, 2-wk sampling; and (F) Banana River winter planting, 2-wk sampling. The /' values included in each plot represent the probability (Mestl that the mean size of clams re- covered on the surface was not significantly different than the mean size of clams that were buried upon recovery. \ P < 0.05 indicates a statistically significant difference in the mean size of surficial vs. bur- ied clams. amount of sperm sufficient to ensure fertilization of all eggs as determined by microscopic examination. Spawning occurred in three "batches" beginning at 1200 EDT and ending at 1600 EDT. Transport from Harbor Branch (1800 EDT departure) to the Ba- nana River release site (2030 EDT release) required approximately 2.5 h, so the clams ranged in age from 4.5 to 8.5 h at the time of release. Larval Release Larvae were released at a site in (he Banana River lagoon between SR 520 and SR 528 (28°23.32()'N latitude, 80°37.95rw longitude) at 2030 EST on May 16, 20(.)0 (Fig. 2). During the next 24 h. the drifters that tracked the water mass within which the larvae were released were transported towards the west until they approached the western shore of the lagoon (Fig. 9). As the drifters approached the shoreline, they gradually swung around to the north, but four of the five drifters contacted the bottom, hung up, and were retrieved. The fit'th drifter passed through a small bridge at the western end of the SR 528 causeway and was retrieved to prevent its loss. Analysis of water samples collected on May 16, before the release of cultured larvae, detected no evidence of naturally oc- curring hard clam larvae in the study basin. On May 17. after our larval release during the evening of the 16th, we detected larvae at only one sampling station (Fig. 9), probably because the larval mass remained tightly constrained in the area around that station. By May 23, the larval mass had spread throughout the basin (with the exception of the southeast comer), although several peaks of abundance were detected. Two of these peaks were in the north- west comer of the basin, where larval density exceeded 7 veligers L^' (Fig. 10). DISCUSSION We tested three strategies for enhancing the abundance of har- vestable hard clams in the Indian River Lagoon. Florida. The first strategy involved harvesting adult clams from low-density popu- lations and concentrating them in high-density patches in an effort to increa.se fertilization success and the production of viable larvae. This strategy does not appear to be cost-effective because most of the clams that we transplanted did not survive the 1 2-mo moni- toring period. Furthermore, the vast majority of the clams that we transplanted were infected with gonadal neoplasia, a disease that progressively reduces fecundity and probably proceeds to a fatal outcome (Yevich & Barry 1969, Hesselman el al. 1988, Eversole & Heffernan 1995). The second strategy involved planting small seed clams at relatively high densities, again with the intention of creating concentrated patches of reproductively active clams. This approach has one anticipated advantage (a longer life span for the spawners) and one unanticipated advantage (avoidance of gonadal neoplasia) when compared with the spawner transplant strategy. However, the first advantage may be offset by the high rates of mortality experienced by most size-classes of the seed clams, and the second advantage may be temporary because the seed clams become increasingly susceptible to gonadal neoplasia, as they grow older (Bert et al. 1993). Although survival rates were rela- li\ely high for the largest size-class of clams (16 mm mean SH), the considerable cost of those clams ($0,036 each) reduces the cost-effectiveness of this strategy. The third strategy involved cir- cumventing the entire process of natural fertilization by releasing already fertilized eggs directly into the lagoon. Our results suggest that this strategy may be effective, but more information is needed. It appears that large numbers of clam larvae survived to an age at which settlement can be reasonably expected (8 days to set in culture conditions during May using Indian River water; B. Leem- ing, personal communication). However, the ultimate fate of those larvae has not been determined, and successful settlement needs to be demonstrated in vivo for this approach to have any validity. The common currency with which to gauge the success of each of these strategies will be an increase in the abundance of hard clams available for harvest by the fishermen. Hard clams in the Indian River Lagoon require approximately 18 months to achieve the legal harvest size of 2.54 cm in shell width (Arnold et al. 1996). Clam Population Enhancement in Florida 667 w E n O o > 0) O) ra C 0) u 0) a. 40 - 30 - B) 20 - ll, 10 0 ■ , -H- --J|- J ^ ,„ II 1 1 II III. .1. , 40 - 30 - C) 1, 20 - „ 1, 1 . 10 - 0 , J . M , ll, ll 1 1 — Jl — -1 , 40 - 30 ■ D) 1 1 10 0 • L i. J J. 1. h., -b^ (f> if> «f> «p fcO 6") -VO ^b 4« 4% ,jO 7 Hard Clam Size-Class (mm) Figure 6. Percentage of live hard clams {Mercenaria spp.) recorded within various size-classes during each seasonal sampling event fol- lowing the (A) fall: (B) winter; (Cl spring: and iDl summer transplants in the Indian River lagoon. For each size-class category within each transplant season, sampling dates are plotted from left to right. Thus, the leftmost bar within each size-class represents the sample collected on the original date of harvest, and the rightmost bar represents the sample collected on the final sampling date. See Table 1 for sampling dates and sample size for each date. Note that samples were collected on only five dates for the winter transplant, four dates for the spring transplant, and three dates for the summer transplant because of the impacts of Hurricane Irene, which destroyed the sample plots on Oc- tober 16, 1999. Otherwise, missing bars indicate that no clams were collected from that size-class on that date. For the adult transplants, it would have been possible to detect adult offspring as early as spring 2000, assuming that clams trans- planted in fall 1998 spawned very soon after transplant. That is a reasonable assumption considering that a fall spawn has been de- scribed for Indian River hard clams {Hesselman et al. 1989) and was similarly indicated by our fall 1999 reproductive data. For the seeding study initiated in fall 1 998, we would have expected that maternally derived offspring would be available for harvest no earlier than fall 2000. Hard clams as small as 27 mm SH are reproductively active in the Indian River Lagoon (Hesselman et al. 1989), but clams are generally male during the first year of life (Loosanoff 1937). Thus, we would not have expected egg produc- tion from the 16 mm size-class of seed clams for at least 6 months post-planting (i.e., spring 1999). For the larval release study con- OT E _2 o > o 0) c 0) o i. V ifs' u<>= 800(X) clams as- suming a 2:1 female:male sex ratio. We did not anticipate the rate of loss of transplanted clams that was actually realized during this study. Similar transplants have 670 Arnold et al. Banana River 1 1 L..iid -^r Release Poinl Larval Distribution IVlay 23, 2000 Larval Concentration 0 (Veligers/L) 2 5 ^'^ 7 c^ — ") I /l 'Merritt / i, N Island^' i\ Oraftge v\-'i~^^ 7 Cap 1 Q^ e averal Osceol; Co Brevard \ Co. V i Atlantic Ocean • i ■ s udy Area \ \ 0.5 0 0.5 Kilometers W A n-" Merritt Island Fijjurt 10. Cimlour plot of the distribution of hard clam {Mercenaria spp.l larvae as estimated bv sampling conducted on Ma> 23, 2000, in the Banana River lagoon, Florida. Data are presented as number of clam larvae 1 ', and larval concentrations are depicted at the location of each sample station. been conducted in other areas throughout the range of Merceiuiria. either for eventual harvest of the relayed clams (e.g.. Rice et al. 2000) or to increase or expand larval production (e.g.. Kassner & Malouf 1982, Ganz 1991). However, the high frequency of go- nadal neoplasia in the clams that we collected for transplant will mitigate against the success of this approach. Gonadal neoplasia is common in Indian River hard clams (Hesselman et al. 1988). par- ticularly in the northern lagoon where we collected clams for trans- plant (Bert et al. 1993). Although gonadal neoplasia has been reported in hard clams collected from northeastern US coastal waters (e.g.. Barry & Yevich 1972), the frequency of occurrence was less than 5% versus greater than 80'7r in our study. That disease appears to substantially reduce the reproductive potential of hard clams (Hesselman et al. 1988) and probably contributes to the relatively short life span of hard clams in Indian River waters (Jones et al. 1990). Considering that the reproductive potential of Mercenaria increases with age (Bricelj & Malouf 1980. Peterson 1983. 1986), the high frequency of gonadal neoplasia in Indian River hard clams appears to render spawner transplants an inef- fective strategy for enhancing the abundance of harvcstable clam populations in the lagoon. In all cases, gonadal neoplasia was more prevalent in the trans- planted clams than in their undisturbed conspecifics, and the dif- ference in the frequency of neoplasia between the transplants and their undisturbed conspecifics was greater in the Indian River than in the Banana River. This difference was minor and may reflect sampling bias related to differences in the frequency of neoplasia that have been reported for various size-classes and genotypes of hard clams (Bert et al. 1993). Nevertheless, gonadal neoplasia was considerably more prevalent in clams collected during our study than in clam collections reported by either Hesselman et al. ( 1988) or Bert et al. (1993). Neoplasia is more common in hybrid hard clams (Bert et al. 1993) and hybrid clams are more common in the northern Indian River Lagoon where we collected our transplant animals (Bert & Arnold 1995). Our spawner transplant study would perhaps have been more successful if we had collected clams from more southerly Indian River waters, but we were lim- ited in our choice of harvest sites by the availability of clams. Seeding as a means of increasing the abundance of hard clams has been attempted in various areas throughout the range of Mer- cenaria. including both coasts of Florida (Men/el & Sims 1962. Menzel et al. 1976. Marelli & Arnold 1996). Georgia (Walker 1985). North Carolina (Peterson et al. 1995). Virginia (Castagna & Kraeuter 1977). New York (Flagg & Malouf 1983). and Rhode Island (Rice et al. 2000). With the exception of the work in North Carolina (Peterson et al. 1995), these efforts have met with limited Clam Population Enhancement in Florida 671 success due to the high rate of loss of seeded clams even when protective measures are used. In North Carolina, relatively large seed clams (14—22 mm shell length) were planted at relatively low density (I m"") in shell hash habitat in late fall, resulting in 35% survival after 14 months (Peterson et al. 1995). However, clam density below 5 m~~ is considered to be inadequate for commercial harvest in Florida waters (Arnold et al. 2000), and increasing the density of planted seed clams might result in a loss of economic viability attributed to this approach. Moreover, achieving even the limited enhancement goals that we have set for our project (50 million harvestable clams) would require seeding almost 15.000 hectares of submerged land with almost 1.5 billion clams. Even if the available clam hatcheries could produce that many clams, the cost of the clams alone would be exorbitant. At least for the 500 hard clam fishermen currently licensed to work Brevard County waters, seeding for direct harvest does not appear to be a cost- effective means of ensuring a minimum annual income, although that approach may be feasible for projects of a smaller scale. We are familiar with only one application of larval release as a means of stock enhancement. Shepherd and colleagues (Preece et al. 1997, Shepherd et al. 2000) released various densities of aba- lone larvae at several sites in South Australia and monitored their survival. They found that because larval and post-larval survival was density-dependent, the releases of relatively low densities of larvae were more successful than were the high-density releases. The overall conclusion of those authors (Shepherd et al. 2000) was that larval release was not a viable strategy for abalone stock enhancement because of the density-dependent nature of larval mortality. Those results, and results from our own analyses of diffusive processes acting on artificially introduced hard clam lar- vae (Hitchcock & Arnold, unpublished data), suggest that a point release of the larvae is not the best strategy. Instead, higher sur- vival rates may be obtained by spreading the larvae throughout the target basin, thereby enhancing diffusive processes that will take place anyway (Hitchcock & Arnold, unpublished data). We will test that approach in future experiments. Efforts to enhance the population abundance of commercially important marine molluscs have been ongoing for decades, and the published record of those efforts indicates that success has been rare if not nonexistent. Clearly, efforts to enhance even moderately dense populations are superfluous, as the reproductive potential of the natural population is sufficient to swamp any directed enhance- ment efforts. Only when population density is very low, such as it would be in an essentially collapsed population, might such efforts yield success. At that point, we shift from an enhancement effort to a restoration effort, and the goals of the project shift from increasing the abundance of an ecologically functional population to restoring reproductive viability in an ecologically dysfunctional population (Arnold 2001 ). Nevertheless, user groups and manage- ment agencies continue to request that population enhancement efforts be undertaken, and we will continue our efforts to deter- mine if or under what conditions we can meet those requests. ACKNOWLEDGMENTS Kate Hagner, Tracy Idocks, Micah Humphrey, and Melissa Harrison of the Florida Marine Research Institute provided invalu- able field and laboratory assistance. Commercial clam harvesters Perry McMahon, Bill Leeming, Peter Barile. Doug Telgen, Mason Bowen. Bill Bowler, John Condos, David Panizzi, Jay Anderson. George Vincent. Allen Ellingham, Richie Luck, George Rotsch. James Horst, Pete Roy. and Riley Bergman assisted with clam collections for the spawner transplant study. Frederico Prahl, Sandy Zeiner, Richard Baptiste. and David Vaughan of the Divi- sion of Aquaculture, Harbor Branch Oceanographic Institution were essential to successful spawning and fertilization of hard clam eggs. Gary Hitchcock of the University of Miami assisted with larval tracking and was instrumental in the design and de- ployment of the shallow-water drifters. Winnie White (FMRI) pre- pared the maps and provided GIS support. Dick Moravec (FMRI) provided assistance with vessels and vehicles, and Gerry Bruger (FMRI) ensured that our electronic database management was seamless and uneventful. This study was made possible by the generous financial support of the commercial clam harvesters of Brevard County. Florida. This is Harbor Branch Oceanographic Institution contribution number 1483. LITERATURE CITED Arnold, W. 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Joiinwl of Shellfish Rcxeanh. Vol. 21. No. 2. 673-676. 2002. ABUNDANCE OF OCEAN QUAHOG, ARCTICA ISLANDICA, ASSESSED BY UNDERWATER PHOTOGRAPHY AND A HYDRAULIC DREDGE STEFAN AKI RAGNARSSON* AND GUDRUN G. THORARINSDOTTIR Marine Research Institute, Skiilagata 4. P.O. Box 1390, 121 Reykjavik. Iceland ABSTRACT Abundance of ocean quahog (Arcrica ishindica) was estimated with underwater photography and a hydraulic dredge at 15 ni depth in Onundarfjordur. NW Iceland. Abundance estimates based on counts of siphons from the underwater photographs were much higher than from analysis of the dredge catches. A total of seven taxa were identified from photographs, of which only ocean quahogs and brittle stars were found m sufficient abundance for further analysis. The large scale spatial distribution of ocean quahogs and brittle stars was contagious, where the data corresponded well with a negative binomial distribution but differed significantly from a Poisson distribution. The Morisita index of dispersion was used to analyze distribution patterns within each photograph. These analyses revealed that even on small spatial scales the distribution of ocean quahog could be contagious. A.£}' WORDS: ocean quahog. distribution, abundance, underwater camera, hydraulic dredge. ArcUca islaiulica INTRODUCTION The ocean quahog, Arcticii islciiulica, is an important commer- cial species in Icelandic (Thorarinsdottir & Einarsson 1996) and US waters (Kennish & Lutz 1995). Stock assessments of ocean quahog have generally been performed using hydraulic dredges. However, accurate quantitative abundance estimates cannot be provided if the efficiency of the dredge is not known. Besides, efficiency may vary with environmental conditions and between different types of hydraulic dredges (Eleftheriou & Holme 1984, Kennish & Lutz 199.5. Anonymous 1998). Abundance of ocean quahog has also been estimated quantitatively from grab samples (Amtz & Weber 1970. de Wilde et al. 1986) but because of the small surface area the grab collects, relative to the low abundance of ocean quahogs in sediments, this laborious method requires a very large sample size to obtain reasonable quantitative estimates. Many studies have been performed in the last decade using underwater photographic techniques to estimate abundance of epi- faunal bivalves, such as scallops (Langton & Robinson 1990. Stokesbury & Himmelman 1993. Goshima & Fujiwara 1994) and deep burrowing crustaceans, based on counts of burrows (Chap- man et al. 1975. Hughes & Atkinson 1997). However, to our knowledge only one photographic study has estimated abundance of infaunal bivalves (Wigley & Theroux 1970). Estimation of abundance from photographs is more difficult for infaunal bivalves than for epifaunal bivalves because identification depends solely on siphon characteristics and the protruding parts of the shell. However, siphon characteristics can be very distinctive and infau- nal bivalves have been identified to species and genus level by using these features (Siferd & Welch 1992). The stock size of ocean quahogs off Iceland has been estimated using a hydraulic dredge (EiriTcsson 1988, Thorarinsdottir & Einarsson 1996). The stock size in Onundarfjordur NW Iceland was assessed by this method in 1994 (Thorarinsdottir & Einarsson 1996). Assessments conducted with dredges provide only infor- mation about the larger scale (>10 m") distribution patterns of ocean quahogs. However, variation in distribution patterns may occur on much smaller spatial scales (O.I-IO m"^). At these scales, distribution of ocean quahogs could be influenced by small scale variation in sediment grain size, bottom topography, and the pres- ence of other infaunal bioturbators. *Corresponding author. E-mail: steara@hafro.is Assessments of ocean quahog stocks using underwater photog- raphy has the clear advantage that quantitative information on abundance of ocean quahogs can be assessed more rapidly and cheaply compared with surveys conducted with hydraulic dredges. The aim of this study was to assess whether underwater photog- raphy can be used to estimate the abundance of ocean quahogs and secondly to assess how reliable these estimates are by comparing them with estimates obtained with hydraulic dredge within the same area. Furthermore, large and small scale distribution patterns of ocean quahogs and brittle stars were investigated. MATERIALS AND METHODS The study was performed in July 1999 in Onundarfjordur NW Iceland (66°02'N-23°34'W) at a depth of 15 m. A 35 mm verti- cally oriented underwater camera (Photosea lOOOA) loaded with a roll of 10 m (250 exposures) of 200 ASA filtn (KODAK) and an obliquely oriented 150 watt strobe (Photosea I500S) were placed on a steel frame. A videocamera directly connected to a monitor was also attached to the frame. While the vessel was drifting, the frame was repeatedly (at 3()-sec intervals) lowered until the trigger weight touched the bottom, activating the camera and the flash to take a photograph (at a speed of l/lOO sec). Because the flash from the strobe could be clearly seen in the video monitor, the camera equipment was hauled up =2 m immediately after each photograph was taken. Photographs were taken at a constant distance (0.9 m) from the bottom (the distance from the camera to the trigger weight) and therefore the area of seabed covered in each photo- graph was always the same (0.46 m"). The position of the start and the end of each photographic transect was located by a global positioning system. To analyze the photographs, they were displayed using a slide projector onto a board ( 144 cm x 100.5 cm), the size of the dis- played image matching the size of the board. The board was di- vided into 24 squares (i.e., sampling units), each of 0.019 m" and all animals on the photographs were counted and identified to the lowest taxonomic level possible. To estimate numbers of ocean quahogs per photograph, all siphons were counted. The siphon of the ocean quahog is very short and consists of inhalant and exhal- ant apertures, which lie close to each other on the inner fold of the mantle. These are the only parts of the bivalve that can be seen on the sediment surface (Fig. 1 ). Only two species were found in sufficient abundance for sta- tistical analysis, ocean quahogs (Arcticci islaiulica) and brittle stars 673 674 Ragnarsson and Thorarinsdottir Figure 1. Underwater phulugruph of the seabed showing siphons of ocean quahogs. (Ophiiiroidea). The large scale distributions of the ocean quahog and brittle stars were compared witli Poisson (random) and nega- tive binomial (contagious) distributions using a chi-square test (Elliot 1977). To calculate the goodness-of-fit (chi-square) of the Poisson distribution, only variance and average abundance statis- tics are required. The negative binomial distribution is based on two parameters, arithmetic mean and the exponent k, which is related to the spatial distribution, and is calculated iteratively using the maximum-likelihood equation (Elliot 1977). In the negative binomial distribution, the variance is greater than the mean, indi- cating that the distribution of individuals is patchy, whereas if it is the same as the mean, the distribution is random (Poisson). The significance of departures from random and negative binomial distributions were tested using chi-square tests. The extent of con- tagion in the distribution of ocean quahog was furthermore as- sessed using the variance to mean ratio (Elliot 1977). The abundance of ocean quahogs was in general sufficiently high to allow analysis of small-scale distribution patterns within each photograph using the Morisita index of dispersion (Elliot 1977). This index is ideal for this analysis because it is indepen- dent of the mean and total number of individuals per photograph and the number of sampling units (i.e., photographic squares) was always the same. The index is greater than one for a contagious distribution, less than one for a regular distribution, and equal to one for a random distribution. Data for analysis were based on abundance of individuals within each of the 24 squares of the photograph. Photographs with less than 20 individuals were not considered suitable for this analysis. A single cage hydraulic dredge was used to make a compara- tive investigation of the abundance of ocean quahogs in the same area where the underwater photography took place. The dredge had an overall dimension of 590 x 103 x 230 cm. with a 150 cm wide cutting blade. The bar spacing in the dredge was 34 mm. To determine ocean quahog densities, the distance covered by the dredge was calculated. Ocean quahogs from the catch were weighed, and the total catch weight was divided by the size of the area covered in the tow (5 min duration) to give biomass caught in kg m"". Individuals in a subsample of 25 kg were counted, mea- sured and weighed. The towing speed was 2.4 nautical miles per hour. RESULTS Six invertebrate taxa were identified from a total of 180 seabed photographs. Of these only ocean quahogs [Aixlica isUimlicu) and brittle stars (Ophiuroidea) were found in high numbers (Table 1). The brittle stars could not be identified down to species level from the photographs. The remaining species, Asterias rubens (starfish), Biiccinum undatum (whelk), Cucttmaria frondosa (sea cucumber), and Hyas araneus (spider crab) were all found with mean abun- dance less than O.I m"". Ocean quahog was present in 97% of the photographs (max = 148 m~"; Fig. 2a) and brittle stars in 71% of photographs (max = 252 m~~; Fig. 2b). The large scale distribu- tion patterns of ocean quahogs and brittle stars agreed with the negative binomial distribution but differed significantly from the Poisson distribution (Table I ). Furthermore, the variance-to-mean ratio exceeded unity for both ocean quahog and brittle stars (Table I ). The findings from both these analyses suggest that the large scale distribution of ocean quahogs and brittle stars were patchy. The Morisita index of dispersion ranged greatly between pho- tographs. In 90% of photographs, the Morisita index ranged from 0.68 to 1.62 for ocean quahogs and from 0.46 to 2.58 for brittle stars. These results indicate that dispersion patterns within photo- graphs can be very variable i.e., in some photographs the disper- sion patterns of ocean quahogs were highly contagious whereas in others more regular (Fig. 3). The estimated mean biomass of ocean quahogs in the dredge catch was 1.6 kg m"". equivalent to 14 indiv. m"- (mean live weight of an individual caught by the dredge was 1 18 g). Because of the selectivity of the hydraulic dredge, 85% of the clams caught ranged in shell length between 70 to 95 mm, the mean shell length was 82 mm, and no clams <30 mm were observed. Ocean quahog was the only bivalve species found in the dredge samples. DISCUSSION This study shows that underwater photography can be used to estimate the abundance of infaunal bivalves, although it may be limited to areas dominated by a single species because identifica- tion depends solely on siphon characteristics. Bivalves of size comparable to ocean quahog have not been found in Onundarf- TABLE 1. Analysis of the distribution patterns of ocean quahogs and brittle stars. Poisson Negative Binomial Mean V/M df (n = 1) Chi-square df k Chi-square Ocean quahog Briltle star .«;2.s 43..'^ 13.5 28.9 177 128 1343.64* 4682.8* 4 5 3.68 l.OS 6.74 10.37 The goodness of fit of the observed frequency (Jistrihiitioiis compared wilh the negative binomial distribution and the Poisson distribution were assessed with the chi-square test. * P < 0.005. mean, mean number of individuals nr'; V/M. variance to mean ratio. Abundance of Ocean Quahog 675 Ocean quahog CM E 0) E 3 0 20 40 60 80 100 120 140 160 180 Brittle star ':W^UjiyL 0 20 40 60 80 100 120 140 160 180 Photograph frame number Figure 2. Abundance of ocean quahogs (a) and brittle stars (bl. per each successive photographic frame. jorSur in previous surveys (Thorarinsdottir & Einarsson 1996). suggesting that all siphons seen on the photographs belonged to ocean quahog. Several smaller sized species have been recorded in Onundarfj6r9ur (Gardarsson et al. 1980) but the siphons of these are likely to be too small to be seen on the photographs. The abundance estimates of ocean quahog based on dredge catch (14 m"~) were about four times lower than based on the photographs (53 m""). The proportion of individuals (i.e.. effi- ciency) caught by the dredge relative to the total number of indi- viduals in sediments (based on counts from photographs! was therefore 21%. Although this can be regarded as a crude method to calculate efficiency, Thorarinsdottir and Ragnarsson (2001) ob- tained a very similar estimate (19%) using the same hydraulic dredge in a different area. Dredge efficiency may vary with sediment type, current speed, the ratio warp length:water depth, towing speed, hydraulic pres- sure of the jet, vertical distribution of the clams, and the size of the dredge. This may explain why efficiency estimates can differ so greatly between different studies (Medcof & Caddy 1971. Anony- mous 1998). Ocean quahogs are known to periodically burrow deeper in sediments until the siphons can no longer be seen on the sediment surface (Taylor 1976). Prolonged absence of food (e.g.. during winter) has been suggested as one of the factors thai triggers them to bury deeper in sediments (Oeschger 1990). Ocean quahogs are able to reduce their metabolic rate to 1% of the aerobic rate, enabling them to remain deeply buried without feeding over long periods of time (Theede et al. 1969, Oeschger 1990). Clearly, if a large proportion of the ocean quahogs are deeply buried, stock assessment with underwater photography or hydraulic dredges will always underestimate abundance. Recent evidence suggests that the vertical movements of ocean quahogs within sediments are seasonal in Icelandic waters. Si- phons were seen on the sediment surface from April to September 2000 but not between the period of November 2000 to February 2001 (Thorarinsdottir, unpublished data). These findings suggest that ocean quahogs can burrow to greater depths during winter but may emerge to the sediment surface and start feeding when sea- water temperature rises and the levels of phytoplankton biomass X 0) ■D C 2.0 - 1.5 ^ 1.0 0.5 Ocean quahogs Brittle stars Figure 3. Small-scale distribution patterns (within each photograph) of ocean quahogs and brittle stars assessed with the Morisita index of dispersion. The box plot shows the range in the distribution of the Morisita index in all photographs with more than 20 individuals. The box and whiskers represents 50 and SO'/r of the data, respectively. Mean (dotted line) and median (solid line) are shown as well. increases during summer (Thordardtittir & Eydal 1996). If this proposition remains true, this may explain why catches of ocean quahog per hour towed tend to be much lower in winter than in summer as the catchability of this bivalve is likely to be lower while deeply burrowed in sediments (Thorarinsdottir unpublished data). It is therefore likely that only a small proportion of indi- viduals were deeply burrowed in sediments when this study was performed. The present study demonstrates that ocean quahog and the brittle stars were contagiously distributed. Analysis of the distri- bution patterns within each individual photograph revealed that even on such small scales, the distribution of ocean quahogs and brittle stars can be highly contagious. In some photographs this was caused by the presence of physical features such as stones, which limited their distribution, whereas on other photographs, the underlying reasons for an aggregated distribution were not clear. Patchy distribution of brittle stars is a common feature widely reported in other studies (e.g.. Warner 1971, Piepenburg & Juterzenka 1994). Density-dependent effects (Fujita & Ohta 1990. Summers & Nybakken 2000). predation pressure (Aronson 1989). and local heterogeneity in environmental conditions (Summers & Nybakken 2000) have been suggested to explain contagion of brittle stars. Stock assessments performed with underwater photographic techniques may provide more accurate quantitative estimates of ocean quahog abundance than using dredges. However, such in- vestigations performed during winter months may greatly under- estimate the abundance of ocean quahogs because a large propor- tion of individuals may be buried deeply in sediments. Further- more, the small surface area covered by each photograph requires a relatively large sample size to obtain reasonable quantitative estimates. This method is not without limitations and may only be useful in areas where no large infaunal bivalve other than ocean quahog occurs, as identification depends solely on siphon charac- teristics. Future research should focus on investigating those factors that can influence stock assessment of ocean quahog such as vertical movement in sediments and the efficiency of hydraulic dredges. ACKNOWLEDGMENTS We would like to thank Karl Gunnarsson. Elena Guijarro Gar- cia, James Begley, Loma Taylor, and Astj^or Gi'slason for provid- ing comments that improved this manuscript and Loma Taylor for her help with the statistics. We also want to thank Elena for her assistance in the field and the crews of the vessels Stundvis and Skel IS. 676 Ragnarsson and Thorarinsdottir LITERATURE CITED Anonymous. 1998. 27"' Northeast Regional Stock Assessment Workshop (27'" SAW). Northeast. Fish. Sci. Center. Ref. Doc. 98-15. Amtz. V. W. E. & W. Weber 1970. Cyprina islandica L. 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Arctica islandica. using a hydraulic dredge and underwater photography. ICES CM 2001/P:24. Warner. G. F. 1 97 1 . On the ecology of a dense bed of the brittle-star Ophiotrrx fragdis. J. Mar. Biol. Assoc. U.K. 51:267-282. Wigley, R. L. & R. B. Theroux. 1970. Sea-bottom photographs and mac- rohenthos from the continental shelf off Massachusetts. US Dept of the Interior, US. Fish and Wildl. Sen: Rep. no. 613. de Wilde. P. A. W. J.. E. M. Berghuis & A. Kok. 1986, Biomass and activity of benthic fauna on the Fladen ground (Northern North Sea). Nelh. J. Sea. Res. 20:313-323. Journal of Slwllfhh Research. Vol. 21, No. 2. 677-684, 2002. TETRAPLOID INDUCTION BY MEIOSIS INHIBITION WITH CYTOCHALASIN B IN THE DWARF SURFCLAM, MULINIA LATERALIS SAY: EFFECTS OF TEMPERATURE STEFANO PERUZZI AND XIMING GUO* Haskin Shellfish Research Ltiboralory. Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue. Port Norris, New Jersey 08349 ABSTRACT Tetraploidy, which is useful for the production of all-triploids. can be induced in marine hival\es by manipulating meiosis. In this study, we tested the effects of temperature on cytochalasin B (CB)-induced meiosis inhibition in a model bivalve, the dwarf surfclam, Mulinia lateralis Say. Newly fertilized eggs of the dwarf surfclam were treated with CB (0.75 mg/L) at proper times to block the release of polar body I ( PB 1 ) or polar bodies I and 11 ( PB 1 &2) at 19'C, 24°C, and 28'C. Inhibition of PB I produced largely triploid and aneuploid-tetraploid (3.7n to 3.8n) larvae, and sometimes small percentages of pentaploids. Inhibition of PB1&2 produced primarily pentaploids and occasionally triploids and aneuploid-tetraploids. Most of the larvae with ploidy levels higher than triploid developed as trochophores only and died a few days after fertilization. At Day 25 post-fertilization, four tetraploids (29!-) were detected among 196 juvenile clams in one of the eight PB1&2 groups. No tetraploids were observed in the eight PBl groups produced. In general, higher (28'C) temperatures did not improve tetraploid production measured as survival to juvenile stage. Low temperature (19'C) made CB treatment less effective, producing all diploid juveniles by Day 17. This work indicates that tetraploidy can be tolerated in M. lateralis and scope for further work on this species therefore exists. KEY WORDS: tetraploidy, triploidy. cytochalasin, temperature, clam. Mulinia lateralis INTRODUCTION Tetraploid induction in bivalves has been widely investigated because of its potential to provide an ideal approach to triploid production. All-triploid Pacific oysters have been produced by mating tetraploids and diploids (Guo et al. 1996). Triploids are of interest in shellfish aquaculture for their superior growth and itn- proved meat quality. Since the original work on triploidy by Stan- ley et al. ( 1981 ) with the American oyster (Crassostrea virginica). triploids have been produced in over 20 bivalves by manipulation of polar body I (PBI) and polar body 2 (PB2) as reviewed by Beaumont and Fairbrother ( 1991 ) and Guo (1999). Viable tetraploids have been produced with variable success in fish (reviewed by Pandian & Koteeswaran 1998). and in amphib- ians (Humphrey & Fanhkauser 1949, Fischberg 1958. Reinschmidt et al. 1979. Nishioka & Ueda 1983). In bivalves, tetraploid larvae have been produced by several methods, including meiotic and mitotic inhibition, meiotic inhibition of gynogenetically activated eggs, and cell fusion (Beaumont & Fairbrother 1991. Guo 1991). However, tetraploid embryos rarely survived beyond metamorpho- sis in bivalves. In the Pacific oyster Crassostrea gigas. tetraploid gynogenesis produced high percentages (95*^) of tetraploid em- bryos, but no viable juveniles were later obtained (Guo et al. 1993), Similar results were obtained by inhibition of mitosis I (Guo et al. 1994), inhibition of PBl (Stephens 1989, Guo et al. 1992a. 1992b). and blastomere fusion (Guo et al. 1994). There are probably many other failed attempts that have not been reported. A small number of viable tetraploids have been obtained by manipulating meiosis in blue mussel (Scarpa et al. 1993). Manila clam (Allen et al. 1994). and zhikong scallop (Yang et al. 2000). These sporadic successes have been difficult to reproduce and no breeding populations of tetraploids have been established by ma- nipulating meiosis in eggs from diploids. A different method of tetraploid induction was developed in the Pacific oyster in 1993, when inhibiting PBl in eggs from triploids produced 2000 viable tetraploids (Guo and Allen 1994). This method produced small *Corre.sponding author. E-mail: xguo@hsrl.rutgers.edu nuinbers of tetraploids in the pearl oyster Pinctada martensii (He et al. 2000) and the eastern oyster (Supan et al. 2000). Large numbers of tetraploid spat were subsequently obtained in the Pa- cific (Eudeline et al. 2000) and eastern (Guo et al. 2002) oysters, demonstrating the reproducibility of the Guo-Allen method. Al- though the Guo-Allen method is reproducible, it is limited to species in which triploids produce significant numbers of eggs. Therefore, effective methods for direct induction of tetraploids from diploids are needed in some species. In the search of new candidate species for tetraploid induction and of conditions that might favor its production, we examined the suppression of PBl alone and both PBI&2 in the dwarf surfclam. Mulinia lateralis, by use of cytochalasin B (CB) at different tem- peratures. Different levels of tetraploids have been produced by inhibiting PBl at different temperatures in the Pacific oyster (Stephens 1989. Guo 1991 ). We expect that higher or lower treat- ment temperatures might affect cellular properties and chromo- some segregation patterns, possibly enhancing the effectiveness of the chemical and finally improve survival of tetraploids. Several characteristic of the dwarf surfclam make it suitable as model for genetic investigations in inarine bivalves: a short gen- eration time, a gametogenic activity that occurs over much of the year under cultured conditions, a relatively high reproductive rate, a sex differentiation easily discernible through the shell of sexually ripe specimens, easy conditioning, and small space requirements (Calabrese 1969), MATERIALS AND METHODS Broodstock and Gamete Collection Adult M. lateralis were collected from wild populations of Rhode Island, They were conditioned in recirculating seawater at 20°C (salinity 30 ppt) for 2-4 wk before use. During this period, they were fed daily with Isochiysis fitdhana (C-ISO) at densities of 80-100.000 cells/niL. For natural spawning, ripe clams were se- lected, air-dried for 1 h and induced to spawn in individual beakers containing filtered (1 |j,m) and UV-sterilized seawater at 24°C. Clams that did not respond to thermal stimulation were dissected 677 678 Peruzzi and Guo for gamete collection. Eggs were passed through a lOO-fxm-nytex screen to remove any tissue debris, collected, and rinsed on a 20-jjim screen. Sperm were passed through a 20-|i,m screen, and their motility was checked under microscopy before fertilization. The total number of eggs per female was estimated under micro- scope by counting small aliquots, and the eggs checked for spon- taneous development or possible contamination. Artificial Fertilization, Chemical, and Thermal Treatments Sperm were added to egg suspension at about 3-10 sperm/egg. Fertilization and incubation were conducted using filtered seawa- ter with a salinity of .^0 ppt at 19-28°C according to the experi- mental design. Fertilized eggs were treated with CB. dissolved in dimethyl- sulfoxide (DMSO) at a final concentration of 0.75 mg/L. All CB treatments started approximately at 5-7 min post-fertilization (PF). The control group was used to gauge the timing of the treatment at all times. For PBl inhibition, treatments lasted approximately 15- 20 inin or until about 75'7f of the untreated eggs released PBl in the control group. For the retention of both PB1&2, CB treatments were extended for an additional period of 15-20 min, or until the majority of untreated eggs in the control group were ready for the first mitosis (as indicated by the formation of the first polar lobe). After each treatment, the eggs were separated from the chemical by passing them onto a 20-|xm screen and gently rinsed with 0.5% DMSO in seawater. Finally, they were suspended in fresh seawater and left undisturbed. Experimental Design Experiments were performed using pooled eggs from 4-6 fe- males and pooled sperm from three males. Soon after fertilization the gametes were divided into three groups: in the first group, fertilized eggs were allowed to develop as controls, and in the second and third group the eggs were treated with CB to block PB 1 or both PB1&2. Treatments were tested at normal (24°C). high (28-C). and low ( 19°C) temperatures. All experiments were replicated three times except for the experiment at low temperature that was conducted twice. Control groups were exposed to DMSO only at the same temperature as the treated groups and for as long as the longest treatment lasted. Lanal Culture and Growth-Out Larvae were reared in 20-L buckets at 22-24°C and fed daily a diet of Isocliiysis galbana at densities of 100-120,000 cells/mL starting at 24 h PF. Larvae were cultured at a maximum density of 20 larvae/mL and reached metamorphosis in approximately 8-10 days at a shell length of 180-250 jjim. Culture water was com- pletely changed every 48 h, and larvae were collected and washed gently on screens of proper size. D-stage larvae were separated from trochophores by using a 44-jjim screen placed on top of a 25-|j.m screen. At Day 2, the percentages of development (D- larvae and/or trochophores) were calculated from at least 100 in- dividuals per group. Larval survival was determined for all groups at each water change. After metamorphosis, M. lateralis specimens were cultured in upwellers and then in trays contained in a well-aerated recirculat- ing seawater system at a temperature of 19-20°C. Changes of culture water (approximately 1/5 of total volume) were performed every other day. Determination of Ploidy Ploidy status of larvae (Day 1-14 PF) and ju\eniles (Day 17- 55 PF) was determined by flow cytometry with DAPI staining according to Allen and Bushek (1992). For larvae, several hundred were sampled after concentrating on a nytex screen and mixed with a DAPI/DMSO stain (Guo et al. 1993). Samples were kept frozen at -80°C and thawed to room temperature before analysis. They were vortexed, passed three times through a 26-gauge needle, and then filtered through a 25-|jim-mesh filter. Larvae were analyzed in pooled samples (/; = 500-1000). whereas juveniles (/; = 100) were analyzed individually. Juvenile samples were pre- pared by mincing the whole body in a 1.5-mL test tube. Haploid sperm and diploid cells from untreated clams were used as stan- dards at all times. Statistical Analysis Fertilization level was determined as percentage of divided eggs at 2— f h PF. Cumulative survival at Day 2 (D-stage), Day 7. Day 17. and to juvenile was calculated relative to the number of fertilized eggs. Statistical analyses were performed using SYSTAT 10 (SYSTAT Inc.). Percentage data for fertilization and survival were arcsine-transformed and analyzed by ANOVA followed by Tukey's HSD post-hoc comparison. All differences were accepted as significant when P < 0.05. RESULTS Meiotic and Mitotic Events At temperatures of 24°C. control eggs started to release PB 1 around 12 to 15 min PF, and PB2 at 30-35 min PF. At higher (28°C) or lower (19°C) temperatures, timing was respectively shortened or delayed by 10-15 min. Nevertheless timing of PBl and PB2 release varied among replicates according to egg quality. In CB-treated groups, no release of PBs was observed during treatments, and meiotic events were restored 3 to 5 min after complete removal of the chemical. Untreated eggs reached mitosis 40-50 min PF except at low temperatures ( 19°C), where eggs were slightly retarded in development and started cleaving at 55-65 min PF. As expected, mitotic events in CB-treated eggs, particularly in PBI&2 groups, were delayed compared with their control. Survival and Development Treatment temperatures did not significantly affect fertilization level within any of the experimental groups, and there was no difference among treated or control groups at any given tempera- ture. Overall, fertilization level ranged between 75% and 99% and varied among replicates (Table 1 ). At Day 2, no differences in survival were found between con- trol and treated groups at 24°C or 28°C. At 19°C. the percentage development in the PB1&2 group was 21%. which was signifi- cantly lower than in control (53%. P = 0.009) and PBl (51%. P = 0.01) groups. At 28°C, eggs in most of the PB1&2 groups survived as trochophores only and did not develop any further, unlike most of the control or PBl groups where variable propor- tions of D-larvae (ainong all larvae) were found (Table 2). At this temperature, survival in the PBl group was generally low (13- Tetraploid Induction in the Dwarf Surfclam 679 TABLE 1. Inhibition of PBl or PB1&2 in M. lateralis under different temperatures: number of eggs used; fertilization level; percent survival of fertilized eggs to Day 2, 7 and 17; and the number of juvenile obtained. TABLE 2. Inhibition of PBl or PB1&2 in M. lateralis under different temperatures: percentages of D-larvae and trochophores observed in experimental groups at Day 2. Group D-larvae ( % ) Trochophores ( % ) Eggs Fertilization xlOOOl {9c) Day 2 Day 7 {%) Day 17 (%) Juvenile (/I) Group ( Low temperature (19°C) LTl-C 100 0 Low temperature (19X) LT2-C 100 0 LTl-C 88 90 58 35 6 5.000 LTl-PBl 81 19 LT2-C 103 84 49 25 0.7 600 LT2-PB1 84 16 LTl-PBl 86 86 51 -)2 1.6 1,200 LT1-PB1&2 73 27 LT2-PB1 70 89 52 32 0.8 480 LT2-PB1&2 75 25 LTI-PBI&2 136 90 21 10 0.2 300 Normal temperature I24''C) LT2-PB1&2 83 93 21 4 0.2 L50 NTl-C 100 0 Normal temperature (24°C) NT2-C 100 0 NTl-C 251 97 100 63 56 50.000 NT3-C 100 0 NT2-C 278 99 90 20 0 0 NT 1 -PBl 51 49 NT3-C 158 94 87 66 0.2 350 NT2-PB1 74 26 NT 1 -PBl 1,045 94 21 8 4 2,000 NT3-PB1 85 15 NT2-PB1 500 99 98 48 20 8,000 NT1-PB1&2 26 74 NT3-PB1 144 90 40 43 0.6 102 NT2-PB1&2 41 59 NT1-PB1&2 1,105 94 15 -) 0.8 800 NT3-PB1&2 71 27 NT2-PB1&2 405 99 63 24 0.1 300 High temperature (2 8°C) NT3-PBI&2 194 89 40 5 0.3 104 HTl-C 0 100 High temperature (28°C) HT2-C 22 78 HTl-C 323 91 S" 0 0 0 HT3-C 92 8 HT2-C 113 84 29 0.8 0.3 218 HTl-PBl 25 75 HT3-C 265 94 69 53 4 1.000 HT2-PB1 23 77 HTl-PBl 420 89 13 0.7 0.3 200 HT3-PB1 35 65 HT2-PB1 91 84 34 1 0.1 59 HTI-PB1&2 17 83 HT3-PBI 163 91 15 3 0.3 78 HT2-PB1&2 0 100 HT1-PB1&2 280 156 90 75 IS" 0 0 0 0 0 0 HT3-PB1&2 0 100 HT2-PB1&2 HT3-PB1&2 182 93 4" 0 0 0 ' Trochophores only. 34%) and extremely variable in the control {5-69'7c). In one con- trol group (HTl-C). all the larvae developed as trochophores only. Overall, percentages of D-larvae at 24°C were significantly higher in control groups (100%) than in PBl (70%, P = 0.01) and PB1&2 (46%, P = 0.002) groups, whereas no differences were found between treated groups. At lower temperatures (19°C), again control groups had higher percentages of D-larvae (100%) than PBl (82%, P = 0.001) or PBI&2 (74%. P < 0.001) groups. D-larvae percentages were not analyzed for the high- temperature experiment where 100% trochophores were observed in all PB1&2 groups and one control. Trochophores in the CB- treated groups appeared to be deformed and/or swam in a cir- cular motion. Larvae in all groups metamorphosed between Day 7 and Day 10. At Day 7 and until Day 17. survival of larvae did not differ among treated and control groups at any temperature and varied among replicates (Table 1 ). Percentage survival to juvenile was generally low within the high-temperature groups, averaging 2.2% and 0.2% in the control and PBl groups, respectively. An unex- pected and complete mortality was encountered after Day 7 in one of the control groups (NT2-C). In general, the number of surviving juvenile and juvenile clams (Days 16-54) varied greatly among replicates but equally among groups (Table 1 ). Ploidy of Larvae and Juveniles Ploidy of larvae was analyzed on Day 1 and Day 2 to detect possible difference due to differential mortality. No differences in ploidy composition were found between Day 1 and Day 2, and only data from Day 2 are presented. Ploidy of 2-day-old larvae from treated groups varied greatly among treatments and replicates, ranging from diploidy to penta- ploidy (Fig. lA-D). In particular. PBl inhibition produced vari- able proportions of triploid. aneuploid-tetraploid and some penta- ploid larvae (Fig. lA and B), whereas pentaploids were most com- monly observed and dominant ploidy in PB1&2 groups. The aneuploid-tetraploid peaks were between 3.6-3.8n, sometimes overlap with 4n. One PB1&2 group had distinctive aneuploid (3.7n) and pentaploid peaks (Fig. IC), whereas others had clear diploid, triploid, tetraploid and pentaploid peaks (Fig. ID). After separation of D-stage larvae and trochophores, the flow cytometric analysis of isolated larvae, showed that all or nearly all tetraploid and pentaploid larvae developed as trochophores only (Fig. 2B and D), whereas virtually none or small proportions (Fig. 2C and E) were detected among the D-stage larvae. Control groups from treatments at 19°C and 24°C contained diploid larvae only, unlike controls at 28"C that showed variable proportions of diploid, triploid and pentaploid larvae in two of the three rep- licates (HTl and HT2). indicating some influence of tempera- ture on meiotic events. In general, there was no clear pattern that higher or lower temperatures affected the proportions of poly- 680 Peruzzi and Guo 300 ■ count 1 ^" 2*0 ■ 2n ll JOO ■ llll 160 ■ ll i too \ i 1 [ 1 50 - Q ■ lll>ll»mw>H II 11*11*11 i-iM^Ha ^ ^Si 1 Figure 2. Flow cytometry analysis of 2-day old Mulinia larvae from I'B1&2 inhibition: A, a sample containing both trochophores and D-larvae; B, trochophores only; C: D-stage larvae only; D: a representative abnormal trochophore; and E: a representative D-stage larva. 682 Peruzzi and Guo tion that tetraploid oysters may revert to triploids or triploid/ tetraploid mosaics (Guo et al. 2002). However, reversion is thouglit to be rare event in triploid and tetraploid oysters (Allen et al. 1997). Secondly, it is possible and even likely that the CB treatment for the inhibition of PBI&2 was not 100% effective and that only PBl was inhibited in some of the eggs (Guo et al. 1992b, Scarpa et al. 1993). In fact, treatments targeting specific meiotic events are rarely 100% effective because of unsynchronized de- velopment of zygotes, which is why triploid induction is rarely 100% effective (Lu 1986. Allen et al. 1989). Therefore, the tetra- ploids we obtained might be from accidental inhibition of PB 1 alone. Interestingly, however, treatments specifically targeting PBl did not produce viable tetraploids in this study. Inhibition of PBl is known to result in a complex segregation pattern that leads to the production of triploids, tetraploids and variable proportions of aneuploids larvae (Guo et al. 1992a, 1992b). These findings were later confirmed in zhikong scallop by Yang et al. (2000). Our study showed that PBl inhibition had similar effects producing a comparable spectrum of nuclear DNA content in 2-day-old trochophores and D-larvae from CB-treated eggs. Triploids and aneuploids were most commonly observed unlike tetraploids that were rarely detected by flow cytometric analysis. The pentaploid larvae were most likely produced from the incidental inhibition of both PBl and PB2. Although their ploidy status was not confirmed by chromosome counting, the putative aneuploids (approx. 3.7n) were either hypertriploid or hypotetraploid and possibly produced through tripolar segregation patterns under CB treatment (Guo et al. 1992b). Aneuploids were not found among control groups. Variable proportions of aneup- loids were also observed by tlow cytometry after PB 1 inhibition in zhikong scallop (Yang et al. 2000) and confirmed by chromosome counting. Similar results were obtained on other mollusks by pre- vious workers (Guo et al. 1992a, 1992b, Scarpa et al. 1993, Allen et al. 1994). Our results show that the higher ploidy levels observed by flow cytometry were mainly represented by populations of tro- chophores, which declined rapidly over the same period. Clear- ly, few if any tetraploid embryos survived to D-stage in most groups. The proportion of aneuploids and pentaploids declined abruptly during the first week, and only diploids and triploids were detected among the surviving juvenile. A similar decrease in the proportion of pentaploids and increase in triploids in CB groups was observed in other studies (Scarpa et al. 1993, Guo & Allen 1994). Generally, aneuploidy probably causes imbalance of gene dosage and is often lethal in mollusks with survival de- pending on specific tolerance to chromosome loss or gain (Guo & Allen 1994, Wang et al. 1999). The failure of tetraploid and pentaploid larvae to survive beyond early developmental stages and metamorphosis has been explained by the unbalanced cytoplasm/nucleus ratios or the cell-number (egg-volume) defi- ciency hypothesis (Guo 1991, Guo & Allen 1994, Guo et al. 1994). Guo's hypothesis states that the cleavage of eggs of a given size with large tetraploid (or pentaploid) nuclei results in reduc- tions in cytoplasm/nucleus ratio or cell number, both fatal for further development in bivalves. Accordingly, high percentages or large numbers of tetraploids (up to 100%) have been pro- duced using larger eggs from triploids in the Pacific (Guo & Allen 1994, Eudeline et al. 2000) and eastern (Guo et al. 2002) oysters. Supan et al. (2000) also reported the production of tetraploid eastern oysters, but specific numbers and percentages were not available. Under our experimental conditions, high or low temperature did not improve tetraploid induction as measured by viable tetra- ploids produced. Effects on ploidy of early larvae were not clear partly because of the tremendous variation within treatments. The presence of large proportions of aneuploids made ploidy determi- nation by flow cytometry difficult. We did not do chromosome counting in this study and had no accurate estimates what chro- mosome numbers were actually induced under different tempera- tures. Nevertheless, ploidy of surviving juveniles indicates that high (28°C) and normal (24°C) temperatures produced similar percentages of triploids and that low ( 19'C) temperature made CB treatment ineffective. Low temperatures did not produce viable polyploids in this study. This result would suggest that a lower temperature might influence the cellular properties and chromo- some segregation patterns in this species. In a previous study in the Pacific oyster, high levels of tetraploids were produced under low temperatures (Stephens 1989). High temperatures (28 C) nega- tively affected the development of larvae deriving from eggs treated to block both PB1&2 and finally provoked complete inor- tality. It is likely that this result was induced by a combination of long chemical treatment and high temperature. Generally, control groups exposed at the same temperature and for the same duration did produce viable larvae. However, the same temperature was partially effective in blocking PB release and induced 3% triploidy in one control group. Poor egg quality might have favored these TABLE 3. Inhibition (if PBl or PB1&2 in M. lateralis under different temperatures: age (in days), number, and ploidy composition of Mulinia juveniles as determined b) flow cytometrj. Age Juvenile 2N 3N 4N Group (days) in) (%> (%) (%) Low temperature (19"C) LTI-C 17 100 100 0 0 LT2-C 16 100 100 0 0 LTI-PBl 17 100 100 0 0 LT2-PB1 16 100 100 0 0 LT1-PB1&2 17 100 100 0 0 LT2-PB1&2 16 100 100 0 0 Normal temperature (24°C) NTI-C 25 100 100 0 0 NT2-C na" 0 (1 0 0 NT3-C 27 100 100 0 0 NT 1 -PBl 25 100 84 16 0 NT2-PB1 25 100 64 36 0 NT3-PB1 27 102 100 0 0 NTI-PBI&2 25 1% 89 9 -) NT2-PBlcS:2 25 100 83 17 0 NT3-PB1&2 27 104 99 1 0 High temperature (28°C) HTI-C na 0 0 0 0 HT2-C 54 100 97 3 0 HT3-C 51 100 100 0 0 HTI-PBl 55 44 31 69 0 HT2-PBI 54 59 88 12 0 HT3-PBI 51 50 98 1 0 HT1-PBI&2 na 0 0 0 0 HT2-PBI&2 na 0 0 0 0 HT3-PB1&2 na 0 0 0 0 ' na, not available because of complete mortality. Tetraploid Induction in the Dwarf Surfclam 683 300 count 250 200 150 100 50 B - i ^ iH#w,. j 1 ■■.■'i. -f .Xir-i -t f 50 100 150 DNA contenl Figure. 3. Flow cytometry analysis of 25-day-old Mulinia clam: A, representative juvenile clams analyzed; B: analysis of a diploid (1) and a tetraploid clam (2). conditions too. Heat shocks in the range of 32^0°C are most commonly required in blocking the release of PBs in other mol- lusks (Quillet & Panelay 1986, Yamamoto & Sugarawa 1988, Guo et al. 1994). In summary, this study indicates that temperature has little effects on the final outcome of tetraploid induction in M. lateralis. Temperature affects the liming of meiotic segregation and there- fore should affect tetraploid induction frequency. We could not delect such effects in this study probably due to random variation in treatment and eggs quality. Considering the low induction ef- ficiency (2%), manipulating meiosis may not be a viable approach to tetraploid production in this species. Nevertheless, this study provides the first evidence that tetraploids are viable in M. lateralis and offers encouragement for further research on tetraploidy in this species. ACKNOWLEDGMENT We are grateful to Dr. Huiping Yang for assistance with clam culture and sampling and Dr. Standish K. Allen Jr. for constructive comments. The authors thank Dr. Timothy Scott for providing clam broodstock. This work is supported by a grant from the New Jersey Sea Grant Consortium (R/BT-20()1) and by a grant from New Jersey Commission on Science and Technology's R&D Ex- cellence Program (No. 00-2042-007-20). This is IMCS/NJAES Publication No. 2002-18 and NJSGC No. 02-495. 684 Peruzzi and Guo IJTERATIRE CITED Allen, S. K.. Jr. & D. Bushek. 1992. Large seale proJuLtion ol Iriplmd Crassostrea viri;iinca (Ginelin) using "stripped" gametes. Aquacullure 103:241-251. Allen. S. K.. Jr.. S. L. Downing & K. K. Chew. 1989. Hatchery manual h>r producing tripliiid oysters. 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Induced triploidy m the mussel. Mylilus edulis. by temperature shock. .Aquaculture 72:21-29. Yang. H.. F. Zhang & X. Guo. 2000. Triploid and tetraploid Zhikong scallop. Chlaniys farreri Jones et Preston, produced by inhibiting polar body I. Marine Biotech. 2:466-475. Joiirniil of Shellfish Research. Vol. 21. No. 2. 685-690. 2002. CYTOGENETIC STUDY OF OSTREA CONCHAPHILA (MOLLUSCA: BIVALVIA) AND COMPARATIVE KARYOLOGICAL ANALYSIS WITHIN OSTREINAE ALEXANDRA LEITAO,' - RAQUEL CHAVES," SARA SANTOS," PIERRE BOUDRY,' HENRIQUE GUEDES-PINTO," AND CATHERINE THIRIOT-QUIEVREUX'* ^ Laboratoire de Genetiqiie et Pathologie. Station de I'lnstitut Frangais pour la Recherche et I' Exploitation de la Mer (IFREMER). B.P. 133. 17390 La Tremhlade. France: ~Departamento de Genetica e Biotecnologia. ICETA-UTAD. Vila Real. Portugal: ' Oh.se rvatoi re Oceanologique, Universite Pierre et Marie Curie, Centre National de la Recherche Scientifique. B.P. 28. 06230 Vdlefranche-sur-Mer. France ABSTRACT Chromosome preparations of the Olympia oyster Ostrea conchaphila Carpenter were studied using conventional Giemsa. silver staining, and C-banding techniques. The karyotype consists of six metacentric (1. 2. 4, 6. S. and 10) and four submetacentric (3. 5, 7, 9) chromosome pairs. The silver-stained nucleolus organizer regions (Ag-NORs) were terminally located on the short arms of the submetacentric pair 5 (569f of cases) and on the long arms of submetacentric pair 7 (6% of cases). Constitutive heterochromatin was observed as telomeric C-bands on the short arm of the NOR-bearing chromosome pair 5 and as centromeric blocks of several chromosome pairs. Comparative analysis of patterns of karyotype, Ag-NORs, and C-bands of this species and of five other flat oysters, Ostrea angasi. O. cliilensis. O. denselamellosa. O. ediiiis. and O. piielchaiui. for which data have been previously published, were performed, allowing the inference of cytota.\onomic relationships within Oslreinae. KEY WORDS: Oslrea conchaphila. cytogenetics. cytota,\onomy. Ostreinae INTRODUCTION Studies on oyster cytogenetics have been performed so far on 26 species of Ostreacea (see Nakaniura 1985, leyama 1990, Thiriot-Quievreux 2002). The first data only concerned chromo- some number and gross morphology (Ahmed & Sparks 1967, Menzel 1968). Later, morphometric measurements of chromo- somes enabled the comparison among karyotypes at the interspe- cific and intraspecific level (e.g., Ladron de Guevara et al. 1996. Li & Havenhand 1997). During the last decade, the development of banding techniques has allowed the fine characterization of indi- vidual chromosomes (e.g.. Leitao et al. 1999a). According to the morphologically based classification of Harry (1985), which is currently used, the family Ostreidae includes three subfamilies, that is, Lophinae, Ostreinae, and Crassostreinae. These oysters are sequential hermaphrodites and contain both broadcast spawners (Crassostreinae) and brooders (Lophinae and Ostreinae). Recent techniques such as inolecular phylogenetic analysis provided novel insights into oyster evolution and system- atics (Littlewood 1994, Jozefowicz & 6 Foighil 1998, 6 Foighil & Taylor 2000). Karyological analysis among cupped oysters, the Crassostreinae (Leitao et al. 1999b). has proven complementary to these approaches and has provided additional evolutionary infer- ences. Among the flat brooding oyster species, the Ostreinae, five species have been previously karyologically investigated: Ostrea edulis (Linne) (Thiriot-Quievreux 1984). O. denselamellosa (Lis- chke) (Insua & Thiriot-Quievreux 1991), O. puekluma (Orbigny) (Insua & Thiriot-Quievreux 1993), O. chilensis (Philippi) (Ladron de Guevara et al. 1994), and O. angasi (Sowerby) (Li & Haven- hand 1997). The Olympia oyster, O. cimchaphila (Carpenter 1857). previ- ously known as O. hirida (Carpenter 1864). has been studied by Ahmed and Sparks (1967) and Ahmed (1973) using squash tech- niques and tentative grouping of chromosomes. Ostrea con- *Conesponding author. E-mail; thinotteobs-vlfr.fr chaplula. native to the western United States and Canada, ranges from the southeast Alaska to Baja California (in tidal channels, estuaries, bays, and sounds). Commercially important in the late 19th century, this species was cultured in the state of Washington until near-collapse of the industry in the 1950s (Baker 1995). In the present work, the karyotype, nucleolus organizer regions (NORs), and constitutive heterochromatin distribution were stud- ied in Ostrea conchaphila (Carpenter 1857) and a comparison with previously published karyological data on the five other flat oyster species mentioned above was performed to analyze cytotaxonomi- cal relationships within Ostreinae. MATERIALS AND METHODS Specimens of the Califomian Olympia oyster Ostrea con- chaphila (GO) were imported from the Pacific Institute (Olympia, WA). Oysters were strictly confined to the quarantine facilities of the IFREMER hatchery of La Tremhlade, Charente-Maritime, France, according to international recommendations. After repro- duction, the progeny (Gl ) used in this experiment was reared in the same quarantine facilities for at least 5 mo before sampling. Whole juvenile animals (ca. 2.5 cm length) were incubated for 7-9 h in a 0.005% solution of colchicine in seawater. The gills were then removed by dissection and treated for 30 min in 0.9% sodium citrate in distilled water. The material was fixed in a freshly prepared mixture of absolute alcohol and acetic acid (3:1 ) with three changes of 20 min each. Fixed pieces of gill from each individual were dissociated in 509J: acetic acid with distilled water solution. The suspension was dropped onto heated slides at 44'C and air-dried (Thiriot-Quievreux & Ayraud 1982). For conventional karyotypes, gill preparations were stained with Giemsa (4%, pH 6.8) for 10 min. The silver-staining method for NORs was performed on unstained slide preparations accord- ing to the procedure of Howell and Black (1980). This method only detects those NORs that were active at the precedent inter- phase (Miller et al. 1976). Chromosomal Ag-NORs can serve as characters for inferring phylogenetic relationship (e.g., Amemiya 685 686 Leitao et al. & Gold. 1990). Constitutive heterochromatin regions (C-bands) were revealed using the method of Sumner ( 1972) with the coun- terstain propidium iodide. The evolutionary significance of the heterochromatin has previously been discussed in vertebrates (e.g.. Hsu & Arrighi 1971. Saffery et al. 1999. Chaves et al. 2000). Images of Giemsa-stained metaphases and C-banding were ac- quired with a CCD camera (Axioplan, ZEISS) coupled to a ZEISS Axioplan microscope. Digital images were processed using Adobe Photoshop 5.0 (Windows) using functions affecting the whole of the image only. Microphotographs of Giemsa stained metaphases and C-banding were taken with a ZEISS Axioplan microscope. Digital images were processed using Adobe photoshop 5.0 (Win- dows). Microphotographs of suitable NOR-stained metaphases were taken with a ZEISS III photomicroscope. After karyotyping, chromosome measurements of 10 suitable metaphases were made with a digitizer table (Summa Sketch II) interfaced with a Macintosh. Data analysis was performed with an Excel macro-program. Relative length was expressed as 100 times the absolute chromosome length (in (xm) divided by the total length of the haploid complement. Centromeric index was calcu- lated by dividing 100 times the length of the short arm by the total chromosome length. The arm ratio was determined (length of short arm divided by length of long arm). Both centromeric index and arm ratio are given because each expresses centromere position and allows comparison with other karyological studies. Terminol- ogy relating to centromere position (m: metacentric, sm: submeta- centric) follows that of Levan et al. (1964). To elucidate similarities between Ostreinae species, a hierar- chical agglomerative flexible clustering program was used (Lance & Williams 1966). Both NOR and centromeric index information of O. conchaphila and five previously studied Ostreinae species were used to cluster species. The Manhattan metric was used to discriminate and then to associate individual species. Manhattan distance appears appropriate to this kind of combination of quan- titative (centromeric index values) and qualitative (NORs posi- tions) data and to measure an association between individual ob- jects (species) (Legendre & Legendre 1998). RESULTS Analysis of 60 mitotic metaphase spreads from 15 individuals of O. ctmchaphila confirmed the diploid chromosome number of In = 20. scored by Ahmed and Sparks (1967). For karyotyping. the chromosomes of 21 well-spread metaphases were paired on the basis of chromosome size and centromere position. From these, the 10 best spreads were used for chromosome measurements and classification (Table 1). The karyotype (Fig. I A) consists of ten chromosome pairs. Pairs I, 2, 4, 6. 8. and 10 were metacentric. Pairs 3. 5. 7. and 9 were submetacentric. The Ag-NORs were examined in another 122 metaphases from 10 animals. A variable number of one to three Ag-NOR chromo- somes were identified (Fig. IB). The NOR site was located ter- minally on the short arms of the submetacentric pair 5 and on the long arms of the submetacentric chromosome pair 7. The most frequent case (56% of observed silver-stained metaphases) was one active silver-.stained NOR chromosome in pair 5. The Ag- NORs located on pair 7 occurred in few cases (6%). Constitutive heterochromatin was observed in 31 karyotypes made from well-spread C-banded metaphases from 13 animals. Telomeric C-bands were always observed on the short arm of the NOR-bearing chromosome pair 5. In addition, centromeric blocks were also found in chromosome pair 2 in 84% of observed metaphases. pairs 1, 4. and 5 in 68%. pairs 6 and 8 in 58% of the C-banded karyotypes and in fewer cases in pairs 3. 7. and 9 (35%), and in pair 10 (26%) (Fig. IC). To compare the karyological data from O. concluiphlki and from the other five flat oyster species previously studied, ideo- grams (Fig. 2) were constructed from relative length and centro- meric index values of O. conchaphila (see Table 1 ). O. ediilis (after Leitao 2000. French population of La Tremblade hatchery. Charentes Maritimes. France), O. aiigusi (after Li & Havenhand 1997). O. chilensis (after Ladron de Guevara et al. 1994), O. clenselamcllosa (after Insua & Thiriot-Quievreux 1991), and O. pnelchana (after Insua & Thiriot-Quievreux 1993). The location of Ag-NORs was also included because chromosomal NOR have been used as characters for inferrmg hypothesis of cytotaxonomic relationships (e.g.. Amemiya & Gold 1990, Leitao et al, 1999 b). The comparison of the relative length and centromeric index of the 10 chromosomes pairs of the studied species showed that pair 1 was similar among all species, pair 2 was also similar except for O. puelchuna, pair 3 and 4 were similar except for O. conchaphila. but taking into account the close relative length and the standard deviation of pair 3 and 4 of O. conchaphila. they may be inverted. Pair 5 was variable among species, pairs 6 and 7 were identical except for O. denselamello.sa. but in this case, the pairs 6 and 7 cannot be inverted because of their different relative length and the TABLE 1. Chromosome measurements and classification in U) cells of Ostrea conchaphila. Chromosome Pair No. Relative Length Arm Ratio Centromeric Index Mean SD Mean SD Mean SD Classification 1 12.77 0.99 2.-W 0.21 42.12 1.78 111 2 1 1 .60 0.35 2..'i4 0.18 43.87 1.89 m 3 10.64 0.57 1.26 0.20 27.76 2.86 sm 4 10.54 0.50 2.37 0.27 41.94 2.81 m 5 10.47 0.87 1.72 0.16 34,05 2.28 sm 6 9.88 0.78 2.48 0.25 42.81 2.67 m 7 9.46 0.38 1.26 0.16 27.56 2.59 sm 8 9.30 0.58 2.53 0.39 43.59 3.92 m 9 8.79 0.63 1.52 0.25 31.27 3.37 sm 10 6.56 0.65 2 4.^ 0.26 42.18 2.60 ni Cytogenetics of Ostrea conchaphila 687 St l\ M H U 1 2 3 4 5 V Ki 10 ■». j^ 3 K>, ¥ ■.>i». ; aK iX 10 y 1 1 y< t^ n 10 Figure 1. Karyotypes of Ostrea conchaphila. A, Conventional Gicmsa staining; B, silver-stained nucleolus organizer regions (Ag-NORs); C, C-banding. Note the simultaneous presence of Ag-NOR and C-bands in a telomeric position on the short arms of pair 5 (arrows) and the centromeric heterochrnmatic blocks on chromosome pairs 1, 2, 4, 5, and 9. Scale bar = 5 )im. C-bandiiig of pair 6. Pair 8 was variable among species. Pair 9 was identical except for O. aiif;asi and pair 10 was variable. A statistical analysis based on Ci and NORs (Fig. 3) high- lighted the clustering of O. ediiUs and O. angasi and of O. dense- lamellosa and O. ehilensis with O. conchaphila placed near this cluster. O. puekluma is separated from the other species by the highest dissimilarity. DISCUSSION This is the first report on karyotype after chromosome mea- surements and NORs and C-banding patterns of the Olympia oys- ter. The diploid chromosome number In = 20 observed is char- acteristic of the genus Ostrea and is common throughout the Os- treacea (Nakamura 1985, Thiriot-Quievreux 2002). The karyotype. 688 Leitao et al. Relative length edulis 120 100 80 60 40 20 7 8 9 10 Chromosome pair Figure 2. Ideograms of six flat oysters constructed from relative length and cenlromeric index values. Stippled chromosome: metacen- tric, white chromosome: suhmetacentric. striped chromosome: suhte- locentric, hiack chromosome: telocentric. Circles indicate Ag-NORs, dark circles the most frequent case. O.den O.Chi O.con O.pue O.edu O.ang Figure 3. Hierarchical agglomerative flexible clustering of Ostrea spp. O. den: Ostrea deiiselamellosa; O. chi: O. chileiisis; O. con: O. con- chaphila: O. pue: O. piiekhana; O. edu: O. edulis; O. ang: O. angasi. including six metacentric and tour sulimetacentric cliromosome pairs, and the NOR and C-band distribution differ from the other ostreinid species studied. The comparison of the relative length and centromeric index of the 10 chromosome pairs of the studied species shows that, if one postulates that shared chromosome pairs with the same relative length and centromeric index may be con- sidered as primitive, pairs I, 3. and 4 are primitive and pairs 5. 8. and 1 0 the most derived. However, these chromosome homologies should be confirmed by other banding techniques. The comparison of karyotypes and location of Ag-NORs among species highlighted first the chromosome similarity be- tween the European species O. edulis and the Australian and New Zealand species O. angasi. already pointed out by Li and Haven- hand (19971. Their karyotypes differ slightly {5m. 5 sm in O. edulis and 5m. .^ sm. 2 st in O. angasi). but the phenomenon of variation in the number of submetacentric and subtelocentric chro- mosomes have been reported in French populations (Thiriot- Quievreux 1984). More striking is that the most frequent Ag-NOR patterns are similar in both species. The isolated karyotype of O. puelchana is remarkable because of the single telocentric chromosome. The occurrence of telocen- tric chromosomes has been only seen in one other species of Os- trcidae. Dendrostrea folium (Lophinae) (leyama 1990). The three other flat oysters bear high karyotype resemblance, that is. .seven metacentric and three submetacentric pairs for O. denselamelosa and O. chilensis and six metacentric and four sub- Cytogenetics of Ostrea conchaphila 689 metacentric pairs for O. conchaphila. Their NOR chromosomal location revealed that there is a higher resemblance between the NOR patterns of O. chilensis and O. conchaphila than between these two species and O. denselameUosa. O. chilensis and O. con- chaphila showed terminally located NORs on the short arms of one chromosome pair and on the long arms of another chromo- some pair. On the contrary, in O. denselamelosa. Ag-NORs were always terminally located on the short arms of chromosome pairs. Data on constitutive heterochromatin distribution only con- cerned three species, O. denselameUosa (Insua & Thiriot- Quievreux 1991), O. angasi (Li & Havenhand 1997), and O. con- chaphila (this study). Centromeric C-bands were observed in chro- mosome pairs 3. 6, 8, 9, and 10 in O. angasi and in pairs 6. 8, 9, and 1 0 in O. denselameUosa. Occasional C-bands were seen on the centromere of pairs 4 and 7 in O. angasi and on telomeres of pairs 3, 5, 6, 8, 9, and 10 in O. denselameUosa. A substantial proportion of the eukaryote genome consists of constitutive heterochromatin. This genomic fraction includes, among other repetitive sequences, satellite DNA. Sequence analysis of these repeats suggests that the sequences are rapidly evolving, and hence they are valuable as evolutionary markers; consequently, constitutive heterochromatin analysis can give insights about the phylogeny relationships of related species (Saffery et al. 1999. Chaves et al. 20001. The ob- servation in O. denselameUosa and O. conchaphila of the simul- taneous presence of Ag-NORs and C-bands on telomeric position in the same chromosome pair, that is, pairs 3 and 8 in O. dense- lameUosa and pair 5 in O. conchaphila. might corroborate the close karyological relationship between these two species noted above. The cytotaxonomic relationships pointed out here are incon- gruent with the morphologically based classification of Harry (1985), who stated that O. chilensis and O. angasi were junior synonymous of O. puelchana in the subgenus Eoslrea of the genus Ostrea and that O. edulis and O. denselameUosa were included in the subgenus Ostrea ss. The species O. liirida was considered as a junior synonymous of O. conchaphila in the genus Ostreola. Li and Havenhand ( 1997) have also previously disagreed with Harry (1985), placing O. angasi as a separate species, very close to O. edulis. Our results show greater congruence with molecular phyloge- netic analyses of the Ostreinae, based on partial mitochondrial 16S rDNA (Jozefowicz & 6 Foighil 1998) and nuclear 28S rDNA (6 Foighil & Taylor 2000) datasets. This is most evident for O. edulis and O. angasi. where a sister species relationship for these Euro- pean and Australian flat oysters is strongly supported by both karyological and gene tree data. The ostreinid mitochondrial gene trees place the six karyologically-characterized flat oysters into two clades: one containing (among other taxa) O. puelchana. O. conchaphila. and O. denselameUosa. the other composed of O. edulis. O. angasi. and O. chilensis. With the exception of posi- tioning of O. chilensis. which in our study is closer to O. dense- lameUosa, these results are in broad agreement with the topology generated by our statistical analysis based on Ci and NORs. All Ostreinae species are of the brooding type with an extended planktotrophic larval development with the exception of O. chil- ensis, which shows a greatly abbreviated pelagic phase (Walne 1963). This peculiarity is not reflected at the karyological level. However, O. puelchana is the only brooding oyster with a distinct dwarf male and it shows a unique phenomenon of settling the larvae on an expansion of the anterior shell margin (Pascual et al. 1989). These unique morphologic features could be related to the karyological isolation of O. puelchana. ACKNOWLEDGMENT This work was partially supported by a Ponuguese grant from the Ministry of Science and Technology (FCTI): SFRH/BPD/ 1582-2000. We are grateful to S. Lapegue and D. Cheney for supplying live oysters. The authors thank S. Sabini and S. Heu- rtebise for excellent technical assistance, R. Ben Hamadou for statistical analysis, V. Thiriot for collaboration in Fig. 2, P. 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New Jersey 08087 ABSTRACT To develop a quantitative stock assessment for the New Jersey oyster (Cra.v.vo.sv/ca virginica) seed beds, oyster dredge efficiency was measured on 10 different oyster beds in the Delaware Bay. Depending on size class and location, mean dredge efficiency for market-size oysters varied from 7.8 to >8,'i'}j-. The sampled beds could be allocated into two groups, one characterized by low dredge efficiency and the other characterized by high dredge elTiciency. The low-efficiency group. Group 1. had mean dredge efficiencies for market-size oysters that ranged from 10.9 to 19.5%. The high-efficiency group. Group 2. had mean dredge efficiencies for market-size oysters that always exceeded 45%. A strong tendency existed for market-size oysters to be captured with higher efficiency than smaller oysters. In addition, live oysters tended to be captured with higher efficiency than boxes (articulated valves). Although a conclusion cannot be reached unequivocally, the differential in dredge efficiency observed between Group 1 and Group 2 beds may represent the difference between dredge efficiencies on beds routinely fished and those not routinely fished. An effect of salinity regime cannot be excluded as a possible explanation, however. The differential in dredge efficiency between the two bed groups, about a factor of 4.5 for market-size oysters indicates that variations in bed consolidation may have a large influence on dredge efficiency and may significantly bias estimates of abundance if not taken into account in stock assessments. KEY WORDS: oyster, dredge efficiency, stock assessment, survey, fishery INTRODUCTION Dredges are frequently used survey tools. Knowing the effi- ciency of the dredge is, therefore, paramount in a quantitative estimate of stock abundance. Dredge efficiencies have been evalu- ated for a number of bottom-dwelling commercial species, includ- ing .scallops. Placopecten magellanicus and Zygochlamys patag- onica (Giguere and Brulotte 1994, Lasta and Iribame 1997), surf clams, Spisula solidissiina (NEFSC 2000a), ocean quahogs, Arc- tica islandica (NEFSC 2000b), and blue crabs, Callinecies sapidiis (Volstad et al. 2000). The stock assessment for the New Jersey oyster seed beds uses a standard 1.27-m oyster dredge (Fegley et al. 1994). Quantifica- tion of this survey depends upon knowing the efficiency of the dredge under survey conditions. Oyster dredges are not among the most efficient of sampling gear. Estimates of dredge efficiency range between 2 and 32% in survey mode (Chai et al. 1992). As used by the industry in the normal routine of fishing, the dredge efficiency consistently falls into the lower portion of this range (Banta et al. in press). To develop a quantitative stock assessment for the New Jersey oyster seed beds, we performed a series of measurements of dredge efficiency for a standard oyster dredge (Fig. I ). Because previous estimates had varied over a wide range, we conducted these mea- surements on a number of oyster beds covering a range of salinities and degrees of fishery impact to evaluate whether changes in bed environment and fishing history affect dredge efficiency. METHODS Field Program Dredge efficiency measurements were conducted in summer 1999 and summer 2000 on 10 different oyster beds in the Delaware Bay. Eight of these were in New Jersey waters and two were in Delaware waters (Fig. 2). Normally, three separate experiments were conducted on each bed. Time constraints limited the number to less than three in *Corresponding author. E-mail: eric@hsrl.rutgers.edu several cases. Each experiment was conducted in an identical man- ner in the following way. The oyster boat F/V Howard W. Sockwell carried out a l-miii dredge tow using a standard 24-tooth 1.27-m dredge (Fig. I). Tooth length was approximately 44 mm, and the mouth opening was 1.27 m X 31 cm. The bag consisted of 17 rows of 50.8 mm rings. During the dredge tow, a data logger recorded DGPS posi- tion and time at 5-s intervals. A second boat, the RA' Zephyrus, ran immediately parallel but about 5 m off the oyster boat. A buoy was dropped from the R/V Zephyrus at the point immediately opposite dredge deployment and another immediately opposite dredge retrieval as the tow progressed. The dredge haul was brought on board and u one-bushel sample taken for analysis. The remainder of the haul was measured volu- metrically and discarded overboard. A full oyster dredge holds about 12 bushels of material. Presumably, dredge efficiency de- clines as this volume is approached. In this study, dredge volumes exceed 8.5 bushels at only two sites, Bennies and Arnolds, where dredge volumes routinely fell between 9 and 10 bushels. However, efficiency estimates tor these sites did not materially diverge from efficiency estimates for other sites, suggesting that tow distances were short enough so that dredge capacity did not influence mea- sured efficiency. A buoyed 23-m transect line was dropped from the R/V Zephyrus near the first buoy and the line payed out towards the second buoy. Limitations in visibility prevented divers from rou- tinely sampling within the tow path. As a consequence of the need to maintain a consistent sampling protocol, the diver line was positioned about 5 m from the dredge path so that all diver samples were taken near to and in parallel with the tow path. Twelve collection sacks were affixed at equal intervals along this line. Divers were then deployed to sample along the transect line using an 0.5- X 0.5-m quadrat (0.25 m'). A random 0.25-m" sample was taken at the location where each collection sack was affixed. Divers attempted to retrieve all of the loose bottom material, in- cluding all live oysters and boxes (dead, articulated valves). Col- lection was facilitated by the use of small hand-held scratch rakes. The divers were instructed to take only the loosely consolidated 691 692 Powell et al. DELAWARE RIVER Figure 1. A standard 1.27-m New Jersey oyster dredge. material on the surface that would normally be taken by the dredge. In most cases, quantitative retrieval was simple because the consolidated portion of the bed was near the surface. In a few cases, unconsolidated shell extended downwards for some dis- tance. In these cases, the diver took the upper portion of the shell until the collection sack was filled. Luhoratory Analyses Each bushel sample and each diver sample was sorted into live oysters, bo,\es, and shell and other debris and the respective vol- umes measured. The longest dimension of each oyster and box >2() mm was measured. Swept area was calculated for each dredge tow from the 5-s position logs and the dredge width. Population abundance (A') is normally related to survey abun- dance UiJ by a catchability coefficient: A' = qii^. The conversion requires four parameters: A. the area of the survey; il. the distance traveled per tow; ir the cross-section sampled by the dredge; and ('. the efficiency of capture by the dredge (NEFSC unpubl.). Thus, (/ = A/dw Me. Becau.se, in this study, the dredge boat and diver samples were individually normalized to a 1 nr area {Aldw = 1 ). catchability q becomes the reciprocal of efficiency e. and was calculated as follows; / ,Z, number of oysters or boxes {diver sample) number of diver samples * 0.23 m' '' ( luimber of oysters or boxes (busheiy * # bushels in' dredge swept area NEW JERSEY OVER THE BAR LOWER MIDDLE DELAWARE Figure 2. Location of the 10 oyster beds where dredge efficiency mea- surements were conducted in survey mode. Banta et al. (In press) obtained estimates of dredge efficiency under commercial use from New Beds. Statistical Analysis For statistical analyses, we used Spearman's rank correlation and ANOVA on ranked data. When appropriate, differences within the ANOVA were resolved with comparisons that used the least squares means. For some statistical analyses, live oysters and boxes were split into three size classes: juvenile (20-63.5 mm), submarket (6-^..'>-76.2 mm), and market (>76.2 inm). RESULTS Mean values of q for each seed bed are provided in Table 1. Values of q for live oysters ranged from 1.54 (an efficiency of 64.9%) to 11,27 (an efficiency of 8,9%). With one exception, all the size classes of oysters and boxes were correlated with one another (Table 2), The efficiency of collection of shell debris (disarticulated shell and shell fragments), however, was much more rarely correlated with the efficiency of collection of live oysters or boxes and the correlation coefficients were consistently lower. Divers had difficulty determining when to stop digging out shell and shell fragments on some beds, and this uncertainty in diver collection resulted in xariation in the computed catchability for debris among diver samples. Visual observation of Table 1 suggests that the sampled beds can be divided into two groups, those with relatively high values of q (low dredge efficiency) and those with relatively low values of q (high dredge efficiency). This observation was confirmed by ANOVA analysis comparing the efficiency of collection of mar- Inherent Efficiency of Oyster Dredges 693 TABLE 1. Mean values of q (the reciprocal of dredge efficiency! for eacli of the size classes of live oysters and boxes, total live oysters, total boxes, and debris. Live Oysters Boxes Oyster Bed Juveniles Submarket* .Markets Total Live Juveniles Submarkets Markets Total Box Debris Group 1 Arnolds 10.30 2.22 6.46 9.26 10.05 8.81 10.04 9.74 7.47 Cohansey 12.80 7.96 7.81 11.27 17.62 50.54 1 1 .40 18.83 37.23 Over the Bar 9.63 5.88 5.14 7.61 6.81 12.58 5.58 7.33 6.93 Lower Middle 9.33 7.69 6.23 8.80 8.09 7.92 13.40 8.31 15.26 Ship John 10.16 12.06 9.15 10.40 12.79 9.91 13.17 11.88 50.12 Group 2 Shell Rock 4.16 2.84 1.99 3.70 5.37 4.55 2.44 4.71 12.45 Nantuxent Pt. 3.86 1.98 - 3.30 2.32 2.48 - 1.98 6.06 Bennies 2.57 2.31 1.17 2.32 6.70 4.01 5.48 5.58 5.20 New Beds 2.04 3.12 0,64 2.13 2.88 2.08 - 2.71 9.65 Egg Island - - in 1.54 15.41 6.00 20.31 16.99 14.47 Debris includes disarticulated shells and other shell debris. Dash indicates situations where diver sampling did not provide an adequate catch of that variable to permit an estimate of dredge efficiency. ket-size oysters between oyster beds (Table 3). Note that, in gen- eral, the beds Arnolds. Cohansey. Over the Bar. Lower Middle, and Ship John, hereafter termed Group 1 , are usually significantly different from the beds Shell Rock. Bennies. New Beds, and Egg Island, hereafter termed Group 2, in this analysis (Table 3). Mar- ket-size live oysters were not collected at Nantuxent Point, but perusal of the remaining data in Table 1 suggests that, had they been, Nantuxent Point would have fallen within the latter group of beds. The average values of q for Groups 1 and 2 are shov\ n in Table 4. With the exception of market-size boxes and debris, the effi- ciency of collection of live oysters and boxes on Group 1 beds is significantly lower (a higher q) than the efficiency of collection of live oysters and boxes on Group 2 beds. The efficiency of capture of market-size oysters was higher (lower q) than for submarket-size (P = 0.04) and juvenile oysters P = 0.003). The latter two were not significantly different. The averages recorded in Table 4 also suggest that the efficiency of capture of live oysters is somewhat higher than boxes. In fact, values of q were significantly lower for all live oysters relative to all boxes (P = 0.002), live submarket oysters relative to submar- ket boxes (P = 0.04), and juvenile live oysters relative to juvenile boxes (P = 0.006). Market-size live oysters and boxes did not differ significantly, although the mean of the former falls well below the mean of the latter, especially for Group 2 beds. Accord- ingly, boxes were collected with a lower efficiency than live oys- ters overall. The two groups of beds differ in aveiage salinity. Group I beds are upbay of Group 2 beds. However, the intensity of fishing also follows the salinity gradient. Visual inspection of samples showed that oysters were tnuch more clumped in samples from Group 1 beds as a consequence of the much lower frequency of dredging that has historically occurred on these beds. Clumping and reef consolidation might decrease dredge efficiency. If so, a correlation might exist between the amount of dredging on the bed during the preceding year and our measurement of dredge efficiency. We evaluated the significance of dredging using Spearman's rank cor- relations between the number of bushels taken per bed in 1 999 and 2000 versus the measured value of ^. The number of bushels taken is a reasonable surrogate for the total swept area of dredging (Banta et al., in press). All correlations were negative in accor- dance with the hypothesis that a higher value of q (lower dredge efficiency) should coincide with lower harvest rates. However, only the correlation with market-size live oysters was significant. DISCUSSION Oyster dredge efficiency varied over a wide range among the oyster beds sampled in the Delaware Bay. The range measured TABLE 2. P-values from Spearman's rank correlations between the efficiency of capture of the various groupings of live oysters, boxes, and debris. Submarket Market Total Juvenile Submarket Market Total Live Oysters Live Oysters Live Oysters Boxes Boxes Boxes Boxes Debris Juvenile live oysters 0.0013 0.0001 0.0001 0.0001 0.0001 0.0048 (.1.000 1 0.1289 Submarket live oysters 0.0022 0.0005 0.0007 o.ooos 0.0073 0.0002 0.0003 Market live oysters 0.0001 0.0001 0.0001 0,0019 0.0001 0.0167 Total live oysters 0.0001 0.0001 0.0047 0.0001 0. 1 1 28 Juvenile boxes 0.0001 0.0003 0.0001 0.0231 Submarket boxes 0.0030 0.0001 0.0136 Market boxes 0.0001 0.3216 Total boxes 0.0174 694 Powell et al. TABLE 3. /"-values from comparisons of least squares means for the efficiency of capture of market-size live oysters among the sampled beds. Over Ijower Ship Shell New Egg Cohansey the Bar Middle John Rock Bennies Beds Island 0.5529 0.5360 0.6912 0.2775 Arnolds 0.0310 0.0057 0.0207 0.1967 Cohansey 0.2597 0.3290 0.5651 0.0100 0,0019 0.0093 0.0972 Over the Bar 0.7895 0.1312 0.1478 0.0356 0.0645 0.4315 Lower Middle 0.1.590 0.0645 0.0121 0.0351 0..3017 Ship John 0.0061 0.0014 0.0057 0.0539 Shell Rock 0.3778 0.3680 0.7253 Bennies 0.7784 0.3337 New Beds 0.3111 No market-size oysters were collected at Nantuxent Point, hence this bed is not included in the table. Boxed area delineates the comparisons between Group 1 and Group 2 beds discussed in the text. encompasses dredge efficiencies higher than those recorded by Chai et al. (1992) in the Chesapeake Buy. In that study, dredge efficiencies varied from 2 to 32%. In this study, depending on size class and location, mean dredge efficiency for market-size oysters varied from 7.8 to >85%. Such high efficiencies are achieved when the dredge is used in survey mode, with short one minute tows that do not result in the complete filling of the dredge. The oyster fishery, as it routinely fishes, rarely achieves a dredge efficiency above 5% (Banta et al., in press) because the tows are longer and the dredge is routinely full when retrieved. The range of efficiencies measured is large. Extreme values, whether high or low, probably are due to patchiness in the sampled area. Diver samples were not taken from the dredge tow path, but rather along a transect run parallel and close to the dredge tow path. Nevertheless, the sampled beds could readily be allocated into two groups, one characterized by low dredge efficiency and the other characterized by high dredge efficiency. The low- efficiency group, Group 1 , had mean dredge efficiencies for mar- ket-size oysters that ranged from 10.9 to 19.5%. The high- efficiency group had mean dredge efficiencies for market-size oys- ters that always exceeded 45%. A strong tendency existed for market-size oysters to be cap- tured with higher efficiency than smaller oysters. Presumably, a greater tendency exists for the smaller oysters to pass between the dredge teeth or through the rings of the collection bag and, thus, not be collected. Dredge efficiencies were particularly low for juveniles, many of which may be attached to smaller pieces of shell that are poorly sampled. Very likely, dredge samples rou- tinely result in a significant bias against juveniles. In this study. market-size oysters were captured with about twice the efficiency of juveniles. In addition, live oysters tended to be captured with higher efficiency than boxes. The difference was highly significant, par- ticularly in Group 2 beds where boxes tended to be captured with an efficiency of about one-third the efficiency of living oysters. Two possible reasons exist for the lower capture efficiency of boxes: I ) collection by dredge may result in disarticulation. This possibility is not supported by experiments designed to evaluate this source of disarticulation, however (Powell et al. 2001); 2) some boxes taken by divers may be deeper in the reef than the dredge normally samples. Efficiency of collection of debris was significantly correlated with efficiency of collection of total boxes in accordance with this hypothesis (Table 2); however, the effi- ciency of collection of debris was also correlated with some live oyster variables. Thus, a conclusive explanation for the variation in efficiency of capture between boxes and live oysters is not pro- vided by the present analyses. Box counts are routinely used as a method to estimate mortality rates in shellfish populations (Merrill and Posgay 1964, Fegley et al. 1994, Christmas et al. 1997). The differential in dredge effi- ciency measured in this study could result in a significant bias in the live:dead ratio and a significant underestimation of mortality rate from box counts if diver collections are unbiased. On the other hand, Christmas et al. (1997) observed that disarticulation often took more than one year, whereas mortality estimates from box counts usually assume that boxes were produced in the current year. This bias would tend to counterweigh the potential bias im- posed by dredge efficiency. However, Powell et al. (2001) ob- TABLE 4. Mean values of q (the reciprocal of dredge efficiency) for each of the size classes of live oysters and boxes, total live oysters, total boxes, and debris for two groups of beds. Live Oysl ers Boxes Oyster Bed Juveniles Subniarkets Markets Total Live Juveniles Subniarkets Markets Total Box Debris Group 1 Group 2 P value 10.46 3.33 0.0009 6.89 2.57 0.04 6.93 1.54 0.0001 9.40 2.83 0.0002 11.26 6.78 0.04 18.98 4.03 0.0008 11.00 8.85 NS 11.47 6.50 0.02 21.49 9.55 NS Group 1 contains Arnolds. Cohansey. Over the Bar, Lower Middle, and Ship John. Group 2 contains Bennies. Shell Rock, Nantuxent Point, Egg Island, and New Beds. Debris includes cultch and other debris. P values record the results of ANOVA analysis comparing the two groups with respect to the variable listed as the column heading. NS. not significant (a = 0.05). Inherent Efficiency of Oyster Dredges 695 served that disarticulation rates might be higher in the Delaware Bay than observed in Chesapeake Bay by Christmas et al. ( 1997). Consequently, the potentially offsetting biases in the interpretation of box counts cannot, as yet, be fully evaluated. Why Group 1 beds yielded such low dredge efficiencies in comparison to Group 2 beds cannot unequivocally be identified. Group 1 beds are all upbay of Group 2 and, thus, exist at lower average salinities. Unfortunately, Group I beds also have been impacted less over the long term by dredging because effort in the Delaware Bay oyster industry also follows the salinity gradient, with lower effort on the lowest salinity beds (HSRL 2000, Fegley et al. 1994. Banta et al., in press). We attempted to assess the influence of dredging using catch data for 1999 and 2000, without much success. However, whether the catch data for the year before sampling is the correct estimator of the effect of dredging is ques- tionable. Visual observation, for example, shows that oyster clumps are larger and contain more living oysters on these upbay beds. These clumps, very likely, are more firmly attached to the underlying bed than are the oysters on Group 2 beds. Greater bed consolidation on the upbay beds (Group I) would reduce the ef- fectiveness of the teeth in scraping shell material up into the dredge and, consequently, reduce dredge efficiency. Oyster catchers normally report that catch rates are lower on beds that have not been fished for a time and that catch rates improve after repeated dredging over a few days. Very likely, this repeated dredging breaks the bottom up and results in a substantial increase in the efficiency of capture. Although the conclusion can- not be reached unequivocally, it seems most likely that the differ- ential observed between Group 1 and Group 2 beds represents the difference between dredge efficiencies on beds routinely fished and those not routinely fished. The differential is large, about a factor of 4.5 for market-size oysters, indicating that variations in bed consolidation may have a large influence on dredge efficiency and may significantly bias estimates of abundance if not taken into account in stock assessments. ACKNOWLEDGMENTS Special thanks to Larry Hickman, Captain of the FA^ Howard W. Sockwell and to Bivalve Packing for providing the F/V How- ard W. Sockwell and logistical support for this study. Special thanks also to Royce Reed, Captain of the RA' Zephyrus and to the New Jersey Department of Environmental Protection for providing vessel support for the dive team and help in sample collection. We particularly thank the dive team. Jack Keith. Dave Andrews. Geoff Graham, Bill Dixon, and Jessica Vanisko, who collected the 324 diver samples that constituted the quantitative collections for this study. LITERATURE CITED Banta, S. E., E. N. Powell & K. A. Ashton-Alcox. in press. Evaluation of dredging behavior by the Delaware Bay oyster fishery. North Am. J. Fisheries Management. Chai, A-L., M. Homer. C-F. Tsui & P. Goulletquer. 1992. Evaluation of oyster sampling efficiency of patent tongs and an oyster dredge. North Am. J. Fisheries Management 12:825-832. Christmas, J. P., M. R. McGinty, D. A. Randle, G. F. Smith, & S. J. Jordan. 1997. Oyster shell disarticulation in three Chesapeake Bay tributaries. J. Shellfish Res. 16:115-123. Fegley, S. R., S. E. Ford, J.N. Kraeuter, & D. R. Jones. 1994. Relative effects of harvest pressure and disease mortality on the population dynamics of the Eastern oyster [Crassostrea virginica) in Delaware Bay. Final Report # NA26FL0588 to the National Oceanic and Atmo- spheric Administration, Rutgers University, Haskin Shellfish Research Laboratory. Giguere, M. & S. Brulotte. 1994, Comparison of sampling techniques, video and dredge, in estimating sea scallop iPUicopecten mageltaniciis. Gmelin) populations. / Shellfish Res. 13:25-30. Lasta, M. L. & O. O. Iribame. 1997. Southwestern Atlantic scallop (Zy- gochlamys patagonica) fishery: assessment of gear efficiency through a depletion experiment. J. Shellfish Res. 16:59-62. Merrill, A. S. & J. A. Posgay. 1964. Estimating the natural mortality rate of the sea scallop {Placopecten magellanicus). ICNAF Res. Bull. 1:88- 106. NEFSC. 2000a. 30"' northeast regional stock assessment workshop (30'" SAW): Stock assessment review committee (SARC) consensus sum- mary of assessments. Northeast Fisheries Science Center Reference Document 00-03. 477 pp. NEFSC. 2000b. 31"' northeast regional stock as.sessment workshop (31"' SAW): Stock assessment review committee (SARC) consensus sum- mary of assessments. Northeast Fisheries Science Center Reference Document 00-15. 400 pp. NEFSC. unpubl. 34"' northeast regional stock assessment workshop (34''' SAW): Stock assessment review committee (SARC) consensus sum- mary of assessments. Northeast Fisheries Science Center Reference Document 02-06. 346 pp. Powell, E. N., K. A. Ashton-AIcox. S. E. Banta & A, J. Bonner. 2001. Impact of repeated dredging on a Delaware Bay oyster reef / Shellfish Res. 20:961-975. V0lstad, J. H., A. F. Sharov, G. Davis & B. Davis. 2000. A method for estimating dredge catching efficiency for blue crabs, Callinectes sapi- diis. in Chesapeake Bay. US Fish Wildlife Service Fisheries Bull. 98: 4I0-f20. Joiinwl of Shellfish Research, Vol. 21. No. 2. 697-705, 2002. NOVEL REPEAT ELEMENTS IN THE NUCLEAR RIBOSOMAL RNA OPERON OF THE FLAT OYSTERS OSTREA EDULIS C. LINNAEUS, 1758 AND O. ANGASI SOWERBY, 1871 E. KENCHINGTON,'* C. J. BIRD," J. OSBORNE," AND M. REITH" ^ Department of Fisheries & Oceans. Bedford Institute of Oceanography, 1 Challenger Drive. P.O. Bo.x 1006. Dartmouth. NS. B2Y 4A2. Canada; 'Institute for Marine Biosciences. National Research Council of Canada. Nil Oxford Street. Halifax. NS. B3H 3ZI. Canada ABSTRACT The nucleotide sequence of the ribosomal RNA operon. from the 18S rRNA gene through ITS-1. 5.8S rRNA gene, and ITS-2, wa.s determined for single clones of the flat oysters Ostrea edulis from Europe and O. angasi from Australia. The genie regions, the 18S and 5.8S rDNAs, were Identical between the two species and displayed a high degree of similarity with available sequence from Crassostrea gigas, C. nippona, Saccostrea commercialis. and 5. cucullala. In lTS-1. the Ostrea species had a characteristic repeat region, with subrepeats of 54 base-pairs (bp) (a) flanking a 27-bp fragment {b). where h remained constant while a changed by 1 bp through successive Iterations ia' . a"). In O. edulis. the pattern was a. b, a', b. a", whereas In O. angasi It was a. b. a". Apart from the difference in repeats, the ITS-1 of the two Ostrea species was identical except for a single substitution and two 2-bp Insertions/ deletions. lTS-2 was Identical between the two Ostrea species except for a single substitution and a 3-bp insertion/deletion, and contained a neariy complete repeat of a near the 5' end. These ITS sequences were markedly dissimilar to available sequence from the other oyster species examined, and the repeat region was unique to Ostrea. with possible functionality In transcription. On the grounds of sequence similarity. It Is suggested that O. edulis and O. angasi are conspeclfic. KEY WORDS: rDNA sequence, ITS. Oslrea INTRODUCTION The taxonomy and systematics of oysters is far from being resolved (Carriker & Gaffney 1996), and confusion over the rec- ognition of species persists, although the recent application of genetic techniques has been very successful in providing valuable characters to address phylogenetic relationships of the major spe- cies groups and families (Buroker et al. 1979a, Buroker et al. 1979b, Brock 1990, Banks et al. 1993. Anderson & Adlard 1994, Littlewood 1994, Boudry et al. 1998, Jozefowicz & 6 Foighil 1998, 6 Foighil et al. 1999, 6 Foighil & Taylor 2000). Among the remaining contentious issues is the question of the phylogenetic relationship between Ostrea edulis Linne, 1 758, the European flat oyster, and O. angasi Sowerby, 1871, the Australian flat oyster. In the most recent comprehensive revision of the living oysters, Harry ( 1 985 ) reduced O. angasi to a synonym of the Southern Hemisphere species O. piielcluina D'Orbigny, 1841, placing it in a different subgenus from O. edulis based on larval morphological and developmental characters. However, this classification has not been universally accepted (e.g., Jozefowicz & O Foighil 1998), and further data have since become available to suggest a closer relationship between O. angasi and O. edulis than had been sup- posed. Comparison of karyotypes, nucleolus organizer regions, and constitutive heterochromatin from chromosome preparations of O. angasi and O. edulis shows a very high degree of similarity, which is not seen between O. angasi and its supposed Southern Hemisphere conspecifics O. puelchana and O. chilensis Philippi, 1845, or between O. angasi and the Indo-West Pacific O. dense- lamellosa Lischke, 1869 (Li & Havenhand 1997). Partial mito- chondrial 16S rRNA gene sequences position O. edulis and O. angasi in a well-supported clade with a low degree of DNA se- quence divergence relative to the other conspecifics (Jozefowicz & O Foighil 1998). These results are also supported by partial 28S rRNA gene sequences (O Foighil & Taylor 2000). Furthermore, the concept of O, puelchana (previously thought to be native to *Corresponding author. E-mail; Kenchlngtone@mar.dfo-mpo.gc.ca Argentina) as a single circumglobal species in the Southern Hemi- sphere has been convincingly challenged by partial mitochondrial 16S rRNA (Jozefowicz & 6 Foighil 1998) and 28S rRNA gene sequences (O Foighil & Taylor 2000). These data support resur- rection of the regional taxa in the southern hemisphere, namely O. chilensis in Chile and New Zealand, O. angasi in Australia and O. algoensis Sowerby. 1871 in South Africa. To date, genetic information on the living oysters has focused on the rRNA gene (rDNA) array. In the Bivalvia, and in many other taxa, the rRNA genes have been useful in inferring phylo- genetic and taxonomic relationships (e.g.. Field et al. 1988, Ghis- elin 1988, Nakamura 1989, Rice et al. 1993, Kenchington et al. 1994. Kenchington et al. 1995, Sterner & Muller 1996. Campbell et al. 1998, Frischer et al. 1998). These genes are co-transcribed, producing a single transcript consisting of the 18S, 5.8S. and 28S rRNA coding regions (or their homologues), separated by two internal transcribed spacers (ITS-! and ITS-2) with an external transcribed spacer (ETS) located upstream of the 18S rRNA gene (Fig. 1). The transcribed precursor rRNAs are separated by inter- genic spacers (IGS or NTS) consisting of transcribed and non- transcribed spacer sequence. This rRNA cistron is tandemly re- peated head to tail at one or more chromosomal sites termed nucleolus organizer regions (NORs) (Hadjiolov 1985). The pri- mary transcript is cleaved via a series of processing reactions into the 18S. 5.8S and 28S rDNAs, which along with approximately 85 ribosomal proteins and 5S rDNA, form the 40S and 60S pre- ribosomal subunits (Hadjiolov 1985). Regions of the rRNA cistron are under varying degrees of functional constraint and therefore provide a wide range of phy- logenetic resolution. The IGS, a noncoding region, is under the least selective constraint and is therefore expected to show the greatest sequence divergence, rendering it useful for intraspecific comparisons. The ITS regions are also rapidly evolving, but to a lesser degree than the IGS. In contrast, the coding region of the rRNA repeat unit, that is. the I8S. 5.8S. and 28S genes, is highly conserved within and among species both in sequence and in sec- ondary structure, with the 18S being the most conservative over a 697 698 Kenchington et al. ITS-2 ETS 18S 5.8S 28S ribosomal RNAs transcribed regions; 40 ribosomal proteins Figure 1. A schematic of the ribosomal RNA (rRNAl cistron indicat- ing the genie regions in black and the noneoding regions in white. broad taxonomic range (Appels & Honeycutt 1986). However, within the IBS rRNA gene, there are both conserved and variable regions associated with its secondary structure. Although some regions of this gene are conserved across all eukaryotes. other regions (loops) can be highly variable. Therefore, it has been sug- gested that this gene is valuable for phylogenetic investigations at a variety of taxonomic levels (Sogin et al. 1986). particularly when the time since divergence of the taxa is not known. A similar argument has been made for the much larger 28S rRNA gene (Hillis & Dixon 1991). Here, we examine the nuclear rRNA cistron for sequence dif- ferences in the 18S. ITS-1, 5.8S. and ITS-2 regions to provide additional support for the level of differentiation between O. an- gasi and O. edulis. In the course of our studies, novel repeat elements were identified in the ITS which are described and dis- cussed in context of their possible evolution. MATERIALS AND METHODS Sample Location Specimens of O. angasi were collected from Cloudy Bay La- goon, south end of Brany Island. Tasmania. Australia on June 23. 1993 by Dr. John Thomson and shipped alive to our laboratory. Specimens of O. edulis were obtained from Department of Fish- eries and Oceans research stock, originally imported from Europe via the United States to Canada during the late I97()s and used as broodstock at a hatchery in Port Medway, Nova Scotia. Animals were dissected and voucher specimens of the shells were archived at the Bedford Institute of Oceanography. Dartmouth, Nova Scotia. DNA Extraction and Purification High-molecular-weight DNA was extracted from the adductor muscle of a single animal of each species according to the protocol of Rice and Bird ( 1990). Briefly, tissue was ground in liquid ni- trogen, proteins were removed by mixing with an equal volume of phenol:chloroform:isoamyl alcohol, followed by a final wash with an equal volume of chloroform:isoamyl alcohol to remove the phenol residues, and DNA was precipitated with sodium acetate in cold ethanol. The DNA was further purifled on a cesium chloride gradient by high-speed centrifugation. DNA Amplification The 18S rRNA gene, ITS-1, 5.8S rDNA. and ITS-2 regions of the rRNA cistron were amplified as two fragments, one approxi- mately 1825 base pairs (bp) and corresponding to the 18S rRNA gene, and the second approxnnately 1220 bp and containing the ITS spacers and 5.8S sequences. Synthetic oligonucleotides cor- responding to conserved regions of the 18S rRNA gene and ap- pended to restriction endonuclease sites were used as amplification primers for the first fragment (Bird et al. 1992). Deoxy-uracil monophosphate (dUMP) primers, with four trinucleotide repeat at the 5' ends and complementary to the coding strand of the 18S (dUMP 18SF: 5'-CALICAUCAUCAUGGTTTCCGTAGGTGAA- CCTGC-3') and the 28S rDNAs (dUMP 28SR: 5'-CUACUACU- ACUAATATGCTTAAGTTCAGCGGGT-3'). were used to am- plify the second fragment. The change in protocol to the dUMP primers was purely a matter of convenience because these primers were available in our laboratory at the time for another project (i.e.. Patwary et al. 1998). Amplification was performed in a Perkin- Elmer (Irvine, CA) DNA Thermal Cycler programmed for an ini- tial cycle of 5 min. at 94°C followed by 30 cycles of 2 min at 37°C. 3 min at 72°C, and 1 min at 94'C. plus a final cycle of annealing for 2 min at 37°C and one of extension for 5 min at 72"C. The same amplification conditions were used for each of the primer pairs and a negative control (no DNA template) was included in each run. Cloning Polymerase Chain Reaction Products Amplified DNA containing the 18S rRNA gene was ligated directly into the plasmid sequencing vector pCR 1000 using the TA Cloning'"''^ kit, following manufacturer's instructions (Invitro- gen Corp.. Carisbad, CA). Recombinant plasmids were maintained in the OneShot'^' Escherichia coli INVaF' cells provided in the kit. Transformants were grown on YT agar plates supplemented with kanamycin (100 p-g mL"') and were detected by blue/white selection in the presence of X-gal and IPTG (Gold Biotechnology, St. Louis. MO). Amplification products for the second fragment containing the ITS spacers and 5. 88 gene generated with the dUMP primers were purified using the Geneclean If" elution kit (BIO/CAN Scientific. Mississauga. ON). The purified DNA was directionally cloned into Epicurean competent cells (Stratagene. La Jolla. CA) using the CLONEAMP"' System (Life Technologies, Rockville, MD) for rapid cloning of amplification products. The size of the inserts was screened by protoplasting (Sekar 1987), followed by digestion with Noll (New England Biosystems, Mississauga, ON) and Kpn\ (Pharmacia, Peapack, NJ). A single colony was inoculated into 2xYT medium, following the protocols for a large-scale plasmid preparation (Maniatis et al. 1982). DNA Sequencing and Alignment Single clones of purified plasmid DNA containing the I8S rRNA gene were subjected to dideoxy sequencing according to the USB Sequenase version 2.0 protocol (USB Corporation, Cleve- land, OH). Amplification primers plus the oligonucleotide primers described in Bird et al. ( 1992) were used to sequence the complete I8S rRNA gene in both directions. dITP was substituted for dGTP in the sequencing protocol to resolve compressions caused by sec- ondary structure. Purified DNA containing the ITS spacers and 5.8S rRNA gene was sequenced on an ABI 373 Automated DNA Sequencer, using the PRISM '^' Dye Terminator Cycle Sequencing Kit (PE/ABI; Applied Biosystems. Foster City. CA) according to the manufac- turer's instructions. M13 universal forward (5'-GTAAAACGA- CGGCCAGT-3') and reverse (5'-TTCACACAGGAAACAG-3') primers (LISB) were used for initial sequencing. The DNA was sequenced completely in both directions by using a set of internal Repeat Elements in Ostrea ITS Sequence 699 18S (end) Fl ITSl F2 F3 F4 5.8S F5 F6 ITS2 F7 F8 ITS I Rl R2 R3 5.8S R4 R5 1TS2 R6 R7 TABLE 1. Forward (F) and Reverse (R) Primers used to sequence the ITS-1, 5.8S, and ITS-2 regions of the rRNA cistron. -GGTTTCCGTAGGTG AACC-3 ' -CTCCGTCGCCTTAAATGC-3' -GATTCCGAGCTCACGGAC-3' -CGTTTCACGTACATCAATAGT-3' ' -TCTAAGTGGTGG ATC ACTCG-3 ' ■CATTGAACATCGACATCTTG-3' -CTCCGTCGCCTTAAATGC-3' (m repeat points of identical sequence and iiyphens indicate points of sequence deletion. Numbers refer to the number of nucelolides from the 5' end. The 54-bp repetitive element (a) is indicated in the O. edulis sequence by shading, and nucleotide changes in its second («') and third (a"» iterations are underline: the 24-bp subrepeat (/)) is outlined. GenBank (U88709). differed from our O. edulislangasi sequence because of numerous insertions downstream from the repeat region in If) sites and was identical with the other oysters al 8 of those (Fig. 5). For ITS-2. both Ostrea sequences were shorter than that sjtes. of Crassostrea gigiis. which was 61 1 bp (Fig. 4). Overall sequence Similarly, the 159-bp 5.8S iRNA genes of O. edulisi angasi similarity of the ITS-2 region between the Ostrea species and C. showed a high degree of similarity with the Crassostrea gigas gigas was on the order of 82%: conserved regions at the 5' and 3' sequence, which differed at only four positions (Fig. 2; 97.5% ends were identified. In C. gigas. the area corresponding to a similarity). Partial sequence from C nippona and Saccostrea com- contained six differences from a apart from the absent terminal merciatis was also nearly identical with that of O. edulislangasi. nucleotides, including only one of the two point mutations in the By contrast, considerable variability was evident among the O. edulis sequence. A BLAST search (Altschul et al. 1997) on the spacer sequences of the oyster genera (Figs. 4 and 5). None of the repeat elements could not identify similar sequence in other or- other ITS-1 sequences showed repeat elements, and similarity was ganisms. low in the region corresponding to the a and /' elements. However. the sequences were more conservative in an area coincident with DISCUSSION the a" repetitive element in O. edulis and O. angasi. Lacking repeats, the sequences from Cra.^sostrea and Saccostrea were Nuclear ribosomal sequences are often assumed to be homog- shorter (445-520 bp) than in O. edulis (546 bp). although the enized within individuals and populations ot a species through length of the C. nippona sequence approached that of O. edulis concerted evolution processes (Hillis & Davis 1988). Homogem- Repeat Elements in Ostrea ITS Sequence 701 O.edulis GAAACTATCCATCGGACA CCTGGGTTTGTCCGCCTTGGGCCGTCGTAGCCGAAA 54 O.angasi GAAACTATCCATCGGACA CCTGGGTTTGTCCGCCTTGGGGCGTCGTAGCCGAAA 54 C.gigas GAAACTATCAATCGACTAAAATTTCATTATTCTAGTCGCCTTGGGCCGTCGAAGCCTCTC 6 0 ********* r * * r****** ***** **** 0 . eduli s A-GGCTCCGTCGCCTTAAATGCAGACCGACGCCC GCTCGA 93 O . angasi A-GGCTCCGTCGCCTTAAATGCAGACCGACGCCC GCTCGA 93 C. gigas ACGGCTCCGTCGCCTTAAATGCAGACCGATGACCAAAGAAACGGCTCTATTGGTGACTCG 120 * *■■*■■*■■*■* + *■*■****■*■ + *■* + ***■*■** + * + + * ** t * + * 0. edulis GAGCGTCGCCGG-TCTCACCGCCTATGGGTTTCC CCCACGGTAGAAGGCTT--CTCG 147 O.angasi GAGCGTCGCCGG-TCTCACCGCCTATGGGTTTCC CCCACGGTAGAAGGCTT--CTCG 147 C.gigas GTGCCTCGGCGGGACGAAACACGTCTCGACTTCCACTCTTTTCGCTAAACTCTTATCCCG 180 0. edulis AACGTCTGCTCGGACGGATGGCGA- -GAGGAT-CGGGG AGGGCGTTGA CG 194 O . angasi AACGTCTGCTCGGACGGATGGCGA- -GAGGAT-CGGGG AGGGCGTTGA CG 194 C. gigas AACGGCTGCTCGGTCGGACGGCGAAGGAGGACGCGGGGTGCGAGGGCGCAGAAAAAGACG 240 + **■*■ ******** **** ***** ***** ***** ****** ** ** 0 . edulis GCAACGCGGTCTGGTCT TGACGACCGA ACCGA 22 6 O. angasi GCAACGCGGTCTGGTCT TGACGACCGA ACCGA 22 6 C. gigas GCAACGCGGTCTGGCCACATACGCGAAGCTCCGGCTATAGCGGCGGGTCTCGACCATCAA 3 00 ************** * * ** * * * * * 0. edulis GAGCGTGCCCGAAGCCC CTTACACCAAACT CGAACCTCGCCATTCGCTTT 276 O.angasi GAGCGTGCCCGAAGCCC CTTACACCAAACT CGAACCTCGCCATTCGCTTT 27 6 C. gigas AAGCGTGCCCGAAAGTGATGCAAACTCGCAACAAACTTCCCCGGACTCTCCAT-CGCTTT 3 59 ************ ** ** ****** * *** **** ****** O . eduli s GTCCTTGC - -GGTGCAAGTACGGACGGCGACG TACCGAGG GGGGAGCCGGGG 32 6 O. angasi GTCCTTGC --GGTGCAAGTACGGACGGCGACG TACCGAGGAGGGGGGAGCCGGGG 329 C.gigas GGCCTTGCCTGGCCTTAGTACGGATAACAAAGGCGGTTAAAGAAGGAGATCGTTTCGCTG 419 * ****** ** ******** * * * ** ** * * + * O . edul i s CGGA TCGAGAACGGC GCAAGTTGGAGC AGGTGTCGCGAG 3 65 0. angasi CGGA TCGAGAACGGC GCAAGTTGGAGC AGGTGTCGCGAG 3 68 C.gigas CAGAGTCCTGCCATTCAAAAAGGGGCTGAAGAGTCAACAGGGGCTCAAGGGCATCGCGAG 47 9 *** ******* * * **** *+ ******* 0 . eduli s GAGGGCTCGCCG GCCGCCTCGTACAACACTAATCATGC CTCA 4 07 O.angasi GAGGGCTCGCCG GCCGCCTCGTACAACACTAATCATGC CTCA 410 C.gigas GGATCCGCGTTTTAAGAAAAAAAGCGCGGACCTCGATAAAACCAAAAACGGGACAACCAA 53 9 ****** **** ********* ** 0. edulis TGATTCCTCAA CTCCGACCTCAGATCAGGCGAGACTACCCGCTGAACTTAAGCATAT 4 64 O.angasi TGATTCCTCAA CTCCGACCTCAGATCAGGCGAGACTACCCGCTGAACTTAAGCATAT 467 C. gigas TTTTTTTTCAATCACTCCGACCTCAGATCAGGCGAGACTACGCCCTGAACTTAAGCATAT 599 * ** **** *************************** * **************** O. edulis O.angasi C . gigas TAGTAGTAGTAG TAGTAGTAGTAG CACTAAGGGCAG 476 479 611 Figure 4. Nucleotide sequence of the ITS-2 region from tlie oysters Ostrea edulis, O. angasi. and Crassostrea gigas (GenBank Accession Number F28((610). Asterisks identify points of identical sequence and hyphens indicate points of sequence deletion/insertion. Numbers are the number of nucleotides from the 5' end. The segment that corresponds to the repetitive element (a) from the ITS-1 region of Ostrea is indicated by shading of the O. edulis sequence, and the two substitutions within it are underline, as is a third substitution of O. angasi. zation is most likely to occur if the rRNA genes occur in a single tandem array on one chromosome. In both O. cdiiUs and O. angasi. two NORs are located terminally on chromosome pairs 9 and 10 (Thiriot-Quievreux & Insua 1992, Li & Havenhand 1997). Addi- tionally, intraspecific variation in the number of NORs per cell and the size (copy number) has also been identified in O. edulis (Thiriot-Quievreux & Insua 1992). From this, one would predict that sequence variation would exceed homogenization. However, sequence divergence in the rRNA genes is very low between O. angasi and O. edulis. The sequences for the coding regions of the I8S and 5.8S rDNAs were identical between the Ostrea species. This similarity 702 Kenchington et al. O. edulis O. angasi C.gigas C.nippona Saccostrea ATTAACAAAACAAGGAAAC - - AACCAAGGTCGTTGAGACGTGT- -CTCTCTCGTCATAGC 5 6 ATTAACAAAACAAGGAAAC- -CACCAAGGTCGTTGAGACGTGTGTCTCTCTCGTCATAGC 58 ATTAACAAAACAAAATCGG-GGCTCGTTGGCAAGAGCGACCGAGAGGCTAACGCAA--AA 57 ATTAACAAAAAAAGC ATCG -GGCTCGTCGGC A - GAGC ACCGAGGACGGC AACGAAACGAA 5 8 ATTAAC AAAC AAAATGGGGCAAACCATTGGC AAACCATCTCATGGTCG AGATGTAACGGT 6 0 O . edu lis O. angasi C. gigas C.nippona Saccostrea CCGCGACC AGGTCGTGTGGAACGGGCGAGAGAAGGCC AATCGCCGTCCCCGCGGCGCCTT 116 CCGCGACCAGGTCGTGTGGAACGGGCGAGAGAAGGCCAATCGCCGTCCCCGCGGCGCCTT 118 ACACAGCCGAGG-G-TTGTTGTT AAAGTCG- -GATCGACCTCG-AACGCCCA 104 ACACAGCCGAGG-GGTTGCTGCTGTTTGGCAAGTGTCG--TGTCGACCTCT-AACGCCGA 114 CCAAACCCCGGA-GCCCGTGACC AGGTCG- -CGCGGA- -TGG-GGTGCGGA 105 O. edulis 0. angasi C. gigas C. nippona Saccostrea gggccgtcgtagccgaacaggctccgtcgccttaaatacagacg»1atgggaaactcgtcg| 1 7 6 gggccgtcgtagccg aac aggctccgtcgccttaaatac ag acgaatggg aaactcgtcg 1 7 8 GCG ACC AGGTCGCTAGGCCGAGGG - ATCC AGGCGAACTCGCTGGGGGGGTTTA 156 GCGACCAGGTCGCAAGGCCGAGGG-ACAC-GGCGA-CTCGCTTGGGGGGGTCGGTTCAAA 171 GAGAAAACGCTATCTCTTTCGGTC - GTAC CTCGTTGGCAGAGGCG 149 o. edulis |ccgtcttcccga|cggcgccttgggcagtcgtagccgaacaggctccgtcgccttaaatac 0. angasi CCGTCTTCCCGA C.gigas TTTCCTCT C.nippona GCCTTTCCCCCC Saccostrea CAACCGA 236 190 164 183 156 O. edulis 0 . angasi C. gigas C. nippona Saccostrea agacgaIatgggaaactcgtcgccgtcttcccgaIcggcgccttgggcagtcgtagccgaac cggcgccttgggc agtcgtagccgaac cagcgccttgggccgtcgaagccttcc cagcgccttgggccgtcgaagcctttt gagagccttgggcagtcgcacccgaac 296 217 191 210 183 O. edulis O. angasi C. gigas C.nippona Saccostrea j^ GGCTCCGTCGCCTCAAATACAGACGAGCTCGACTTTCGGTGATT 341 ft GGCTCCGTCGCCTCAAATAC AGACGAGCTCGACTTTCGGTGATT 262 T GCTCCGTCOCCTTAAGTACAG ACGAGCTCGACC GCGACC 231 TTTCTTCGGAACAAAGGGCTCCGTCGCCTTAAGTACAGACGAGCTCGACC GCGACC 266 - GGCTCCGTCGCCTTAAATTC AG ACGAGCTCGACC - --GCGACC 22 4 O. edulis 0. angasi C. gigas C.nippona Saccostrea CCGAGCTCACGGACACGGCATTTGGTCACCGAATAT GACGCCAGGCATCGGCGG- - 3 9 5 CCGAGCTCACGGACACGGCATTTGGTCACCGAATAT GACGCCAGGCATCGGCGG - - 316 CCGGGCTTCCGGAACGGTGGGTCGCTAAAAC AAAAC AAAAAGC ACTCGGC ATCGTCGGGG 291 CCGGGCTTCCGGAACGGTGGGTCGC- -AAACAAAACTCATAGCACTCGGCATCGTCGGGA 324 C-GGGCTTT-GACGGGGAAGGTCGC AAAAC ACCGCTCGGCATCGTCGG- - 270 O. edulis TCGTGGGGCCGACGTTATAAGCACCCTGGTATGAACGTT-TCACGTACATCAATAGTTTT 454 O. angasi TCGTGGGGCCGACGTTATAAGCACCCTGGTATGAACGTT-TCACGTACATCAATAGTTTT 375 C. gigas TGGAAACCTCGACGTTATAAGCACCCGAGTATGTACTTTCTCACGTATACCAACAGATTT 3 51 C. nippona GGTTGGCCTCGACGTTATAAGCACCCGAGTATGTACTTTCTCACGTATACCGATAGCTTT 384 Saccostrea TC ATTGTTCCGATGTTGTAA - CGCCCGAGTATGTAC ATTCTCACGTATACGAAATCCTTA 329 O. edulis O . angasi C. gigas C .nippona Saccostrea GATCTTGGCGCGGGG GGAAAAA C 477 GATCTTGGCGCGGGG GGAAA C 396 CT-TACACAAAACCCGGGAAGGCGGAGTCGGCAAG GCTCTTG 3 92 TTAC ACACAAN AACCCGGGGGGCGGAGTCGGGAGGCAGGCAATTAAGTCCTC AACTCTGC 444 TTTTA - ACGAGGA - - AGGGGTCGGGAGCAGGCAAAG TCTCTCGGC 3 71 0. edulis 0. angasi C. gigas C.nippona Saccostrea ACTCCTCGC GGTCG-CGTCCGGCGT-TCTTGTTGCGCGCCGAGACCGGCCGAATTT- 531 ACTCCTCGC GGTCG-CGTCCGGCGT-TCTTGTTGCGCGCCGAGACCGGCCGAATTT- 4 50 CCTTTTTTC GGGGGTCGGCCGGCGTACCTCGTTGCGCGCCGCGACCG ACCGAATT- - 447 TTTTTTTTCTCGGGGGGTCGGCCGGCGTACCTCGTTGCGCGCCGAGACCGACCGAATT-- 502 TTTTCTTTCCTCGGTCG-CGTCAGGCGTACTTCGTTGCGCGCCTCGACCGGCCGAATCAC 4 30 O. edulis 0. angasi C . gigas C .nippona GGCATC-T-CT-TTTTGA 546 GGCATC-T-CT-TTTTGA 465 GGCATCATACTATTTTGA 465 GGCATCTTATTTTTTTGA 520 Saccostrea TTTGGCATC- -TAT--TGA 445 Figure 5. Nucleotide sequence of the ITS-2 region from the oysters Ostrea edulis. O. angasi. and Crassoslrea gigas (GenBank Accession Number F28n60<)l. Asterislis identify points of identical sequence and hyphens indicate points of sequence deletion/insertion. Numbers refer to the number of nucleotides from the 5' end. The 54-bp nucleotide repetitive (a) elements in (>. edulis are indicated by shading of the O. edulis sequence. The 27-bp nucleotide repeat (ft) is outlined on the same sequence. Repeat Elements in Ostrea ITS Sequence 703 O.angasi CTTTCGCTCTCGTCCTTGCGTGCACGTCTCGACCTGTGGAGACTGCTCTCTGGGCAGTGG 720 O. edulis CTTTCGCTCTCGTCCTTGCGTGCACGTCTCGACCTGTGGATACTGCTCTCTGGGCAGTGG 720 O.edulis (Littlewood 1994) 6 O.angasi CCGCTAACTGCGAGTGCGCACGGGGTGGGAGGCGTGGCGGTGCCCGATGGTCAGTGGCGA 7 80 0. edulis CCGCTAACTGCGAGTGCGCACGGGGTGGGATGCGTGGCGGTGCCCGATGGTCAGTGGCGA 780 O.edulis (Littlewood 1994) 6 ATCGGTCGGGCGTCCACCCGACCCGTCTTGAAACACGGACCAAGGAGTCTAACATGTGCG ATCGGTCGGGCGTCCACCCGACCCTTCTTGAAACACGGACCAAGGAGTCTAACATGTGCG (Littlewood 1994) G 840 840 O. angasi O. edulis 0. edulis Figure 6. Partial nutk'otide sequence of the 28S rRNA gene showing variable positioning between the oysters Oslrea angasi (GenBank Accession Number AF137()46) and O. edulis (GenBank AF137047) as used by 6Foighil and Taylor (2(l()(ll and as determined by Littlewood (1994). prompted a re-examination of the partial 28S rRNA gene se- quences produced for O. edulis by Littlewood (1994) and those cited by 6Foighil and Taylor (2000) for O. edulis and O. angasi (deposited in GenBank). The sequences by 6 Foighil and Taylor (2000) show three variable positions between the taxa over 911 nucleotides. However, the Littlewood ( 1994) sequence for O. edu- lis does not differ from O. angasi at those positions (Fig. 6). Therefore, it would appear that O. angasi and O. edulis may have identical nucleotide sequences for the coding regions of the rRNA cistron given that the partial data from the 28S rRNA gene were taken from the variable domains (Dl. D2, D3). The 10-bp differ- ence between the O. edulis 1 8S rDNA sequence of this study and U88709 from GenBank is not considered further as the latter is not published and the methodology used to infer it is not known. The I8S rRNA gene was expected to be the most conserved of the coding regions and therefore is unlikely to differ so greatly within a species. Variation was delected in the non-coding internal spacer re- gions. The ITS-1 and ITS-2 both show sequence divergence be- tween O. edulis and O. angasi irrespective of the repeat elements in ITS- 1. However, the differences between O. edulis and O. an- gasi in ITS-1 are small compared with those observed between congenerics Crassostrea gigas and C. nippona (Fig. 5). The major difference between the Oslrea species is in the repeat unit of the ITS-1. Repeat elements in the ITS-1 spacer have been identified pre- viously (Long & Dawid 1980), including those with the same subrepeat pattern Ui. h. a), (van Herwerden et al. 1999). However, it is not known if the repeating elements in these oysters have a function. Those found in the IGS region of both Drosophila niela- nogaster (Kohorn & Rae 1982) and Xenopus laevis (Moss 1983) have an influence on the level of transcription (Busby & Reeder 1983. Grimaldi & Di Nocera 1988). The ITS-1 region has an ability to form ribonucleoprotein complexes with cellular proteins and this domain is critical to the efficient maturation of the pre- cursor RNA (Lalev et al. 2000). It is likely that the repeat element in Oslrea will have some effect on transcription. The secondary structure formed in O. edulis is more thermally stable than that of O. angasi. Further, the conserved nature of the base repeat and the repetition of the a element in the ITS-2 strengthens the case for functionality of these eleiuents. In other parts of the genome, repeat regions commonly arise through a preferential gain of a few repeat units at one end of the tandem repeat array, generating a minisatellite sequence (Jeffreys et al. 1994). Slip-stranded mispairing (Levinson & Gutman 1987) is commonly proposed as a model for VNTR repeat formation, expansion and contraction. Chance mutations are necessary for repeat expansion and these produce a few tandem repeats that facilitate the first strand slippage event (Messier 1996, Taylor & Breden 2000). However, different mechanisms may be responsible for the formation of repeats with motifs longer than 5 bp (Jeffreys et al. 1994). Taylor and Bi-eden (2000) have described a general model for minisatellite birth that results in the formation of a locus with long repeats flanked by one unit of the original noncontiguous repeat. The appearance of differently sized repeats in the ITS-1 of O. edulis and O. angasi can be most expeditiously explained by unequal or misaligned crossing over. Depending on the ancestral configuration, the a' . b pair has been either lost or gained in one of the two taxa since their geographic separation. The implication of these data is that O. edulis and O. angasi are conspecific as suggested by Jozefowicz and O Foighil (1998). These findings parallel the example of Saccostrea connnenialis and S. glomerata (Anderson & Adlard 1994), in which the taxa are morphologically similar, yet geographically separated, with iden- tical flanking 18S. ITS-1. and 5.8S regions. Although O. angasi and O. edulis have identical coding regions, some variation was detected in the ITS-1 and in the ITS-2, primarily through the deletion of repeat elements in O. angasi. However, this level of .spacer variability is minor compared with that observed among the Crassostrea species. As with the Saccostrea species studied by Anderson and Ad- lard ( 1994), the species in this study are also geographically sepa- rated. Ostrea angasi is found in Australia, from Western Australia along the southern coast to Tasmania and north along the east coast to the Clarence River in New South Wales (Thomson 1954). Os- trea edulis is mainly European, occurring from Norway to Moroc- co, including the Mediterranean Sea and the Black Sea (Carriker & Gaffney 1996). As put forward by Jozefowicz and O Foighil (1998). this incongruity can be explained by undocumented an- thropogenic transport. Although O. angasi is widespread in Aus- tralia, it is not common throughout its range (Thomson 1954) and its distribution is centered around the area first colonized by Eu- ropeans. Such a scenario would suggest that the ITS- 1 of O. anga.'ii underwent a deletion of the a', b region in the approximately 200-300 y after the transfer of O. edulis to Australian waters. 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ULTRASTRUCTURAL AND HISTOCHEMICAL CRITERIA FOR DETERMINING NORMALITY IN MATURE OOCYTES OF THE PACIFIC OYSTER CRASSOSTREA GIGAS MARIA EUGENIA VALDEZ-RAMIREZ,'* ANNE DONVAL,' AND MARCEL LE PENNEC^ ^Cenlro de Investigaciones Biologicas del Noroeste, S. C. (CIBNOR) - Programa de Acuicidtiini Marina. P. O. Box 128. La Paz. B. C. S.. 23000. Me.xico: -Institiit Universitaire Eiiropeen de la Mer ilUEM) - UMR CNRS 6539 Technopole Brest-Iwise. 29280. Plouzaue. France ABSTRACT In hatcheries of bivalve molluscs, female gametes are assessed according to the moiphologic stages of oocytic devel- opment. Three stages of oocytes immature, mature, and overripe were studied. We identitled each category, based on ultrastructural features of the vitelline coat, cytoplasmic membrane, and organelles. Vitelline mclusions in the ooplasm were identified by ultra- structural and histochemical methods. Cytologic alterations involved in the typical degenerative process of mollusc oocytes were observed, including initial damage in the cytoplasmic membrane, rupture and degeneration of the vitelline coat, and damage in ooplasm and vitelline globules. All of these characteristics seem to be associated with the natural process of oocytic degeneration, but they can also be the consequence of handling during reproduction by anitlcial means. KEY WORDS: oocyte, ultrastructure, histochemistry. Cnissoslrea gigas, reproduction INTRODUCTION Poor quality of gametes is believed to be one of the main causes of irregularities of embryonic and larval development in bivalve mollusc hatcheries (Raven 1966, Galtsoff 1964, Dohmen 1983, Wilson et al. 1996, Gerard et al, 1997, Le Pennec et al. 1998). In aquaculture, the quality of the gametes is defined by its capacity to be feitilized and consequent development of a viable larva (Kjors- vik et al. 1990). Morphologic criteria are commonly used for iden- tification of female gametes of good quality. Three types of oo- cytes can be identified at spawning: mature oocytes with a round form and healthy appearance, immature oocytes with a pear shape and heterogeneous appearance, and overripe or atretic oocytes (Dorange & Le Pennec 1989. Paulet et al. 1992), This is a simple, quick, and inexpensive method. Nevertheless, these criteria are not sufficient to assess the viability of spawnings of Crassostrea gigas (Valdez-Ramirez et al. 1999). Results obtained from hatchery pro- duction reveal great variations that do not correlate with the amount of mature or normal oocytes. Germ cells maturation can be disturbed by multiple endogenous and exogenous factors in bi- valve molluscs (Pipe 1987, Dorange & Le Pennec 1989, Paulet et al. 1992). A visual inspection does not confirm that mature oocytes will develop after fertilization. Daniels et al. (1973) described the normal and abnormal char- acteristics of female gametes of Crassostrea virginica in the cy- toplasm components, in particular, yolk granule distribution. This study suggests that knowledge of the gamete cytology is signifi- cant to evaluate the reproductive capability of the species. For Pecten maximus, Dorange and Le Pennec (1989) described, by means of transmission electron microscopy (TEM), the ultrastruc- tural features in the stages of oocyte development and the natural process of oocytic degeneration. These authors showed that, at the ultrastructural level, cytologic alteration could be observed in the plasmic membrane, nuclear envelope, and mitochondria that were not detectable by gross morphologic observations. The main purpose of this study is to address some questions related to the quality of the oocytes of C. gigas. Specifically, we tried to identify and describe the cytologic and cytochemical *Corresponding author. E-mail: gvaldez(8'cibnor.mx anoinalies that most commonly affect the viability and quality of female gametes. MATERIALS AND METHODS Samples of Crassostrea gigas (Thunberg 1793) were obtained from the Bay of Brest, Brittany, France during its natural spawning period. June and July. Oocytes were obtained by draining of go- nads. The oocytes were kept in seawater for one hour to recover their shape, as they are compressed in the gonads. Some gametes were fertilized to obtain embryos and larvae. Fragments of gonads of different specimens v\'ere also used in this study. Sample Preparation for Transmission Electron Microscopy Samples (oocytes, embryos or fragments of gonads) were fixed for one hour in 2.59^ glutaraldehyde buffered with 0.2 M sodium cacodylate adjusted to 1 100 mOsm and pH 7.2 (Cross & Mercer 1993). Postfixation was completed in 1% OsOj using the same buffer for 60 min at 4°C. The samples were rinsed, followed by progressive dehydration in ethanol baths (70%, 95%, and 100%). They were embedded in resin (SpuiT 1969). Resin polymerization was completed at 60°C for 48 h. Semi-thin (1 |Jim) and ultrathin (60-70 nni) sections were cut with glass and diamond ultramicrotome knives (Ultracut-S Leica) according to the method of Cross and Mercer (1993). The semi- thin sections for study under light microscopy were stained with 0.5% toluidine blue. The ultrathin sections were contrasted with uranyl acetate and lead citrate stains (Reynolds 1963). Observa- tions of ultrathin sections were made with a Transmission Electron Microscopy (TEM) (JEOL 100 Cx). In addition, a few semi-thin sections were also treated using techniques for the identification of vitelline globules. Detection of Lipids Before postfixation, some samples of oocytes were treated in a solution of methanol-chloroform (1:1 ) at 60X for 12 h to extract the lipids (E samples). They were postfixed using the same pro- cedure described above. The identification of the lipid globules was made by comparing sections from the same batch of oocytes from which lipids were not extracted (NE samples). 707 708 Valdez-Ramirez et al. Detection of Proteins The study of basic proteins was made on semi-thin sections treated with the 1.5% periodic acid at 40°C for one hour. They were stained by Ponceau 2r at 40°C for 3 h (Gori 1977). The protein globules revealed by staining were compared with the in- clusions observed in ultrathin sections of samples from the same batch, by size, form, and their location in the cytoplasm of the oocyte. Detection of Carbohydrates The characterization of carbohydrate reserves in the oocytes was obtained by a PAS reaction (Periodic acid-Schiff) (Gabe 1968). Negative "glycogen" controls were prepared with amylase at 37°C for 3-6 h. After several rinses, these sections were treated at the same time as the positive glycogen controls. RESULTS Mature Oocyte Mature or normal oocytes, measuring from 60 to 65 p.m. con- tain a bulky nucleus (Fig. 1: 1). The germinal vesicle, in the prophase of first meiotic division, shows regular contours, ap- proximately 25 |j,m in diameter. The nucleolus, when visible, is generally eccentrically located. Nuclear pores are sometimes ob- served. The cytoplasmic membrane bear numerous regular mi- crovilli embedded in a vitelline coat (oolemma) (Fig. 1:2). The fibrillar structure of the vitelline coat appeared highly electron- dense at the periphery (Fig. 1: 2). This was observed in all the samples of unfertilized (Fig. I: 2) oocytes and in those still in place in the gonad (Fig. 1: 1). Observations made of the vitelline coat ultrastructure in oocytes before fertilization (Fig. 1: 2), at the mo- ment of fertilization (Fig. 1: 3). and 3 h after fertilization (Fig. 1: 4) showed no real differences in electron-density. The perivitelline space is sometimes visible between the vitelline coat and the plas- mic membrane (Fig. 1: 5). In the cytoplasm, mitochondria, endoplasmic reticulum, and many vitelline inclusions are visible (Fig. 1:5). Mitochondria are very common and are distributed homogeneously. Their size is variable, the longest measuring approximately 0.5 |a.m. The lamel- lae of the endoplasmic reticulum are not abundant in the cyto- plasm. Cistemae of endoplasmic reticulum are sometimes visible in the vicinity of the mitochondria. Some dictyosomes are also present. Cytoplasmic inclusions accumulated during vitellogenesis are numerous and are varied in types. They occupy the larger part of the cytoplasm, differing in size. form, and density. By coupling ultrastructure study with cytochemical observations, the chemical nature of the inclusion was identified. Three types of inclusions were detected (Fig. 2: 1 to 8) and are described in the following three sections. Type /, Lipid Inclusions On semi-thin sections of the NE samples, the lipid inclusions (stained with Sudan black) occupy the larger part of the cytoplasm, and their distribution is homogeneous (Fig. 2: 1 ). At the ultrastruc- ture level, comparison of the samples with or without lipid extrac- tion (Fig. 2; 2 and 2: 3). shows that these compounds correspond to the largest inclusions, reaching a diameter of approximately I (j,m. Their contour is regular and. in most cases, they are enclosed by the granular endoplasmic reticulum. A higher electron density in the center than at the periphery characterizes these inclusions (Fig. 2: 2). In several NE samples, the lipid globules were partially or completely absent (Fig. 2: 4 and 5). Type II, Glycoprotein and Carbohydrate Inclusions Round organelles, from 0.2 to 1 \^.m in diameter, are sur- rounded by a membrane. The homogeneous contents are visible in ultrathin sections. They may be dispersed in the cytoplasm, but they are mainly present at the periphery (Fig. 2: 5). The distribu- tion and location of these reserves in ultrathin sections correspond to positive PAS reaction (Fig. 2: 6) and to the globules stained pink by Ponceau 2r (Fig. 2; 8) on the semi-thin sections. This suggests that type II inclusions are glycoproteinaceus in nature. Glycogen detection by comparison between PAS staining in semi-thin sections (Fig. 2: 6) and the negative control (Fig. 2: 7) does not allow clear observation of this compound in the mature oocyte. However, we note a slightly more intense staining on oocytes untreated with amylase, which suggests the presence of glycogen. Type III, Lysosome Inclusions Distribution and occurrence of the lysosome inclusions de- tected by Ponceau 2R (Fig. 2: 8). are comparable way as type II inclusions. Certain differences were observed in MET. Type III inclusions are an irregular structure, and their location is unusual. They are provided with a single external membrane. They are heterogeneous in size. form, and electron density (Fig. 1 : 2 and I: 5) and always abundant, with a maximum size comparable to the lipid inclusions. Their heterogeneity contrasts with other inclu- sions of relatively homogeneous forms. Ultrastructure Anomalies of the Oocytic Degeneration Different degenerative alterations are observed (Fig. 3: I to 5). Degeneration in the female gamete can present a large increase of the perivitelline space (Fig. 3: I to 3) and a dilatation of the base of the microvilli. In some cases, the vitelline coat is detached and no microvilli are seen at the periphery (Fig. 3: 4). In the cytoplasm, mitochondria seem to be the first oocytic organelles involved in the degenerative processes (Fig. 3: 3). Other vitelline inclusions can also deteriorate, but lysosome bodies stay intact (Fig. 3: 4 and 5). The most severe damage observed in the cytoplasm include large vacuoles enclosing the vitelline bodies and cytoplasmic com- ponents, which are degraded to a lesser or greater level and form- ing dense globular masses. At this stage, intact lysosomal bodies are common (Fig. 3: 5). Other Cytologic Alterations In some cases, mainly in free oocytes, the vitelline coat can be partially or completely detached from the plasmic membrane (Fig. 4: 1 and 2). In addition, the rupture of the plasmic membrane is sometimes seen (Fig. 4: 3), but the contents of the mature oocyte are not degraded. DISCUSSION Mature oocytes of C. gigas are comparable to those in other bivalve mollusc species, such as Spisula solidissima (Longo & Anderson 1970a. Longo & Anderson 1970b). C. virginica (Daniels et al. 1973). and Pecten maximus (Dorange & Le Pennec 1989. Devauchelle et al. 1997), In oocytes of C. gigas, the vitelline coat Criteria for Determining Normality in Mature Oocytes of the Pacific Oyster 709 Figure. 1. Ultrastructural characteristics of the normal oocytes (1) Mature Crassoslreu nigus oocyte prior to breal-) after staining with the PAS. Scale: Id fim. (7| Negative control in glycogen detection by reaction with amylase for 3-5 h al 37 C. The less dense bottom suggests the presence of glycogen. Scale: III fim. (8| Glycoprotein inclusions, type II, (>■) and probably type III (^) on semi-thin sections stained by Ponceau 2r for 3 h at 41) C. Scale: 10 (jm. Legend: L, Lipid inclusions; GI, Glycoprotein inclusions; Ger, Granular endoplasmic reticulum; Lge, Lipid inclusions after extraction; La, Lipid absents. Criteria for Determining Normality in Mature Oocytes of the Pacific Oyster 711 w* 'mm < , ^ " # 1 Ps Vc h- / * "'' . w Figure 3. Ultrastructural anomalies of the oocyte; oocytic degeneration (1) Vitelline coat deterioration (^), normal vitelline coat in another oocyte (^1. Scale: 1 (im. (2) Vitelline coat, advanced stage of degeneration (^). Perivitelline space increases. Ooplasm and organelles may be modified at the periphery of the oocyte. Normal structure of vitelline coat (-*). Scale: 1 pm. (3) Degeneration in the ooplasm. Only mitochondria are damaged (^1: their shape is modified, the cristae disappear, and their content becomes clearer. Scale: 1 ym\. (4) Advanced degeneration in the ooplasm. The altered organelles are more abundant in the cytoplasm. Deteriorated vitelline coat is observed separated at some places from the plasmic membrane (-»). Scale: 1 pm. (5) Advanced oocytic degeneration. Plasmic membrane is not visible. Vitelline coat surrounds the contents of the oocyte ( -> ). In the cytoplasm, organelles are often grouped in vacuoles ( >• ). Degeneration of the organelles is important ( ^ 1. Intact type III inclusions are abundant. Scale: I pm. Legend: Vc, Vitelline coat: Pm. Plasmic membrane; Vi, Vitelline inclusions; M, Mitochondria; Ps, Perivitelline space; Mv, Microvilli; L, Lipid inclusions; Ly, Lysosome type inclusions. tion membrane. The precise mechanism for preventing polyspermy is not clearly understood (Dohmen 19831. The formation of this membrane in oocytes of C. gigas could be explained as a specific reaction (Thierry & Rambourg 1974). Three types of vitelline inclusions were identified in the mature oocytes of C. gigas. Lipid inclusions, type I are abundant and easily locatable globules in the cytoplasm because of their homo- geneous form. We observed a partial or total vacuolization of the contents of these globules in some oocytes. Vacuolization is not the result of extraction from an extended period of dehydration in elhanol because, in the same batch of oocytes, one finds normal oocytes. If a technical problem is excluded, the assumption is that this is a variable state in the maturity of oocytes. Steele (1998) shows comparable inodifications of these globules that coincide with other deteriorations in the cytoplasm. Dorange (1989) shows that the density of lipid inclusions can be correlated to the stage of 712 Valdez-Ramirez et al. ©. Pm Vc 7 Figure 4. Other ultrastructural anomalies of tlie oocytes ( I ) Rupture of vitelline coat in mature oocytes. Plasmic membrane is not brol4 in 5 min) in all cases (h = 3/3), as expected. Furthermore, when freshly isolated sperm were snap-frozen on dry ice. thawed, and assayed, the thawed sperm were still capable of stimulating a series of shell closures (range: 2-4 in 5 min) in all cases (/; = 4/4). Oyster Spawning Pheromone 717 Importantly, these observations suggest that the factor(s) thai stimulate shell closure activity during spawning are resistant to at least one cycle of freezing and thawing. This suggests that male gonads could be dissected and stored at -7()°C during the repro- ductive season and thawed for experiments at a later date. After selective removal of extrinsic membrane proteins from freshly isolated sperm, aliquots of resuspended high-speed P3 pellets con- taining intrinsic membrane proteins were assayed. The P3 material (1 niL = 10<7r of total sample) stimulated shell closure activity (range: 3^ in 5 min) in 7 of 9 oysters (78%). whereas aliquots of high-speed S3 supernatants (10% of total volume) containing ex- trinsic membrane proteins stimulated shell closures in only 2 of 1 1 assays (18%). When male gonad tissue that had been frozen at -70°C for more than six months was subjected to the same puri- fication procedure, that is. extraction and selective removal of extrinsic membrane proteins followed by differential centrifuga- tion, similar results were obtained: resuspended P3 pellets ( 1 mL = 10% of total) stimulated vigorous, repetitive shell closures (range: 3-10 in 3 inin) in 8 of 9 assays (89%). The data indicated that the activity was due to an intrinsic membrane factor(s) and not to extrinsic membrane proteins. In control experiments, the solu- tion that was used to strip extrinsic proteins off of membranes (0. 1 M sodium carbonate, pH 11.5) did not stimulate shell closure responses in any oysters (;i = 21). A 20-minute bioassay period was used in all subsequent shell closure bioassays. The number of shell closures in control assays using ASW was 1 .2 ± 0.35 (mean ± S-: ii = 32 oysters tested; Fig. 2A). After selective removal of extrinsic membrane proteins from male gonad membranes, 1-mL aliquots of resuspended P3 pellets (12 mg protein/mL: 10% of total sample) were found to stimulate an average of 10.8 ± 3.8 (mean ± S^: n = 60 oysters tested) shell closures within 20 min (Fig. 2B). After resuspended P3 pellets ( 12 mg protein/mL) were heated at 68°C for 30 min, the number of shell closures was not significantly different (1 1.4 ± 3.4: mean ± S-; P = 0.82; n = 5 oy.sters tested; Fig. 2B). However, when resuspended P3 pellets (12 mg protein/mL) were heated at 100°C for 10 min, the number of shell closures decreased by 41% (6.4 ± 1.7; mean ±S-:P< 0.05; /; = 10 oysters tested: Fig. 2B). To test whether the active factor(s) was a protein. P3 pellets were resus- pended in denaturation buffer, reduced and alkylated, dialyzed. and digested with trypsin; in this case, the number of shell closures decreased by 68% (3.5 ± 0.8; mean ± S-: P < 0.0001; n = 58 oysters tested) (Fig. 2B). Additional experiments that were performed outside of the re- productive season, when the majority of animals had presumably already released most or all of their spawn, examined whether P3 pellets also stimulated detectable spawning in addition to shell closure. After selective removal of extrinsic membrane proteins from male gonad membranes, aliquots of resuspended P3 pellets were observed to stimulate spawning in three males (;; = 3 of 80 experiments). In each case, sperm were released through the ex- current siphon and were visualized microscopically. These results suggested that an intrinsic sperm membrane protein(s) stimulated spawning as well as shell closure activity. DISCUSSION Mass spawning of oysters in an aquaculture setting requires at least one male in the tank to stimulate other male and female oysters to spawn. In some instances, the spawning operation is delayed for several hours or is not possible at all due to lack of a o o A B No Membranes Membranes 14 - 12 ■ 10 . 8 T 6 - 4 - T 2 - n 1 " 1 ASW Control Untreated Heat (68 C) Heal(100 C) Red/Alk/Tr Treatment Figure 2. An intrinsic factor in sperm menihrane stimulates spawnin;; in Crassostrea \irf;iiiica: spawning is accompanied by a series of shell closures (shell clapping) that have been used to assess the presence of the activity. The graph shows the average number of closures (± .S-) in animals during a 2(l-min period following exposure to either: artificial seawater (ASW) alone: or ASW to which treated or untreated mem- brane fractions (resuspended P3 pellet) were added. (A) The average number of shell closures is low when oysters are incubated in ASW alone. (B) The average number was increased when untreated sperm membranes were added. Heating the membrane fraction before it was bioassayed reduced the a\erage number. The effect was statistically significant at UMl t (P < (UI5), but not at 68 f iP = 0.82). Membranes were also reduced and alkylated to break disulfide bonds, dialyzed, and then digested with trypsin. The reduction in the mean number of closures is significant (/' < (l.tKMII), supporting the suggestion that the factor is an intrinsic protein in the sperm membrane. single spawning male to trigger general spawning of a tank of brood oysters. In the British Isles, there is a need for a synthetic or recombinant pheromone for routine 48-h exotoxicity tests that are in widespread use there. Presently, commercial hatcheries in the British Isles purify fresh sperm from C. iiifiii.y Thunberg to stimu- late spawning for exotoxicity tests, but would prefer to avoid hav- ing sperm from another oyster in the water for the assays (S. Steele, Royal Holloway Univ. of London, pers. comni.). The avail- ability of a synthetic or recombinant oyster spawning pheromone would: (1) avoid introducing sperm into the water during exotox- icity tests: (2) allow for a pathogen-free method for inducing spawning in oysters; (3) allow males and females to be induced to spawn separately, providing pure gametes for use in genetic ma- nipulation for selective breeding of oysteis with disease resistance, fast growth, etc.: and (4) simplify and standai'di/e oyster hatchery operations. As a first step toward characterizing the sperm-associated pheromone responsible for stimulating spawning in oysters, we used a method for selectively stripping extrinsic proteins off mem- branes that does not affect the disposition of integral components such as transmembrane and lipid-anchored proteins (Fujiki et al. 1982). It is the procedure most widely used to extract extrinsic (or adsorbed) proteins from organelle membranes, and has enabled investigators working on integral membrane proteins to efficiently remove soluble contaminating proteins, and to distinguish periph- eral membrane proteins (those desorbed in the presence of sodium 718 Rice et al curhon;ite) from integral menibrune proteins (those that remain membrane-associated following treatment) (Coligan et al. 1997). Following sodium carbonate extraction (Fujiki et al. 1982) of freshly isolated oyster sperm membranes, the purified membranes (P3 pellet) enriched with intrinsic membrane proteins caused vig- orous, repetitive shell closures in 78% of oysters tested; in con- trast, the S3 supernatant, which was enriched with extrinsic mem- brane proteins, stimulated shell closures in only 18% of oy.sters tested. Similar results were obtained using male gonad tissue stored at -70"C for more than 6 mo. These initial data suggested that the sperm-associated pheromone was retained in sperm mem- branes, and that sodium carbonate treatment did not remove the shell closure-stimulating activity from sperm membranes. More extensive experiments were conducted to test the heat and trypsin sensitivity of the candidate oyster sperm pheromone (Fig. 2). After removing extrinsic proteins from sperm membranes, vigorous and repetitive shell closure activity was still present in purified sperm membranes (P3 pellet fractions). Incubation of these P3 membranes at lOO^C resulted in a significant reduction in this activity. Likewise, breaking disulfide bonds in intrinsic sperm membrane proteins followed by trypsin digestion resulted in a significant reduction of this activity, suggesting that the active factor(s) is a transmembrane or lipid-anchored membrane protein in the sperm membrane. Little is known about the identity of any invertebrate or verte- brate water-borne peptide/protein pheromones or their receptors. Painter and colleagues (1998, 1999, 2000) were the first to deter- mine the structure and biologic activity of a water-borne peptide pheromone in invertebrates; the 58-residue attractant ("attractin") in Aptysia has been cloned (Fan et al. 1997) and its 3-D structure predicted (Schein et al. 2001). Water-borne peptide pheromones (e.g., E/--1, Er-2) and their receptors have been best characterized in the protozoan Eiiplotes raikovi; the Er receptors, which prob- ably arise by alternative splicing from the same gene for the Er pheromones, each contain a copy of a pheromone sequence se- creted by the cell (reviewed in Luporini et al. 1996). In contrast with soluble peptide/protein pheromones. oyster spawning presumably involves the direct binding of an intrinsic sperm membrane-associated protein(s) with membrane-associated receptors in neighboring oysters. This is based on the observation that oysters initiate a series of strong, rhythmic contractions of the adductor muscle in response to sperm entering the mantle cavity via the incurrent siphon (Galtsoff 1938a, Galtsoff 1938b). Detailed information regarding the nature of the sperm membrane protein(s) and its receptor are lacking, however. Therefore, a complete un- derstanding of the molecular mechanisms underlying spawning activation will not be achieved until the sperm-associated mem- brane protein(s) and its receptor are structurally characterized and cloned. A molecular mechanism for spawning has been proposed for other mollusks. It has been demonstrated in abalone that hydrogen peroxide causes gravid male and female abalones to spawn and that this effect may result from a direct activation of the enzyme- catalyzed synthesis of prostaglandin endoperoxide. The latter is a direct precursor of prostaglandins and thromboxanes: all three may be involved in regulating spawning in abalone (Morse et al. 1977). Hydrogen peroxide also induces synchronous spawning in male and female mussels {Mytihis) and in other molluscan species (Morse et al. 1977). Lastly, regardless of the molecular mechanisms underlying sperm-induced spawning in oysters, the response of female oysters to sperm is not species-specific; sperm from C. virginica. C. gigtis. or C. angulata Lamarck have been shown to stimulate spawning in females of all three species (Nelson 1931, Galtsoff 1932, Galtsoff & Smith 1932). This suggests that a synthetic or recombinant C virginica spawning pheromone could be used in the aquaculture industry to induce spawning in several conmiercially important species, ACKNOWLEDGMENTS The authors thank B. Clough. S. Black, and C. Binz for tech- nical assistance and an anonymous reviewer for helpful comments. Supported by Texas Advanced Technology Program grant ()04952-(M02-1999 to G.T.N. , S.D.P., and S.M.R. LITERATLl Coligan. J. E.. B. M. Dunn. H. L. Ploegh, D. W. Speicher & P. T. Wing- field. 1997. Current Protocols in Protein Science. New 'I'ork. NY: John Wiley & Sons Inc. Fan, X., B. Wu, G. T. Nagle & S. D. Painter. 1997. Molecular cloning of a cDNA encoding a potential water-bome pheronional attractant re- leased during Aplysia egg laying. Mol. Brain Res. 48:167-170. Fujiki, Y., A. L. Hubbard, S. Fowler & P. B. Lazarow. 1982. Isolation of intracellular membranes by means of sodium carbonate treatment: Ap- plication to endoplasmic reticulum. J. Cell Biol. 93:97-102. Galtsoff P. S. 1930. The role of chemical stimulation in the spawning reactions of Ostrea virginica and Ostrea gigas. Proc. Ncitl. Acad. Sci. 16:555-559. Galtsoff P. S. 1932. Spawning reactions of three species of oysters. J. Wa.'ih. Acad. Sci. 22:65. Galtsoff, P. S. 1938a. Physiology of reproduction of Ostrea virginica. I. Spawning reactions of the female and male. Biol. Ball. 74:461—486. Galtsoff, P. S. 1938b. Physiology of reproduction of Ostrea virginica. II. Stimulation of spawning in the female oyster. Binl. Bull. 75:286-307. Galt.soff P. S. 1940. Physiology of reproduction of Ostrea virginica. III. Stimulation of spawning in the male oyster. Biol. Bull. 78:117-135. Galtsoff, P. S. & R. O. Smith. 1932. Stimulation of spawning and cross- fertilization between American and Japanese oysters. Science 76:371. RE CITED Luporini. P.. C. Miceli. C. Ortenzi & A. Vallesi. 1996. Ciliate pheromones. Prog. Mol. Subccll. Biol. 17:80-104. Morse. D. E.. H. Duncan, N. Hooker & A. Morse. 1977. Hydrogen per- oxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase. Science 196:298-300. Nelson. T. 1 93 1 . Stimulation of spawning in the American oyster by sperm of the Portuguese oyster. Anal. Rec. 51:48. Painter. S. D., B. Clough. R. Garden, J. V. Sweedler & G. T. Nagle. 1998. Characterization of Ap/y.s/a attractin, the first invertebrate peptide pheromonal attractant. Biol. Bull. 194:120-131. Painter, S. D., B. Clough. D.-B. G. Akalal & G. T. Nagle. 1999. Charac- terization of attractin. a pheromonal attractant in .Xplvsin. Invert. Re- prod. Devel. 36:191-194. Painter. S. D.. D.-B. G. Akalal. B. Clough. A. J. Susswein. M. Levy & G. T. Nagle. 2000. Characterization of four new members of the attractin family of peptide pheromones in .Aplysia. Soc. Neurosci. Ahstr. 26: 1166. Schein. C. G. T. Nagle. J. Page. J. V. Sweedler, Y. Xu. S. D. Painter & W. Braun. 2001. Aplysia attractin: Biophysical characterization and mod- eling of a water-bome pheromone. Biophxsical J. 8 1 :463-472. JoiinuU of Shellfish Research. Vol. 21, No. 2. 719-723, 2002. FACTORS AFFECTING SPERM MOTILITY OF TETRAPLOID PACIFIC OYSTERS QIAOXIANG DONG,' BENOIT EUDELINE," STANDISH K. ALLEN, JR.,' AND TERRENCE R. TIERSCH'* ^Aqiiacuhure Research Station. Louisiana Agricultural Experiment Station. Louisiana State University Agricultural Center, Baton Rouge. Louisiana 7OH03: 'Whiskey Creek Shellfish Hatcheiy. Tillamook. Oregon 97141: ^Aquaculture Genetics and Breeding Technology Center, Virginia Institute of Marine Science, Gloucester Point, Virginia 23062 ABSTRACT Factors such as osmotic pressure, extender solution, addition of caffeine, and pH have been shown to affect sperm motility in aquatic species. We evaluated the effects of 18 osmotic pressures, two extender solutions, seven caffeine concentrations, and a pH range of 3 to 14 on motility of sperm from tetraploid Pacific oysters, Crassostrea gigas. Motility was highest at 1000 mOsmol/kg (mean ± SD: 83 ± 14%). Calcium-free Hanks" balanced salt solution yielded significantly higher sperm motility than did artificial seawater. Sperm motility increased with caffeine concentrations to 20 niM (81 ± 12%) and decreased when concentrations were higher than 50 mM (55 ± 20%). Highest motility was obtained at a pH range of from 4 to 12; values outside this range yielded no motility. Addition of 10 mM caffeine to the different pH treatments also enhanced motility significantly. Overall, calcium-free Hanks' balanced salt solution at 1.000 mOsmol/kg. the addition of 10 mM caffeine, and a pH of around 10 could he used to enhance speiTn motility of tetraploid Pacific oysters. Our findings would assist the use of motility assays to evaluate the effectiveness of various refrigeration or cryopreservation procedures, especially outside of the peak spawning season, when sperm motility can be low and variable. KEY WORDS: Crassostrea gigas. sperm motility. pH. caffeine, osmotic pressure, tetraploid. cryopreservation INTRODUCTION MATERIALS AND METHODS Factors such as osmotic pressure (Bates et al. 1996), extender composition, pH, temperature (Lahnsteiner et al. 1997, Sunitha and Jayaprakas 1997), and additives such as caffeine (Scheerer and Thorgaard 1989, Tiersch et al. 1998) have been shown to affect sperm motility in aquatic species. Most studies have been con- ducted on teleosts (e.g.. Moiisawa et al. 1983a. Morisawa 198.^b). and studies on invertebrates mainly focus on sea urchin (Morisawa et al. 1990), polychaetes (Pacey et al. 1994), and ascidians (Yoshida et al. 1992, Yoshida et al. 1994). Little is known about the effects of these factors on oyster sperm, especially on sperm from tetraploid oysters, which possess four sets of chromosomes instead of the noimal diploid two sets. Tetraploid oysters create opportunities for genetic improve- ment, including direct production of triplnid (sterile) seedstocks by crossing with normal diploids. Refrigerated and frozen storage of tetraploid oyster sperm will be a critical tool for commercial-scale application of tetraploid stocks and for developing tetraploid breeding programs. Although subjective, motility estimation is the technique used most cotnmonly to assess sperm quality of fish and shellfish (Piironen 1993, Tiersch et al. 1994, Koupal et al. 1995). Motility has been used to assess the sperm quality of oysters (Pa- niagua-Chavez et al. 1998), but its application with tetraploid oys- ter sperm is unexplored. The objective of this study was to develop procedures for evaluation of sperm quality to assist the overall goal of sperm storage for tetraploid Pacific oysters, Crassostrea gigas. Specifically, we evaluated the effects on sperm motility of: (1) osmotic pressure; (2) extender solution; (3) caffeine, and (4) pH. Our findings indicate that these factors can alter the motility of tetraploid oyster sperm collected late in the spawning season. To our knowledge, this is the first study to systematically characterize sperm motility of tetraploids of an aquatic species. ♦Corresponding author. E-mail: ttiersch@agctr.lsu.edu Tetraploid oysters were obtained in September and October 2001 from Whiskey Creek Shellfish Hatchery (WCSH) (Tilla- mook, Oregon) and were shipped chilled by overnight delivery to the Louisiana State University Agricultural Center, Aquacullure Research Station (ARS). Water samples from WCSH had an os- molality of 873 mOsmol/kg as measured by vapor pressure o.s- mometry (model 5500, Wescor Inc.. Logan, UT) at the ARS. Sperm were collected by dry stripping of the gonad (Allen & Bushek 1992). Undiluted nonmotile sperm were equilibrated in 30 |j.L of test solutions at 23'^C for 2 min before assessment of mo- tility. Sperm motility was estimated at 200x magnification using darkfteld microscopy (Optiphot 2, Nikon Inc.. Garden City, NY) and was expressed as the percentage of cells actively moving in a forward direction. Throughout the experiments, two extender solutions were used: artificial sea water (ASW) (Fritz Super Salt, Fritz Industries, Inc. Dallas. TX) and calcium-free Hanks" balanced salt solution (C-F HBSS) (Paniagua-Chavez et al. 1998). All chemicals (except ASW) were of reagent grade (Sigma Chemical Corporation., St. Louis, MO). Osmolality was measured with a vapor pressure os- mometer. In our first study, the effect on sperm motility of ASW of 18 different osmolalities ranging from 30 to 1400 mOsmol/kg was evaluated with a total of 20 oysters in four trials, for which oysters were received on August 24, August 30, September 19, and Sep- tember 26. The second study compared ASW and C-F HBSS at 13 different osmolalities with five oysters, which were received on October 16. In the third study, the effect of caffeine was evaluated at seven concentrations (2 to 100 mM) with eight oysters from two shipments received on October 10 and October 16. In the fourth study, a pH range of from 3 to 14, with and without caffeine, was evaluated with three oysters that were received on October 10. Sperm from individual oysters was used for all studies (samples were not pooled). Within this manuscript, extender solutions at 719 720 Dong et al. specific osmolalities such as ASW at 1 .000 iiiOsriiol/kg are abbre- viated as ASW 1 ()()(). Data were analyzed using one-way or two-way analysis ot variance. Tukey's honestly significant difference procedure was used to test for differences (a = 0.05) among results for osmo- lalities, caffeine concentrations and pH levels (.SA.S Institute 1991). RESULTS Among the osmolalities tested, sperm Irom tetraploid Pacific oysters remained immotile when diluted with A.SW below 51)0 mOsmol/kg (Fig. I). Motility increased from 12 ± 12% (mean ± SD) in ASW500 to 50 ±11% in ASW670. There was no signifi- cant difference among sperm motilities activated with ASW at 670, 700. 750, and 800 mOsmol/kg {P > 0.05). Although sperm motility was not significantly different among ASW at 900, 950, 1,000. and 1.100 mOsmol/kg. ASWIOOO elicited the highest mo- tility 83 ± 14% (Fig. 1). Motility decreased significantly when osmolality was greater than 1,100 mOsmol/kg. Motility in C-F HBSS was significantly higher than that in ASW (P < 0.0001) (Fig. 2). Each extender showed the highest motility at 1,000 mOsmol/kg, which agreed with the results of the previous experiment. But motility in C-F HBSS 1000 (81 ±9%) was double that in ASWIOOO (40 ± 22%). Other osmolalities of C-F HBSS also enhanced spemi motility. For example, motility in C-F HBSS670 (50 ± 10% I was six times greater than that in ASW670 (8 ± 13%) (Fig. 2). Because the oysters used in this experiment were received later in the spawning season than those used for the first study, lower motilities overall were observed for ASW. Based on the results of the second experiment. C-F HBSS at 1 ,000 mOsmol/kg was used for caffeine assessments. Motility in- creased with caffeine concentrations of from 2 mM (60 ± 13%) to 20 niM (81 ± 12%) and decreased when concentrations were higher than 50 niM (55 ± 20%) (Fig. 3). The lowest motility (49 ± 10% I was in the control treatment: C-F HBSS 1000 without the addition of caffeine, but additions of 2, 4. and 6 niM caffeine were 100 100 o 80 60 40 20 ghi ■-■^■1 gh ooooooooooor^oooooo cooooooor^oiflor^omoooo Osmolality (mOsmol/kg) Figure 1. Percent motility (mean ± SI)) of tetraploid Pacific oyster sperm activated in artificial seawaler at 18 different osmotic pressures. Bars sharing a letter were not significantly dilierent (/' > 0.(15). O Osmolality (mOsmol/kg) Figure 2. Percent motility (mean ± SD) of tetraploid Pacific oyster sperm activated in .\S\\ (filled bars) and C-F HBSS (open bars). Bars with an asterisk indicate a significant difference {P > (1.05) between ASW and C-F HBSS. not significantly different from the control (P > 0.05). Motilities in 10 and 20 niM caffeine were significantly higher than in other concentrations (P < 0.05), but they were not different from each other iP > 0.05) (Fig. 3). Based on these lesults, C-F HBSS 1000 with and without 10 mM caffeine were used for pH assessment. Motility was highest at a pH lange of from 4 to 12: values outside this range yielded no motility (Fig. 4). The highest motility was in pH 10.5 (67 ± 6%), followed by pH 10 (63 ± 6%). The addition of 10 mM caffeine to these pH treatments enhanced motility significantly (P < 0.001) 100 80 g 60 I" '^ O S 40 20 a 3 H H I I 4 6 10 20 Caffeine (mM) 50 100 Figure 3. Percent motilit> (mean ± SD) of tetraploid Pacific oyster sperm activated with the additicm of caffeine into C-F HBSS at 1.0(10 m()smol/kg. Bars sharing a letter were not significantly different iP > ().()5). Motility of Tetraploid Oyster Sperm 721 o 00 80 * * II T . ih\] 60 1 Jll f- * * 40 • 20 n T CO oi^o^T^^*^^ pH Figure 4. Percent motility (mean ± SDl of tetraploid Pacific oyster sperm in C-F HBSS at 1,(100 mOsmol/kg with different pH treatments. Open bars, C-F HBSS without addition of caffeine; filled bars, C-F HBSS with lU mM caffeine. Bars with an asterisk indicate a significant difference (P > 0.05) between presence and absence of caffeine. (Fig. 4). Motility was 90 ± 0% in pH II. 87 ± 6% in pH 10.5. and 88 ± 3% in pH 10. However, there was no significant difference in sperm motility across the pH range of from 7 to 12 (P > 0.05). DISCUSSION Sperm activation is a complex process in which roles are played by many factors, including osmotic pressure, extender com- position, membrane structural changes, and extracellular and in- tracellular pH. Previous studies have suggested that different spe- cies have different sperm activation mechanisms. Basically, in fish there seem to be two major factors influencing sperm activation: changes of osmotic pressure and changes of ionic concentration. For instance, studies on muskellunge Esox inasqiiinongy (Lin & Dabrwoski 1996), channel catfish ktalunis piinclatiis (Bates et al. 1996), and Asian catfish Clarias macrocephalus (Tan-Fermin et al. 1999) suggested that sperm motility was initiated by a reduction of osmotic pressure. However, activation of sperm from salmonids and cyprinids was caused by changes in concentrations of ions such as potassium and calcium (Morisawa et al. 1983a, Billard & Cosson 1992). The mechanism of initiation of sperm motility has been most studied in salmonids although much remains unknown, especially at the intracellular level. Motility studies in other taxa such as invertebrates are limited and the associated mechanisms remain largely unknown. There are less than 40 references addressing oyster sperm motility in any way since 1970. Most use motility as a criterion to evaluate fer- tilization or in toxicological assays to evaluate waste eftluents or heavy metals. Factor that affect sperm motility such as osmolality, extender composition. pH, and temperature are briefly mentioned in these studies and others on sperm cryopreservation, and there- fore the information is fragmented and dispersed. The present study was designed to address factors affecting the sperm motility of tetraploid Pacific oysters and provided a more detailed and systematic approach. Osmolality, as mentioned above, plays an important role in the activation of fish sperm. Osmolalities of -1000 mOsmol/kg (the full-strength salinity of sea water. 32 ppt) and 670 mOsmol/kg were previously used for sperm activation and extender solutions for C. gigas (Yankson & Moyse 1991. Kurokura et al. 1990). Other species were studied with sea water of ambient environmen- tal salinity. The first investigation of sperm motility across a wide range of osmolalities was with the diploid eastern oyster. C. vir- ginica (Paniagua-Chavez et al. 1998). High sperm motility (90%) was observed across a range of 600 to 1,500 mOsmol/kg (Pa- niagua-Chavez & Tiersch 2001). In the present study, sperm mo- tility of tetraploid Pacific oysters was also observed across a wide range of osmotic pressures (500-1400 mOsmol/kg). but the high- est motility was limited to 1.000 mOsmol/kg. although the oysters were conditioned in seawater at 873 mOsmol/kg. Whether osmotic pressure plays the major role in oyster sperm activation remains unknown: however, motility was suppressed in tetraploid sperm of C. gigas at osmolalities of lower than 500 mOsmol/kg. Suppres- sion was reported to occur at less than 22 mOsmol/kg in C. vir- ginica (Paniagua-Chavez et al. 1998). This might correspond to the habitat of these species in natural environments. Crassostrea gigas prefers higher and more stable salinities (15 to 33 ppt) (Kusuki 199! ) than does C. rirginica. which normally occurs from 5 to 40 ppt (Galtsoff 1964, Wallace 1966). Natural seawater and ASW are most commonly used as ex- tender solutions for spenn of marine organisms including estuarine organisms like oysters. Other extenders such as DCSB4 (Bougrier & Rabenomanana 1986), HBSSI990 (Zell et al. 1979), and C-F HBSS640 (Paniagua-Chavez et al. 1998) with specific ionic com- positions have also been successfully used for oyster sperm. The removal of calcium from HBSS was found to enhance motility in sperm of diploid eastern oysters (Paniagua-Chavez et al. 1998) and tetraploid Pacific oysters (this study) and was also superior to ASW. Contrary to the situation in salmonids. in which sperm motility is initiated by a decrease in potassium concentration upon release into fresh water (Morisawa et al. 1983a). an increase of potassium concentration (200 mM) was found to have an activat- ing effect on diploid Pacific oyster sperm (Faure et al. 1995). The same study showed no effect of the increase of potassium on sperm of the king scallop Pecten nui.xinuis. but an increase in motility was observed with media lacking sodium. These results may indicate a species-specific response for ionic effects on bivalve sperm mo- tility. Also, changes in ion concentration, rather than absolute con- centration, may act as the trigger of initiation of sperm motility. Caffeine has been used as motility stimulant to optimize the recovery and quality of thawed spermatozoa in mammalian species (Correa & Zavos 1996. Park & Sirard 1996). Few studies have addressed use of this chemical in sperm of aquatic species and there are no reports for oysters. The present experiment showed a significant increase in sperm motility with the addition of 10 mM caffeine, while concentrations above 50 niM reduced motility, A previous study in the razorback sucker Xyraiichen texamts showed increased motility in refrigerated sperm after the addition of 5 mM caffeine, but not in thawed sperm (Tiersch et al. 1998). We did not evaluate the addition of caffeine with thawed sperm in this study, but fertilizing capacity of cryopreserved semen of rainbow trout Oncorhynchus mykiss was improved when eggs were fertilized in a buffered saline solution containing 5 mM theophylline, a chemi- cal relative to caffeine used to prolong and intensify sperm motility (Scheerer & Thorgaard 1989). Alkaline pH has been found to be conducive to sperm activa- tion in aquatic species (Thorogood & Blackshaw 1992. Sunitha & 722 Dong et al. Jayaprakas 1997. Ciereszko et al. 2001 ). and pH value.s between 7 and 9 have been used in most studies for oyster sperm (e.g.. Zell et al. 1979, Paniagua-Chavez et al. 1998). Study of the king scallop (Faure 1996) found that gonadal pH was more acidic than seawater pH and suggested that the acidity of the genital tract maintained the spermatozoa in a quiescent state. A reduction of sperm motility was reported in P. maximus and C. gigas upon decrease of pH in seawater (Faure 1996). In the present study, pH values below 7 induced a significant reduction in spenn motility although there was variation among individual oysters. The highest sperm motil- ity was observed at a pH of 10, which agrees with findings for the Japanese pearl oyster Pinctada fucata (Yu et al. 1998). although that study found an interaction between salinity and pH. In that study, sperm motility could not be activated by seawater at a salinity of 30 ppt and a pH of 8.0, but motility was greater than 80% when pH ranged from 9.0 to 11.5. Sperm from tetraploid Pacific oysters showed motility over a wider pH range in the present study. It appears that relative influence of most factors affecting sperm motility of bivalves are species-specific. Finally, the main practical interest in tetraploidy is for the production of triploids by mating with diploids. The induction ot tetraploidy in oysters was first reported in 1994 (Guo and Allen. 1994). Studies of the factors affecting sperm motility of tetraploid oysters have only now begun as reported here. Our experiments indicate that use of C-F HBSS at 1,000 mOsmol/kg as an extender, the addition of 10 mM caffeine, and a pH of around 10 can be used to enhance sperm motility of tetraploid Pacific oysters. This would assist the use of motility assays to evaluate the effectiveness of various refrigeration or cryopreservation procedures, especially outside of the peak spawning season when sperm motility can be low and variable such as in this study. The effectiveness of these conditions in improving fertilization rates requires further study. Future research is required to evaluate differences between sperm of diploid and tetraploids within and among aquatic species. ACKNOWLEDGMENTS The authors thank Y. Li for laboratory assistance and J. Dock- stader for help with data analysis. This study was supported by funding from the USDA-SBIR program and the Louisiana Sea Grant College Program. This work was approved for publication by the Director of the Louisiana Agricultural Experiment Station as number 02-66-04.37. LITERATURE CITED Allen, S. K. & D. Bushek. 1992. Large scale proJuction tit triploid Cras- sostrea virginica (Gmelin) using "stripped'" gametes. AqiiaaiUure 103: 241-251. Bales. M. C. W. R. Waynian & T. R. Tiersch. 19%. Effect of osmotic pressure on the activation and storage of channel catfish sperm. 7"ra/i\. Am. Fish. Soc. 12.'i;798-802. Billard, R. & M. P. Cosson. 1992. Some problems related to the assessment of sperm motility in freshwater fish. / E.xp. Zool. 261:122-131. Bougrier. S. & L. D. Rabenomanana. 1986. Cryopreservation of sperma- tozoa of the Japanese oyster, Crassostrea gigas. Aiiuaculture 58:277- 280. Ciereszko. A.. K. Dabrowski. B. Piros, M. Kwasnik & J. Glogowski. 2001. 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Influence of pH. temperature, sa- linity and media on activation of motility and short term preservation of Motility of Tetraploid Oyster Sperm 723 spermatozoa of an estuarine fish. Mystus giilio (Haniiltonl (Siluridae- Pisces). Indian J. Mar. Sci. 26;361-365. Tan-Fermin. J. D.. T. Miura. S. Adachi & K. Yamauchi. 1999. Seminal plasma composition, sperm motility and milt dilution in the Asian catfish Clarias marcincephalus (Gunther). At/iiacKlniie 171:323-338. Thorogood, J. & A. Blackshaw. 1992. Factors affecting the activation, motility and cryopreservation of the spermatozoa of the yellowfin bream, Acanthopagnis aiistrali.s (Guenther). Aijtuiciilt. Fisli Manage. 23:337-344. Tiersch, T. R., C. A. Goudie & G. J. Cannichael. 1994. Cryopreservation of channel catfish sperm: Storage in cryoprotectants, fertilization Inals and growth of channel catfish produced with cryopreserved sperm. Trans. Am. Fish. Soc. 123:580-586. Tiersch, T. R., C. R. Figiel Jr, W. R. Wayman, J. H. Carmichael & O. T. Gorman. 1998. Cryopreservation of sperm of the endangered razorback sucker. Tran.';. Am. Fish. Soc. 127:95-104. Wallace, D. H. 1966. Oysters in the estuarine environment. A symposium of estuarine fisheries. Am. Fish. Soc. Spec. Piibl. 3:68-73. Yankson. K. & J. Moyse. 1991. Cryopreservafion of the spermatozoa of Crassostreo tulipa and three other oysters. Aquacuhure 97:259-267. Yoshida, N., K. Inaba & M. Morisawa. 1992. Partial purification of sperm- activating and sperm-attracting substance in the ascidian, Ciona intes- tinalis. Zool. Sci. 9:1206. Yoshida, N.. K. Inaba. K. Ishida & M. Morisawa. 1994. Calcium and cyclic AMP mediate sperm activation, but Ca"* alone contribute sperm che- motaxis in the ascidian, Ciona savignyi. Dev. Growth Differ. 36:589- 595. Yu, D.. J. Chen, T. Su, H. Zhu & S. Jiang. 1998. A primary study on experimental biology of spermatozoa oi Pinctada fucata (Gould). Trop. Oceanol./Redai Haiyang 17:83-87. Zell, S. R., M. H. Bamford & H. Hidu. 1979. Cryopreservation of sper- matozoa of the American oyster Crassostrea virginica Gmelin. Cryo- biology 16:448-460. .fnurnal of Shcllfi'ih Rfsi-arch. Vol. 21. No. 2. 725-732. 2002. AN IMPROVED PROCEDURE TO COUNT PERKINSUS MARINUS IN EASTERN OYSTER HEMOLYMPH AMY D. NICKENS,' - JEROME F. LA FEYRE,'* ERIC S. WAGNER,' TERRENCE R. TIERSCH' ^ Cooperative Aquatic Animal Health Research Proi^ram. Department of Veterinary Science. Louisiana State University- Agricultural Center. Ill Dalrymple Building. Baton Rouge. Louisiana 70803: 'Acjuaculture Research Station. Louisiana Agricultural. Experimental Statiim. Louisiana State University Agricultural Center. 2410 Ben Hiir Road. Baton Rouge. Louisiana 70820 ABSTRACT Perkitisiis marimis infeL'tion intensity in Crassostrea virginica can be quantified without killing of oysters by deter- mining parasite density in hemolyniph samples incubated in fluid thioglycollate medium (FTM). The goal of this study was to improve existing protocols for counting of P. inarimis in oyster hemolymph. Specifically, the objectives were to examine the effects on parasite number and diameter of: 1) adding supplements to FTM such as lipid and oyster extract; 2) incubating with various FTM preparations with and without agar or beef extract; 3) incubating with various hemocyte densities (lO*^. 10'', and 10' hemocytes/mL of FTM) in a constant FTM volume; 4) incubating with different volumes of FTM (0.2 mL, 1 .0 mL, 5.0 mL. and 25.0 mL); and 5) sodium hydroxide digestion of cellular debris. From these results, an improved hemolymph protocol was developed. The diameters and numbers of enlarged parasites or hypnospores in hemolymph of 20 oysters measured by the improved protocol and the standard FTM hemolymph assay of Gauthier and Fisher were compared. Finally, the standard and improved protocols were compared with the FTM body burden assay. The diameter of hypnospores from samples processed with the improved protocol (26 ± 13 |a.m) was significantly greater than the diameters from samples processed with the standard protocol ( 10 ± 4 (jim). The number of hypnospores in samples processed with the improved protocol (8.6 x 10' + 3.3 x 10') was significantly greater than the numbers in samples processed with the standard protocol (1.9 X 10' ± 3.4 x 10'). Results of the body burden assay were significantly correlated with results of the standard hemolymph assay and with results of the improved hemolymph assay. The coefficient of determination (r = 0.7602) and slope (O.yi 189) of the regression of the FTM body burden assay against the improved FTM hemolymph assay was improved from the coefficient of determination (0.5543) and slope (0.61257) of the regression of the FTM body burden assay against of the standard FTM hemolymph assay. KEY WORDS: dermo. Perkinsus iiuiriim.s. FTM hemolymph diagnostic assay. Crassosuea virginica INTRODUCTION Use of a protociil Id quantify the presence of the pathogenic protozoan Perkinsus marums by determining the number of en- larged parasites, or hypnospores. in hemolymph samples of eastern oysters. Crassostrea virginica. allows estimation of infection in- tensity without killing oysters (Gauthier & Fisher 1990). There- fore, infection intensity in the same animal can be monitored over time. In the existing protocol, hemolymph from the adductor muscle sinus is removed, centrifuged. and the cell pellets contain- ing oyster hemocytes and parasites are incubated in fluid thiogly- collate medium (FTM) for 1 week. After incubation, cellular de- bris is digested with sodium hydroxide (NaOH). The hypnospores are stained with LugoFs solution and their numbers per mL de- termined. This FTM hemolymph assay has been useful to follow the progression of the disease in individual oysters under different environmental conditicnis (Fisher et al. 1992. Ragone Calvo & Burreson 1994). Gauthier and Fisher ( 1990) proposed additional advantages of the FTM hemolymph assay, including the detection of early in- fections and the measurement of systemic infections rather than localized infections. In their initial study, the FTM hemolymph assay detected many infections misdiagnosed as negative by Ray's FTM tissue assay. In Ray's FTM tissue assay, a piece of oyster tissue (e.g.. mantle tissue in Gauthier and Fisher's 1990 study) is incubated in FTM for about a week, the tissue is then smeared on a slide, and the parasites stained with Lugol's solution and the intensity of infection are estimated using a semi-quantitative scale ♦Corresponding author. E-mail; jlapeyre(a>agctr.lsu.edu (Ray et al. 1953, Ray 1954a, Ray 1954b). Bushek et al. (1994), however, found no evidence that the FTM hemolymph assay was more sensitive than the FTM tissue assay in detecting low P. inarimis infections when both mantle and rectal tissues were used in the tissue assay. Moreover, correlations between FTM tissue and fTM body burden assays were always higher than correlations between FTM hemolymph and FTM body burden assays. The body burden assay measures the number of parasites in the whole oyster and is considered the most sensitive and accurate diagnostic assay for P. marinus (Bushek et al. 1994, Fisher & Oliver 1996, Oliver et al. 1998). A quantitative competitive polymerase chain reaction (QCPCR) assay for P. marinus was recently shown to be more sensitive than the FTM hemolymph assay, detecting infections in 24 oysters compared with 22 infections with the FTM hemolymph assay (Yamall et al. 2000). Diagnosis of P. marinus in oyster hemolymph by QCPCR detected as many infections as with the FTM body burden assay, suggesting that hemolymph is suitable for determining P. marinus infection in oysters. Whereas QCPCR is an effective assay and specific for P. marinus. it is also expen- sive and requires technical expertise and equipment in molecular biology to perform as indicated by Yarnall et al. (2000). The ITM hemolymph assay in contrast is easy and inexpensive to perform. There is thus a need to improve the sensitivity and accuracy of the FTM hemolymph assay. Potential problems with FTM assays have been previously noted and include insufficient parasite enlargement, parasite clumping, and parasite adherence to the walls of centrifuge tubes, each of which can lower parasite counts (Bushek et al. 1994, Fisher & Oliver 1996). Poor parasite enlargement may be caused 725 726 NiCKENS ET AL. by exhaustion of necessary nutrients in FTM or tissue (Ray 1954a, Bushek et al. 1994, Fisher & Oliver 1996). In addition, the pro- tocols used in FTM hemolymph assay varied greatly among re- searchers in sample preparation (e.g., centrifugation speed), sample processing, and in parasite counting protocols (Gauthier & Fisher 1990, Ragone Calvo & Burreson 1994, Bushek et al. 1994. Oliver et al. 1998). It is likely that these recognized problems and differences in protocols account for the poor performance (i.e., sensitivity and accuracy) of the FTM hemolymph assay. The goal of this study was to improve the protiicol for counting of P. marinus in oyster hemolymph through systematic examina- tion of components of the standard protocol described by Gauthier and Fisher (1990). The objectives were to examine the effects of: ( 1 ) adding supplements to FTM, including lipid and oyster extract; (2) adding various FTM preparations (with and without agar or beef extract); (3) incubating with various hemocyte densities (10\ 10", and 10^ hemocytes/mL of FTM) in a constant FTM volume; (4) incubation of hemocytes in different volumes of FTM (0.2 mL, 1.0 niL, 5.0 mL, and 25.0 mL); and (5) NaOH digestion of cellular debris on hypnospore diameter and number. MATERIALS AND METHODS Oysters Eastern oysters were collected from Hackberry Bay |29°23'54"N, 90°28"W) in the spring of 1999, placed in 16 mm mesh shellfish cages (Aquatic Eco-Systems, Inc., Apopka, FL). and hung in the water from docks at the Louisiana Sea Grant Oyster Hatchery (29°12'30"N, 90°02'30"W) in Grand Isle, Louisiana, an area en- zootic for P. marinus. In the summer and fall of 1999, oysters were transported to the Department of Veterinary .Science at Louisiana Slate University, Baton Rouge, and were placed in an indoor re- circulating system equipped with 1 p.m and 10 jxm cotton filters in polypropylene filter cartridges and an ultraviolet light. Water was maintained at 15 ppt with hw-Marinemix Professional sea salts (Hawaiian Marine Imports Inc., Houston. TX) and 25"C. General Procedures All chemicals were from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. A notch was ground into the edge ot oyster shells near the adductor muscle and 3 mL of hemolymph were withdrawn from the adductor muscle sinus. Hemolymph samples were transferred to 3 mL snap-cap tubes and immediately placed on ice to prevent hemocyte clumping. The number ot hemocytes per mL of hemolymph was determined using a Neu- bauer Bright-Line hemocytometer (Reichert, Buffalo, NY). Hemolymph samples from each oyster, containing 10" hemocytes, were added to 1.5 mL microcentrifuge tubes, centrifuged at 800 x g for 10 min, and the supernatant was discarded. All samples were prepared in triplicate. Pellets were resuspended in 1 mL of alter- native fluid thioglycoUate medium (AFTM) (Sigma number A 0465) prepared according to the manufacturer's instructions and supplemented with 16 g/L of hw-Marine Professional sea salts and 50 jjLg/mL chloramphenicol. This solution, referred to as AFTM throughout this report, did not contain agar, unlike FTM (Sigma number T 9032), which contains 0.75 g/L of agar. Samples were layered with 10 ixL of nystatm (5,000 Units/niL) to prevent fungal growth. Test tubes were stored in the dark at room temperature for seven days, allowing parasites within hemocytes to enlarge to hypnospores. After incubation, samples were centrifuged at 1500 x g for ten min and the AFTM supernatant was discarded. Pellets were resuspended in 1 mL of 2 N NaOH and incubated in a 60°C water bath for 1 to 2 h to digest hemocyte debris. Samples were centrifuged to remove NaOH and hypnospores were rinsed three times with 0.1 M phosphate-buffered saline (PBS) containing 0.5 mg/niL bovine serum albumin (BSA). In a previous study, it was found that BSA decreased parasite clumping and improved para- site recovery by reducing the number of parasite that adhered to the test tube walls (Coates et al. 1999). Samples were stored at 4''C in 1 mL of PBS supplemented with BSA and 2 mg/mL of sodium azide. At the time of counting, samples were centrifuged, and 900 p,L of supernatant were removed. Each 100-p,L sample was trans- ferred to a separate well of a 96-well tissue culture plate. Fifty microliters of each sample was transferred to wells containing 50 |xL of PBS with BSA to form a 1 ; I dilution. Samples were serially diluted in this manner until a 1;64 dilution was reached. Lugol's solution (50 |jiL; 0.012 g/mL of potassium iodide and 0.008 g/mL of iodine in water) was added to each well to stain the samples. Tissue culture plates containing samples were centrifuged at 200 x g for 5 min to form a monolayer of hypnospores on the plate bottoms to facilitate counting. Numbers of hypnospores were re- corded at 200-X magnification using an inverted microscope (Cari Zeiss, Inc., Thornwood. NY) from wells containing 100 to 400 hypnospores. Cells were counted at 400x when they were too small to identify at 200x. The diameter (jji.m) of 50 hypnospores from each sample was measured with an ocular micrometer, and the number of hypnospores per 10" hemocytes was calculated. Experiments Effects of Supplemented AFTM on Hypnospore Diameter and Number Hemolymph was collected from five oysters as described above. AFTM, supplemented with 5% lipid concentrate (Gibco, Gaithersburg, MD), 5% oyster extract, or 5% oyster saline, was added to 10" hemocytes from each oyster. Oyster extract was prepared by homogenizing whole oyster tissue in oyster saline at a concentration of 0.2 g wet tissue per mL, centrifuging at 10,000 x g for 1 5 min, removing the oyster extract (supernatant), and storing h at -20°C. Oyster saline (0.95 g/L CaCl,-2H,0, 1.46 g/L MgSO^, 2.18 g/L MgCU-6H,0, 0.67 g/L KCl, 11.61 g/L NaCl, and 0.35 g/L NaHCO,) was included as a control. Samples were incubated, processed, counted, and measured as de.scribed above. Lipid was found to increase hypnospore diameter and was added to FTM in all further experiments. Effect of FTM Types on Hypnospore Diameter and Nunilier Hemolymph was collected from 15 oysters as described above and five formulations of FTM, supplemented with 16 g/L of hw- Marine Professional sea salts, 50 |j.g/mL chloramphenicol, and 5% lipid concentrate, were added to 10" hemocytes samples. The for- mulations compared were Bacto tluid thioglycoUate medium (Bee- ton Dickinson and Company, Franklin Lakes, NJ. Difco number 0256154), Bacto thioglycoUate medium with K agar (Difco num- ber 0607 178), Bacto fluid thioglycoUate medium with beef extract (Difco number 0697179), alternative fluid thioglycoUate medium (Sisimu number A 0465), and thioglycoUate medium (Sigma num- Improved Hemolymph Procedure to Count P. marinus 727 ber T 9032) (Table 1). Samples were incubated, processed, mea- sured, and counted as described above. Effect of Heniocyte Density on Hypnospore Diameter and Number Hemolymph was collected from 15 oysters as described above and 10^, 10'\ and 10^ hemocytes from each oyster were incubated in I mL of AFTM supplemented with 5*^^ lipid concentrate. For this experiment, the number of hypnospores per hemocyte was calculated, and hypnospore diameter was measured as described above. Effect of AFTM Volume on Hypnospore Diameter and Number Hemolymph was collected from 15 oysters as described above. Hemocytes (10") were incubated in 0.2. 1.0, 5.0, or 25.0 mL of AFTM supplemented with 5% lipid concentrate. Samples were processed, measured, and counted as described above. Effect of NaOH Digestion of Samples on Hypnospore Diameter and Number Hemolymph samples from 15 oysters were processed with and without NaOH digestion after incubation in AFTM supplemented with 5% lipid concentrate. Samples processed without NaOH di- gestion were centrifuged at 1500 x g for 10 min to remove the AFTM supernatant. Cell pellets were rinsed three times with sterile artificial seawater (hw-Marinemix Professional) at 15 ppl and con- taining 0.5 mg/niL BSA. Samples processed with NaOH digestion were treated as described in the General Procedures section. Effect of Protocol on Hypnospore Diameter and Number From the results of the above experiments, an improved pro- tocol for counting of P. marinus in oyster hemolymph was devel- oped and compared with the "standard" protocol of Gauthier and Fisher (1990). Major differences between the standard and im- proved protocols are summarized in Table 2. Briefly, hemolymph samples were collected from 20 oysters infected with P. marinus. Hemolymph samples were divided into two equal aliquots, one aliquot to be processed with the standard protocol and the other to TABLE 2. Differences between the standard protocol and the improved protocol to count Perkinsus marinus in oyster hemolymph. Component Standard Improved Type of FTM FTM Supplement Rinsina solution FTM (with agar) None Distilled water Alternative FTM (without agar) 5% lipid concentrate Phosphate-buffered saline (0.1 M) with 0.5 mg/mL bovine serum albumin be processed with the improved protocol. Hemocytes (10") from samples processed with the standard protocol were incubated for seven days in I mL of FTM (Sigma number T 9032) supplemented with 16 g/L of hw-Marinemix Professional sea salts, 50 p.g/mL of chloramphenicol, and layered with 10 p.L of nystatin (5000 Units/ ml). Hemocyte debris was digested with 1 mL of 2 N NaOH, and hypnospores were rinsed three times with 1 mL of distilled water. Samples were centrifuged, 900 p,L of supernatant were removed, and each 100-p.L sample was transfened to a separate well of a 96-well plate, where it was serially diluted in distilled water and stained with LugoPs solution. Standard protocol centrifugation speeds were increased to the centrifugation speeds of the improved protocol to exclude their effects on parasite recovery. Hemocytes (10") from samples processed with the improved protocol were incubated for seven days in 1 ml of AFTM (Sigma number A 0465) supplemented with 5% lipid, 16 g/L of hw-Marinemix Pro- fessional sea salts, 50 p.g/mL of chloramphenicol, and layered with 10 p-L of nystatin (5000 Units/mL). Hemocyte debris was digested with 1 niL of 2 N NaOH and hypnospores were rinsed once with 1 mL of distilled water supplemented with 0.5 mg/mL BSA and twice with I mL PBS supplemented with 0.5 mg/mL BSA. Samples were centrifuged, 900 p,L of supernatant were removed, and each 100-p,L sample was transferred to a separate well of a 96-well plate, where it was serially diluted in PBS supplemented with BSA and stained with Lugol's solution. After processing samples with both protocols, the number of hypnospores was counted 200x magnification in wells containing 100 to 400 hyp- TABI.E 1. Components of different brands of fluid thioglycollale media. Difco" Sigma" Bacto FTM With Potassium Agar Bacto FTM With Beef Extract Component Bacto FTM Alternative FTM FTM Beef extract 5g — — Yeast extract 5g 5g 5g 5.00 g 5.0 g Casein digest 15 g 15g 15g 15.00g 15.0 g Dextrose 5.5 g 5g 5.500 g 5.50 g 5.5 g Sodium chloride 2.5 g — 2.5 g 2.50 g 2.5 g Potassium chloride — 2.5 g — — — L-Cystine 0.5 g 0.5 g 0.5 g 0.50 g 0.5 g Sodium thioglycollate 0.5 g 0.3 mL" 0.5 g 0.50 g 0.50 g Agar 0.75 g 0.45 g 0.75 g — 0.75 g Resazurin 0.001 g 0.00! g 0.001 g — 0.001 g ' Decimal places reported as suggested by manufacturer. ' Thioglycollic acid. 728 NiCKENS ET AL. nospores after dilutions. The diameter of 30 hypnospores was mea- sured from each hemocyte sample. Comparison of Standard and Improved FTM Himolymph Assay Against FTM Body Burden Assay The standard and improved FTM protocols for counting P. marinus in oyster hemolymph (as described above) were compared with the FTM body burden assay for determining the number of parasites in whole oyster and a regression line was calculated for each comparison. The regression lines were compared with deter- mine if infection intensities obtained from the improved hemolymph protocol were more closely correlated with infection intensities obtained from the body burden assay than those ob- tained from the standard hemolymph protocol. The 20 oysters from which hemolymph was removed for com- parison of the standard and impriived hemolymph protocols, were processed using modification of the body burden assay of Fisher and Oliver (1996) (Coates et al. 1999). Brietly. each oyster was removed from the shell, blotted dry. and homogenized with a hand-held Biohomogenizer, a stainless-steel rotor/stator emulsify- ing instrument. {Biospec Products, Inc.. Batlesville. OK. catalog number 1 28 1 ) in 20 niL of sterile artificial seawater prepared at 15 ppl with hw-Marine Professional sea salts. One milliliter of ho- mogenate was transferred to 9 mL of AFTM (Sigma number A 0465) supplemented with 16 g/L of hw-Marinemix Professional sea salts, 50 jjig/mL of chloramphenicol 5% lipid, and layered with 100 jiL of nystatin (5000 Units/mL), and incubated for seven days. Samples were centrifuged. the supernatant removed, and 10 mL of 2 N NaOH added for 4 to 5 h at 60°C to digest the oyster tissue. Hypnospores were rinsed once with 10 mL of distilled water supplemented with 0.5 mg/mL BSA and twice with 10 mL PBS supplemented with 0.5 mg/mL BSA. Samples were centrifuged, 5 niL of supernatant were removed, 50 (jiL of each sample were transferred to a separate well of a 96-well plate, where it was serially diluted in PBS supplemented with 0.5 mg/mL BSA and stained with Lugol's solution. The number of hypnospores was counted (200x magnification) in wells containing 100 to 400 hyp- nospores after dilutions. The number of hypnospores per gram of oyster tissue was calculated for each oyster and compared with the number of hypnospores per 10** hemocytes determined by the stan- dard and improved hemolymph protocols using linear regression analysis. Slalislical Analysis Statistical analysis was pertoniied using SAS Version 8.0 soft- ware (SAS Institute, Inc. Gary, NC). Only hemolymph samples containing at least 50 hypnospores (per triplicate) were used data analysis. Data were log transformed and analyzed with a random- ized block design (blocked by oyster). Tukey's post-ANOVA test was used to examine differences among treatments. Differences were considered significant at P < 0.05. All data were reported as mean ± standard deviation. To compare the standard and improved FTM hemolymph assays against the FTM body burden assay, two linear regressions were calculated using log-transformed data. RESULTS Effects of Supplemented AFTM on Hypnospore Diameter and Sumher The diameter of hypnospores from samples incubated in AFTM supplemented with lipid concentrate (27.3 ± 11.5 (Jtm) was sig- nificantly greater (P < 0.0001 ) than the diameter of hypnospores from samples incubated in AFTM supplemented with oyster ex- tract (18.8 ±9.4 (Jim) or with saline (16.5 ± 10.2 jim) (Fig. I). No significant difference in hypnospore diameter was found between samples incubated in AFTM supplemented with oyster extract or with saline. No significant difference was found in the hypnospore numbers between samples incubated in AFTM supplemented with lipid concentrate (1.9 x 10^ ± 7.1 x lO"*), oyster extract (1.8 x 10^ ± 4.7 X 10^) or the saline control (2.2 x 10'^ ± 6.6 x 10"*). Effeet of FTM Types on Hypnospore Diameter and Sumher No significant differences were found for h\ pnospore diameter or hypnospore number among samples incubated in different for- mulations of FTM (Table 3). The absence of agar in the Sigma alternative fluid thioglycollate medium facilitated sample process- ing because the viscous layer that generally collected over hypno- spore pellets after centrifugation when samples were incubated in FTM with agar was eliminated. Effect of Hemocyte Density on Hypnospore Diameter and Sumter The diameter of hypnospores from samples that received 10' hemocytes per ml of AFTM (19.9 ± 8.8 p.m) was significantly greater (P < 0.001 ) than the diameter of hypnospores from samples that received 10" hemocytes per ml (15.3 ± 8.7 p.m) and 10^ hemocytes per ml (15.2 ± 12.1 p-m) (Fig. 2). No significant dif ference was found in hypnospore diameters between samples that received lO*" hemocytes per ml and lO' hemocytes per ml. No significant difference was found in the number of hypnospores per hemocyte among samples that received 10'' hemocytes per ml (0.06 ± 0.15 hypnospores/hemocyte), 10'" hemocytes per ml (0.67 ± 2.26 hypnospores/hemocyte) and 10^ hemocytes per ml (0.03 ± 0.07 hypnospores/hemocyte). Effect of AFTM Volume on Hypnospore Diameter and Number The diameter of hypnospores from samples that were incubated in 5.0 mL (24 ± 12 (jim) and 25.0 mL (28 ± 13 fj.m) of AFTM was significantly greater (P < 0.0001 ) than the diameter of hypnospores from samples incubated in (1.2 mL ( 13 ± 6 p.m) and 1.0 mL ( 16 ± 8 |jim) of AFTM (Fig. 3A). However, the number of hypnospores in samples incubated in 25.0 mL of AFTM (4.5 x K)-* ± 6.9 x lO^*) was significantly lower iP < 0.0002) than the number of hypno- spores in samples incubated in 0.2 mL (9,3 x lO"* ± 1.1 x 10"), 1.0 Saline Lipid Oyster Extract Figure 1. Hypnospore diameter (mean ± SD. »! = 5) after incubation in AFTM supplemented with lipid concentrate, oyster extract, and saline (control). Hemocytes (10") were incubated for 7 days in 1 niL of supplemented AFTM. Treatments sharing a letter were not signifi- cantl> different. Improved Hemolymph Procedure to Count P. marinus 729 TABLE 3. Average values (mean + SD, n = 9| of hypnospore diameter and number of hypnospores per 10" hemocytes after incubation in different types of FTM. Type of FTM Hypnospore Diameter (^ml Number of Hypnospores Difco Bacto FTM Difco Bacto FTM with K agar Difco Bacto FTM with beef extract Sigma alternative FTM Sigma FTM 21 + 11 S.3 X lo-* ± 1.1 X nr ■o 20± 12 1.8 X 10' ± 3.3 X 10' in 19+11 18± 11 22 ± 13 1.2 X 10' ± 1.7 X 10' 2.0 X 10' ± 4.0 X 10' 1.8 X 10' ±3.1 X 10' a ai O c a >. niLd.Sx 10'±2.7x 10'). atid 5.0 niL ( 1.2 x lO' ± 1.5 x 10') of AFTM (Fig. 3B). No signitlcant difference was found in the num- ber of hypnospores among samples incubated in 0.2. 1.0, and 5.0 niL of AFTM. Effect of NaOH Digestion of Samples on Hypnospore Diameter and Number No significant difference was found in hypnospore diameter between samples processed with NaOH digestion (24 ± 17 (xm) or without it ( 19 ± 9 ixm). No significant difference was found in the number of hypnospores between samples processed with NaOH digestion (1.8 x 10"* ± 2.6 x lO"*) or without it (2.6 x 10"" ± 3.8 x 10''). Sodium hydro.xide facihtated counting by reducing cellular debris. Effect of Protocol on Hypnospore Diameter and Number Hypnospore diameter from samples processed with the im- proved protocol (26 ± 13 (j.m) was significantly greater (P < 0.0001: ;; = 20) than the diameter of hypnospores from samples processed with the standard protocol (10 ± 4 (j.m). Hypnospore numbers in samples processed with the improved protocol (8.6 x 10' ± 3.3 X 10') were significantly greater (P < 0.0001) than the 3: 30 £ o E 5 20 o a o 10 c Q. T ^ b 1x10* 1x10* Hemocytes/ml 1 xlO' Figure 2. Effect of hemocyte density ( KF, 10'', or lO' hemocytes per ml of AFTM) on hypnospore diameter (mean ± SD, n = 13). Hemocytes were incubated in 1 mL of AFTM supplemented with 5% lipid for seven days. Means sharing a letter were not significantly different. 50 — 40 E E. I 30 « 20 10 400 w S o a (A O c >; 200 H |_g_y _L 0.2 25 1 5 FTM Volume (ml) Figure 3. Hypnospore diameter (.A) and number (B) (mean ± SD. n = 10) after incubation in 0.2. 1.0. 5.0. and 25.0 niL of AFTM. Hemocytes (10'') were incubated for seven days in 1 niF of AFTM supplemented with 5% lipid. Means sharing a letter in each panel were not signifi- cantly different. number of hypnospores in samples processed with the standard protocol (1.9 x 10' ± 3.4 x 10'). Hypnospores from hemocytes processed with the improved protocol readily settled to the plate bottom and could be easily counted. Hypnospores processed with the standard protocol were difficult to count because they re- mained suspended in the wells and the media column had to be scanned vertically as a consequence (Fig. 4). Comparison of Standard and Improved FTM Hemolymph Assays with FTM Body Burden Assay Using linear regression, comparison of the infection intensity determined by the body burden assay (log of hypnospores per gram of oyster tissue) and the infection intensity determined by the standard hemolymph assay (log of hypnospores per 10'' hemocytes) showed that the correlation was highly significant [P < 0.0001) with a coefficient of determination (r) of 0.5543 and a slope for the regression line of 0.6126 (Fig. 5A). Using linear regression, comparison of the infection intensity determined by the body burden assay and the infection intensity determined by the improved hemolymph assay showed a highly significant correla- tion (P< 0.0001 ) with an improved coefficient of determination {r = 0.7602) and slope (0.91 19) (Fig. 5B). This increased coefficient of determination indicated a reduction in variance with the im- proved FTM hemolymph assay. 730 NiCKENS ET AL. • * • • • • • • • » • • •. ' *• ^ ,' Jk • • ## • •# # ♦ • • « # • « • • ^ t • • • ♦ Figure 4. Light micrographs of Perkiitsiis marinus hypnospores after incubation and processing with the standard and improved protocols at 200x magnification. Bar represents 30 pm. A, Hypnospores were small (7-13 pm) and not clearly visible after incubation and processing with the standard protocol. B, Hypnospores were larger (15—44 pni) and more visible after incubation and processing with the improved protocol. DISCUSSION Use of the improveiJ protocol to count P. marinus in heniolymph resulted in a 167% increase in hypnospore diameter and a 358% increase in hypnospore number over the standard protocol. The improved protocol also facilitated sample processing and counting. These improvements were caused by modifications of the standard hemolymph protocol, such as the addition of lipid concentrate to AFTM. the use of PTM without agar, and the ad- dition of BSA to rinsing solutions. These modifications resulted in larger hypnospores, decreased sample viscosity, increased hypno- spore recovery, and reduced cellular debris. Because processing. identification, and counting of hypnospores were facilitated, the improved protocol was simpler and more accurate than the stan- dard protocol for counting of hypnospores in hemolymph. As a result, the coefficient of determination {r) of the linear regression between P. marinus log,,, numbers in oyster body and P. marinus login numbers in hemolymph increased from 0.554 to 0.760 when the improved hemolymph protocol instead of the standard hemolymph protocol was used to count P. marinus in hemolymph. Results from previous studies have suggested that nutrients from FTM and oyster tissues are used by enlarging parasites and that the ratio of FTM to oyster tissue may be critical for optimal hypnospore enlargement (Ray 1954a. Bushek et al. 1994. Fisher & Oliver 1996). The failure of parasites in highly infected oyster tissue to fully enlarge in FTM was proposed to be a result of exhaustion of necessary substances in medium or tissue (Ray iy54a). Hypnospore enlargement was also reported to be greatest in the FTM tissue assay, intermediate in the FTM hemolymph assa\ and least in the FTM body burden assays and therefore related to the volume of oysters sampled per ml of FTM (Bushek et al. 1994). Moreover, parasite enlargement was inversely related to infection intensity. Using P. marinus cultured in vitro, parasite enlargement in FTM can be significantly increased by addition of various nutrients including lipids (Wagner et al. 2001). Despite these observations, the effects of supplementing FTM with nutri- ents such as lipid or oyster extract and the effects of the ratio of FTM to oyster nutrients (e.g.. from hemocytes) on parasite en- largement and numbers had not been studied and therefore needed to be investigated to improve the FTM hemolymph assay. Addition of lipids to AFTM significantly increased hypnospore diameter by 66% over the saline control, and facilitated sample processing and counting because larger hypnospores were more easil\ pelleted during centrifugation. Lipids were added because the accumulation of numerous lipid droplets in hypnospores is characteristic of this life stage and may be critical to enlargement (Perkins & Menzel 1966. Perkins 1996). It was recently confirmed that lipids are taken up and stored in hypnospores (Soudant et al. 2000). This study found that fluorescent lipid analogs were pri- marily stored in cytoplasmic lipid droplet after 24 h of incubation but that after 24 h. fluorescence appeared in the membrane and cytosol of hypnospores. The mean diameter of hypnospores in AFTM supplemented with 5% oyster extract was only 14% greater than the diameter of hypnospores in AFTM supplemented with oyster saline. It was surprising that the addition of oyster extract did not induce sig- nificant cell enlargement over saline as oyster extract would be expected to contain high concentrations of nutrients. Although the components of the oyster extract were not examined, it is likely that the water-soluble extract contained only small amounts of oyster lipids, which may account for the minimal enlargement of hypnospores. Determining the effects of the ratio of infected hemocyte num- ber to AFTM volume on hypnospore formation helped formulate the optimal combination of nutrients, from oyster and AFTM, to promote maximal parasite enlargement. In this study the number of infected hemocytes incubated in a con.stant volume of AFTM was adjusted, as was the volume of AFTM that had a constant number of infected hemocytes. Hypnospores from the lowest hemocyte density ( 10"' cells per ml of AFTM) had the greatest enlargement indicating hypnospores in this treatment had more nutrients avail- able from AFTM. Unfortunately, this low density may not provide an adequate sample size for determining parasite infection inten- sity. Using a higher number of hemocytes while maintaining hemocyte density (e.g.. 10* per 10 mL) requires a larger volume of FTM. which in turn may affect the assay. To address this potential effect of volume on the assay. 10'' hemocytes were incubated in various volumes of AFTM. Hypnospores from 10'' hemocytes in- cubated in the largest AFTM volumes (5 and 25 mL) had the greatest enlargement, but there was also a significant decrease in the number of hypnospores from samples incubated in 25 mL of AFTM. indicating that smaller cells may be lost during processing of these volumes. Moreover, large volumes of AFTM requires the purchase of larger amounts of media and antibiotics, which in- creases cost. The most practical and optimal ratio was set at 10'' hemocytes to 1 mL of AFTM. At this ratio of infected hemocytes to AFTM volume, parasites had adequate nutrients available for Improved Hemolymph Procedure to Count P. marinus 731 a> 3 M (0 *^ E re « I. o a « o c a >. X o o y = 0.61 26x + 4.4344 ♦ ♦ |6 LOG Hypnospores/10 Hemocytes y = 0.9119x + 2.8232 R^ = 0.7602 3 5 0 B LOG Hypnospores/10 Hemocytes Figure 5. Linear regression lines, formulas, and coefficient of determinations for the comparison of the log of the numher of hypnospores per 10'' hemocytes determined hy the standard FTM hemolymph protocol and the log of the numher of hypnospores per gram of oyster tissue determined by the body burden assay (A), and for the comparison of the log of the number of hypnospores per 10'' hemocytes determined by the improved FTM hemolymph protocol and the log of the number of hypnospores per gram of oyster tissue determined by the body burden assay (B). enlargement in a small AFTM volume while minimizing protocol cost and hypnospore loss. Perkinsus marinus in hemolymph has exclusively been re- ported in numbers of parasite per ml of hemolymph (Gauthier & Fisher 1990. Bushek et al. 1994. Oliver et al. 1998. Yamall et al. 2000). The volume of hemolymph collected per oyster for use in the assay in these past studies varied by as much as a factor of 1 0. affecting the assay performance. Most parasites, however, are found within the phagosomes of hemocytes (Ray 1954a, Perkins 1996) and the density of hemocytes in hemolymph of bivalves can vary considerably with factors such as temperature, disease and feeding (Feng et al. 1977, Ford & Tripp 1996). Perkinsus may. therefore, be better expressed in number of parasites per number of hemocytes (e.g.. per 10'' hemocytes). The coefficient of determination (?~) of the linear regression of P. marinus log,,, numbers in oyster body on P. marinus logm numbers expressed per 10^ hemocytes was 0.760 (n = 20). When the numbers of P. marinus for the same hemolymph samples were expressed per ml of hemolymph instead of IC hemocytes. the coefficient of determination was 0.776 (n = 20) and higher than all. except for one previously reported r. An r of 0.53 (n = 12) for Texas oysters sacrificed 24 h after collection and an r of 0.89 (/? = 12) for animals held for ten days at high temperature and salinity before sampling were reported by Gauthier and Fisher (1990). An r of 0.675 (n = 25) was reported by Yamall et al. (2000). An r of 0.241 (n = 100) for oysters from Apalachicola Bay, Florida and an /' of 0.771 in = 100) and 0.738 (/i = 100) for oysters from Virginia and New York were reported by Oliver et al. (1998). The use of FTM hemolymph assay is not recommended for predicting the number of parasites in whole oyster with light P. marinus infections (cystidium marnnnn. Biol. Hull. Mar. Sci. Gulf Carihh. 3:6-33. Ray. S. M. 1954a. Biological studies of Derntocysticlium mariiium. a fun- gus parasite of oysters. Rice Institute Pamphlet. Special Issue. Houston: The Rice Institute. Ray. S. M. 1954b. Studies on the occurrence of Dermocystidinm m young oysters. Natl. Shellfisheries Assoc. Conv. Add 1953:80-88. Soniat. T. M. 1996. Epizootiology of Perkinsns marinus disease of eastern oysters in the Gulf of Mexico. / Shellfish Res. 15:35-43. Soudant. P., F.-L. E. Chu & Y. Marty. 2000. Lipid class composition of the protozoan Perkinsns marinus. an oyster parasite, and its metabolism of a fluorescent phosphatidylcholine analog. Lipids 35:1387-1395. Yarnall. H. A.. K. S. Reece. N. A. Stokes cS: E. M. Burreson. 2000. Quan- titative competitive polymerase chain reaction assay for the oyster pathogen Perkinsns marinus, J. Parasitol. 86:827-837. Wagner. E.. S. Casas & J. F. La Peyre. 2001. Induction of hypnospore formation and /oosporulation of Perkinsns marinus cells from long term cultures. Ac/uaculture. 2001. Book of Abstracts, page 670. Journal of Shellfi.'.li Research. Vol. 21. No. 2, 733-741. 2002. OYSTER BIOMASS, ABUNDANCE, AND HARVEST IN NORTHERN CHESAPEAKE BAY: TRENDS AND FORECASTS STEPHEN J. JORDAN,'* KELLY N. GREENHAWK,' CAROL B. MCCOLLOUGH,' JESSICA VANISKO,' - AND MARK L. HOMER' ^Maiylond Department of Natural Resources, Paul S. Sarbanes Cooperative Oxford Lxiboratory. 904 South Morris Street. Oxford, Maryland 21654; 'University of Maryland. Chesapeake Biologic Laboratory. P.O. Box 38ni Solomons, Maryland 20688 ABSTRACT We applied time series of fishery-dependent and fishery-independent data to develop indices of relative biomass and estimates of absolute abundance and biomass for the Maryland oyster population. The principal objectives for this work were to specify a baseline and determine trends relative to the Chesapeake Bay Program's goal to achieve a 10-fold increase in the standing stock of oysters in the Bay from a 1994 baseline. Population biomass varied by 3-fold from 1986-2001. with most of the variation caused by changes in the stock of market-sized oysters. There were also important spatial differences in population structure and trends over a gradient of salinity. Lagged correlations between small oyster biomass and either niarket-si,^ed oyster biomass or landings were not statistically significant but suggested that most of the market-si/ed oysters and those harvested were four to five years old. The index of relative biomass for market-sized oysters proved to be a useful predictor of annual harvests in Maryland. We used this relationship to estimate absolute abundance and biomass of oysters for the time series. The latter estimates may be biased negatively with respect to true abundance and biomass because of biases in both the fishery and the fishery-independent monitoring program. The index of relative biomass will be a suitable measure of population status relative to the oyster restoration goal only so long as the population remains at relatively low levels. Because the index has a theoretical maximum of about 75'7r of the 10-fold goal, a quantitative estimator will be required for higher population levels. KEY WORDS: oysters, Crassostrea virf^inica. stock assessment, biomass. Chesapeake Bay INTRODUCTION MATERIALS AND METHODS Oysters once supplied the most valuable fishery in Chesapeake Bay (Christmas & Jordan 1991), but after the mid-1980s, a de- clining oyster stock could no longer suppoil historical levels of harvest. The principal reasons for reduced harvests from 1987 to 2001 were (1) high mortality rates of oysters caused by the para- sitic diseases MSX [Haplospuridiitm nel.soni) and Dermo (Per- kinsus marinus) and (2) low rates of spat settlement throughout most of this period (Krantz undated. Maryland DNR 2001). In addition to economic losses to the fishery, the depletion of oyster standing stocks has been thought to ( 1 ) reduce the quantity and quality of shell-bar habitats for oysters and associated epifauna (MacKenzie 1996) and (2) impair water quality by loss of the oyster population's capacity to filter particulate matter (Jordan 1987, Newell 1988. MacKenzie 1996). In June 2000. the interjurisdictional Chesapeake Bay Program established a goal to increase oyster populations 10-fold by 2010, from a 1994 baseline. Our objectives for the work presented here were to (I) quantify the 1994 baseline, (2) compare the current population with recent and historical populations, (3) develop methods to measure consistently the status of populations with respect to the goal, and (4) examine relationships between long- term fishery-independent surveys and annual harvests. In addition to meeting these objectives, we have developed methods for esti- mating absolute abundance, biomass and fishing mortality from these data. Only data from the Maryland portion of Chesapeake Bay were used in our analysis. We are working with scientists in Virginia on a combined, bay-wide assessment of the oyster popu- lation. *Corresponding author. E-mail: sjordan@dnr.state.mo Oyster Surveys Size frequency, disease, mortality, and spat density data were collected from oyster dredge samples at 43 sites (Fig. 1 ) in October to November from 1990 to 2000 (in a few cases, 42 or 44 sites were sarnpled). The standard dredge has an opening 80 cm wide and a capacity of approximately 2.5 bushels (the volume of a Maryland oyster bushel is =46 L, or about 475 oysters at the minimum market size of 76 mm). Dredge tows were taken from areas of the oyster bars with relatively high concentrations of shell, as detennined by the sampling crew with a sounding pole. Our observations indicated that tow times and boat speed (hence the area swept) tended to be consistent, but these variables were not formally controlled. From 1990 to 1996, five replicate dredge tows were made at each site; data were collected from a 0.2 bushel subsample of all material in each dredge sample. From 1997 to 2000, two tows were made at each site, with a 0.5 bushel sub- sample taken from each tow. All live oysters in the subsamples were counted and measured, with measuretiients recorded in 5 mm size classes. Boxes (articulated oyster shells without tissue remain- ing) also were counted and measured. All spat were counted in each sample. Sample data were standardized to a fixed volume (one bushel, as defined above) of all material retained by the dredge. A complete description of this survey, known as the Modi- fied Fall Survey (MFS) can be found in Smith and Jordan ( 1993). A more extensive survey of 300 to 400 sites has been con- ducted in the fall of each year since the 1970s. In this survey, typically only one dredge tow is tnade; all live oysters, spat, and boxes from 0.5 bushel of material are counted. The oysters are not measured individually but are classified as markets (>76 mm), smalls (age l-l- and older oysters <76 mm), or spat. Means and ranges of shell heights for each class are estimated visually by an experienced technician and recorded (Homer et al. 1996). 733 734 Jordan et al. Low ■-^i^ + Medium ^%^i^ ,> High /5^' J ^. Figure 1. Maryland MFS sites in Chesapeake Bay and tributaries. Symbols identify classification of sites by long-term mean salinity: Low 14 ppt. Length-Weight Analysis A relationship between dry tissue weight and shell height was developed to convert size-frequency data to biomass. During the 1999 fall survey. 10 oysters were collected from each of 42 sites (one of the 43 survey sites did not have sufficient live oysters) and returned to the laboratory for processing. The oysters were selected to be representative of the size range encountered at each site. In the laboratory, the oysters were measured (bill to umbo curved shell height in mm), then shucked carefully, retaining all meat and shell liquor. Each oyster was put into a tared aluminum weighing boat, weighed, dried to constant weight at 85"C. and then re- weighed. Wet and dry tissue weights were recorded in g. to the nearest mg. The relationship between dry tissue weight and shell height was quantified by linear regression of the log|(,-transfornied variables. Indices of relative biomass were calculated by applying the length- weight regression equation to each 5 mm size class of live oysters (excluding spat), then multiplying the number of oysters in that size class by the predicted weight. The midpoint in mm of the size class was used as the nominal shell height for the estimated weight. The weights were summed over all size classes within each rep- licate subsample. resulting in 1629 individual observations of the index over 43 sites and ten years. Indices of relative abundance were calculated similarly using total counts of oysters (excluding spat) within each subsample. Indices of biomass and abundance were calculated separately for small (<72 mm) and market (>72 mm) oysters, and for all oysters combined. The legal minimum size for harvested oysters in Maryland is 3 inches (76 mm), but the nearest size class in which survey data are recorded is 72 to 77 nmi. We examined the underlying variation in the indices by two- way analysis of variance, accounting for the fixed effects of site, year, and site-by-year interaction. Relationships Between Fishery-Dependent and Fishery-Independent Data We u.sed linear regression to determine whether the fishery- independent data could predict commercial harvests. Harvest data were obtained from the Maryland Department of Natural Re- sources. Because the oyster season is open from October to March. landings are reported for the year the season closes, whereas the MFS data applicable to the annual harvest are collected in October through November of the previous year. For this reason, the analy- ses reported here are indexed to the harvest year, that is. the 198,'i MFS corresponds to the 1986 harvest, and the biomass index for 1986 is derived from data collected in the fall of 1985. However, when referring to the fishery-independent data per se. we use the year in which the data were collected. Annual harvest totals, re- corded in bushels, were regressed against the annual mean biomass index for market oysters and the annual abundance index for mar- ket oysters. Nine years of data, from harvest years 1991-1999, made up the initial calibration data set. We then used dredge survey data from 1985-1989 for the bars sampled by the MFS to calculate the biomass indices and perform a hindcast test of the harvest prediction model. The 1985-1986 harvest was 1.56 million bushels, nearly 20 times greater than the 1993-1994 harvest. Also, the index previous to 1990 was based on mean sizes for market oysters rather than individual measurements. Prediction of the 1985-1986 and subsequent harvests, therefore, would provide a stringent test of the robustness of this simple model. Once the model was calibrated and validated by hindcasting, we recalibrated over the entire time series, and forecast landings for the 1999-2000 and 2000-2001 harvest seasons. Quantitative Estimation of Standing Stocks The success of the market oyster biomass index in predicting annual landings suggested that we could use this relationship to quantify the oyster population. We made the key assumption that differences between predicted and reported landings were entirely caused by differences in rates of fishing mortality. Smith and Jordan (1993) estimated a mean exploitation rate of 0.53 (53% of the market stock harvested) for Maryland oysters for the 1990 to 1991 season. They projected size-frequency data from the 1990 MFS to 1991, assuming a mean growth rate of 20 mm per year and subtracting 1991 observed natural mortality, estimated from box counts as [boxes/(boxes -i- live oysters)], from each size class. The exploitation rate then was estimated by difference between the projected relative abundance (without exploitation) and that ob- served from the 1991 survey. Because the ratio of reported to Oyster Biomass in the Chesapeake Bay 735 predicted harvest for that year was approximately unity, we com- puted the instantaneous rate of fishing mortahty (F) for the time series as F = -logf ■ 0.53 where H = reported harvest and H = predicted harvest. Estimates of harvestable stock size (S) for each year were computed as S = H ■ e^ (in units of bushels). Estimates of total population abun- dance (M excluding spat) were computed as N, = N,„ + N,„[ — where the subscripts i. in. and .v indicate total, market, and small oysters, respectively. R refers to the relative abundance estimates from the fishery-independent survey, and N.., a,S where a, = lo^-"^"'"" '^"-'\ /?_ js the relative abundance of market oysters in each size class, R, is the total relative abundance of market oysters, and L, is the nominal length for each size class of market oysters. The last equation estimates the number of oysters of a given size per bushel, derived by linear regression from tabu- lated counts (unpublished data. Maryland Department of Natural Resources). Absolute population biomass was estimated by mul- tiplying the proportion of oysters in each size class from fishery- independent size frequency distributions by total absolute abun- dance iN^ calculated as above) and the nominal weight for each size class: «, = 2 R, N,W,\ - where B, is total population biomass. W, is the nominal weight in g for each 5 mm size class. /?, is the relative abundance in each size class, and R, is the total relative abundance over all size classes. Recruitment Spat (young of the year oysters) are counted in each sample taken during fall oyster surveys. Although annual mean spat counts have been used for many years as an indicator of year class strength (Meritt 1977, Homer et al. 1996, Krantz undated), they are not always reliable indicators of subsequent recruitment to the population because of high, variable rates of early juvenile mor- tality (Newell et al. 2000) and the impacts of diseases on submar- ket oysters. Counts of small oysters (>1 y old and <76 mm in shell height) may be better indicators of recruitment to adult and ex- ploitable stocks than spat counts. Therefore, we examined the po- tential of small oyster counts and biomass to predict landings, as well as biomass of market oysters in subsequent years, by regress- ing landings and market oyster biomass against 1- to 5-y lags of the small oyster indices. InPuence of Salinity Recruitment of oysters to the population and disease-related mortality are positively correlated with salinity in northern Chesa- peake Bay (Jordan 1993. Calvo et al. 1996). To examine the effects of .salinity on the biomass index, we assigned each of the MPS sites to one of three salinity zones based on 1 990 to 2000 mean salinity measured during the fall at the time oyster samples were taken (Fig. 1). These zones were defined as low (mean salinity 14 ppt). At salinity <12 ppt. H. nelsoiii infections occur rarely, if ever; Perkinsus marinus infec- tions, although chronic in this zone, are associated with low to moderate mortality rates: and recruitment rates of both small and market oysters are typically very low, except in areas where natu- ral recruitment has been supplemented by transplanted seed oys- ters. In the medium salinity zone. H. nelsoni epizootics are spo- radic, occurring only in drought years, mortality associated with P. marinus is moderate to high, and recruitment is variable. In the high-salinity zone. H. nelsoni infections tend to be enzootic, mor- tality rates associated with P. marinus infections are consistently high, and recruitment, although variable, tends to be higher than in the lower salinity zones. RESULTS Length-Weight Relationship The linear relationship between the log of dry tissue weight and the log of shell height was quantified as log,o(dry tissue weight) = 2.06[log|„(shell height)] - 3.76. with tissue weight in g and shell height in mm; /; = 410. r" = 0.50, P < 0.0001 (Fig. 2). Removal of a few outlying observations or weighting the regression for uneven distribution of the dependent variable on the independent variable made only trivial differences in the parameter estimates and the coefficient of determination. Therefore, population-scale estimates of biomass should be accurate, even though the relation- ship does not have satisfactory precision for estimates at smaller scales (individuals or small samples). Variability of Biomass Indices In the two-way analysis of variance models, sites, years, and site-by-year interactions explained 85 to 919^ of the variation in index values for small oysters, market oysters, and the total popu- lation; all effects were significant (Table I). The residual 9 to 15% of overall variation was caused by differences between replicate samples. T \ 1 r 40 50 60 80 100 150 SHELL HEIGHT (mm) Figure 2. Relationship of oyster dry tissue weight to shell height. 736 Jordan kt al. TABI.E 1. Analysis of variance results for examining sources of variability in bioniass indices of total, market, and small oysters, 1990 to 1999. hindcast Source of Variation Degrees of Freedom P Value Total Biomass (Mean = 1 14.4 g dry weight per bushel, r Site 43 Year 9 Site X year 378 Total 1628 Market Oyster Biomass (Mean = 63.7 g dry weight per bushel, )' = Site 43 Year 9 Site X year 378 Total 1630 Small Oyster Bioniass (Mean = 44.8 g dry weight per bushel. /•" = Site 43 Year 9 Site X year 378 Total 1630 = 0.91. II = 1629) S.'i.96 <0.(10()1 89.10 <0.0001 18.73 <0.0001 28.10 <0.0001 0.85. II = 1631 46.54 <0.0001 94.46 <0.0001 8.88 <0.0001 16.24 <0.0001 0.89, 11 = 1631) 64.60 <0.0001 23.31 <0.0001 15.81 <0.0001 22.74 <0.0001 Relationships Between Fishery-Dependent and Fishery-Independent Indicators There was a significant linear relationship between the mean relative biomass of market oysters sampled in October through November of each year and the quantity of oysters harvested dur- ing the commercial season (generally October I through March 3 1 . with minor variations). The resulting regression equation was log,o(y) = 1.64(log,oX) + 2.39. where y = predicted annual har- vest in bushels and x = mean relative biomass of market oysters ((' = 0.89; Fig. 3). This regression was developed from 10 y (harvest years 1991-2000) for which we had replicate samples and size frequency data. A hindcast of this model for 1986 to 1990 landings, using only counts and mean sizes of market oysters, yielded reasonable estimates. Over the 15 harvest years from 1986 to 2000. the mean absolute difference between predicted and re- ported landings was IS.S^'r (minimum 3.7%. maximum 57.6%. standard deviation 17%). The poorest prediction was for 1987, a period of sharply declining relative biomass associated with an epizootic of MSX disease. An earlier validation of the model used O Si < o _l o 500 400 300^ 200 100 calibration • ■■ predicted — reported fore- cast 35 40 50 60 70 80 90 MEAN MARKET OYSTER BIOMASS gdw/bushel Figure 3. Relationship between annual reported oyster landings and annual mean market oyster biomass index (g dry weight of oyster tissue per bushel of dredged material). Both axes are logarithmic, r- = 0.89. 1990 1992 1994 1996 HARVEST YEAR Figure 4. Predicted and reported oyster landings, 1986 to 2001. a regression of nine years of data along with biomass from the 1990 Fall .Survey to predict 1999 to 2000 oyster landings at 370,000 bushels; reported landings were less than 1% gi-eater (380.(J00 bushels). The prediction for 2000 to 2001 landings was 331.000 bushels, about 5% less than reported (348,000 bushels). With the model validated by hindcasts and forecasts, it was ap- propriate to use the entire time series to estimate model parameters (Fig. 4). Biomass as an Indicator of Population Size The biomass index for the total population showed more than 3-fold variation over a 15-y period (Fig. 5). Most of this variation was caused by changes in the market oyster index, which varied by more than 6-fold. Small oyster biomass was more stable, varying by a factor of 2.4. The 1 994 baseline index for the total population small oysters I market oysters 300 n 200 ? O) CO (O < 100 o CD 86 88 90 92 94 96 98 00 YEAR Figure 5. Biomass indices (g dry weight of oyster tissue per bushel of dredged material) for small, market, and total oysters, 1986 to 2001. Oyster Biomass in the Chesapeake Bay 737 — abundance -•- biomass [260 220 200 180 160 140 120 100 80 a. CD ^ > 1985 1987 1989 1991 1993 1995 1997 1999 2001 SURVEY YEAR Figure 6. Annual means of relative biomass and relative abundance, 1985 to 2001. (84.4 g dry weight of oyster tissue per bushel of dredged material) was the lowest in the time series, largely because of a very low index of market oysters (34.8), yet small oyster biomass in 1994 (49.6) was close to the 13-y mean (51.4). Although there were similarities between trends in relative bio- mass and relative abundance from 1986 to 2001, there were im- portant divergences (Fig. 6). The sharp rise in abundance from 1 997 to 1 998, for example, reflects the large spat set observed in 1997, followed by a sharp increase in the abundance of small oysters in 1998. The biomass also increased from 1997 to 1999. but more slowly. Sharp declines in biomass from 1985 to 1989 and from 1990 to 1992 were associated closely with disease epizootics. Trends in biomass varied considerably over three salinity zones (Fig. 7). In the low-salinity zone, population biomass was moder- ate and relatively stable, whereas in the midsalinity zone, biomass declined almost steadily for nine years, followed by partial recov- ery from 1995 to 1999. In the high-salinity zone, biomass de- creased sharply from 1986 to 1989 and did not show sustained recovery. Differences between the zones were even more evident when expressed as mean biomass per individual over the full time series (Fig. 8). Estimates of Fishing Mortality, Total Mortality, Absolute Abundance, and Absolute Biomass Estimated instantaneous annual rates of fishing mortality varied from 0.23 in harvest year 1997 to 1.29 in 1989. Instantaneous annual total mortality for the market oyster stock ranged from 0.57 in 1997 to 1.67 in 1989 (Table 2, Fig. 9). These ranges corre- 400 (0 < o CQ z < UJ S ^ 300 in E 200 ■o 100: 4 1 . 1 .ow i Medium High s12 1 12-14 >14 I 1 L " 1 1^ ,:■.■ r i i . ;l 1 ■ ' m 86 00 86 00 86 00 HARVEST YEARS 1986-2000 Figure 7. Mean relative biomass by .salinity zone, 1986 to 2001. SALINITY ZONE Figure 8. Mean relative biomass per individual oyster by salinity zone. Error bars are standard errors. sponded to annual exploitation rates of 21 to 72'7r and annual total mortality rates of 43 to 92% for the market stock. Our estimates of total oyster abundance from harvest years 1991 to 2001. for which size-frequency data were available, ranged from 2.66 to 6.29 x 10** individuals, with a mean of 4.78 x 10**. Total population biomass over the same time period ranged from 2.41 to 8.64 x 10" g dry tissue weight, with a mean of 5.74 X lO'^ g (Fig. 10). Mean biomass per individual oyster (biomass divided by abundance) was 1.18 g. ranging from 0.82 to 1.42 g over the time series (Fig. 11). For reference, the predicted dry weight of a minimum-sized market oyster (76.2 mm) is 1.31 g. Recruitment Lags of small oyster biomass (0-5 y) were not significantly correlated (Spearman /■, P > 0.05) with market oyster biomass or annual oyster harvests from 1986 to 2001. The strongest positive correlation (r = 0.58, P = 0.06) was between harvest and a 4-y lag of small oyster biomass (Fig. 12). DISCUSSION We estimated total oyster population abundance and biomass for Maryland's portion of Chesapeake Bay from 1986 to 2001. Although these estimates appear to represent population trends accurately, there are several sources of uncertainty and potential bias, which are discussed below. Until these uncertainties can be reduced by further assessment and analysis, we recommend use of the indices of relative biomass reported here as more reliable mea- TABLE 2. Summary statistics for instantaneous rates of Tishing mortality (F), natural mortality (M), and total mortality (Z) for harvest years 1986 to 2001 (H = 16). Variable Minimum Maximum Mean Standard Deviation F M Z 0.23 0.11 O.-'i? 1.29 0.91 1.67 0.71 0.39 I. II 0.2.^ 0.22 0.^2 738 Jordan et al. 86 88 90 92 94 96 98 00 HARVEST YEAR Figure 9. Estimated instantaneous annual mortality rates, 1986 to 2001; F = Fisliing mortality, M = natural mortality. sures of changes in the oyster population than our estimates of absolute abundance and biomass. Indices of relative biomass and abundance rely on the principal assumption that the fishery-independent monitoring program ac- curately represents trends in the oyster population as a whole. The MFS. the source of data for index calibration, was designed spe- cifically for consistent assessment of trends in oyster relative abun- dance, population structure, mortality and disease status (Smith & Jordan 1993). For these purposes, fixed sites, sampled consistently Figure oyster. 1993 1995 1997 1999 HARVEST YEAR II. Estimated annual means of absolute biomass per individual 1981 to 2002. 3 ^ 9UU,UUU : ; 700,000 500,000 < O QQ 300,000 ': 100,000 over time, are appropriate. The inonitoring sites are widely dis- tributed throughout the oyster-producing areas of Maryland, and evidence presented here suggests they provide data representative of real trends in the population. Unbiased, quantitative estimates of population size generally require spatially randomized sampling, a condition not satisfied by the data used in our assessment. Our estimates of absolute biomass and abundance also depend on the untested assumption that dif- ferences between predicted and reported oyster landings are caused solely by variations in fishing mortality. Both of these considerations introduce bia.ses and inaccuracies of unknown mag- nitude to the quantitative assessment. Preliminary estimates of population abundance we derived entirely from fishery-inde- pendent data sources resulted in higher estimates of population abundance than those derived from fishery-dependent estimates. The fishery-independent estimates ranged from 1.1 to 1.7 x 10"^ oysters in 2000 to 2001 . compared with 0.6 x \(f oysters estimated for the same period by the methods reported here. We conjecture that neither the fixed-station monitoring program nor the fishery landings data adequately represent potentially large numbers of oysters scattered at low densities in marginal habitat areas. Areas with low densities of oysters would not be targeted by the fishery because the catch per unit of effort would be very low. Similarly, the fixed monitoring sites were selected to represent typically pro- ductive areas of high quality habitat. We plan to collect quantita- tive data from a variety of habitat types during 2002 to get better 1991 1993 1995 1997 1999 2001 HARVEST YEAR <0 0.31 cx: LL 0.2- o' -0.1^ 12 3 4 5 12 3 4 5 SMALL OYSTER BIOMASS LAG (YEARS) Figure 10. Estimates of absolute biomass and abundance, 1991 to Figure 12. Correlations between lags of small oyster biomass and (A) 2002. reported landings and tB) market oyster biomass. Oyster Biomass in the Chesapeake Bay 739 estimates of oyster densities, especially in areas of marginal hiabi- tat. Until further refinements can be made to the quantitative as- sessment, we view it as yielding minimal estimates of oyster abun- dance and biomass. The equation relating shell height to dry tissue weight used to compute the biomass index explained 50% of the variation be- tween individual oysters. Morphology, growth, and physiologic conditions can vary greatly in C. virginica, depending on specific habitat conditions, health, and other factors (Carriker 1996, Ford & Tripp 1996). The condition of each oyster weighed and measured for this study was assessed both by visual inspection (scored 1-8. "watery" to "fat," according to standard procedures used in our laboratory) and by calculating the percentage of water in the tis- sues from the difference between wet tissue weight and dry tissue weight. Inclusion of either of these condition indicators as a co- variate in the length-weight regression improved the total /•" to > 0.70. Because oyster condition is not measured routinely in popu- lation surveys, we could not account for this source of variation in biomass indices. We conclude that the length-weight equation has low precision for predicting the biomass of individuals or small groups of oysters, but has sufficient accuracy for estimating bio- mass at population or sub-population scales. Harvest Predictions, Fishing Mortality, and Recruitment The close relationship between reported annual landings and the biomass index for market oysters has provided a method for predicting total annual harvest early (mid-November) during each oyster season (October-March). This capability is helpful to man- agers of the oyster fishery, who are often asked by the press and other interested parties to make these predictions. This relationship also has provided a quantitative link between relative and absolute abundance and biomass of oysters, and a way to estimate fishing mortality rates. Because natural mortality (the ratio boxesdive oys- ters plus boxes) is observed directly during the annual monitoring program, we now have estimates of total mortality for market- sized oysters. These relationships are being used, along with other parameters, to calibrate a population dynamics model for Mary- land oysters. Ulanowicz et al. ( 1980) developed a multiple regression model to predict Maryland oyster harvests four years into the future from data on spat settlement and management effort (transplantation of seed oysters) in previous years. The time series of data used in their model was from the 1960s and 1970s, a period when parasitic diseases were not major influences on oyster population dynamics. During the period of the present study, variations in annual land- ings have depended strongly on oyster mortality rates, which in turn have depended primarily on variations in disease intensity (Gieseker 2001). Instantaneous annual rates of fishing mortality (F) varied from 0.23 to 1.29 from 1986 to 2001, corresponding to exploitation rates of 2 1 to 72% of the market oyster stock harvested each year. The mean exploitation rate of market-sized oysters over 16 y was 51%- (F = 0.71). This value of F contrasts with that estimated by Rothschild et. al. (1994) at 1.3 (73% exploitation rate) for 1990; our estimate of F for that year was 0.76 (53%). Their estimate of F apparently included some natural mortality (M) because their estimated value of M was 0.15, compared with our measured value oi M = 0.22 for 1990. The resulting total mortality of the market stock for 1990 was 0.94 (61%) in this study, compared with 1.45 (77%) reported by Rothschild et al. (1994). Besides underestimat- ing natural mortality. Rothschild et al. (1994) estimated mean age at first capture at 2.6 y (legal ininimum length 76 mm), and the mean age of the harvest (85 mm mean length) at 3.0 y. Growth curves we derived from size-frequency data for thousands of Maryland oysters collected during surveys from 1990 to 2000 predicted that the average 76 mm oyster was 4.0 y old, and the average 85 mm oyster was 4.5 y old. This disparity in age-at- length determinations could explain the higher rates of total mor- tality and fishing mortality estimated by Rothschild et al. (1994) using a modified Beverton-Holt formulation (Ehfhardt & Ault 1992), because overestimation of the growth coefficient would yield a higher rate of total mortality. Our recruitment analysis suggested that oysters are most likely to be harvested at ages of four to five years in Maryland, consistent with our growth-based predictions of age at first capture and mean age of capture, and with findings by Ulanowicz et al. (1980). Our estimates of total mortality assume that instantaneous rates of fishing mortality and natural mortality are additive, that is, these sources of mortality do not occur simultaneously within a harvest year. This assumption cannot be strictly true, but mortalities asso- ciated with P. imiriiins and H. nelsoni dominate the natural mor- tality term and generally occur from May to October (Ford & Tripp 1996). Because the fishery operates from October to March, interaction between the two mortality terms should not be a major source of error, but probably is a source of positive bias in the total mortality estimates. We plan to test this assumption more rigor- ously in our continuing stock assessments. Among the uncertain- ties in estimating mortality, we note the questionable accuracy of determining annual rates of natural mortality from box counts. For example, experimental data presented by Christmas et al. (1997) indicated that roughly 50% of boxes remained intact for more than two years in northern Chesapeake Bay, suggesting that our surveys overestimated annual mortality. Counteracting biases might in- volve (1) mechanical disarticulation of boxes by the dredge used for sampling, or (2) pre-mortem deterioration of the hinge ligament in diseased and dying oysters, which could hasten disarticulation compared with the chemically-killed oysters used in the Christmas et al. (1997) study. Effects of Salinity on Population Structure and Biomass The sharp differences between trends in oyster biomass in high-, medium-, and low-salinity zones partially reflect different rates of recruitment and natural mortality. In high salinity areas, infections with H. nelsoni and P. nuiriniis are enzootic, with chronically high rates of mortality. Relatively few oysters survive to market size in this zone, but higher rates of spat settlement and recruitment to the sub-market stock tend to maintain populations of small oysters. Populations in the medium salinity zone undergo epizootics of H. nelsoni and attendant mortality episodes during extended dry periods, when salinity rises to within the tolerance range for the parasite (13-15 ppt; Paraso et al. 1999). Infections with P. marinus are enzootic in this zone except during very wet periods; mortality can be high even in the absence of H. nelsoni. but oysters generally survive to larger sizes than in the high- salinity zone. Spat settlement is moderate and episodic in these mid-salinity areas. In the low-salinity zone, H. nelsoni is absent or very rare, and although P. marinus can reach high prevalence in these populations, oyster mortality rates are lower than in high- and mid-salinity zones. Spat settlement and natural recruitment are sporadic and must be supplemented by transplants from other re- 740 Jordan et al. gions of the Bay or from hatcheries to sustain these harvested subpopulations. Occasionally, freshets cause significant oyster mortality in the areas of lowest salinity. The net long-term results of these dynamics are as follows: (1) relatively stable subpopula- tions of larger oysters in the low-salinity zone; (2) subpopulations that tluctuate in size and abundance in the mid-salinity zone; and (3) subpopulations in the high-salinity zone that are typically com- prised mostly of submarket oysters, which can reach high abun- dance in some areas. Jordan (1995) used multivariate analysis to classify Maryland oyster bars into six groups based on several population attributes; salinity was the most important independent variable associated with population structure in that analysis. Oyster Biomass and Abundance Relative to the Chesapeake Bay Restoration Goal In terms of the relative biomass index, the 1994 baseline Mary- land oyster population (from which a 10- fold increase is to be gained by 2010) was 84.4 g dry tissue weight per bushel of dredged bottom material. An index of 844, therefore, would indi- cate achievement of the goal. In 2001. the index was 1 17. about 1.4 times the baseline, or 14% of the goal. By combining the length- weight equation and the equation for estimating the number of oysters per bushel, we estimate that a Maryland bushel cannot contain more than about 640 g dry weight of oyster tissue. For this reason, the index of relative biomass cannot realize the desired value of 844 with current sampling methods. Although the biomass index is a satisfactory indicator of trends as long as the oyster population remains at low levels, quantitative assessment will be essential for determining attainment of the goal. Working with Virginia scientists, we expect to establish a bay-wide baseline and annual assessments of absolute abundance and biomass by the end of 2002. These analyses would not have been possible without relatively long time series of consistent monitoring data. Earlier publications based on MFS data (Smith & Jordan 1993, Jordan 1995) charac- terized the first 2-4 y of the data set ( 1990 — 1993), but relation- ships such as those depicted in Figures 3, 4 and 12, along with our estimates of absolute oyster abundance and biomass, would be difficult to elicit without data spanning many years. Significant temporal trends in ecosystems as large and variable as Chesapeake Bay can be difficult to detect without decades of consistent data (Vaas & Jordan 1991, Jordan & Vaas 2000), an important consid- eration in the design and maintenance of large-scale monitoring programs. In turn, such monitoring programs, including the re- sources to manage, analyze and interpret the data, are essential wherever society has established specific goals for rehabilitation of natural resources and ecosystems. ACKNOWLEDGMENTS This work was supported in part by a grant from the NOAA Chesapeake Bay Stock Assessment Committee (NA07FU0539), and by the Maryland Department of Natural Resources (DNR). Many people from DNR's Shellfish Division, especially Roy Scott, John Hess, Mickey Astarb, Mitchell Tamowski, and Cap- tains John Collier and Lee Daniels contributed to field data col- lection. Connie Lewis of DNR provided oyster landings data. Jim Uphoff s thoughtful review of a draft of this article was most helpful. The authors thank our colleagues from Virginia, Roger Mann and Jim Wesson, for their enthusiastic collaboration in our stock assessment efforts, and Kennedy Paynter from the University of Maryland for his support and encouragement. Copies of the following literature are available from the first author of this ar- ticle: Christmas and Jordan ( 1991 ), Gieseker (2001 ), Homer et al. ( 1496). Krantz (undated), Maryland DNR (2001 ), and Smith and Jordan (1993). LITERATURE CITED Calvo, G. W., R. J. Fagan, K. N. Greenhawk, G. F. Smith & S. J. Jordan. 1996. Spatial distribution and intensity of Perkinsus marinits infections in oyster recovery areas in Maryland. J. Shellfish Res. 15:381-389. Carriker. M. L. 1996. The shell and ligament. In: V. S. Kennedy, R. I. E. Newell & A. F. Eble, editors. The Eastern oyster Crassosrrea virginica. College Park. MD: Maryland Sea Grant College Publ. UM-SG-TS-96- 01. pp. 7.5-168. Christmas, J. F. Jr. & S. J. Jordan. 1991. Choptank River Oyster Mortality Study. Annapolis, MD: Maryland Dept. of Natural Resources CBRM- HI-91-1. Christmas. J. F.. M. R. McGinty, D. A. Randle. G, F. Smith & S. J. Jordan. 1997. Oyster shell disarticulation in three Chesapeake Bay tributaries. J. Shellfish Res. 16:115-123. Ehrhardt, N. M. & J. S. Ault. 1992. Analysis of two length-hased mortality models applied to bounded catch length frequencies. Trans. Am. Fish- eries Soc. 121:115-122. Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In: V. S. Kennedy, R. I. E. Newell & A. F. Eble, editors. The eastern oyster Crassoslrea virginica. College Park. MD: Maryland Sea Grant College Publ. UM-SG-TS-96-01, pp. 581-A60. Gieseker, C. 2t)OI. Year 2()00 Maryland Oyster Disease Status Report. Oxford, MD: Maryland Department of Natural Resources Technical Report FS-SCOL-01-1. Homer, M. L., R. Scott & G. E. Krantz. 1996. Maryland Oyster Population Status Report: 1995 Fall Survey. Oxford, MD: Maryland Department of Natural Resources Report MDDNRSPl-96. Jordan. S. J. 1987. Sedimentation and remineralization associated with biodeposition by the American oyster Crassoslrea virginica (Gmelin). Ph.D. Dissertation. College Park, MD: University of Maryland, 2(X) pp. Jordan, S. J. 1995. Population and disease dynamics of Maryland oyster bars: a multivariate classification analysis. / Shellfish Res. 14:459- 468, Jordan, S. J. & P. A. Vaas. 20()0. An inde.\ of ecosystem integrity for Northern Chesapeake Bay. Environ. Sci. Policy 3:S59-S88. Krantz. G. E. undated. Oyster recruitment in the Chesapeake Bay 1939 to 1993. Oxford, MD: Maryland Department of Natural Resources, 15 pp. MacKenzie. C. J. Jr. 1996. Management of Natural Populations. In: V. S. Kennedy. R. I. E. Newell & A. F. Eble. editors. The eastern oyster Cras.wsirea virginica. College Park. MD: Maryland Sea Grant, pp. 707-721. Maryland DNR. 2001. Maryland Oyster Population Status Report, 1996- 2000 Fall Surveys. Annapolis, MD: Maryland Dept. of Natural Re- sources, Annapolis. Meritt, D. W. 1977. Oyster spat settlement on natural cultch in the Mary- land portion of the Chesapeake Bay (1939-1975). UMCEES Special Report No. 7, Cambridge. MD: University of Maryland, Horn Point Laboratory. Newell. R. I. E. 1988. Ecological changes in Chesapeake Bay: are they the result of over-harvesting the American oyster. Crassoslrea virginica! In: M. Lynch & E. C. Krome. editors. Understanding the estuary: advances in Chesapeake Bay research. Solomons. MD: Chesapeake Research Consortium, pp. 536-546. Newell, R. I. E., G. S. AKspach Jr., V. S. Kennedy & D. Jacobs. 2000. Oyster Biomass in the Chesapeake Bay 741 Mortality of newly metamorphosed eastern oysters {Crassosm-a vir- ginica) in mesohaline Chesapeake Bay. Marine Biol. 136:665-676. Paraso. M. C. S. E. Ford. E. N. Powell. E, E. Hofmann & J. M. Klinck. 1999. Modeling the MSX parasite in Easter oyster iCrassostrea vir- ginica) populations. 11. Salinity effects. / Shellfish Res. 18:501-516. Rothschild. B. J.. J. S. Ault. P. Goulletquer & H. Heral. 1994. Decline of the Chesapeake Bay oyster population: a century of habitat destruction and overfishing. Marine Ecol. Prog. Series 1 1 1:29-39. Smith. G. F. & S. J. Jordan. 1993. Monitoring Maryland's Oysters: a Comprehensive Analysis of Modified Fall Survey Data 1990-1991. Oxford. MD: Maryland Department of Natural Resources Report CBRM-OX-93-03. Ulanowicz. R. E.. W. C. Caplins & E. A. Dunnington. 1980. The forecast- ing of oyster harvest in central Chesapeake Bay. Esniarine. Coastal Shelf Sci. 11:101-106. Vaas. P. A. & S. J. Jordan. 1991. Long term trends in abundance indices for 19 species of Chesapeake Bay fishes: reflection of trends in the Bay ecosystem. In: New perspectives in the Chesapeake System: a research and management partnership. Proceedings of a Conference. Chesa- peake Research Consortium Publ. 137. Journal of Shellfish Research. Vol. 21, No. 2. 743-747, 2002. EXPERIMENTAL EFFECTS OF WATER TEMPERATURE ON THE GAMETOGENIC DEVELOPMENT OF BROODSTOCK IN THE OYSTER, OSTREA CHILENSIS ANDREW G. JEFFS,' ' B. J. DUNPHY." AND R. M. G. WELLS" ^ National Insliliile of Water and Atmospheric Research. P.O. Bo.x 109-695. Auckland. New Zealand: 'School of Biological Sciences. University of Auckland, Private Bag 92019 Auckland, New Zealand ABSTRACT A protocol for reliably conditioning broodstock of the Chilean oyster, Osirea chilensis. is required because the commercial aquaculture of this species has been constrained by the low and variable number of naturally spawning adults. Therefore, the gametogenic response of hatchery conditioned O. chilensis broodstock under differing temperature regimes ( 10°C, 15°C, and 20°C) was investigated, and compared with changes in a wild population over the same time. The overall loss of gametes, particularly ova. through spawning and phagocytosis was significantly higher in oysters held at 20°C and these oysters tended to increase early oogenesis and decrease early spermatogenesis. There was also greater variability in the abundance of reproductive products, particu- larly female products, among oysters at the higher experimental temperature. At IO°C and 15°C. oysters had similar levels of gametogenic products to wild oysters. Over the experimental period when natural water temperatures remained low the wild oysters also increased early spermatogenesis and oogenesis, but there was little loss of gametes or phagocytosis. The results indicate that warmer water temperatures may be involved in synchronizing and initiating oogenesis, and could be a useful tool for resetting the gametogenic cycle in broodstock. Unlike many other species of cultured oysters, it appears that conditioning broodstock of O. chilensis may best be undertaken at low water temperatures. KEY WORDS: Chilean oyster, Ostrea chilensis. conditioning, gametogenesis, reproductive cycle. New Zealand INTRODUCTION The lack of a large and reliable supply of larvae is a major impediment to commercial aquaculture of tfie Chilean oyster. Os- irea chilensis (Philippi 1845), in a number of countries including South America and New Zealand (Lepez 1983. Utting 1987, Va- lencia Camp 1990, Hickman 1992. Jeffs 1995). The development of hatchery techniques for artificially conditioning and inducing larval production in broodstock at any time of the year would overcome this obstacle. However, the factors influencing the re- productive cycle of the Chilean oyster are poorly understood and consequently the development of effective hatchery techniques remains in their infancy (Jeffs & Creese 1996). There are only three published reports of preliminary attempts in Chile to condi- tion broodstock of this oyster, all of which have produced mixed results (Ramorino 1970, Chaparro 1990, Wilson et al. 1996). The first unsuccessful attempts to condition and spawn Chilean oysters in a laboratory were made with minimal control over water tem- perature and feeding (Ramorino 1970). A preliminary study by DiSalvo et al. ( 198.3a), DiSalvo et al. (1983b) induced some oys- ters to spawn earlier than in the wild by using artificially raised temperatures. Similar results were obtained with control over both water temperatures and feeding by Chaparro (1990). He found that some broodstock held at 17°C and 20"C matured and spawned spermatozoa and eggs, however, oysters held at 14°C matured and spawned spermatozoa, but did not spawn eggs. Broodstock held at I4°C and given a higher feeding allocation was the only experi- mental treatment in which oysters maintained their reproductive condition. Field research in New Zealand has suggested that low winter water temperatures (50 mm shell height) were collected randomly from the Manukau Harbour and processed for histology. Ambient water temperature in the harbor was recorded at each sampling time. Histologic Processing Each oyster was carefully opened and a note made of the pres- ence of larvae in the brood chamber. A 5-mm thick section of tissue was excised from each oyster parallel to the anterior- posterior axis between the labial palps and the posterior adductor muscle and then fixed in Bouin's solution. Tissue sections taken in this manner for this species are known to contain gametes repre- sentative of the whole gonad (Jeffs 1998). Permanent microscope slide mounts were made of the gonad material once it had been stained with haemotoxylin and eosin to differentiate the reproduc- tive tissues. Histologic Analysis Each slide was examined under a compound microscope at up to xlOO magnification and the presence of different types of re- productive products were analyzed by a semi-quantitative method developed and verified specifically for this oyster (Jeffs 1998. Jeffs 1999). The method relies on assigning a score of 0-3 (0 = absent. 3 = abundant) to the abundance of reproductive features found that are associated with different aspects of the gametogenic cycle. Five stages of spermatogenesis (spermatogonia, primary and sec- ondary spermatocytes, spermatids, and spermatozoa) and three stages of oogenesis (oogonia, oocytes, and ova) were scored. Ad- ditionally, the release of ova and spermatozoa from the lumen of the follicles ( = gamete loss or spawning) and the abundance ot reabsorption of unspawned ova and spermatozoa ( = phagocytosis or reabsorption) were scored in the same manner that has previ- ously been verified (Jeffs 1998. Jeffs 1999). The amount of con- nective tissue surrounding the follicles was also scored, providing an inverse measure of the overall quantity of reproductive material present in the follicles (Jeffs 1998. Jeffs 1999). Finally, a visual estimate was made of the percentage of male reproductive prod- ucts over the entire gonad section. Statistical Analyses Statistical comparisons were made for the scores of the game- togenic attnbutes of oysters among the three wild sampling events, the three temperature treatments and between the replicate pairs of treatment tanks using Kruskal-Wallis tests (Sokal & Rohlf 1995). The 1 2 response variables consisted of those reproductive attrib- utes measured under the microscope. Bonferroni corrected signifi- cance levels were used for the tests to control for inflated type 1 errors caused by multiple significance testing. RESULTS Water Temperatures Recorded water temperatures in Manukau Harbour were 13.1"C at the initial collection of broodstock. 1 1.6°C at the day 28 of the experiment, and 1 1 .9 'C at the completion of the experiment. After the initial water temperature change over 48 h the water temperatures in the experimental tanks did not fluctuate more than ± 1 -C from the treatment temperatures for the remaining duration of the experiment. Brooding At the completion of the experiment five oysters (6.9'7c) were brooding larvae in the 10=C treatment (3 in one tank and 2 in the other). Three oysters (4.2%) were brooding in the I5°C treatment (3 in one tank and none in the other). No oysters were brooding in the 20-C treatment. Samples of oysters taken from the wild population found 5 out of 70 oysters (7.1%) were brooding larvae at the outset of the experiment, 5 out of 73 oysters (6.8%) were brooding at day 28 and 6 out of 73 oysters (8.2%) were brooding at the end of the experiment. Statistical Analyses Analyses showed that there was no difference in the reprtxiuc- tive attributes of oysters held in the two tanks used in each ex- perimental treatment. Therefore, the results for the two tanks were pooled for each experimental treatment. Statistical comparisons were made of the scores for the reproductive attributes of oysters among the three experimental temperature treatments, and among those sampled from the wild over the duration of the experiment (Fig. 2 and Fig. 3). For the experiment, significant differences were found among the scores for connective tissue (P < 0.000001 ) and phagocytosis iP < 0.000005). For oysters sampled from the wild, significant differences were found among the scores for per- centage'^male (P < 0.001). spermatogonia iP < 0.0005). primary spermatocytes (P < 0.0001 ) and oocytes (P < 0.0001 ). The overall trends in the data were explored by plotting mean values for the variables with their standard errors (Figs. 2 and 3). Spermatogenesis Over all of the oysters examined the male reproductive prod- ucts were generally more abundant than female reproducti\e prod- Effect of Temperature on Chilean Oyster Gametogenesis 745 100- 80- S? 40- 20- 3- I 2- CO E I 1- ■5. S. 1- peO.001 p<0 0005 p<0 0001 Wild Experimental 0 28 50 Day of Experiment 10 15 20 Water temperature ( °C ) Figure 2. Mean scores (± standard error) of male gametogenic char- acteristics and percent male of Chilean oysters plotted against either time of sampling (wild population), or experimental water tempera- ture (experimental population). Signincant ditTerences between scores as identified by Kruskal-Wallis tests are indicated (ns = not signifi- cant). ucts. In the wild population of Chilean oysters there was a signifi- cant increase in the proportion of male reproductive material ac- cumulating in the gonad {P < 0.001). This was associated with a trend for male reproductive attributes, especially for the early sper- matogenesis stages (spermatogonia (P < 0.0005), primary sperma- tocytes ^P < 0.0001 ) and secondary spemiatocytes) to increase in abundance over the 50-day experimental period as the natural wa- ter temperatures decreased. At the end of the experimental period oysters in the three temperature treatments contained a similar abundance of male reproductive products as oysters in the wild population. However, there was a general trend for the abundance of male reproductive products, especially the early spermatogen- esis stages, to decrease slightly and for the variability in the abun- dance of reproductive products to increase with increasing experi- mental temperature. Oogenesis On average ova were more abundant among all of the experi- mental and wild oysters than oogonia or oocytes. These earlier stages of oogenesis tended to increase in the wild population of Chilean oysters over the study period. For example there was a significant increase in oocytes among wild oysters over the period (P < 0.0001). At the end of the experiment the oysters in three temperature treatments contained a similar abundance of female reproductive products as oysters in the wild population. The abun- dance of the early stages of oogenesis (oogonia and oocytes) in oysters tended to increase with higher experimental water tem- perature. The abundance of ova in the 20^C treatment, however, tended to be lower than for the other temperatures, and for the wild population. The variability in abundance of female reproductive products was also greatest in the 20°C treatment. 8 p<0.0001 p<0.000001 Wild p<0,C00005 Experimental 28 Day of expenment 15 Temperature ( C.) 20 Figure 3. Mean scores (± standard error) of female gametogenic of Chilean oysters plotted against either time of sampling (wild popula- tion), or experimental water temperature (experimental population). Significant differences between scores as identified by Kruskal-Wallis tests are indicated (ns = not significant). 746 Jeffs et al. Gamete Loss and Phagocytosis The recent loss of gametes, the amount of connective tissue and phagocytosis of reproductive products was at similar lovv* levels among the three wild samples of oysters suggesting that gamete materials were accumulating in the gonads over this period. How- ever, for the experimental oysters the recent loss of gametes, and especially the amount of connective tissue and the amount of phagocytosis of reproductive products all tended to increase with increasing temperature and especially at 20°C. DISCUSSION Low water temperatures have been widely implicated in con- trolling the production of female gametes in Ostica chilensis (Westerskov 1980. Winter et al. 1984. Jeffs et al. 1997a. Jeffs et al. 1997c. Jeffs 1999). Field studies within New Zealand surmised that water temperatures of < 1 2''C acted to increase the gonad con- dition and the development of ova in local Chilean oyster popu- lations (Westerskov 1980. Jeffs et al. 1997b. Jeffs 1999, Jeffs & Hickman 2000). The results of our laboratory study revealed that the effect of temperature on gonad development was consistent with the patterns observed in wild populations of this oyster (Jeffs & Cree.se 1996, Jeffs et al. 1997a). However, a considerable amount of variability remained in the gametogenic response of individual oysters to experimental temperatures, a pattern that ap- pears to be a feature of this species (Jeffs et al. 1997b). In this study male and female gametes were accumulated in the gonads of wild oysters experiencing cold winter water tempera- tures and there was little overall loss or phagocytosis of gametes. The accumulation of reproductive products in winter/spring pre- ceding a minor spring/summer peak in spawning has been ob- served in other wild populations of this oyster (Jeffs et al. 1996. Jeffs 1999. Jeffs & Hickman 2000). The experimental oysters ex- hibited a similar response to lower water temperatures, with oys- ters held at 10°C and 15°C also accumulating similar amounts of developing reproductive material to wild oysters. In both the wild and experimental oysters at 10°C and 15°C early spermatogenesis had increased. In wild oysters there were signs that early oogenesis also increased. A similar pattern has also been observed in the Olympia oyster. Ostrea lurida. where experimental broodstock held at I2"C increased their gonad condition, while those at I8°C and 2r"C lost condition through the gonad cycle being rapidly advanced (Santos et al. 1993). Similar results have also been found for Chilean populations of O. cliileiisis maintained in hatcheries over a range of temperatures (Chaparro 1990, Toro & Morande 1998). Such a response is common to many other species of bi- valves held at elevated temperatures (Utting & Spencer 1991, Ut- ting & Millican 1997). This rapid advance of the gonad cycle would also explain the absence of brooding oysters among the broodstock oysters held at 20°C in our study because these oysters would have already spawned or reabsorbed their gametes, whilst those at lower temperature were still developing. This was evi- denced by the increases in the loss of gametes, connective tissue and phagocytosis observed amongst our experimental oysters at 20°C. and previously identified among.st oysters taken from wild populations during periods of similar warm water conditions (Jeffs et al. 1996. Jeffs et al. 1997a. Jeffs et al. 1997c. Jeffs et al. Jeffs 1998. Jeffs et al. 1999). The results for these two Ostrea species are in contrast to the widely cultivated Pacific oyster. Crassostrea gigas. the European oyster. O. edidis, and the American oy.ster. C. virginica. which are known to undergo gonad development at warmer water tempera- tures, (e.g.. 18"C to 24°C) and commercial broodstock condition- ing relies on using these warmer temperatures (Aboul-Ela 1960, Quayle 1969. Mann 1979. Muranaka & Lannan 1984. Dinamani 1991. Santos et al. 199.3, Castagna et al. 1996). Within this tem- perature range the experimental O. chilensis in this study lost condition through increased gamete loss and phagocytosis and decreased early spermatogenesis. There was also a trend for early oogenesis to increase, but this has previously been associated with rapid initiation of early gametogenesis after the extensive loss of gametes and subsequent rapid phagocytosis of remaining gamete material at warm water temperatures (Jeffs 1998, Jeffs 1999, Jeffs & Hickman 2000). All of these patterns of gonad change observed in our study are consistent with those previously observed in popu- lations of wild oysters in warm water temperatures during sum- mer-autumn (Jeffs 1998, Jeffs & Hickman 2000). The results of this study suggest that manipulating water tem- peratures has the potential to be an effective method of controlling broodstock development in O. chileiisis. Unlike other species of commercial oysters, cold-water temperatures appear to be impor- tant in stimulating early spermatogenesis and oogenesis and the accumulation of developing gametes much needed for effective conditioning of broodstock. Warm water temperatures appear to rapidly advance the development and loss of developing gametes (through spawning and phagocytosis) already held in the gonads, and this is followed by the rapid increase in oogenesis. Therefore, warm water temperatures have the potential to be used for advanc- ing the development of gametes in broodstock conditioned at cold temperatures toward spawning and for restarting the gametogenic cycle from early gametogenesis in broodstock in attempt to create greater synchrony among individual broodstock. ACKNOWLEDGMENTS The authors thank Geoff McAlpine for sagely oyster advice. Jo Evans and Simon Hooker for helping to establish the algal cul- tures, and Beryl Davy for the histologic preparations. Logistic support from Bob Creese and the Leigh Manne Laboratory made this research possible. Martin von Randow undertook the statistical analyses. This work was supported by the New Zealand Founda- tion for Science. Research and Technology. Aboul-Ela. I. A. 1960. Conditioning Ostrea ediilis from the Limfjord for reproduction out of season. Mcddelelser Fret Danmarks Fiskeri - Of; Huvuuders0gelser 2: 1-15. Brown. M. R.. S. W. Jeffrey & C. D. Garland. 1989. Nulrilional aspects of microalgae used in maricultuic; a literature review. CSIRO Mar. Udi. Rept. Ser. 205:1-44. Castagna, M., M. C. Gibbons & K. Kurkowski. 1996. Culture: application.. In: V. S. Kennedy. R. 1. E. Newell & A. F. Able, editors. The Eastern LITERATURE CITED Oyster. Cnissoslreci virginicti. Maryland: Maryland Sea Grant Publi- cations, pp. 675-690. Chaparro. O. R. 1990. Effect of temperature and feeding on conditioning of Ostrea chilensis Phillipi 1845 reproductors. Aqua. Fish. Manag. 21: 399^05. Dinamani. P. 1991. The Pacific Oyster Cras.soslrea gigas (Thunberg. 179.^). in New Zealand. In: W. Men/el. editor. Estuarine and Marine Bivalve Mollusk Culture. Boca Raton: CRC Press. Effect of Temperature on Chilean O'ister Gametogenesis 747 DiSalvo. L. H.. E. Alarcon & E. Martinez. 1983a. Induced spat production from Oslrea chilensis Phillipi 1845 in mid-winter. Aqiiaciilnire 30: 357-362. DiSalvo, L. H.. E. Alarcon & E. Martinez. 1983b. Progress in hatchery production of seed of the Chilean oyster Oslrea chilensis (Phillipi 1845). Symposium Internacional de Acuacultura, Coquiinho. Chile, pp. 269-280. Hickman, R. W. 1992. Spat production. In: P. J. Smith, G. G. Baird, M. F. Beardsell, editors. Farming the dredge oyster: proceedings of a work- shop. July 1992. N. Z. Fish. Occas. Pub. 7. pp. 7-11. Jeffs, A. G. 1995. The aquaculture and Biology of Bluff oysters. Seafood N.Z. 3:37-39. Jeffs, A. G. 1998. Gametogenic cycle of the Chilean oyster, Tiosirea chil- ensis (Phillipi 1845), in north-eastern New Zealand. Invertebr. Reprod. Dev. 34:109-116. Jeffs, A. G. 1999. The potential for developing controlled breeding in the Chilean oyster. Aquacul. Int. 7:189-199. Jeffs, A. G. & R. W. Hickman. 2000. Reproductive activity in a pre- epizootic wild population of the Chilean oyster, Oslrea chilensis, from southern New Zealand. Aijiuicullure 183:241-253. Jeffs, A. G. & R. G. Creese. 1996. Overview and bibliography of research on the Chilean oyster Tiosirea chilensis (Phillipi 1845) from New Zealand waters. / Shellfish Res. 15:305-31 1. Jeffs, A. G., R. G. Creese & S. H. Hooker, 1996. Annual pattern of brooding in populations of Chilean oysters, Tiosirea chilensis, (Phil- ippi. 1845) from northern New Zealand. / Shellfish Res. 15:617-622. Jeffs, A. G.. R. G. Creese & S. H. Hooker. 1997a. The potential for Chilean oysters, Tiosirea chilensis (Philippi, 1845), from two populations in northern New Zealand as a source of larvae for aquaculture. Aqiiacutl. Res. 28:433^41. Jeffs, A. G„ S. H. Hooker & R. G. Creese. 1997b. Variability in life history characters of the Chilean oyster Tiosirea chilensis (Philippi, 1845). N. Z. J. Mar FW Res. 31:487-495. Jeffs, A. G., S. H. Hooker & R. G. Creese. 1997c. Annual pattern of settlement in populations of Chilean oysters, Tiosirea chilensis, (Phil- ippi. 1845) from northern New Zealand. J. Shellfish Res. 16:91-95. Laing, 1. 1991. Cultivation of marine unicellular algae. Ministry of Agri- culture, Fisheries and Food (G.B.). Lahorulory Leaflet 67:1-31. Lepez. M. 1983. El cultivo de Oslrea chilensis en la zona central y sur de Chile (in Spanish). Mem. Asoc. Latinoam. Acidciilt. 5(2):1 17-127. Mann, R. 1979. Some biochemical and physiological aspects of growth and gametogenesis in Crassosirea gigas and Oslrea edulis grown at sus- tained elevated temperatures. / Mar. Biol. Assoc. U.K. 59:95-110. Muranaka, M. S. & J. E. Lannan. 1984. Broodstock management of Cras- sosirea gigas: environmental influences on broodstock conditioning. Aquaculture 39:217-228. Quayle, D. B. 1969. Pacific oyster culture in British Columbia. Fish. Res. Bd Canada, Bull. 169 pp. Rainorino, L. 1970. Estudios preliminares sobre crianza de Oslrea chilensis en el laboratorio. Biol. Pesq. 4:17-32. Santos, J. M., S. L. Downing & K. K. Chew. 1993. Studies on the effects of water temperature on the sexual development of adult Olympia oysters, Oslrea lurida. World Aquacult. 24:43^6. Sokal, R. R. & F. J. Rohlf. 1995. Biometry: The principles and practice of statistics in biological research. New York: W. H. Freeman and Co. Toro, J. E. & P. R. Morande. 1998. Effect of food ration and temperature on length of brooding period, larval development, and size of pedive- ligers released in the Chilean oyster Oslrea chilensis. J. World Aquac- ult. Soc. 29:267-270. Utting. S. D. 1987. Fresh prospects for bivalve farming. Fish Farnter 10:17-18. Utting. S. D. & B. E. Spencer 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Ministry of Agriculture. Fisheries and Food (G.B). Udwruioiy Leaflet 68:1-31. Utting, S. D. & P. F. Millican. 1997. Techniques for the hatchery condi- tioning of bivalve broodstocks and the subsequent effect on egg quality and larval viability. Aquaculture 155:45-54. Valencia Camp, J. 1990. Estado actual del cultivo de ostra chilena, Tiosirea chilensis. In: Proc. 6th Taller de Acuacultura-Actualidad. technologia, mercado e interaccion social del cultivo del ostion, ostra y especies potenciales. Universidad catolica del Norte y Associacion de Produc- tores de Ostras y Ostiones. pp. 99-109. Walne, P. R. 1970. Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Oslrea. Crassosirea, Merceimriu and Mylilus. Ministry of Agriculture, Fisheries and Food (G.B.). Fisheries Investigations 2(25): 1-62. Westerskov, K. 1980. Aspects of the biology of the dredge oyster Oslrea lularia Hutton, 1873. PhD thesis. Dunedin: University of Otago. 192 pp. Wilson. J. A., O. R, Chaparro & R. J. Thompson. 1996. The importance of broodstock nutrition on the viability of larvae and spat in the Chilean oyster Oslrea chilensis. Aquaculture 139:63-75. Winter, J., J. Toro, J. Navarro, G. Valenzuela & O. Chaparro. 1984. Recent developments, status, and prospects of molluscan aquaculture on the Pacific coast of South America. Chapt. 14. Vol. 39. In: D. E. Morse, K. K. Chew & R. Mann, editors. Recent innovations in Cultivation of Pacific Molluscs. Developments in Aquaculture and Fisheries Science. Amsterdam: Elsevier Scientific, 95 pp. Joiinwl of Shellfish Reseurch. Vol. 21, No. 2, 749-756. 2002. HISTORICAL CHANGES IN INTERTIDAL OYSTER {CRASSOSTREA VIRGINICA) REEFS IN A FLORIDA LAGOON POTENTIALLY RELATED TO BOATING ACTIVITIES RAYMOND E. GRIZZLE,'* JAMIE R. ADAMS,' AND LINDA J. WALTERS- ^Jackson Estituhne Laboratory, University of New Hampshire, Diirliain. New Hampshire 03S24; ^Department of Biology, University of Central Florida. Orlando, Florida 32816 ABSTRACT Research in the late 19y0s showed that some intertidal eastern oyster (Cra.ssosrrea viiginica. Gmelin) reefs in Mosquito Lagoon within the Canaveral National Seashore. Florida had dead margins consisting of mounded up. disarticulated shells. It was hypothesized that heating activities were the cause of the damage because all the reefs were adjacent to major navigation channels. To investigate this, we characterized the history of the appearance of dead margins and other reef changes using aerial photographs taken between 1943 and 2000. Imagery analyzed included prints (black & white, color, or color IR) from 1943, 1951, 1963, 1975. 1988, and 1995, and digital imagery from 2000 (USGS 1:12,000 digital onho-quarterquads). at scales from 1:6.000 to 1:24.000. Prints were scanned at a resolution sufficient to yield 1-m pixels. After scanning, each set of images was georeferenced to the year 2000 imagery using Arc View and Arclnfo GIS software. All reefs found to have dead margins based on 1995 and 2000 aerials were visited in 2001 and 2002 to confirm the presence and extent of dead areas. This provided ground-truthing for the "signature" (a highly reflective, light-colored area adjacent to darker-colored live reef) to be used to detect the appearance of dead margins in the historical aerials. The earliest appearance of dead margins was in the 1943 aerials on one reef adjacent to the Intracoastal Waterway (ICW). a major navigation channel. The total number of reefs affected and areal extent of dead margins steadily increased from 1943 through 2000. In 2fK10. 60 reefs (of a total of -400 in the Park) had dead margins, representing 9. 1'^f of the total areal coverage by oyster reefs in the Park. Along the ICW. some reefs migrated away from the channel as much as 50 m and in 2000 consisted mainly of empty shells mounded up a meter above the high water mark. In contrast, many reefs in areas away from navigation channels showed little change over the 57-year period. This historical analysis provides strong (although correlative) evidence that boating activity has had dramati- cally detrimental effects on some oyster reefs in the study area. Ongoing studies are aimed at further testing this hypothesis and elucidating the causal mechanisms involved. KEY WORDS: eastern oyster. Crassosirea virginica, reefs, aerial photographs, remote sensing, boating activities INTRODUCTION The eastern oyster, Crassosirea virginica. forms extensive reefs subtidally and intertidally along the eastern US coast (Bahr & Lanier 1981. Burrell 1986). Intertidal reefs make up the dominant form from North Carolina to the northern end of the Mosquito Lagoon in east central Florida. Most of Mosquito Lagoon in this area is within the boundaries of Canaveral National Seashore (CANA). the present study area. Here, the intertidal oyster reef constitutes a major habitat type (Grizzle 1990). Within CANA. reefs are managed for their ecological importance and are har- vested commercially and recreationally (Walters et al. 2001). Previous research within CANA characterized reef distribution and abundance patterns (Grizzle 1990) and focused on manage- ment issues (Grizzle & Castagna 1995). Based on an analysis of 1995 aerial imagery and subsequent field surveys, it was discov- ered that numerous reefs had dead margins consisting of disarticu- lated shells mounded up several decimeters above the adjacent living reef (Grizzle, pers. obs.). This pattern differed from the well-documented, long-term growth pattern of a dead middle area surrounded by living oysters (the "senescent stage" of Bahr & Lanier 1981) because the dead zones were along the margins of the reefs. Also, the dead margins consisted mainly of well-packed shells instead of a shell/sand/mud mixture as typically found in the dead middle area of senescent reefs. Further analysis showed that all reefs with dead margins occurred adjacent to channel areas that were heavily used by boats, including the federally maintained Intracoastal Waterway (ICW) that runs along the western edge of CANA. It was hypothesized that boating activities may be respon- sible, at least in part, tor the dead margins. This study was initiated as part of a larger project aimed at assessing the potential impacts of boating activities on oysters. Emphasis in the present report is on historical changes in oyster reefs based on aerial imagery, and the results of associated studies will be reported in future publications. The major objectives of this report are: (1) describe the present (2000) distribution of oyster reefs in CANA; (2) characterize historical ( 194.3 to 2000) changes in the CANA reefs based on aerial photographic imagery, with an emphasis on those reefs with dead margins in 2000; and (3) relate historical reef changes to environmental factors, with an emphasis on boating activities. METHODS Study Area *Corresponding author. E-mail: ray.grizzle@unh.edu The Study area was in northern Mosquito Lagoon in east- central Florida (Fig. I ) and restricted to the Canaveral National Seashore (CANA). a National Park unit that is an example of a relatively stable barrier beach/lagoonal ecosystein (Grizzle 1990, Walters et al. 2001 ). Most of the Lagoon within CANA is a com- plex system of shallow open water areas and nearly 100 mangrove (Rhizophora mangle a.n(X Avicennia genninansydommaied islands. Oyster reefs occur in intertidal areas, often adjacent to seagrass (mainly Halodide wriifhtii) beds that are extensive in some areas (Morris et al. 2000). Water depths are <1 m in most areas and the annual salinity range is typically between 25 and 35 psu. occa- sionally increasing to 40 psu (Grizzle 1990). Mosquito Lagoon is the northernmost body of water in the Indian River Lagoon system (IRL) that has been described as the richest and most diverse estuary in the continental United States (see references in 1995 special issue of Bulletin of Marine Science. Vol. 57; also see Walters et al. 2001). The ecological importance of this area has 749 750 Grizzle ht al. ; _ ,., •'; ' ( > 4 5 > - -^^ HwT 7 9 . o o ELDORA Figure 1. Northern portion of Canaveral National Seashore, showing most ol 40 quantification of them (see reviews by Bahr & Lanier 1981. and Kennedy & Sanford 1999: see additional discussion later). Bahr and Lanier (1981) suggested that vertical growth rate and morphologic form of intertidal reefs were strongly affected by wave action. Waves (and currents) transport sediment to and from the reef, and in some cases eventually result in sedi- ment buildup and loss of oysters in the central region (see more discussion in the Introduction section). Bahr and Lanier aLso noted (p. 57) that "■. . . a water current or wave energy regimen above a certain threshold level will prevent the development . . ." of a reef. And they showed (p. 34) a photograph of "shell debris character- istic of high energy beach shores . . .'" that resembled the mounded up dead margins observed in the present study. Wave energies of some magnitude are clearly capable of destroying an existing reef. However, we are not aware of research that quantifies the rela- tionships involved, nor the actual mechanisms involved. Hypothesized inhibitory mechanisms for reef development in- clude; vertical accretion of the reef surface to an elevation too high in the intertidal caused by waves (Churchill 1920; Bahr & Lanier 1981). smothering and substrate instability caused by excessive sediment transport (Marshall 19541. and inhibited larval settlement caused by sediment ("grit") movement (Gunter 1979). Analysis of historical aerials for reefs along the ICW in this study also suggest that a reef can be physically moved by wave energies, resulting in a range of responses from slow migration to total destruction, much in the fashion that barrier islands migrate shoreward (termed "'transgression") under increased wave energies (Davis 1996). As already discussed, this study was not designed to test particular 756 Grizzle et al. causal mechanisms for observed reef declines. They are the topic of ongoing studies by Linda Walters and colleagues (e.g.. Wall et al. 2002. 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JoKi-iwI of Shellfish Research. Vol. 21, No. 2. 757-762. 2002. BIOCHEMICAL COMPOSITION OF SPONDYLUS LEUCACANTHUS BRODERIP, 1833 (BIVALVIA: SPONDYLIDAE) AND ITS RELATIONSHIP WITH THE REPRODUCTIVE CYCLE AT ISLA DANZANTE, GULF OF CALIFORNIA, MEXICO SONIA RODRIGUEZ-ASTUDILLO,* MARCIAL VILLALEJO-FUERTE, FEDERICO GARCIA-DOMINGUEZ, AND RAFAEL GUERRERO-CABALLERO Institute) Poliieciiico NacioiuiL Centra Intenlisciplinaiio de Ciencius Marinas. La Paz. Baja California Stir. Mexico, A.P. 592 C.P. 23UUU ABSTRACT The monthly variation of the biochemical composition was studied for one year in relation to the reproductive cycle in a population of S/xiiulxlus leucacmuhus. a commercially important species in the Gulf of California. Carbohydrate, lipid, and protein concentrations were determined in the adductor muscle, gonad, and digestive gland. Protein was the main constituent in the gonad, digestive gland, and adductor muscle. Lipids were important in the digestive gland, whereas in the gonad they were moderately represented and in the muscle they showed low and virtually invariable concentrations during the study period. Carbohydrates were found largely in the adductor muscle and the digestive gland, whereas the gonad showed lower concentrations. S. leucacantlms has a seasonal reproductive cycle with a reproductive inactivity period at the end of the autumn and throughout the winter. Gametogenesis starts at the end of the winter, continuing during springtime, and ending in the summer, where 90% of organisms are ripe, coinciding with the highest lipid concentration in the gonad. Spawning takes place in early autumn. It is concluded that this species has a conservative reproductive strategy because it largely uses energy reserves for gamete development, in addition to energy obtained from food, to supplement its energetic expenditure. KEY WORDS: Spondylus leucucunthiis. reproductive cycle, biochemical composition. Gulf of California INTRODUCTION In the Gulf of California, Spondylus leucacanthus and other bivalve species, including Megapitaria aurantiaca, Argopecten ventricosus. Lxropecten nodosus. and Spondylus calcifer are of great importance because of the high commercial value of soft parts, adductor muscle, and shell. (Villalejo-Fuerte & Muneton- Gomez 1995, Skoglund & Mulliner 1996. Villalejo-Fuerte & Garcia-Dominguez 1998, Muneton-Gomez et al. 2001). The ex- ploitation of commercially important bivalve populations in the Gulf of California has fostered a number of studies focused on reproductive and feeding aspects (Baqueiro et al. 1982, Villalejo- Fuerte & Ochoa-Biiez 1993, Villalejo-Fuerte & Cebailos-Vu/quez 1996. Felix-Pico et al. 1997, Villalejo-Fuerte & Garcia Dominguez 1998. Villalejo-Fuerte et al. 2002). In general, reproduction in marine bivalves is associated with food availability and nutrient reserve storage in specialized organs (Gabbott 1975, Bayne 1976. Barber & Blake 1981. Boadas et al. 1997. Claereboudt & Himmelman 1997). When organisms reach reproductive maturity, growth slows down as a result of the re- productive effort, and the biochemical composition may change according to the reproductive requirements iLodeiros et al. 2001 ). Those species where energy storage and gainete production cycles overlap temporally are considered "opportunistic"; in contrast, those in which both cycles are clearly separated in time because of the use of previously stored energy reserves for gonad develop- ment are known as "conservative species" (Bayne 1976). Gonad development implies an intense metabolic activity, in- cluding the storage of large amounts of lipids in the developing eggs, either at the expense of glycogen reserves previously accu- mulated in storing tissues, at the expense of food supply, or from both sources (Gabbott 1975), A decrease in glycogen and protein contents coupled with a rise in lipid content during gonad devel- opment has been described for the pectinids Chlamys septemra- diaki (Ansell 1974), Pecten maximus (Comely 1974), Argopecten *Corresponding author. E-mail: srodrig@ipn.mx irradiims (Hickey 1978. Barber & Blake 1481). Chlumys opcrcu- laris (Taylor & Venn 1979). Plucopectcn imigellanicus (Robinson et al, 1981 ), and Himiites giganteus (Lauren 1982). The influence of temperature on nutrient-transfer regulation from reserve-storing organs to the gonad has also been described for A. irradians and Patinopecten caurinus (Sastry & Blake 1971. Barber & Blake 1981. MacDonald & Bourne 1987). The potential relationship be- tween morphophysiologic indices and the storage and use of nu- trient reserves during reproduction was described for wild popu- lations of A. circularis (Villalejo-Fuerte & Ceballos-Vazquez 1996) and S. calcifer (Villalejo-Fuerte et al. 2002), In the Gulf of California. S. leucacanthus banks have been overexploited at depths of less than 15 m (Baqueiro et al. 1982), but beyond 40 m there are banks that have not been affected by commercial fisheries. Studies of this population focused on the reproductive cycle (Villalejo-Fuerte & Garci'a-Domi'nguez, 1998) and gut content have been conducted in relation to reproduction and phytoplankton abundance (Muneton-Gomez et al. 2001). The lack of infonnation on protein, lipid, and carbohydrate content in storage organs and its potential association with the reproductive period in this species, analyzed from a seasonal perspective, were the reasons for the development of this research. MATERIALS AND METHODS An average of 36 specimens of S. leucacanthus with a range in shell height from 40 to 94 mm (mean of 60 mm) were collected each month, from June 1 996 to May 1 997. south of Isla Danzante t26°55'-26°30'N and 112-1 1 1"40'W) in the Gulf of California (Fig. 1 ). using a net at 40 m of depth. For the biochemical analysis. 16 specimens were used each month. In the laboratory, specimens were cleaned, eliminating water from the pallial cavity, and were washed with distilled water. Then, soft parts were separated from the shell and weighed, fol- lowed by the dissection of the adductor muscle, gonad, and diges- tive gland. Each organ was weighed separately and oven-dried at 100°C for 24 h. The dry tissue was pooled and homogenized and 757 758 RODRl'GUEZ-ASTUDILLO ET AL. \ la L I? G U L % Puerto S U F O -"T A 26° 49 r > : A L \ I I \ ^^ I F I O 1 R J a J \ N / n /^ \ 1 1 * r I A \ X X ■^ » \ \ ^— .. t \j X ^ • 1S'4S- \ Sampling Area RESULTS Hiochemical Coiiiposilidii .11.20 '"°'*' Fifjurt 1. Location of the sampling area in the \icinity of Danzante island, (iulf of California. then was ground into a powder. Subsaniples ol this homogenate were taken for the biochemical determination. Protein determination was conducted using the method by Lowry et ai. (1951). Carbohydrates were determined by Hewitfs method (1958). Lipid extraction was performed using the method by Folch et al. (1957) and a modification of Bligh and Dyer's method (1959). Energetic substrate concentration mg/g dry weight curves were interpreted as follows: An increase was considered as indicative of accumulation and a decline as indicative of substrate use in the corresponding organ. The reproductive cycle analysis included an average of 20 specimens per month. The degree of gonad development was de- fined from histologic section (7 jxm thick) stained with the hema- toxylin and eosin technique (Humason 1979), assigning one of the following categories proposed by Villalejo-Fuerte and Garci'a- Domingue/. (1998) for this species, undifferentiated, developing, ripe, spawning, and spent. Botlom water temperature was recorded at the time of collections. A Spearman's rank conelaiion analysis irj was used to deter- mine the relationship of the gametogenic cycle stages with protein, lipid, and carbohydrate concentrations in the adductor muscle, di- gestive gland, gonad, and water temperature. Before performing the correlation analysis, logarithmic transformations were per- formed for energetic substrates, and arc-sin transformation were conducted for percentages of (he reproductive cycle stages, aimed at obtaining data normality and homoscedasticity (Sokal & Rohll 1995). Only tho.se results where a significant correlation was found are reported below. The trend in the protein concentration curve is similar for the three organs analyzed. During the summer, protein concentration reaches high values and remains relatively unchanged. In autumn, there is a noticeable decline, with low concentrations remaining until the end of this season. In the winter, protein concentration in the gonad and digestive gland shows a recovery, reaching high values in mid-winter and decreasing by early spring. In the muscle, proteins show a steady increase that lasts until mid-spring, reach- ing high values also in the digestive gland and gonad, then de- creasing at the end of the spring (Fig. 2A). Lipid concentration in the adductor muscle did not show no- ticeable variations during the study period. In the digestive gland, high concentrations were found at the beginning of the summer, which declined throughout this season and remained low, although with some variations, during the autumn. In winter, there is a clear recovery, reaching the peak value in mid-winter and then declin- ing, with some fluctuations, afterwards and during the spring. In the gonad, the highest lipid concentration was recorded during the summer, with lower values in all other seasons (Fig. 2B). During the summer, carbohydrate concentration drops in the three organs analyzed (August), \Mth drops being more pro- nounced in the muscle and gonad. In late summer and early au- tumn, a considerable rise occurs in both gonad and muscle, re- maining unchanged in the gland. During autumn, there is a decline in the muscle and gonad, remaining low in early winter whereas there is a considerable increase in the digestive gland (November); at the end of the autumn, carbohydrates decrease, reaching low \alues at the beginning of the winter. During the winter, carbohy- drates in the muscle and digestive gland rise steadily, reaching peak values during the spring. In the gonad, there is a slight in- crease in mid-winter, decreasing afterwards until mid-spring, then recovering at the end of this season (Fig. 2C). Temperature During the study period, bottom water temperature ranged from 17 to 24.5'C. the highest values recorded in July (24.5 C) and October 1996 (24°C). The lowest temperature occurred in January 1997 ( 17'C; Fig. 2D). Spawning took place during a temperature rise from 22.5 to 24°C. and gametogenesis occurred when tem- perature rose from 17 to 18"C (Fig. 2D). Reprodiicliie Cycle The bivalve population enters a reproductive inactivity stage at the end of the autumn (undifferentiated stage near lOO'/f in No- vember), high values being maintained (around 70%) during the winter. The reproductive activity (gametogenesis) starts at the end of the winter, and in springtime 100% of the population is under- going this stage. The first ripe individuals appear at the end of the spring, and in the summer 90% of the population is ripe. The population spawns in the autumn (90% in October). Gamete re- sorption is rapid because the post-spawning stage only takes place in early winter with low percentages (Fig. .^A-D). DISCUSSION 111 general, proteins were the main constituent of the gonad, digestive gland, and adductor muscle. Lipids were important in the digestive gland, whereas in the gonad they were highest betore Biochemical Composition of Spondylus leucacanthus 759 500 A -m- Muscle -^ Gonad -*- D Gland 400 B O N -^ Gonad -^ D Gland D Figure 2. Variation of biochemical components in Spondylus leuca- canthus. A. proteins: B, lipids; C carbohydrates in muscle, digestive gland, and gonad: and D, bottom water temperature at Danzante is- land, (iulf of California. JJASONDJFMAM Figure 3. Reproductive cycle stages in Spondylus leucacanthus. A, inac- tivity; B, gametogenesis; C, maturity; D, spawning; E, post-spawning. spawning and in the muscle they showed low and virtually un- changed concentrations during the study period. Carbohydrates were found chiefly in the adductor muscle and digestive gland, whereas the gonad showed lower values. These same trends in energetic substrate concentration were found in Pcclen ituLxiiiiKs (Strohmeier et al. 2000). During gonad maturation of many marine bivalves, an increase of biochemical compounds is observed in the female gonad, followed by a drop during spawning (Ansell 1974, 760 RODRIGUEZ-ASTUDILLO ET AL. Taylor & Venn 1979. Barber & Blake 1981. Suntlel & Vahl 1981. Pazo.s et al. 1996). The slight variation of proteins durini; the summer, when the population is ripe, suggests a low demand of this energetic sub- strate during this stage. The trend in the lipid curve for the diges- tive gland suggests that lipids are used during the ripe stage: how- ever the correlation obtained is low (r^ = 0.55) and nonsignificant. Lipid transfer from the digestive gland to the ovary has been demonstrated in Chlamy.s Iwricia (Vasallo 1973). Furthermore, digestive gland carbohydrates show a significant correlation with the ripe stage (i\ = 0.83, P < 0.05). indicating its use during this stage. It is likely that during the last gamete growth stage, food is also used as an energy source. In this same population, ripe organisms have been found in July, coinciding with a peak in food availabil- ity, which may provide a portion of the nutrients required for the gonad's final ripening (Mufteton-Gomez et al. 2001). Similar be- havior was reported for Pkwopecten magellanUiis. which reside southeast of Terranova (Thompson 1977). In other bivahe species like Doiui.x villatiis and Telliua tenuis, the accumulation of reserve glycogen seems to be related with the season when the highest phytoplankton biomass occurs (Ansell & Trevaillion 1967. Ansell 1972). American oysters (Crassostrea virginica) held at 14 to 19°C were able to exploit a large phytoplankton bloom and syn- thesize glycogen (Ruddy et al. 1975). In S. leucacanthus. spawning is an event that demands a heavy energy expenditure, requiring the use of protein and carbohy- drate from the adductor muscle, the gland, and the gonad. How- ever, it is in this latter organ that a significant correlation is found (,-^ = -0.63, P < 0.05). Lipids do not appear as an important source in this stage. Based on the analyses of morpho-physiologic indexes, gut content, and phytoplankton abundance in the envi- ronment, the required energy expenditure during spawning in this species had been suggested to be partially supported by re- serves stored in the muscle, the digestive gland, and the gonad (Villalejo-Fuerte & Garci'a-Domi'nguez 1998, Muiieton-Gomez et al. 2001). When spawning ends, lipids and proteins remain low whereas carbohydrates are stored in the digestive gland, providing the en- ergy required for the resorption of residual gametes during the stage known as spent that occurs at the end of the autumn. In Crassostra virginica. the concentration of neutral lipids decreased considerably during the spawning (Trider & Castell 1980) in C. giga.^ was observed an increased in lipid during the gametogenesis phase (Deslous-Paoli & Heral 1988). When the population enters reproductive inactivity (undiffer- entiated stage) in early winter, there is a reorganization in the gonad (Villalejo-Fuerte & Garci'a-Dominguez 1998); during this stage, carbohydrates from the digestive gland are used in the first place (i\ = 0.59, P < 0.05), followed by proteins stored in the digestive gland and the gonad. Gametogenesis starts at the end of the winter and in early spring. Energy for the initial gamete development is provided by lipids from the digestive gland, an event occurring similarly in Spondylns culcifer (Villalejo-Fuerte et al. 2002). Proteins from the three organs analyzed are used afterwards, most probably during the gamete final development stages. In this respect, this species has been shown to undergo rapid gamete growth at the beginning of gametogenesis. followed by a slowdown at the end of this stage (Villalejo-Fuerte & Garcia-Dominguez 1998). Temperature has been reported to influence the gametogenic cycle through the regulation of nutrient transfer from reserve- storing organs (Sastry & Blake 1971. Barber & Blake 1981, Mac- Donald & Bourne 1987). Our findings suggest that nutrient trans- fer and use takes place when temperature varies as a result of the summer to autumn (spawning) and winter to spring (developing) seasonal changes. This had been previously suggested for this species by Villalejo-Fuerte and Garcia-Dominguez (1998) and for A. circidaris (Villalejo-Fuerte & Ceballos-Vazquez 1996). The reserve storage process takes place when the lowest temperatures occur. The biochemical composition of marine bivalves is affected by exogenous factors, including food availability and temperature, as well as by endogenous factors such as reproduction (Gabbott 1975, Bayne 1976, Barber & Blake 1981. Boadas et al. 1997, Claere- boudt & Himmelman 1997). "Opportunistic species" use the en- ergy directly obtained from food for gonad development, and en- ergetic substrate storage and gamete production cycles may over- lap temporarily, whereas "conservative species" use previously stored energy reserves (Bayne 1976). Based on the findings of this study, it is confirmed that 5. leiicacantlnis has a markedly seasonal reproductive cycle. This species shows a reproductive inactivity period that is well delim- ited throughout the year, characterized by the accumulation of energy reserves in the adductor muscle, the digestive gland and the gonad. The reproductive cycle stages that demand a higher expen- diture of reserves are developing and spawning. According to these features, it can be concluded that this species has a conservative reproductive strategy, which is a characteristic of iteroparous spe- cies that largely use energy reserves, supplemented by energy obtained from food for energetic expenditures. Similar strategies have been identified in other bivalves. ScupluiiTO broughtonii behaves as a typical conservative species, with gametogenesis taking place during the winter and spring at the expense of reserve glycogen stored during the autuinn. and spav\ning occurring during the summer (Park et al. 2001); by contrast. Cmssosln-a gigcis was shown to behave as an opportu- nistic species, given that gametogenesis starts simultaneously with reserve accumulation and proceeds until gonadic maturity is reached, coinciding with the highest level of biochemical compo- nents followed by a rapid drop of these substances during spawn- ing (Rang et al. 2000). Argopecten ventricosus preferentially uses food available in the environment when it is abundant but makes use of reserves in the adductor muscle when food is scarce (Luna- Gonzalez et al. 2000). In Cerastodenna edide. gametogenesis may occur at the expense of glycogen reserves or simultaneously with glycogen storage, depending on the amount of available food (Na- varro et al. 1989). ACKNOWLEDGMENTS The authors thank the Instituto Politecnico Nacional (National Polytechnic Institute, IPN) for facilitating the economical re- sources to perform this research work (Project 200229), to IPN's Comision de Operacion y Fomento de Actividades Ac- ademicas (Commission for the Operation and Promotion of Aca- demic Activities) for grants given to M. Villalejo-Fuerte and F. Garci'a-Dominguez, and to Maria Elena Sanchez Salazar for her assistance in translating and editing the English version of this article. Biochemical Composition of Spondyws leucacanthus 761 LITERATURE CITED Ansell, A. D. 1972. Distribution, growth and seasonal changes in bio- chemical composition for the bivalve Doiuix vilkinis (Da Costa) from Karnes Bay. Millport. J. Exp. Mar. Biol. Ecol. 10:1.^7-150. Ansell. A. 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A rapid method of total lipid extraction and punfication. Can. J. Biochem. Physiol. 37:91 1-917. Boadas. M. A.. O. Nusetti. F. Mundarain. C. Lodeiros & H. E. Guderiey. 1997. Seasonal variation in the propenies of muscle mitochondna from tropical scallop. Euvola (Pecren) ziczac. Mar. Biol. 128:247-255. Claereboudt, M. & J. H. Himmelman. 1997. Recruitment, growth and production of giant scallops (Placopecten magellaniciis) in Bale des Chaleurs. eastern Canada. Mar. Biol. 124:661-670. Comely. C. A. 1974. Seasonal variations in the flesh weights and bio- chemical content of the scallop Pecren maximus (L.) in the Clyde Sea Area. J. Cons. Int. Explor. Mer. 35:281-295. Deslous-Paoli. J. M. & M. Heral. 1988. Biochemical composition and energy value of Crassostrea gigas (Thumberg) cultured in the bay of Marennes-Oleron. Aqtiut. Living. Resour 1 :239-249. Felix-Pico, E., A. Tripp-Quezada. J. Castro-Ortfz, G. Serrano-Casillas. P. Gonzalez-Ramirez. M. Villalejo-Fuerte. R. Palomares-Garci'a. F. 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Shelfish Assoc. 68:80-81. Humason. G. L. 1979. Animal Tissue Techniques. 4th, ed. San Francisco: W. H, Freeman and Co, 661 pp. Kang, C. K., M. S. Park, P. Y. Lee. W. J. Choi & W. H. Lee. 2000. Seasonal variations in condition, reproductive activity, and biochemical composition of the pacific oyster. Crassostrea gigas (Thunberg). in suspended culture in two coastal bays of Korea. / Shellfish Res. 19: 771-778. Lauren, D. J. 1982. Oogenesis and protandry in the purple-hinge rock scallop. Hinnites giganteus. in upper Puget Sound. Washington. U.S.A. Cure 1. Ijpcation of Mali Slon Bay and four study sites: Bistrina. Soca, Krstac, and Bjeji'xica. of 200 1, surface temperature decreased slowly and was very simi- lar in September and October at all study sites, that was followed by exceptionally low surface temperatures that dropped below 10°C in December and January. In the late spring and early summer periods, due to stratification of a water column, bottom temperature is a few degrees lower than surface temperature. Toward the end of the summer, isothermia was observed in a water column, while during the fall and winter periods near bottom temperatures were higher than surface or mid water column temperatures (Table 1 ). The surface salinity values ranged between 26.6 psu (Bistrina, May 2001) and 39.7 psu (Soca, March 2001). High variations in surface salinity were observed at Bistrina and Soca in a period from October till May and in Krstac and Bjejevica from October till February (Fig. 2). Near bottom layer was characterized with higher salinity values, than surface and mid water column layer, throughout the year. European flat oyster larvae were present in the Bay of Mali Ston in varying numbers throughout the year. Maximal number of larvae recorded in a given month, and average surface temperature recorded in that month, are shown in Figure 3. High numbers of Ostrea I'tlulis larvae were recorded in samples collected from May till September, while larvae were present in some samples, but not abundant, from November till April. Absence of larvae was pri- marily noted in samples collected between January and March 2002 al all study sites. In year 2000, the highest larval numbers were recorded in July at all sites, with the highest value being recorded in the cove of Bjejevica (4875 ind./m') in late July. Minimal, maximal and mean values for number of O. edtili.s larvae recorded in study period are shown in Table 2. In 2001, maximal number of larvae collected in each month appeared to be lower than in 2000 at sites Soca, Krstac, and Bjejevica. At site Bistrina, the highest number of oys- ter larvae was recorded in August 2001 (5029 ind./nr') while the number of larvae observed in other months of that year appears to be similar to values obtained for oyster larvae in year 2000. There was a statistically significant positive correlation (P = 0.05) between number of O. editlis larvae and surface temperature and mid water column temperature (Table 3). Correlation between larvae number and near bottom temperature was significant only at TABLE 1. Water temperature at all four study sites from May 2001 to March 2002. Temperatu re CO Bistrina Soca Krstac Bjejevica Month Sur. Mid. Bot. Sur. Mid. Bot. Sur. Mid. Bot. Sur. Mid. Bot. May 21.0 20.0 18,7 19.9 19.7 19,5 20.6 18.2 18,2 19.7 18.4 17.6 June 23.6 22.8 21.8 23.3 22.8 21,9 22.8 214 19,8 22.7 21.3 19.9 July 23.1 23,0 22.3 23.3 23.0 22.8 234 22.9 21,3 23.3 22.7 21.7 August 26.1 26,0 26.1 26,0 25.8 25.7 26.6 25.7 18.4 26.9 26.1 17.9 September 21.6 21,2 21.1 21 1 21.1 21.0 21.1 20.9 20.7 20.6 20.4 20.0 October 18.8 19.8 20.7 18,7 18.7 20.2 18.7 20.1 20.5 November 154 17.9 17,8 12-2 17.3 17.5 13.8 17.5 18.0 12.8 18.3 17.9 December 18.4 174 17.4 13.8 17.0 17.2 16.8 17.7 17.6 15.3 15.6 16.0 January 12.0 13.7 14.7 114 13.1 15.8 12.4 14,7 15.8 12.8 154 15.5 February 12.0 12,3 13.8 114 12,5 14,3 11.5 14,1 14,3 11,8 13.3 14.5 March 14.4 14.0 14,5 14.5 14,2 14,2 14,1 14,3 14,0 14,0 14,0 14.0 April 14,4 14.9 15.2 14.5 14.7 14.9 14.9 14,7 14,7 14.5 14,4 14.7 May 21.1 17.7 17.5 20.2 184 17.8 18.2 17.0 16,8 18.3 17,1 16.8 June 21.0 20.5 19.8 21.2 21.1 20.5 20.6 20.4 20,1 20,8 20.4 19.9 July 24.4 24.2 22.9 24.3 24.2 22.6 24.5 23.8 19.9 24.6 23.8 18,8 August 25.5 23.9 21.8 25.4 23.8 21.7 25.0 24.7 20.0 25.3 23.7 20.0 September 20.0 20,0 19.9 20.1 19.8 19,7 20.3 20.1 19.6 20.2 19.7 19.3 October 20.0 20.2 20.5 19.9 20.1 20,2 20.0 20,6 20.4 20.0 20.5 20.4 November 13.8 15.6 16.3 13,8 15,2 16 1 15,2 15,6 16 2 14 1 15 3 16,0 December 9.5 9.8 10,6 8.^) 9,0 10,6 1(1.0 10,7 10,6 10 1 10,1 10.3 January 9.3 10.6 11,1 '),! 9,5 11,1 9.3 10,6 11,1 i),() 10,6 11.0 February 11.5 12.6 13.1 114 11.8 12,7 11.8 12.0 12.5 11,6 11,6 12.4 March 13.5 134 13.3 13.4 13.2 13,3 12.8 13,1 13.2 13.0 13,0 134 Seasonal Distribution of Ostrea edlilis Larvae 765 2000 2001 2002 ~Sur. -■— Mid. -*-Bol~| 40 ^ 36 "i 30 " 28 b. Snca '---^^^'^^^''^7^^^-^^ May Jun Jul 1 Aug Sep Oct 1 s ^ 1 1 III ^ II S 1 oM 1 2001 2002 5 ]_._Sur. -.-Mid. -»-Bot, ] d. Bjejevica -Sur. -*-Med, Figure 2. Time trend over a two-year study period for surface (Sur. I, mid water column (Mid.) and near bottom (Bot.l salinity (psu) at four study sites: (a) Bistrina, (b) Soca. (c) Krstac, and (dl Bjejevica. Iwo shallower sites — Bistrina and Soca. Salinity values were not correlatecJ with number of oyster larvae observed in samples, ex- cept at site Soca for near bottom salinity and at site Krstac for mid water column salinity. DISCUSSION Traditionally, the Bay of Mali Ston was well known as an area where the European flat oyster has two spawning peaks per year. one in the late spring (May and June) and other in the autumn (September and October) (Morovic & Simunovic, 1980). How- ever, during the last 15 years, change in spawning season, was observed by local farmers. Results of this research confirm their observations. Although larvae were present throughout the year. 2000 2001 2002 .?»* ^.^ s> ^"cf" cC" ,j'^ o* ■,* **■ ^' ?^' -S** S^ V* ■^'f" *" arches. uga.edu bryos per egg capsule in Chesapeake Bay ranged from 46 to 67 (Harasewych 1982). Castagna and Kraeuter ( 1994) determined the average number of embryos per capsule varied according to posi- tion on the egg string, averaging 52 in the middle section and decreasing in the beginning and terminal portions. The female always begins by anchoring one end of the egg string deep into the substrate by several structurally modified capsules to prevent the string from being washed ashore (Magalhaes 1948). Ram (1977) and Ram et al. (1982) induced spawning in the knobbed whelk by stimulation with nervous system extractions. Spawning always be- gan with a number of unembryonated capsules, a choice dictated by genome. It has not been determined how the female adjusts the number of anchoring capsules. Studies have found that the egg capsules in the anchoring portion are typically thinner walled and closely spaced (Harasewych 1982). The number of unembryonated capsules in this anchoring portion has been reported to range from 8 to 22 (Castagna & Kraeuter 1994). from 13 to 17 (Ram et al. 1982). and to average 21 (D'Asaro 1997). The young pass the veliger stage within the egg capsule and einerge as fully developed miniature "adults." In Virginia. Casta- gna and Kraeuter ( 1994) found that egg strings deposited in the fall hatched from mid-March through early-May in the following year. The average size for hatchling juveniles is -4 mm in length (Ma- galhaes 1948, Castagna & Kraeuter 1994). Most studies that have been conducted were on adult whelks (Morton 1987). Very little information exists about the ecology of post hatchling knobbed whelks. As an adult, the knobbed whelk is a predator of bivalve species. There has been no research published on the habitat and nutritional requirements of juveniles in the wild. In Virginia, juvenile whelks were hatched and raised inside poly- propylene bags in the laboratory to estimate post hatching growth rates (Kraeuter et al. 1989). There was limited information pro- vided on rearing conditions (i.e.. the temperature and diet). Length measurements were taken five times during the first year and one to three times per year for the subsequent nine years. The juveniles were fed live clams when they reached -20 mm in length. Growth 769 770 Power et al. rates averaged 13.2 mm/y over the ten-year period, however ju- veniles grew from 4 to 36.5 mm (32.5 mm/y) in the first year. The only other published growth study on juvenile knobbed whelks was by Magalhaes (1948). She maintained juveniles in the labo- ratory for one month only and observed one individual added 1 .5 mm in length in 22 days. This experiment attempts to determine the optimum food source for juvenile knobbed whelks with respect to growth and survival rates. Temperature is also considered since warmer temperatures foster accelerated growth. MATERIALS AND METHODS Weekly field trips to intertidal sandy-mud fiats around Wassaw Sound, Georgia were carried out from April 2001, when copulat- ing and egg-laying whelks were first observed, to early June 2001 when most egg capsules had opened and the hatchlings had begun to disperse. On each occasion, notes were taken on the behavior (i.e., copulating, depositing eggs, and feeding). If a whelk was in the process of laying, an estimate was made of the number of capsules already produced, her position was taken with a hand held global positioning system, and the location was physically marked with a metal stake. Once the strings were completed, the number of egg capsules per string was determined (excluding the anchor- ing section). The condition of the egg capsules was also monitored regularly to determine the larval developmental period in the field. At the end of May 2001, egg strings were collected and re- turned to the Shellfish Research & Aquaculture Laboratory on Skidaway Island. Ten strings were selected to examine the capsule morphometries, length, width, and thickness, as defined by D'Asaro (1997). The length of unembryonated and embryonated sections of each string was determined with a fish measuring board. Each string was then divided into four portions: the unem- bryonated anchor, and an inner, middle, and outer portion of the embryonated section. Five capsules were randomly selected from each section, the dimensions were measured and the capsules were opened to count the developing embryos. Egg strings were also maintained at the laboratory in an open system raceway until hatching began. Hatchling juveniles were collected, measured for length, and isolated for growth rate ex- periments using diet and temperature variables. Three dietary treat- ments were chosen for the growth study: macroalgae, microalgae, and meat. Sea lettuce. Ulva sp., was harvested from Wassaw Sound and the Skidaway River for the macroalgae series. Benthic microalgae (species unknown) was cultured on mats in a closed system saltwater tank using nylon mesh as a substrate. The third diet consisted of chopped meat: clams, Menenaria mercenaria (L.). brittle stars, Ophiophni^miis wwdemani (Lyman), blood- worms. Glycera dibranchUita (Ehlers). and mussels, Geukensia demissa (Dillwyn), depending on what was available. Temperature was included in the design. At the time of hatching, water tem- peratures averaged 25°C (taken from the nearby Skidaway River), therefore three glass aquaria were set up in temperature controlled rooms, at 20, 25, and 30''C. For the duration of the experiment, the salinities were maintained at 'i5%c. Plastic jars (120.6 x 69.8 mm) were used to contain the juveniles. To help water circulation, thirty 2-mm holes were drilled in the top and bottom of the jars and 15 on each side. There were three replicates for each combination of diet and temperature treatment resulting in 27 containers, each holding 50 haphazardly chosen hatchlings. Jars were labeled and temperatures and salinities in the aquaria were tested and main- tained regularly. The water in the aquaria was aerated, and changed monthly. All the juveniles were provided with an abun- dance of food, which was cleaned out and replaced approximately every three days. Juvenile shell length (to the nearest 0.01 mm) and body weight (to the nearest 0.(MOI g) were recorded every two weeks using an electronic calipers and balance. The animals were first blotted dry by placing on a paper towel. Dead juveniles were removed and counted to determine mortality rates. The experiment was conducted over an eight-week period. Statistical analysis of growth and survival data was pert'ormed using SAS (SAS Institute, Inc.. Gary, NG). The two main effects (temperature and diet) and a nested effect (containers) on growth (length and weight) were analyzed by performing a nested factorial using GLM (General Linear Model) SAS. Survival data were arc- sine transformed prior to analysis [A" = arcsine V (.v/lOO)]. Two main effects were again analyzed (temperature and diet), with repeated measures of both factors using a GLM (SAS). An a level of p = 0.05 was used to determine if significant difference in mean juvenile length, weight, and survival occuned between dit- ferent diets and temperature treatments. At the termination of the eight-week laboratory growth experi- ment, we returned to the sites of egg deposition in Wassaw Sound to search for juveniles that had presumably dispersed on hatching. A circular area with a diameter of approximately 4 m was searched around approximately twenty marked egg cases. The goal was to obtain juveniles to compare the growth obtained in the laboratory with growth in their natural habitat. Methods for searching con- sisted of scooping up handfuls of the surface layer of sediment at low tide and sieving on a 1.40 mm screen. Submerged sites at lower tidal levels were searched by gently feeling the surface of the sediment. RESULTS The abundance of copulating whelks peaked in eariy April 2001 and was observed with decreasing frequency throughout the rest of the month. This period coincided with a dramatic increase in local water temperatures (Fig. 1). Often copulation was ob- served even while the female was in the process of depositing egg capsules (Fig. 2). Typically the larger female lay partially buried in the substrate and was surrounded by several smaller sized males (average of 3, but up to 9 observed), A large variation in the rate of egg deposition was observed: 1 to 23 capsules per day (7.69 ± 2.12. mean ± SE). This is a coarse estimate since the egg strings were observed once per week. An approximate total area of 2.500 m- was searched for whelks and eggs. By the end of April, most egg strings were complete and whelks were less abundant on the sandy-mud flats. From a total of 51 complete egg strings (0.02 m"), the number of embryonated egg capsules per egg string averaged 89.76 ± 3.61, and ranged from 40 to 157. Egg capsules began to soften and deteriorate quickly, the escape aperture was open on all capsules examined on June 4, 2001 (approximately six weeks later). While all capsules were open at this time, only one third of those examined were empty, the remainder still held the fully developed hatchlings. The average length of the unembryonated section tor the ten egg strings selected for morphometric analysis was 199,67 ± 15.21 mm (ranged from 130 to 275 mm). For the embryonated section the average length was 339.86 ±26.17 mm (ranged from 245 to 450 mm). The number of unembryonated capsules per egg string ranged from 6 to 26 (mean = 13.67 ± 1.97). The number of developing embryos per egg capsule ranged from 0 to 99. The Knobbed Whelk Eggs and Hatchlings in Georgia 771 Temperature Salinity Mar-OO May-00 May-01 Jul-01 Sep-01 1-00 Sep-00 Nov-00 Jan-01 Mar- Time (month) Figure 1. The mean ni(mthl> water temperature and salinity of the Skidaway River, Georgia from January 2000 to November 2001 (vertical bars indicate ± 1 standard error from the mean). middle portion of the embryonated section of the egg strings had longer, thicker, and wider capsules with a higher number of em- bryos per egg capsule. The inner and outer portions averaged 23.41 ± 2.77 embryos per capsule, with capsule dimensions of 20.20 ± 0.47 mm in length. 27.26 ± 0.69 mm in width and 5.66 ± 0.09 mm in thickness. The middle portions had an average of 45.86 ± 3.95 embryos per capsule, with capsule dimensions of 22.12 ± 0.41 mm in length, 31.11 ± 0.56 mm in width, and 6.03 ± 0.14 mm in thickness. ^'igure 2. Small male knobbed whelk, Hiisytiin larica. copulating with a larger female that is also in the process of depositing an egg string. In the laboratory, the egg strings maintained in raceways also began to hatch in early June 2001. On hatching the juveniles averaged 5.60 ± 0.02 mm in shell length and 23.10 ± 0.24 mg in weight {n = 540). These were haphazardly assigned to the three different diet (meat, algae and diatoms) and temperature (20, 25, and 30°C) treatments. Figure 3 and Figure 4, present the growth rates of these juveniles for a period of eight weeks, in terms of shell length (mm) and weight (mg), respectively. Figure 5 presents the percentage survival rate at the termination of the experiment for each temperature and diet treatment. Growth in terms of length and weight was significantly affected by diet (P = 0.003 and 0.001. respectively). Duncan's Multiple Range Test revealed no difference in the growth of whelks fed on the macroalgae and microalgae diets (macroalgae; mean length 5.67 mm, mean weight 21.29 mg; microalgae: mean length 5.65 mm, mean weight 21.47 mg). however those fed on the meat diet were significantly larger (mean length 6.35 mm. mean weight 27.81 mg). Survival was not significantly affected by diet (P = 0.2084). There was a signifi- cant (P = 0.001 ) temperature effect on survival. Duncan's Mul- tiple Range Test revealed no difference in survival at temperatures 25 C and 30°C (15.31% and 7.73%). but a much greater survival rate occurred at 20"C (69.50%). Percent survival rates decreased with temperature increase for all dietary treatments (Fig. 5). It proved very difficult to locate juvenile knobbed whelks on the intertidal flats in August and September. The best approach proved to be feeling the surface layer of sediment when a shallow depth of water co\ ered the flats. Even with this approach only nine 772 Power et al. B. 20''C 2 4 6 Time (weeks) as^c 2 4 6 Time (weeks) ° 2 4 6 o 20 25 30 Time (weeks) Temperature ("C) Figure 3. A-C. Ttie mean shell length (mm + SE) of knobbed whelks reared in the laboratory on macroalgae, microalgae and meat diets at temperatures of 20, 25. and 30 C. respectively, for eight weeks post hatching. D: The overall growth rate (shell length, mm) per diet and temperature treatment over eight weeks. juveniles were recovered (Table 1 1, from a total area of approxi- mately 250 m- (-0.04 m""). The wild juveniles located, averaged a much larger size (mean shell length = 21.2 mm) than those reared in our laboratory treatments. DISCUSSION Copulation occurred throughout April 2001 during which av- erage seawater temperatures increased rapidly front a niean ot 15.3°C in March 2001 to 20.8X in April 2001. Walker (1988) observed six pairs of copulating whelks in Wassaw Sound in March 1980 (mean water temperature 14.3°C). We do not believe that whelks commenced spawning earlier than April in 2001 . since intertidal flats were thoroughly searched throughout March 2001 as part of an ongoing whelk mark and recapture study. Another spawning event may take place in Georgia, when seawater tem- peratures drop to a similar range in the fall. Along the eastern coast of the United States, the reported spawning period of the knobbed whelk is progressively later as one moves northwards: March to April in Georgia (Walker 1988. present study) and South Carolina (Stevens 1976). May to June in North Carolina (Magalhaes 1948). and mid August to November in Virginia (Castagna & Kraeuter 1994). In the natural environment, the timing of the reproductive pe- riod is usually synchronized with conditions that are most favor- able for maximization of juvenile survival and continuity of the species (Sastry 1986). Gastropod egg capsules are preyed on by fish, crustaceans, other prosobranchs. polychaete worms, and even shorebirds (Penchaszadeh et al. 2000). Predator activity typically increases in the warm temperatures of the early summer months. However, these months also permit a more rapid embryonic de- velopment of encapsulated larvae that may reduce overall losses. A rapid developmental period of six weeks was observed in the present study, which concurs with findings for the pear whelk in Florida (6 wk: Kent 1983). This is however, significantly lower than the six-month period ( mid summer to following spring) re- ported for the same species in the cooler northern waters of Vir- ginia (Castagna & Kraeuter 1994). Embryonic development of encapsulated knobbed whelks has been noted to temporarily arrest and resume after egg capsules were refrigerated for four days at 0 C (Dr. DeEtte Walker, pers. comni.. UGA Dept. of Genetics). According to Hain and Amaud (1992) embryonic development for related species can be up to thirty times longer in Antarctic than in tropical waters. Although most egg capsules in the field were open in early June, two-thirds of all capsules examined still held hatchlings. A gradual dispersal has also been noted for juveniles of the lightning whelk, with many found within and between the opened capsules (D'Asaro 1997). Juveniles may use the structure as a refugium. Egg strings also provide a substratum for algae, diatoms and even other invertebrates in their juvenile stages and could therefore also serve as a food source for newly hatched whelks. In this study the hatchlings (» = .'540) averaged 5.60 mm in Knobbed Whelk Eggs and Hatchlings in Georgia 773 B. 20X -Macroalgae -Microalgae -Meat 2 4 6 Time (weeks) 25°C 2 4 6 Time (weel E 3 ■ Macroalgae DMicroalgae ZIMeat 20 25 30 Water Temperature (°C) Figure 5. The percentage survival rate of knobbed whelks reared in the laboratory on macroalgae, microalgae and meat diets at temperatures of 20, 25, and 30 C, respectively, for eight weeks post hatching. higher than 35''C (Kinne 19631. Juveniles that hatched in June 2001 would have experienced temperatures up to 30°C on the intertidal flats, but would also have had the ability to bury them- selves in the substrate. Chaitanawisuti and Kritsanapuntu (1998, 1 997 1 found that the highest growth rates of hatchery reared ju- venile spotted Babylon. Babyiim'm areolata (Link 1807) whelks occurred in treatments provided with sand substrate and flow through seawater. while the lowest occurred in those with no sand substrate and static seawater. In our experimental treatments, both water flow and a sand substrate were not provided and may have led to sub-optimal growth and survival rates. The lack of substrate TABLE 1. Juvenile Busycoii carica whelks captured on intertidal flats (previously marked sites of egg string deposition! al Wassaw Island (0.04 individuals m') in August/September, 2001. Weight (mg) 0.2 0.4 0.3 0.4 0.6 0.6 0.5 0.4 1 0.5 Length (mm) Width (mm 17.6 7.5 21.1 9.4 Ig.g 8.3 19.7 8.8 22.7 9.7 22.5 9.7 21.8 9.8 20.2 8.8 26.3 11.5 Means: 21.2 9.3 has been shown to induce interactive energy expenditure by juve- nile queen conchs (Siddal 1984). Catterall and Poiner (1983) describe small juvenile colonies (area of 2-15 m in diameter) of the gastropod Stronibiis lulntanus with between 5 to 100 individuals per meter squared. These colo- nies may persist for up to two years and become larger in area and less dense as members grow and mature. This type of spatial segregation may also occur for juvenile knobbed whelks in the wild. It is likely that juveniles spend a large proportion of time completely buried in the sand, particulariy when the tide retreats, which would explain our difficulty in locating them. We only found juveniles at the surface of the sediment after the tide had advanced over the intertidal flats. In the laboratory the largest sized juvenile at the termination of our rearing experiment (8 weeks later) was approximately 12.5 mm. The average size of the juve- niles found in the wild was much larger at 21.2 mm. These wild juveniles may have resulted from the previous fall spawning event and would therefore be approximately eight months old, which resembles the annual year one growth rate of 34.5 mm reported by Kraeuter et al. (1989) in Virginia. ACKNOWLEDGMENTS This work was supported by the University of Georgia Marine Extension Service and by the National Sea Grant program with funds provided to Savannah State University through the Minority Serving Institute Program. The authors thank Ms. Carolyn Belcher for performing the statistical analysis. Thanks to Mary Sweeney- Reeves and Dodie Thompson for assisting in the field. Knobbed Whelk Eggs and Hatchlings in Georgia 775 LITERATURE CITED Castagna, M. & J. Kraeuter. 1994. Age, growth rate, sexual dimorphism and fecundity of the knobbed whelk Busycon carcia (Gmelin. 1791 1 in a western mid-Atlantic lagoon system. Vii)>inia J. Shellfish Res. 13: 581-585. Catterall. C. & [. Poiner. 1983. Age- and se,\-dependent patterns of aggre- gation in the tropical gastropod Sirombiis luhuwnis. Mar. Biol. 77:171- 182. Chaitanawisuti, N. & A. Kritsanapuntu. 1997. Effects of stocking density and substrate presence on growth and survival of juvenile spotted Babylonia areolata Link 1807 (Neogastropoda: Buccinidae). J. Shell- fish Res. 16:429-433. Chaitanawisuti. N. & A. Kritsanapuntu. 1998. Growth and survival of hatchery-reared juvenile spotted Babylonia areolata Link 1807 (Neo- gastropoda: Buccmidae) in four culture conditions. J. Shellfish Res. 17:85-88. Conklin. E. 1907. The embryology of Fulgur. A study of the influence of yolk on development. Philadelphia: Proc. Acad. Nat. Sci. pp. 320-360. D'Asaro, C. 1997. Gunnar Thorson's world-wide collection of prosobranch egg capsules. Melongenidae Ophelia 46:83-125. Hain, S. & P. Amaud. 1992. Notes on the reproduction of high Antarctic mollusks from the Weddell Sea. Polar Biol. 12:303-312. Harasewych. M. 1982. The evolution and zoogeography of the subfamily Busyconinae (Gastropoda: Melongenidae). Ph.D. Dissertation. Univer- sity of Delaware. 216 pp. Kent. B. 1983. Patterns of coexistence m Busyconine whelks. J. E.xp. Mar. Biol. Ecol. 66:257-283. Kinne, O. 1963. The effects of temperature and salinity on marine and brackish water animals. 1 . Temperature Oceanogr. Mar. Biol. A. Rev. 1:301-340. Kraeuter. J.. M. Castagna & R. Bisker. 1989. Growth rate estimates for Busycon carica (Gmelin. 1791) in Virginia. J. Shellfish Res. 8:219- 225. Magalhaes. H. 1948. An ecological study of snails of the genus Busycon at Beaufon. North Carolina Ecol. Monog. 18:380-407. Miloslavich. P. & L. Dufresne. 1994. Development and effect of female size on egg and juvenile production in the neogastropod Buccinuin cyaneum from the Saguenay Fjord. Can. J. Fish. Aquatic Sci. 51:2866- 2872. Morton, B. 1987. 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Hormonal control of reproduction in Busycon: II. Laying of egg-containing capsules caused by nervous system extracts and further characterization of the substance causing egg capsule laying. Biol. Bull. 162:360-370. Rehder, H. 1981. National Audubon Society Field Guide to North Ameri- can Seashells. New York: Alfred A. Knopf. 894 pp. SAS Institute Inc. 1989. SAS users guide: Statistics: Version 6. Gary: SAS Institute. Inc. Sastry. A. 1986. Pelagic lar\'al physiology and ecology of benthic marine invertebrates in the context of the Indian Ocean. In: M. Thompson. R. Sarojini & R. Nagabhushanam. editors. Biology of benthic miuine organisms: techniques and methods as applied to the Indian Ocean. Rotterdam: A. A. Balkema. pp. 387^01. Siddal, S. 1984. Density-dependent levels of activity of juveniles of the queen conch Stromhus gigas Linn. J. Shellfish Res. A:fn-1A. Spight, T. M. & J. Emlen. 1976. Clutch sizes of two marine snails with a changing food supply. Ecology 57:1162-1 178. Stevens. B. 1976. A study of the seasonal changes in component mdices and lipid contents of the digestive gland, ovary, and fool of Busycon carica. M.S. Thesis (unpublished). College of Charleston. Walker. R. 1988. Observations on intertidal whelk {Busycon and Busycoty- pus) populations in Wassaw Sound. Georgia J. Shellfish Res. 7:473- 478. Weinheimer. D. 1982. Aspects of the biology of Busycon carica (Gmelin. 1791) in waters off South Carolina with emphasis on reproductive periodicity. MS. Thesis (unpublished). College of Charleston. Jimnuil ofSheUfish Research. Vol. 21. No. 2. 111-179. 2002. RAPA WHELK RAPANA VENOSA (VALENCIENNES, 1846) PREDATION RATES ON HARD CLAMS MERCENARIA MERCENARIA (LINNAEUS, 1758) DARIO SAVINI," JULIANA M. HARDING,"* AND ROGER MANN" ' School of Ocean Sciences. Universiry of Wales-Bciniior. Menai Bruli^e. Anglesey. LL59 5EY. Wales, United Kingdom: 'Virginia Institute of Marine Science. Department of Fisheries Science. P.O. Box 1346, Gloucester Point. Virginia 23062: "'Universitd degli Studi di Pavia. Departimento di Genetica e Microhiologica. Via Sunt' Epefanio. 14. 1-27100 Pavia. Italy ABSTRACT The recent discovery of adult veined rapa whelks Rapanu venosa (Valenciennes, 1846) in the Lower Chesapeake Bay, U.S.A. offers cause for both ecological and economic concern. Adult rapa whelks are large predatory gastropods that consume bivalves including commercially valuable species such as hard clams. Mercenaria mercenaria (Linnaeus. 1758). Laboratory feeding experi- ments were used to estimate daily consumption rates of two sizes of whelks feeding on two size classes of hard clams. Large rapa whelks (shell length. SL >10l mm) are capable of consuming up to 2.7 g wet weight of clam tissue daily, equivalent to 0.8% of their body weight. Small whelks (60-100 mm SL) ingest an average of .^.6'7f of their body weight per day. KEY WORDS: rapa whelk. Rapaini venosu. hard clam. Mercemiria mercemirui. prcdation. Chesapeake Bay INTRODUCTION The veined rapa whelk, Rapana venosa. (Valenciennes 1846; Gastropoda: Muricidae) was discovered in the Hampton Roads region of the Chesapeake Bay, USA, in the summer of 1998 (Har- ding & Mann 1999). The species is native to the Sea of Japan. Yellow Sea, East China Sea and the Bohai Sea (Tsi et al. 1983) but was introduced to the Black Sea in the 1940's (Drapkin 1953) and has since spread to the Aegean Sea (Koutsoubas & Voultsiadou- Koukoura 1990) and the Adriatic Sea (Ghisotti 1974). Recently a female specimen together with egg masses was found in the Rio del Plata, an estuary between Argentina and Uruguay in South America (Pastorino et al. 2000). The predatory activity of rapa whelks in the Black Sea is con- sidered by Zolotarev (1996) to be the prime reason for the deci- mation of native Black Sea oyster, scallop and mussel populations. Given this history, there is both ecological and economic concern for the future of shellfish stocks in the Hampton Roads region of the Chesapeake Bay. Hard clam, Mercenaria mercenaria, popula- tions are of particular concern in that the Hampton Roads region supports a substantial local commercial hard clam fishery. Labo- ratory feeding experiments were used to quantify daily feeding rates for two size classes of adult rapa whelks offered hard clams. MATERIALS AND METHODS Twelve adult rapa whelks, collected from the lower Chesa- peake Bay, USA, between March and May 2000, were separated into two different size classes: small (60-100 mm shell length (SL), the inaximum dimension from the apex of the spire to the end of the siphonal canal) and large (101-160 mm SL). Rapa whelks were maintained individually in 60 x 40 x 30 cm plastic net cages submerged in a shallow flume (250 x 70 x 30 cm) with a constant flow of unfiltered York River water as described in Savini (2001 ). The bottom of each cage was covered with 15 cm of clean hard sand substrate. Rapa whelks were starved for 48 hours prior to the addition of hard clams (prey) to each enclosure. Each whelk was given five small (50-70 mm maximum dimension, hereafter shell *Corresponding author. E-mail: jharding@vims.edu; Tel: +1-804-684- 7302; Fax: +1-804-684-7045 height, SH) and five large (71-100 mm SH) hard clams as poten- tial prey. Clams were arranged in the experimental cages so that whelks initially had the same probability of encountering each size of prey (i.e., whelks at the center of a circle with clams of alter- nating size classes spaced evenly around the circumference). The experimental fiume was covered with a fixed plastic net to prevent escape of the whelks and maintained on a 14/10 h natural light/dark schedule. Water temperature and salinity data were col- lected daily from the flume for the 38 day duration of the experi- ment (June 1 1 to July 18, 2000). Experimental cages were exam- ined daily and the empty shells of all prey were removed and measured. Clams that were consumed were replaced daily with clams of similar dimensions thus maintaining constant prey avail- ability. A size range (30-100 mm SH) of fifty hard clams was selected from the pool of potential prey items and used to create size- weight relationships for the prey. Individual hard clams were mea- sured (SH, mm) and weighed (g) prior to the removal of soft tissue. Clam soft tissue was weighed (wet weight, g) to the nearest 0.1 g. Data Analyses Significance levels for all statistical tests were established at P = 0.05 (( priori. Bartlett's test for homogeneity of variance and the Ryan-Joiner test for normality were used prior to analyses. When appropriate, Fisher's test was used for post-hoc multiple comparisons. Feeding Rates The numbers of clams consumed by each size class of whelks during the entire experimental period were compared using a one- way ANOVA with individual whelk as a factor. The number of clams consumed satisfied the assumptions of homogeneity of vari- ance and normality without transformation. Daily feeding rates were calculated for each whelk by dividing the total number of clams consumed during the experimental period by the duration of the experiment (38 days). Consumption on a Weight-Weight Basis Clam wet and dry tissue equivalents consumed by whelks were compared using a two-way ANOVA with whelk size class and 777 778 Savini et al. individual whelk as factors. Tissue equivalent data satisfied the assumptions of homogeneity of variance and normality without transformation. RESULTS Average water temperature during the experimental period was 26°C (±I°C). Average salinity was 21 ppt (±1 ppt). During the 38 day experiment, the six small whelks ate a total of 19 clams while the six large whelks consumed a total of 15 clams. There was no significant difference in the total number of clams eaten by small and large whelks (ANOVA. F = 0.67; P > 0.05). Small rapa whelks did not show any clear size preference when offered hard clams as prey (Fig. I ) although small whelks consumed a total of 1 1 small clams and 8 large clams. It should be noted that 5 of the 1 1 small clams were consumed by one individual. Large rapa whelks consumed large clams more frequently than small clams (4 small clams vs. 1 1 large clams; see Fig. I). Clam Size-Weight Relationships Clam tissue wet and dry weights were plotted in relation to shell height and used to calculate shell height-wet tissue weight relationships for hard clam prey. These relationships were used to calculate wet tissue equivalents for each clam consumed by an individual whelk and were described with the following equations: Loc (CWWgt) = -3.93 + 2.77 * (Log SH); R" 0.96 where CWWgt is clam tissue wet weight (g) and SH is clam shell height. Rapa Whelk Size-Weight Relationships Rapa whelk tissue wet weight was plotted in relation to shell length and used to calculate a shell length-wet tissue weight re- 100 80 60 40 20 0 Percentage of small clams consumed 0 20 40 60 80 100 Percentage of large clams consumed Figure 1. Percentage of small (50-70 mm SH) and large (71-100 mm SH) hard clams eaten in each experimental cage by each rapa whelk during the whole experimental period (June 1 1 to July IS, 2000). .\) N. 1-6 = small whelks (60-100 mm SL), B) N. 7-12 = large whelks (101- 160 mm SL). 1000 100 • Small rapa whelks (60-100 mm SL) O Large rapa whelks ( 10 1 - 1 60 mm SL) 1 10 100 Rapa wtielk body wet weight (g) 1000 Figure 2. Daily Ingested clam wet weight (g) In relation to rapa wet weight (gl observed in laboratory feeding experiments during .lune and .July. 2000. The solid diagonal line represents clam consumption equal to the body weight of the predator (whelk) or a 1:1 consumption relationship on a prey wet weight: predator wet weight basis. Points abo\e the line indicate prey consumption at a rate greater than one while points below the line indicate daily consumption rates less than the body weight of the predator. lationship for whelk predators. This relationship is based on 150 animals (80-165 mm SL) collected from lower Chesapeake Bay. USA between October 1999 and July 2000 (Harding and Mann, unpublished data): WWWgt = 6.4908 * e(0.0229 * SL). R- 0.69 where WWWgt is whelk tissue wet weight (g) and SL is whelk shell length (mm). On the basis of tissue wet weight, large whelks consumed significantly more prey flesh tissue than small whelks (ANOVA. F = 4.45, P < 0.05). Individual small whelks ate proportionately more hard clam tissue on a clam wet weight: whelk wet weight basis than large whelks (Fig. 2). Maximum daily clam consump- tion rates of 5.b7r of body wet weight were recorded for small whelks as compared to 1 .69^ of body wet weight for large whelks. DISCUSSION Large rapa whelks (101-160 inm SL) are able to consume up to 2.7 grams of clam tissue (wet weight) per day or 0.8% of their body weight per day at water temperatures of approximately 26' C. In contrast, small rapa whelks (60-100 mm SL) ingested an average 3.6% of their body weight every day, which is more than four times that observed for larger rapa whelks at similar water temperatures on a weight-specific basis. Edwards and Huebner (1977) suggest that temperature affects feeding rate in the moon snail Piiliuices by increasing predators' metabolic rate, and thus the reqturement for a larger amount of food. The present investi- gation was conducted during warmer months and is probably in- dicative of the maximum feeding activity of rapa whelks. There is Rapa Whelk Predation on Hard Clams 779 considerable variation in reported ingestion rates for predatory gastropods with values up to 25% of its body weight per day reported for the moon snail, Polinices duplicatus (Thorson 1971 ). The hard clam fishery in the lower Chesapeake Bay is already in decline. Hard clam landings during 1999 were less than 10% of landings during 1973 (Virginia Marine Resources Commission. Newport News, VA). The observed decline in hard clam stocks may be related to increased anthropogenic impacts on the Chesa- peake Bay ecosystem in the past 20 years including overfishing, water pollution and disease. Habitat changes are considered the major threats to estuarine ecosystem (Smith et al. 1999). The su- perimposition of a novel invading predator on this already stressed population has clear ecological and economic implications. Vimstein (1977), found that particularly in Chesapeake Bay. densities of infaunal species are not controlled by competitive interactions for food or space but mainly by the action of predators. If the introduction of Rapana venosa into the lower Chesapeake Bay results in a large scale successful invasion, rapa whelks could have a serious negative impact on the density and distribution ot the native hard clam population in the lower Chesapeake Bay. At this time we do not have a good estimate of the resident population of rapa whelks in the Chesapeake Bay but is possible to use our data for a hypothetical calculation to estimate potential impact of the whelk on the clam population. The rapa whelk distribution in the Chesapeake Bay, which extends from the mouth of the Rap- pahannock River in the North, to the Chesapeake Bay Bridge tunnel in the southeast and to the Lafayette River in the south (Harding & Mann 1999, Mann & Harding 2000), is within the historic distribution of M. nierceiiaria (Roegner & Mann 1991). The 1999 summer fishing season for hard clams in the lower Chesapeake Bay produced a harvest of 27388 kg or approximately 3,040,000 individual clams. Based on the predation rates observed in this study, a population of 1000 rapa whelks in the lower Bay could reduce this yield by between 0.3 to 0.9%. ACKNOWLEDGMENTS This work was supported by funds troni the National Oceanic Atmospheric Administration, National Sea Grant Aquatic Nui- sance Species Research and Outreach Program. Virginia Sea Grant, the Virginia Saltwater Commercial Fishing Development Fund, and the Commonwealth of Virginia. It was completed by the senior author during study for a M.S. degree at the University of Wales, Bangor, UK. Financial support for the senior author's visit to the Virginia Institute of Marine Science was provided by the Drapers Company, London. The authors thank Melissa South- worth, Erica Westcott, Stephanie Haywood, Rhonda Howlett, D. Bryn Jones, and Catherine Ware for assistance in general labo- ratory procedures during the study. This is contribution number 2487 from the Virginia Institute of Marine Science, LITERATURE CITED Drapkin, E. I. 1953. Novii molliusc v Cernom more. Priroda 8:92-95. Edwards, D. C. & J. D. Huebner. 1977. Feeding and growth rates of Polinices duplicatus preying on Mya arenaria at Barnstable Harbor, Massachusetts. Ecology 58:1218-1236. Ghisotti, F. 1974. Rapana venosa (Valenciennes) nuova ospite adriatica'.' Conchiglie Milano 10:125-126. Harding, J. M. & R. Mann. 1999. Observations on the biology of the Veined Rapa whelk, Rapana venosa (Valenciennes, 18461 in the Chesapeake Bay. / Shellfish Res. 18, 9-17. Koutsoubas, D. & E. Voultsiadou-Koukoura. 1990. The occurrence of Rapana venosa (Valenciennes, 1846) (Gastropoda, Thaididae) in the Aegean Sea. Bollellino Malacologico. Milano. 26:201-204. Mann. R. & J. M. Harding. 2000. Invasion of the North American Atlantic Coast by a large predatory asian mollusc. Biological Invasions 2:7-22. Pastorino, G., P. E. Penchaszadeh, L. Schejter & C. Bremec. 2000. Rapana venosa (Valenciennes, 1846) (Mollusca: Muricidae): a new gastropod in South-Atlantic waters. J. Shellfish Res. 19:897-899. Roegner. G. C. & R. Mann. 1991. Hard clam [Mercenaria mercenana). In: S. L. Funderburk. J. \. Mihursky, S. I. Jordan & D. Riley, editors. Habitat requirements for Chesapeake Bay living resources. Solomons. Maryland: Chapter 5: 1-17. Chesapeake Research Consortium. Inc. Savini. D. 2001. Predation strategies and feeding rates of the introduced gastropod Rapana venosa (Valenciennes. 1846) in Chesapeake Bay (Virginia. U.S.A.). M.Sc. Thesis. School of Ocean Sciences. University of Wales Bangor. Menai Bridge. Anglesey. Wales. UK. 62 pp. Smith. L. D.. M. J. Wonham. L. D. McCann. G. M. Ruiz. A. H. Hines & J. T. Carlton. 1999. Invasion pressure to a ballast-flooded estuary and an assessment of inoculant survival. Binlog. Inv. 1:67-87. Thorson, G. 1971. Life in the Sea. New York: McGraw-Hdl. 256 pp. Tsi. C X. Ma, Z. Lou & F. Zhang. 1983. Illustrations of the fauna of China (Mollusca). vol. 2. Beijing: Science Press, pp. 1-150. plates I-IV. Virnstein. R. W. 1977. The important of predation of crabs and fishes on benthic infauna m Chesapeake Bay. Ecology 58:1199-1217. Zolotarev. V. 1996. The Black Sea ecosystem changes related to the in- troduction of new mollusc species. P.S.Z.N. I: Marine Ecol. 17:227- 236. J,„anal of Slwlltlsh Research. Vol. 21, No. 2, 781-785. 2002. ECONOMIC ANALYSIS OF A PILOT COMMERCIAL HATCHERY-BASED OPERATION FOR SPOTTED BABYLON, BABYLONIA AREOLATA LINK 1807, JUVENILES IN THAILAND N. CHAITANAWISUTI,* S. KRITSANAFUNTU," AND Y. NATSUKARl' 'Aquatic Resourcfs Research Institute. Cluilaloiigkoni University. Baiii>kuk. Thailand 10330: -Department of Bioproducts, Prince of Songkla University (Suratani campus). Suratani. H4I00: ^Department (if Fisheries. Nagasaki universit^■. I -14. Bunkyo-Machi. 852-8521 Japan ABSTRACT An economic analysis of constructing and operating a pilot hatchery and ntirsery for the commercial production of spotted babylon. Babylonia nreo/aw. juveniles in Thailand was presented on the basis of proven practical techniques and published data from larval culture trials. The total initial investment requirement for the construction of a spotted babylon hatchery producing 1.2 million juveniles per year was 9.310.5 USS. Annual ownership costs were estimated to be 2.497.5 US$. with annual operating costs of 5.31 1.8 USS. Total annual cost for the juvenile production (Hatchery) phase of spotted babylon was 7,809.3 USS, and the co.st of producing 1.2 million juveniles in this hatchery design was estimated at 6.09 USS per 1.000 juveniles. Under the basic assumptions in this study (juvenile production of 1.2 million/year), a selling price of 13.8 USS per 1.000 juveniles results in a positive cash flow by year 2. Price and survival sensitivity resulted in substantial variability in net returns. This pilot hatchery operation is marginally feasible under these conditions if costs can be lowered considerably by improving growth and survival. KEY WORDS: Spotted babylon. Babylonia areolara. economic analysis, hatchery-based operation INTRODUCTION Spotted hahylon. Bahyhmla areolala (Link), are promising a new aquacultuial marine gastiopod in Thailand. Fast growth, high survival, and low feed conversion ratio (FCR) in grow-out culture provide this species with the biologic production and market char- acteristics necessary for a profitable aquaculture venture (Chai- tanawisuti & Kritsanapuntii 1999a. Chaitanawisuti & Kritsan- apuntu 1999b). Considerable interest has developed recently re- garding the commercial culture of spotted babylon in Thailand because of a growing demand and an expanding domestic market for seafood, as well as a catastrophic decline in the natural spotted babylon population in the Gulf of Thailand. An accurate economic assessment of culturing spotted babylon in Thailand is required before producers can make informed decisions regarding the po- tential of this enterprise. A lack of economic data can be an im- portant constraint to the successful development of spotted baby- lon aquaculture in Thailand. A financial investment analysis de- scribes the relationships between yield (growth and survival), market price, fixed and variable costs, and profitability indicators. From 1998 to 2000, Chulalongkom University conducted a col- laborative research and development project with the National Re- search Council of Thailand (NRCT) to develop the land-based aquaculture system for large-scale hatchery and grow-out opera- tions for spotted babylon in Thailand. Thereafter, the methods and techniques are intended to transfer for the economically hatchery- based operations in Thailand. This study was the first attempt to present estimates of the cost of producing juvenile spotted baby- lon, B. areolata. under a successful management scheme. The specific objectives were performed to identify the investment re- quirements, annual ownership and annual operating costs associ- ated with the hatchery, and the net returns, returns on investment and cash flow according to selected survival and prices of juvenile. MATERIALS AND METHODS Hatchery Operation A pilot commercial-scale hatchery in this study is designed to produce a total of 100,000 juveniles in 30-day production cycle for transfer to growout phase of culture. The design and operation of the hatcheiy was based upon the techniques of Chaitanawisuti and Kritsanapuntu (1997). The data necessary to estimate investment requirements for the hatchery were mainly gathered from a pilot research. The broodstock spawned and reared the larvae up through the post-set stage to 1.0 cm juvenile. The process involves manipulating adult snails until natural spawning occurs. The egg capsules are collected and placed in rearing tanks, where they develop into veliger larvae. The set are then placed in rearing units in which algae-enriched water is offered once daily. The algae are cultured from unicellular algal flask cultures of preferred species {Chiietocfros culcltrans and Tetraselmis sp.) in a controlled sys- tem. The average monthly productions of egg capsule and veliger larvae are approximately 8,180 and 7,1 16,600, respectively (Chai- tanawisuti & Kritsanapuntu 1999b). The initial stocking density rate was specified at 500 larvae L"' of water. The entire hatchery process require 14-18 days to go from spawning to newly settled juveniles ready for placement in the nursery system. This method requires a substantial level of capital investment and technical expertise to produce a consistently high quality of algae supply. The nursery is a critical link in the spotted babylon culture process. Placing hatchery juveniles directly into the grow-out system may induce unacceptably high mortality levels. The nursery provides an intermediate step that nurtures hatchery-reared Juveniles to a size less vulnerable to the stress of grow-out operation. In addition, growing juveniles to the size required for the grow-out stage within the intensive hatchery environment would likely not be cost effective. The length of time (30-43 days) required by a nursery to produce 1.0-cm juveniles from newly settled juveniles. Based on this pilot hatchery, an average survival of veliger larvae to 1.0 cm juveniles is approximately LS'/r. Financial Analysis Financial analysis was based on investment cost, production, and marketing data from the pilot-scale trials. The components of the economic analysis include the following. For the initial investment, the building used for hatchery op- erations included a 300-nr space made of concrete floor and sheet zinc roof with no window and wall for reducing construction cost and entrance of natural light. The hatchery design consists of three 781 782 Chaitanawisuti et al. TABLE 1. Initial investment requirements for hatchery production of spotted bab) Ion. li. areolulu. ju\eniles. Number, Investment, Percent of Item (unit) (US$) Total Cost Land 1,149.4 12.35 Buildine (300 nr) 1 3,448.3 37.04 BroodstiK-k lanks (3 x 3 x 0.7 m) 3 229.9 2.47 Lanal reanng tanks (500 Ll 30 689.7 7.40 Nursery tanks (5(H) L) 15 344.8 3.70 Alaal reanng tanks (500 L) 10 229,9 2.47 Mass Algal re;inng tanks (3 ton) 5 689.7 7.40 Aeration system 1 229.9 2.47 Water supply and drainage 1 574.7 6.17 Storage tanks (20 m') 2 459.8 4.94 Algal laboratory 1 804.6 8.65 Hatchery equipment 1 459.8 4.94 Total 9.310 5 100 rectangular broodstock rearing tanks (3.0 x 3.0 x 0.7 m canvas tanks), 30 cylindrical larvae rearing tanks (500-L plastic tanks), 20 cylindrical (nursery tanks (500-L plastic tanks), 15 conical algae culture tanks (500-L plastic tanks), and 4 mass algae culture tanks (4-ton concrete tanks). A small 3.0 x 3.0 m room is also included for algae culture and stock tnaintenance. Sand filter rated at 150 L per minute are specified for filtering water before its initial use in the hatchery. Two sets of 2-horsepo\ver pumps rated seawater used are necessary to transport water through the system. Two 10-ton canvas tanks were used as seawater stocking lanks. The two- horsepower blower is designed to provide a high volume of dis- solved oxygen, low pressure, and uncontaminated air necessary for larval survival and to keep food suspended in the water column after it has been introduced into the larval culture tank. Operating a hatchery requires the regular monitoring of water-quality vari- ables such as pH. salinity, water temperature, ammonia, nitrite, and nitrate levels, and these parameters are maintained at satisfactory levels for optitnum growth and survival. A refractometer is speci- fied for salinity measurements and a seawater test kit is specified for the determination of important water-quality parameters. Other designated equipment includes a refrigerator for the storage of TABLE 3. Estimated annual operatin;; costs (Baht) for hatchery production of the spoiled babylon, B. areulata, juveniles. Item Cost (US$) Percent of Total Cost Repairs and maintenance Hired labors (2 full time) Feed Broodstock purchase Electricity Interest on operating cost Total operating cost 408.0 7.68 2.758.6 51.94 724.1 13.63 413.8 7.79 827.5 15.58 179.6 3.38 5.311.6 100 supplemental feed ingredients and algae stocks. A binocular mi- croscope IS needed for the daily inspection of the quantity of food and growth of the larvae. PVC pipes and fittings are used to transport seawater and air from their sources to the culture system. Additional miscellaneous equipment also is specified for the daily operation of the hatchery. Interest rates for capital cost is based on 2000 bank loan rate of 3.5% for business enterpnse. Annual ownership costs mainly consisted of annual deprecia- tion and interest on investment. These costs are fixed and incurred in the long run regardless of whether the facilities are operated. Annual depreciation was based on the expected useful life of each equipment item. A zero salvage value was assumed on all items constituting the facilities. The hatchery building, canvas tanks, and plastic tanks were assumed to have a useful life of 10 years be- cause of the seawater environment. The life expectancies of equip- ment ranged from 3-5 years. Interest rates are based on 2(.)(;)0 bank loan rates of 3.5% of the original prices for all investment items. Annual operating costs are incurred upon actual operation of the hatchery and include repairs and maintenance, labor, feed, utilities, and interest on operating capital. The annual cost of re- pairs and maintenance for the hatchery was computed as 5.0% of purchase price. Two labor requirements were estimated based on the particular needs for full-time operation of the hatchery. Labor cost for each individual was calculated at a rate of 1 14.9 USS per month, without fringe benefits. Feed cost is based upon the as- sumption that larvae are fed microalgae for 14 days of the cycle. TABLE 2. Estimated depreciation, interest charges, and repairs and maintenance of hatchery production for B. areolala juveniles. Economic Annual Annual Interest Annual Repair and Item Cost (USS) Life (yrl Depreciation (USS) Charges (USS) 40.2 Maintenance (USS) Land 1,149.4 1 1.149.4 - Buildings 3.448.3 10 344.8 120.7 172.4 Broodstock tanks 229.9 10 22.9 8.0 11.5 Larval-rearing tanks 689.7 10 68.9 24.1 34.5 Nursery tanks 344.8 10 34.5 11.9 17.2 Algal tanks 229.9 10 22.9 8.0 11.5 Mass algal tanks 689.7 10 68.9 24.1 34.5 Aeration system 229.9 3 76.6 26.6 11.5 Water supply and drainage 574.7 3 191.5 20.1 28.7 Storage tanks 459.8 10 2.000 16.1 22.9 Algal culture laboratory 804.6 10 80.5 28.2 40.2 Hatchery equipment 459.8 5 45.9 1.6 22.9 Total cost/year 9,310.3 2,153,1 344.4 408.0 Economic Analysis of Spotted Babyxon Hacthery 783 TABLE 4. Estimated annual costs for hatchery production of spotted bahylon. B. areolala. ju\eniles. TABLE 6. Gross return for hatchery production of spotted babylon. B. areolata, juveniles at selected survival rates and selling prices. Item Cost (US$1 Percent of Total Selling Price |IS$ per 1.000 Juveniles) Ownership costs Depreciation 2.153.1 Interest on investment 344.4 Total ownership cost 2,497.5 Operating costs Repairs and maintenance 408.0 Hired labor 2,758.6 Feed 724. 1 Broodstock purchase 413.S Electricity 827.6 Interest on operating capital 179.6 Total operating cost 5,311.8 Total annual cost 7,809.3 27.57 4.41 3 1 .98 5.23 35.33 9.28 5.29 10.59 2.30 68.02 100 Thereafter, feed is principally composed of the fresh meat of fish. The cost of purchasing and caring for broodstock was estimated to be 4.6 US$/kg of spotted babylon. Based on the production sce- nario, the hatchery designed in the present study requires 40 kg of broodstock. This estimate was based on the assumption that ap- proximately 52,200 larvae are produced from one female brood- stock and an average of 1.5% survival of juveniles was expected. Electricity is used for operating the various pumps and lighting units in the hatchery. The average charge of electricity was as- sumed to be 68.9 US$ per month. Interest rates are based on 2000 bank loan rates of 3.5% per year for all depreciable items that compose the hatchery. Return Analysis Net returns and returns on investment for hatchery production were computed at final survivals ranging from 1.0-3.5% and the selling price of juvenile ranging from 9.2-22.9 USS per 1.000 juveniles. Gross return was computed for each level of survival and each selling price. Net return was calculated from the gross return minus the total annual cost. Return to capital and manage- ment was computed for each level of survival and each selling price by subtracting annual operating cost from gross returns. Sub- sequently, return on investment was estimated by dividing returns TABLE 5. Estimated total annual cost for production of spotted babylon, B. areolata. juveniles at selected survival rates. Survival Rate ( % » Annual Production (.Juvenllel Annual Costs (USS) Cost per 1,000 Juveniles (USS) 0.5 1.0 1.5 2.0 2.5 3.0 4.0 426.996 853.992 1.280.988 1.707.984 2,134.980 2.561,976 3,415.968 7,809.3 7,809.3 7,809.3 7.809.3 7.809.3 7.809.3 7.809.3 18.29 9.14 6.09 4.57 3.66 3.05 2.29 Survival rate is calculated from veliger larvae to juveniles of 1 .0-cm shell length with an average monthly egg capsule and veliger production of 8.180 and 7.116,600, respectively. i%) 9,2 13.8 16.1 18.4 22.9 0.5 3.92S.3 5.892.5 6.874.6 7.856.7 9,778.2 1.0 7.856.7 11,785.0 13.749.3 15.713.4 19,556.4 1.5 11,785.1 17,677.6 20,623.9 23,570.1 29,334.6 2.0 15,713.4 23,570.2 27,498.5 31.426.9 39,112.8 2.5 19,641.8 29,462.7 34,373.2 39,283.6 48,891.0 3.0 23,570.2 35,355.3 41,247.8 47,140.3 58,669.3 4.0 31.426.9 47,140.4 54.997. 1 62,853.8 78,225.7 Gross return was calculated for each level of survival and selling price. to capital and management by initial capital investment (Fuller et al. 1992). An annual production budget was developed from the variable and fixed costs, and cash flow budgets were developed to examine profitability in relation to the timing of expenditures and earning. Net cash flow was determined by projecting estimated revenues and costs over a 10-year period because an aquaculture enterprise would be an attractive investment opportunity if it were profitable within this period. The initial investment was charged in the first year, and costs in subsequent year included annual vari- able and fixed costs. (Rubino 1992, Head et al. 1996). RESULTS Total investment requirements for construction of the hatchery were 9,310.5 USS (Table 1). The building was the largest cost component (37.04%) of the hatchery. The rearing tank. land, water supply and storage tanks, and algal culture tanks are the second most expensive items in equipping the hatchery, representing 13.57%, 12.35%. 11.11%. and 9.87% of total investment, respec- tively. These five components of the hatchery represent 83.94% of total investment requirements for production of spotted babylon juveniles. Annual ownership costs were estimated to be 2.497.5 USS with annual depreciation and interest of 2.153.1 and 344.4 USS. respectively (Table 2). The annual operating cost is estimated to be 5,311.8 USS. Hired labor was the largest cost component (51.94%) of the operating cost, followed by electricity, feed, and repairs and maintenance of which comprised 15.58%, 13.63%. and 7.68%, respectively (Table 3). Total annual cost for the juvenile TABLE 7. Net return for hatchery production of spotted babylon, B. arenlata, juveniles at selected survival rales and selling prices. Selling Price (USS per 1,000 Juveniles) (%) 9.2 13.8 16.1 18.4 22.9 0.5 -7,556.3 -19.168 -934.7 47.4 1.968.9 1.0 47.4 3.975.7 5.940.0 7.904.1 11.747.1 1.5 3,975.8 9.868.3 12.814.6 15.760.8 21,525.3 2.0 7,904.1 15.760.9 19,689.2 23.617.6 31,303.5 2.5 11,832.5 21,833.4 26,563.9 31.474.3 41,081.7 3.0 15,760.9 27.546.0 33,438.5 .39.331.0 50,860.0 4.0 23,617.6 .39.331.1 47.187.8 55.044.5 70.416.4 Net return was calculated from the gross return minus total annual cost (7.809.3 USS). 784 Chaitanawisuti et al. TABLE 8. Return to capital and management for hatchery production ol' spotted babylon. B. areolala, juveniles at selected survival rates and selling prices. TABLE 9. Return on in>estnient for hatchery production of spotted babylon, B. areulata, juveniles at selected survival rates and selling prices. Survival (%) Selli ing Price (LIS$ per 1,(100 Juveniles) Selling Price (USS per l.(H)(l .Juveniles) Survival 9.2 13.8 16.1 18.4 •)■) 9 9.2 13.8 16.1 18.4 22.9 (Vr) 0 5 0 \^ 0 06 0 17 0 ""7 0 48 0.5 -1,383.5 580.7 1.562.8 2..544.4 4,466.4 1,0 0.27 0.69 0.91 1.12 1.53 1.0 2,544.9 6.473.2 8.437.5 10.401.6 14.244.6 1.5 0.69 1.33 1.64 1.96 2.58 1.5 6.473.3 12,365.8 15.312.1 18.258.3 24.022.8 2.0 111 1.96 2.38 2.80 3.63 2.0 10,401.6 18,258.4 22.186.7 26.115.1 33.801.0 2.5 1..54 2.59 3.12 3.65 4.68 2.5 14,3.W.0 24,1.50.9 29.061.4 33.971.8 43,579.2 3.(1 1.96 3.22 3.85 4.49 5.73 3.0 18.258.4 26.115.1 30,043.5 41.828.6 35,936.0 49.685.3 41.828.5 57.542.0 53.357.5 72.913.9 4.0 2.80 4.49 5.-34 6.18 7.83 4.0 Return tin investment was calculated b\ ' di\iil ini' return tti cani tal and Return to capital and management was calculated tor each level of survival and selling price by suhtiactmg annual upcrating ci>st (5.31 1.8 US$) frum gross returns. production (hatchery) phase ol spotted babylon culture was 7,809.3 US$ (Table 4). Annual ownership and operating costs accounted for .3 1 .98% and 68.02% of the total annual cost, respec- tively. The major ownership cost item was depreciation on invest- ment representing 27.57% of total annual cost. Hired labors was the highest operating cost item, representing 35.33% of total an- nual cost. The cost associated with producing juvenile spotted babylon is expressed as US.$ per 1.000 juveniles (43.5 Thai Baht is approximately lUSS). The cost of producing 1.200,000 juve- niles in this hatchery design was estimated at 6.09 US$ per 1.000 juveniles. However, as the total number of juveniles produced per year decreases, then cost increases. For example, if 426,996 juve- niles (approximately 0.57( survival) are produced, utilizing the same level of inputs, the estimated cost of production increases to 18.29 USS per 1,000 juveniles. Estimated total annual cost for production of juveniles at selected survivals is presented in Table 5. At 1.5% survival in this study reveals the 13.8 U.S.S Baht break- even price. Thereafter, gross return and net return at these levels are 17,677.6 USS and 9,868.3 USS, respectively (Tables 6 and 7). Return to capital and management and return on investment at these levels are 12.365.8 USS and 1.33. respectively (Tables 8 and 9). Under the basic assumptions in this study (juvenile production of 1 .2 million/year), a selling price of 13.8 USS per I .OOO juveniles results in a positive cash flow by year 2 (Table 10). management by initial capital investment (9,310.5 USS). DISCUSSION Based on juvenile production of 1.5% survival and selling price of 13.8 USS per 1.000 juveniles, the culture system is economi- cally feasible under the assumptions used. The cost of producing B. areolata postlarvae according to the procedures and assump- tions outlined in this study is considerably higher when the sur- vival is very poor. The stand-alone hatchery operation becomes profitable at output level of 1.2 million juveniles. Additional re- search needs to refine the lower limit of profitability and the nature of marginal costs (i.e., the change in variable costs with changes in output) within this range of facility sizes. An underlying assump- tion in this analysis shows that survival rate and market price are sensitive to farm output. However, potential investors must assess the impact to local market prices resulting from large production levels. The analysis assumes a constant market price, which may not be valid as the production volumes from large-scale operations are released onto the market. In general, prices are sensitive to changes in supplies. Prices decrease (increase) as spotted babylon landing increase (decrease). This relationship is not incorporated into the preceding analysis, which focuses primarily on cost changes as output levels vary. Investors in spotted babylon aqua- culture should be aware of the potential negative effects on market prices as output levels increase. Costs presented in this study are based on limited available data. An economic analysis of a pilot hatchery production for spotted babylon would be commercially TABLE 10. Ten-year cash flow of a pilot hatchery production for spotted babylon, B. areolata juveniles. Variable Cost Fixed Cost Investment Total Annual Receipt Net Return Cumulative Year (US$) (USS) (USS) Cost (USS) (USS) (US$) (USS) Year 1 5.311.8 2.497.5 9,310.5 17.119.8 17.677.6 9.868.3 -7.251.5 Year 2 5.311.8 2.497.5 0 7.809.3 17.677.6 9.868.3 2,616.8 Year 3 5.311.8 2,497.5 0 7.809.3 17.677.6 9.868.3 12.485.1 Year 4 5,311.8 2,497.5 0 7.809.3 17.677.6 9.868.3 22.353.4 Year 5 5,311.8 2.497.5 0 7.809.3 17.677.6 9.868.3 32.221.7 Year 6 5,311.8 2.497.5 0 7.809.3 17,677.6 9.868.3 42.090.0 Year? 5.311.8 2.497.5 0 7.809.3 17.677.6 9.868.3 5 1 .958.3 Years 5,311.8 2.497.5 0 7,809.3 17.677.6 9.868.3 61.826.6 Year 9 5.311.8 2.497.5 0 7.809.3 17,677.6 9.868.3 71.694.9 Year 10 5.311.8 2.497.5 0 7.809.3 17.677.6 9.868.3 81. .563. 2 Based on an annual production of 1.2 million juveniles per year and selling price of 13.8 USS per 1,000 juveniles Economic Analysis of Spotted Babylon Hacthery 785 feasible at current selling price at survival rate of 1 .5%. marginally feasible. This study serves as a guideline for understanding the economics of commercial juvenile production. Deviation from the hatchery specifications and management techniques of this study will likely result in altered costs. Costs can be lowered consider- ably by improving growth and survival rate. This economic analy- sis is intended as a guide and must be modified to reflect individual situations. ACKNOWLEDGMENTS We would like to thank National Research Council of Thailand (NRCT) for its support of the project and Professor Piamsak Me- nasveta and Associated Dr. Somkiat Piyatiratitivorakul for their advice and guidance. Last, we would like to thank Soonthorn Thepmoon, Siriwan Kathinniai. Mongkol Maklit. and Sailom Tan- tulvawit for their assistance during the hatchery work. Chaitanawisuti. N. & A. Kritsanapuntu. 1997. Effects of stocking density and substrate presence on growth and survival of juvenile spotted bah- ylon. Babylonia areolata Link. 1807 (Neogastropoda: Buccinidae). ./. Shellfish Res. 16:429-133. Chaitanawisuti. N. & .\. Kritsanapuntu. 1999a. Experimental culture of hatchery-reared juvenile spotted babylon. Babylonia aivolala Link 1807. (Neogastfopoda: Buccinidae) in Thailand. Asian Fisheiy Sci. 12:77-82. Chaitanawisuti, N. & A. Kritsanapuntu. 1999b. Growth and production of REFERENCES hatchery-reared juvenile spotted babylon. Babylonia areohita Link 1807, cultured to marketable sizes in intensive flow-through and semi- closed recirculating water system. Aquaculture Research 31:415— H9. Head, W. D.. A. Zerbi & W. O. Watanabe. 1996. Economic evaluation of commercial-scale, saltwater pond production of Florida tilapia in Puer- to Rico. J. World Aijnaciilture Soc. 27:275-289. Rubmo. M. C. 1992. Economics of red claw (Cherax quadricannatus aqua- culture). J. Shellfish Res. 11:157-162. Joiirmil of Shellfish Research. Vol. 21, No. 2, 787-792. 2002. INCIDENTAL DAMAGE OF BLACKLIP ABALONE (HALIOTIS RUBRA) BY COMMERCIAL DIVERS IN NEW SOUTH WALES, AUSTRALIA P. T. GIBSON,' D. G. WORTHINGTON,' C. BLOUNT,' AND N. L. ANDREW' * ^New South Wales Fisheries, Cronulla Fisheries Centre. PO Box 21. Crunulla. NSW, 2230. Australia and ^National Institute of Water and Atmospheric Research. P.O. Box 14-901, Kilbirnie, Wellington, New Zealand ABSTRACT Rales of incidental damage Ui blacklip abalcme (Hciliotis rubra) were invesligated in a retained, commercial catch in New South Wales, Australia. On average, 10.2% (8.9-1 1.6%, 95% CI) of retained abalone were damaged, but there was no relationship between the level of experience of divens and the degree or frequency of damage. Most damage was relatively minor, such as small abrasions to the foot or cuts to the mantle, and wounds healed rapidly in aquana. Major damage, such as large cuts and abrasions to the foot, occurred in 4.2% (3.6-4.9%) of retained abalone. For individuals with major damage, any mortality was rapid and significantly greater than for undamaged abalone in aquaria. Rates of growth were also lower for individuals with major damage. An average of 19.1% 117.2-21.1%) of abalone removed from the reef were found to be under the minimum legal size and replaced. E.xperienced divers removed and replaced significantly fewer abalone under the minimum legal size (11% vs. 23.8% of those handled) than inexperienced divers. Using the estimated rates described here, approximately 40 tons of abalone are estimated to be damaged by the fishery each year. Most damaged abalone are retained, heal rapidly in aquaria, and can be exported live, but approximately 3 tons of damaged abalone (both retained and discarded) may die. KEY WORDS: abalone, commercial fishing, damage, incidental, mortality INTRODUCTION Incidental damage to species not targeted by a fishery can be a substantial source of mortality (e.g., Broadhurst 2000). Damage to individuals of species that are targeted but subsequently found to be inappropriate for market also can be substantia] (e.g.. Blount & Worthington 20(M ). These types of mortality are most common in fisheries with nonselective collection methods (e.g., prawn trawls), or those with uncertainty about the quality or size of the product (e.g,, sea urchin roe). Despite this, incidental moitality may be an issue in selective fisheries when a minimum size limit requires the handling of individuals (to identify their size) before they are retained. Damage and any consequent mortality to individuals 1 ) above the size limit and retained and 2) below the size limit and returned can be problematic in fisheries where most of the product is sent live to markets. Compared with many other fisheries, commercial tlshing for abalone in New South Wales (NSW) is relatively benign, with minimal direct impact on other species or on abalone not being targeted for collection. In NSW, divers use a blunt, chisel-shaped iron to remove individual abalone from the reef Abalone are then quickly measured and, if larger than the size limit (115 mm in shell length, hereafter referred to as legal size), placed in bags that are sent to the surface for more controlled measuring. Any individuals that are removed from the rock, measured, and found to be below the size limit (hereafter referred to as undersized), either by the diver in the water or on the boat, are returned to the reef by hand. Although this process is relatively simple, the action of levering individuals off the bottom can result in cuts and abrasions to the foot of the abalone. Similarly, damage can occur during handling and storage of the catch on the boat before landing at processing factories. There has recently been a major change in management of the NSW abalone fishery. Before 1995, each of the 37 shareholders was required to harvest the quota they owned, except in excep- tional circumstances such as illness. This resulted in a group of shareholders that entered the fishery in the early 1970s and had *Corresponding author. E-mail: gibsonp@fisheries.nsw.gov.au collected abalone commercially for over 20 y. The change during 1995 enabled shareholders to nominate a diver to collect abalone on their behalf and, as a consequence, there was an intlux of inexperienced divers. This change generated concern within the industry about the impact of increased rates of damage by inex- perienced divers on populations of abalone. In addition, over the same time period, the fishery began to export live almost all aba- lone that were landed. This has also led to concern within the industry over the handling of abalone by divers and the rates of recovery of damaged abalone before export. Different types of damage are likely to have different effects on abalone. ranging from decreased growth and reproduction to death. Even the slightest disturbance, such as tagging, has been known to reduce growth rates and. in some cases, increase mortality (Mc- Shane et al. 1986). There is also some evidence from aquaculture that suggests small disturbances may disrupt reproduction (our unpublished data). Abalone have no clotting agent in their blood. so that damage to (he foot (hat severs any of the major arteries is likely to lead to death (Armstrong etal. 1971 ). Any type of injury may also inhibit their ability to feed or attach to the reef, leaving them more susceptible to predation (Pirker 1992). When disturbed, abalone also clamp down on the reef, making them considerably harder to remove, which may further increase the likelihocxl of damage. In this article, we describe the frequency of different types of damage to abalone by commercial divers in NSW, We estimate rates of damage in the commercial catch and the number of aba- lone that are removed from the rock, measured, found to be un- dersized, and replaced. Finally, to investigate the possible effects of damage to abalone that are not retained, we estimated the rates of recovery, growth, and mortality of individuals with different types of damage when maintained in aquaria. This has implications for marketing because retained abalone are held in tanks at pro- cessing plants before export. MATERIALS AND METHODS Frequency of Damage by Commercial Divers To estimate the frequency of different types of damage by commercial divers, a two-phase sampling design was used. First, 787 788 Gibson et al. divers" daily catches were liapliuzardly selected for samplint; in commercial processing factories from August 1996-December 1997. An individual bin (holding 50-100 abalone) from the di\er's catch was then randomly selected, and the length of all abalone within measured to the nearest mm and weighed to the nearest 5 g. Damage was categorized into several types and magnitudes based on injuries observed in the commercial catch. Categories (in order of increasing severity) were: no damage (ND), abrasions to the edge of the foot or mantle (MA), cuts to the edge of the foot or mantle (MCl, small (<10mm) abrasions to the foot (FAl ), small (<10 mm) cuts to the foot (FCl ), large (>10 mm) abrasions to the foot (FA2), large OlO mm) cuts to the foot (FC2), and major breaks to the shell (BS). These categories were further grouped into minor damage (MA. MC. FAl) and major damage (FCl. FA2, FC2, BS). based on their likely effects on abalone. For the analyses presented here, indi\iduals with more than one type of damage were categorized to the most severe grouping. Confidence limits were calculated using the binomial distribution, and a generalized logit model was used to relate the size of abalone to rates of minor and major damage (see Richards et al. 1994). Although the sampling design described above provided de- tailed information on the frequency of damage, a second sampling design was used to provide less detailed but more comprehensive information. In this design, all abalone from all divers' daily catch supplied to one commercial processing factory were assessed for damage over a four month period (July to August 1999). Abalone were assessed as either damaged, not damaged, or dead. In total 68.900 abalone from 15 divers on 207 diver-days were assessed in this design, compared with only 9.732 in the first design. Although the second sampling design is more comprehensi\e than the first in terms of the absolute number of abalone sampled, it covers only a proportion of the year and only one major processing factory. To estimate the rates of damage to abalone in the nonretained catch, information was collected on the frequency of removing, measuring, and replacing undersized abalone. Commercial divers were supplied with a logbook to record the number of undersized abalone removed and replaced and the number of legal-sized aba- lone retained in their first bag (divers retain harvested abalone in rope catch bags) of the day. Six commercial divers completed this logbook for a period of 15-30 days each between April-December 1999. Recovery. Growth, and Mortality of Damaged Abalone To investigate the effects of damage on individual abalone, two experiments were conducted in aquaria at a commercial processing factory. Experiment 1 began on 31 October 1996 and included 242 damaged abalone selected from the commercial catch. Replicate individuals from each damage category were placed in one of three randomly chosen aquaria. The number of replicate individuals within a damage category was related to the frequency of the category in the commercial catch supplied to the factory on that day (Table I ). Every two weeks, all individuals were briefly re- moved from the aquaria and any damage reassessed. A BS treat- ment was not included because individuals with this type of major damage were often dead before landing or were kniiwn to die rapidly thereafter. In experiment 2, the effects of damage on growth were exam- ined and the damage categories with the highest mortality in ex- periment 1 were further investigated. On 9 April 1997, at least 50 abalone from each of four damage categories (ND, FC 1 , FA2, and TABLE 1. Number of abalont within each treatment of different types of damage, and within each tank for both the first and second experiments. Damage Category Tank ND MA MC FAl FCl FA2 FC2 Total Experiment I 1 16 2 4 3 21 5 9 12 10 21 7 80 6 14 15 9 25 7 80 8 14 12 10 10 7 82 Totiil 41 19 37 39 29 56 21 242 Experiment 2 1 17 — — — 17 17 19 70 2 17 _ _ _ 16 16 19 68 3 17 — — — 17 17 18 69 Total 5! — — — 50 50 56 207 Damage calegcines in order of increasing severity, are no damage (NDl. abrasions to the edge of the loot or mantle (MA), cuts to the edge of the foot or mantle (MC). small abrasions to the foot <10 mm (FAl ). small cuts to the foot 1() mm (FA2) and large cuts to the foot >10 mm (FC2). FC2) were selected at the factory from the commercial catch and placed in one of three randomly chosen aquaria (Table 1). Indi- viduals were not disturbed throughout the six weeks of the experi- ment. All abalone used in both experiments were initially measured (i.e.. maximum shell length, maximum shell width, and wet weight), and a small plastic tag was attached to the shell with glue. Each experiment lasted for six weeks, and the animals were fed fresh drift algae as required. The tanks were checked daily for dead abalone, which were removed immediately. In experiment 1. aba- lone were disturbed every two weeks to investigate recovery of their wounds. This disturbance may have affected growth, so in experiment 2. abalone were not disturbed and weighed at the end of the experiment to investigate growth. Results from both experi- inents were combined to investigate mortality. RESULTS Frequency of Damage by Commercial Divers In total. 9.732 abalone from 29 divers on 123 days were ex- amined for damage. On average. 10.2% (8.9-1 l.O'/r. 95% CD of abalone retained by commercial divers were damaged. There was significant variation among divers in the rate of damage to abalone (nest of highest vs. lowest. P < 0.05). Total rates of damage varied among divers from 1-17% of abalone retained, with little relation- ship to the experience of divers (/?" < 0. 1. P > 0.05, Fig. 1). To some extent, this was cau.sed by large variation in rates of damage among days for individual divers, with daily rates of total damage ranging from 1-30% of abalone retained, and an average coeffi- cient of variation among days of 67%. Most damage was relatively minor, occurring in 6.0% of aba- lone retained (5.2-6.9%, 95% CI), but some divers had signifi- cantly greater rates of major damage than others (; lest of highest vs. lowest, P < 0.05, Fig. I ). For example, rates of minor damage varied among divers from 0-13%, and rates of major damage ranged from 0-12%. Major damage occurred in 4.2% (3.6—1.9%, 95% CI) of abalone retained. Again, there was no relationship between the rates of different types of damage and the experience Incidental Damage of Blacklip Abalone 789 Major damage Minor damage 7 18 I'J :o 21 22 23 24 25 . Diver (rank) Decreasing expenence * Figure 1. Number of abalone \iilh major and minor damage as a percentage of all abalone sampled in the commercial catch of 29 divers. Error is 95% CI on total damage. of divers (both R- <0.\.P> 0.05). MA was the most common type of damage (3.7%) whereas FA2 ( 1 .8%) was the most common type of major damage (Fig. 2). Rates of different types of damage varied greatly among divers and days. For example, rates of FC2 ranged among days from 0-8% and FA2 ranged from 0-16%. There was little relationship between the rates of different types of damage among days (all R~ < 0.07. P > 0.05). but rates of damage were related to the size of abalone. Large abalone were more likely to be damaged than small ones (Fig. 3). For e.xample. individuals of 115 mm were predicted to have rates of minor and major dam- age of 6.9% and 2.4%, whereas individuals of 140 mm were pre- dicted to have rates of 9.1% and 4.7%. respectively. Minor damage Major damage Damage type Figure 2. Number of abalone with different types of damage as a percentage (+95% CI) of all abalone sampled in the commercial catch. Damage categories were abrasions to the edge of the foot or mantle (MA), cuts to the edge of the fool or mantle (MC). small (<10 mm) abrasions to the foot (FAII, small (10 mm) abrasions to the foot (FA2), large (>lt) mm) cuts to the foot (FC2), and major breaks to the shell (BS). Minor damage Major damage 800 150 1-- "l-n J l^m^^r^^ 100 110 150 120 IM 140 Lenglh (mm) Figure 3. Top, number of abalone with major and minor damage as a percentage of all abalone sampled. Bottom, number of abalone sampled in each size class from the commercial catch in NSW. Per- centage damage is shown for those size classes with more than 150 individuals. A generalized logit model was used to fit the solid line for size classes with more than 150 individuals and a dotted line for those size classes with less. The second sampling design provided a significantly greater estimate (f test, P < 0.05) of the total rate of damage at 14.1% (12.6-15.8%, 95% CI). This included 0.7% (0.6-0.8%. 95% CI) of abalone that were dead at landing. Rates of total damage by indi- vidual divers ranged from 7-34% and were related to the size of the daily catch {R- = 0.03. P < 0.05). For example, at an average catch of 140 kg per day, predicted rates of damage were 12%, which was 4.5%' lower than at a catch of 40 kg per day. Despite these differences, there was large variation in rates of damage for all catches, with a standard error of 9.4%'. On average, 19.1% (17.2-21.1%, 95% CI) of abalone removed from the reef were replaced. Rates of replacement varied among divers from 10-31%. with the two most experienced divers having significantly lower rates than others (i.e., 1 1.0% vs. 23.8%, / test, P < 0.05). There was large variation in rates of removal and re- placement for individual divers, with daily rates ranging from 0-34% for the experienced divers and from 3-55% for the inex- perienced, with an average coefficient of variation among days of 51%. Recovery, Growth, and Mortality of Damaged Abalone Recovery of wounds of abalone occurred rapidly, and at a similar rate among treatments. After 6 wk, there was no visible wound in 35.8% (30-42.0%. 95% CI) of previously damaged aba- lone, varying among treatments from 32-54% (Fig. 4). In addition, most remaining damaged abalone were recovering, so that after 6 wk only 21.3% (15.8-27.2%, 95% CI) of all abalone had failed to show any visible recovery. Abalone within the ND treatment re- mained undamaged throughout the experiment. 790 Gibson et al. Li Ll L Figure 4. Number of abalone with different types of damage in four categories of wound progression over a 6-\vk period, as a percentage (+95% CI) of the original number damaged. Damage categories were abrasions to the edge of the foot or mantle ( MA ), cuts to the edge of the fool or mantle 0.05), but followed a consis- tent gradient of greater weight loss with increasing injury. In experiment 1, rates of moilality of damaged abalone were significantly different among treatments after 6 wk in aquaria (Fig. 4). FC2 and FA2 caused the highest mortality of 19% (4.8-38.1%. 95% CI) and 12.5% (5.4-21.4%, 95%. CI), respectively. The re- maining treatments had much lower rates of mortality ranging from 0-5.1%'. One individual within the ND treatment died (i.e., 2.4%), and 88% of all the mortality occurred in the first two weeks of the experiment. In experiment 2, results were similar, with generally lower rates of mortality. The highest mortality again occurred for FC2 with 14.3% (5.4-23.2%, 95% CI), whereas mor- tality in the other treatments ranged from 2-6%. One individual within the ND treatment died (i.e., 2%), and 79% of the mortality occurred in the first 2 wk of the experiment. When rates from both experiments were combined (Fig. 6), FC2 treatments caused the highest mortality averaging 15.6%' (7.8-24.7%, 95% CI) followed by 8.5% for FA2 treatments (3.8-14.2%, 95% CI) and FC! with 5.1% (1.3-10.1%, 95% CI). There was significantly greater mor- 3 1 2 - ' 1 ■ (^ 0 • • 1 - c op -2 ■ . -3 - -4 - ND FC2 FCl FA2 Damage type Figure 5. Change in weight as a percentage of original weight (+95% CI) for abalone with different tvpes of damage after 6 weeks in ex- perimental aquaria. Damage categories are no damage (ND), small (10 mm) cuts to the foot (FC2). tality in the major damage treatments than ND ix' = 5.25. 1 df. P < 0.05). Most of this mortality was attributable to FC2 treat- ments, which were significantly greater than ND treatments (x" = 9.84. 1 df. P < 0.01 ) with all other comparisons not significant. DISCUSSION Frequency of Damage by Commercial Divers Cominercial divers damage a significant proportion of the aba- lone they retain in NSW. Most of the wounds are minor and heal quickly, but over 4% have major injuries that may affect their growth and survival (Fig. 2). Although rates of major damage were variable among divers and days, there was little apparent relation- ship to the experience of divers. Abalone size and size of the daily catch did explain some of the variation in rates of damage. The Damage type Figure 6. Mortality of abalone with different types of damage as a percentage (+95 '7f CD of the original number damaged after 6 wk in an experimental aquaria. Damage categories are no damage (ND), small (<10 mm) cuts to the foot (FCl), large OlO mm) abrasions to the foot (F.\2). and large OlO mm) cuts to the foot (FC2). Incidental Damage of Blacklip Abalone 791 rates of damage were greater for larger abalone and small daily catches, but this effect was small compared with variation among divers and among days for individual divers. Local wind and swell conditions are known to affect catch rates (Worthington et al. 1 998) and are likely to influence rates of damage to abalone due to the difficulty of handling during harvesting and transport in strong winds and large swells. The rate of damage in the commercial catch varied among divers from 1-17%, and averaged 10.2% (Fig. 1). Although the second sampling design provided a greater rate of damage (14.1%), it represented only one major processing factory over a small portion of the year. Rates of damage in NSW were consid- erably lower than those found for other abalone fisheries. Pirker (1992) found rates of damage between 44-78% in the commercial fishery for Haliotis iris in New Zealand, whereas Bruge et al. (1975) found rates above 90% in the recreational fishery for H. rufescens in California. Several differences among the fisheries may explain these differences. For example, in NSW a limited number of commercial divers use surface-supplied air and blunt irons to remove abalone from the reef. Perhaps most importantly, the NSW fishery exports most of the catch live and divers are encouraged by beach price to minimize the damage to their catch. Since this study was completed, further improvements in the han- dling of abalone by commercial divers have been made so that current rates of damage may be even lower. These include recir- culating seawater-holding tanks on board fishing vessels, divers being encouraged by beach price to handle their catch appropri- ately, and an improved knowledge of handling techniques through- out the industry. In contrast to the frequency of damage to retained abalone, the rate of removal and replacement of undersized abalone was related to the experience of divers. Experienced divers removed and re- placed fewer undersized abalone. For a given catch of abalone, this would reduce the rate of damage caused by their removal. The lower rate of removal and replacement by experienced divers could be caused by a variety of factors. For example, more expe- rienced divers may be able to identify undersized individuals or areas where there is little need to remove and measure individuals. Alternatively, less-experienced divers generally have lower catch rates and may be forced to work in times and places more expe- rienced divers can avoid. Some populations are dominated by in- dividuals who are clo.se to legal size (Andrew et al. 1997) and fishing in these areas would require more measuring. Indeed, in some areas few, if any, individuals reach legal size (Worthington et al. 1995). and this can lead to inexperienced divers measuring large numbers of abalone to identify any individuals above the size limit. Damage to undersized individuals at these sites could be a substantial source of mortality (see also Tegner et al. 1989). Recovery. Growth, and Mortality of Damaged Abalone Rates of mortality and growth largely depended on the type of injury sustained. Damage to the foot that results in severing of any of the major arteries is likely to lead to death (Armstrong et al. 1971 ). Minor injuries can be controlled and repaired with the aid of muscle contractions (Armstrong et al. 1971 ). Within the aquaria. major damage resulted in mortality rates of up to 19% and reduced body weight (Figs. 5 and 6). Mortality rates and growth reduction may be much greater in the wild because of increased vulnerability to infection and predators (Bruge et al. 1975, Pirker 1992). For similar reasons, reductions in reproductive ability, such as reab- sorption of eggs (observed when abalone are disturbed in aqua- culture), may also be more intense in the wild. Within aquaria, most mortality occurred within the first two weeks. Recovery of damaged abalone was rapid in aquaria, with most individuals completely recovered or recovering after six weeks (Fig. 4). These results confirm the usefulness of retaining damaged abalone within aquaria until their wounds have recovered and they are appropriate for live export. Most importantly, the rapid time for mortality or recovery is consistent with potential tanking times in the industry within NSW. The estimated rates of damage and mortality can be used to estimate the total weight of abalone being damaged and killed each year by the fishery. During 2000, approximately 1 million abalo- nes were retained and landed by the commercial fishery. With the estimated rate of removal and replacement of undersized abalone from this study (i.e., 19.1%), this suggests almost 1.2 million aba- lone were exposed to potential damage by being removed from the rock. At the observed rate of damage in the commercial catch (i.e., 10.2%), almost 120,000 individuals or approximately 40 tons may be damaged by the commercial fishery each year. Of these dam- aged abalone, approximately 8 tons are undersized and replaced on the reef, and the fishery retains 32 tons. Most of the damaged indi\ iduals probably recover rapidly but, even at the low rates of mortality likely in aquaria, the fishery may kill approximately 3 tons of abalone each year (i.e.. 0.81% of those removed from the reef, estimated from rates of damage and mortality). Of this, ap- proximately 500 kg are undersized abalone that are replaced, sug- gesting such mortality has a relatively minor impact on the pro- ductivity of the population. Furthermore, although many-damaged abalones that are replaced may survive, they are still likely to suffer complications, including reduced growth and lower repro- duction. Damage in the recreational and illegal abalone fisheries in NSW may also have important effects. For example, illegal fishers do not observe the minimum length limit and kill many undersized abalone. Many recreational fishers also handle and retain under- sized abalone (unpublished data) with the potential for high rates of damage because they are not permitted to use scuba and gen- erally have little experience removing abalone. Recreational divers often use unconventional tools to remove abalone, which may cause high rates of damage. During 1997, the recreational catch was estimated at 52 tons (unpublished data), when the commercial catch was over 330 tons. As a result, total rates of damage may be similar between the two sectors. Early in the history of the commercial fishery for abalone in NSW there was no minimum legal size. In 1972, a minimum legal size was introduced at 100 mm, but most abalone were well above this size and almost all individuals removed from the rock were retained. With the progression of size limit upwards to the current 115 mm minimum legal size, and the decline in the size-structure of the population, more removal, measurement, and replacement of under-size abalone has occurred. Current management measures, such as temporal closures, attempt to minimize the disturbance and damage to abalone, particularly during reproductive season. Beach price incentives have also been developed by the industry to en- courage the landing of undamaged individuals and to create an awareness of the damage to the resource caused by injuring aba- lone that are not retained. Incidental damage of undersized indi- viduals in the NSW abalone fishery is small when compared with some other abalone fisheries, but could still be a significant source 792 Gibson et al. of mortality. This i^ obviously iindesirahle in an already hea\ily exploited fishery. ACKNOWLEDGMENTS The authors thank Rowan Chick. Penny Brett. Nokome Bent- ley. Greg Otton. Ross Werner. John Vairy. and the divers who participated with the logbook for their ongoing assistance. We also thank Southern Ocean Seafoods. NSW Abalone. and Australian Shellfish Enterprises for providing us with access to their factories and their abalone. We also thank Steve Kennelly and Doug Ferrell for reviewing the manuscript. The commercial abalone divers of NSW provided funding for this work. LITERATURE CITED Andrew. N. L.. D. G. Wurthington & P. A. Brett. 1997. Size-structure and growth of individuals suggest high exploitation rates in the fishery for blacklip abalone. Hulious nihra in New South Wales. Australia. MoU. Res. 18:27.S-2S7. Armstrong, D. A.. J. L. Armstrong. S. M. Krassner & G. B. Pauley. 1971. Experimental wound repair in the black abalone. Huliotis cracheiddii. ./. Invert. Path. 17:216-227. Blount, C. & D. G. Worthington. 2001. Identifynig nidividuals with high quality roe for the sea urchm Centrosteplninus rodgersii. in NSW. Australia. Fish. Res. Broadhurst. M. 2000. Modifications to reduce bycalch in prawn trawls: a review and framework tor development. Rev. Fish Biol. Fislieries 10: 27-60. Bruge. R.. S. Schultz & M. Odemar. 197.'i. Draft report on recent abalone re.search in California with recommendations for management. Califor- nia: Resources Agency. Department of Fish and Game. 48 pp. McShane. P. E.. M. G. Smith & K. H. H. Beinssen. 1986, Growth and morphomeu-y in abalone {Huliotis rubra Leach) Irom Victoria. Aus. ./. Mar. Freslm-at. Res. .^9:161-166. Pirker. J. G. 1992. Growth, shell-ring deposition and mortality of Paua (Haliotis iris Martyn) in the Kaikoura region. Masters thesis. Univer- sity of Canterbury. New Zealand. Richards. L. J.. J. T. Schnute & J. Fargo. 1994. Application of a general- ized logit model to condition data for trawl-caught Pacific halibut. Hippoglossiis steiiolepi.s. Can. J. Fish. Aqiiat. Sci. ."i 1:357-364. Tegner. M. J.. P. A. Breen & C. E. Lennert. 1989. Population biology of red abalones. Haliotis rufescens. in southern California and manage- ment of the red and pink. Haliotis corrugata, abalone fisheries. US Fish. Bull. 87:31.3-339. Worthington. D. G.. N. L. Andrew & N. Bentley. 1998. Improved indices of catch rate in the fishery for blacklip abalone, Haliotis rubra, in NSW, Australia. Fish. Res. 36:87-97. Worthington. D. G.. N. L. Andrew & G. Hamer. 1995. Covariation be- tween growth and morphology suggests alternative size limits for the blacklip abalone. Haliotis rubra, in NSW. .Australia. US Fish. Bull. 93:551-561. Joiirihil ofSln-llthh Research. Vol. 21. No. 2, 793-798. 2002. THE EFFECT OF NONNUTRITIVE FILLERS ON THE DIGESTIBILITY OF A MANUFACTURED ABALONE DIET MEEGAN E. VANDEPEER,'* PATRICK W. HONE.^ JON N. HAVENHAND,' AND ROBERT J. VAN BARNEVELD^ 'South Australian Research and Development Institute. PO Bo.x 120. Henley Beach. South Australia 5022: 'Fisheries Research and Development Corporation. PO Box 222. Deukin West. Australian Capital Territory 2600: ^Flinders University. GPO Box 2100. Adelaide. South Australia 5001: ^Barneveld Nutrition Pty. Ltd.. 19-27 Coonan Rd. South Maclean. Queensland. Australia 4280 ABSTRACT In this study the effects on nutrient digestibility of adding nonnutntive fillers (kaolin, bentonite. sand and diatomaceous earth) to a research diet for greenlip abalone. Haliolis laevigata Donovan, were investigated. The addition of kaolin significantly improved the gross energy digestibility of the diet with the greatest improvement in gross energy digestibility occurring at the highest level of inclusion investigated (20%). Possible explanations for the positive effect of kaolin on nutrient digestion in abalone include assisting with the breakdown of diets through an abrasive/grinding effect, increasing gut transit time, decreasing digesta viscosity or through supplementation of minerals that may be deficient. KEY WORDS: abalone. digestibility, kaolin, bentonite. sand, diatomaceous earth INTRODUCTION Silicates, more commonly known as bentonite, kaolin, zeolites and other clays, have been used in poultry and swine nutrition for many years (Dias et al. 1998). The reason for their inclusion is due to their binding and lubricating property in the production of pel- leted feeds. There is also suggestion that some clay products may have direct beneficial effects on animal performance, however, results from studies aimed at establishing this have produced mixed results. Experiments with pigs, chickens and fish have shown responses ranging from increases in growth, feed efficiency and nutrient utilization (Kurnick & Reid 1960, Onagi 1966, Kondo & Wagai 1968, Quinsberry 1968, Han et al. 1975, Han et al. 1976, Smith et al. 1980) to negative or no responses (Reinitz 1983, Reinitz 1984. Dias et al. 1998). Because kaolin contributes no protein or energy, it is regularly used as a tiller in research diets for abalone at the South Australian Research and Development Institute's Aquatic Sciences Center. Since the addition of silicates, including kaolin, have been reported to improve nutrient utilization in other animals, it is important that its effects on nutrient digestion in abalone be investigated. The aim of this study is to assess whether four, nonnutritive, fillers (beach sand, diatomaceous earth, kaolin and sodium bentonite) affect the digestibility of a research diet for greenlip abalone, Haliotis lae- vigata. MATERIALS AND METHODS Diets The basal diet used in all experiments was developed by the South Australian Research and Development Institute's Aquatic Sciences Center. The exact diet formulation is confidential; how- ever, semolina constitutes the largest percentage of the diet fol- lowed by soy flour, which is the main source of protein. Chromic oxide was included in the diets at 0.5*7? by dry weight at the expense of semolina, as an indigestible marker for use in calcu- lating the apparent protein and energy digestibility. All diets were initially hand mixed and then mixed in a spiral action dough mixer *Corresponding author: E-mail: vandepeer.meegan@saugov.sa.gov.au ( 'Impastrice', Hill Equipment and Refrigeration. Adelaide. South Australia). The mixture was then fed through a commercial pasta machine (La Prestigiosa mediuin. IPA. Vicenza, Italy) where it was made into 300 mm long strips using a die with slots 18 x 1.5 mm. The strips were dried on mesh trays overnight in a forced draft oven at 55°C. Experiment I — The Effect of Different \onnulriti\e Fillers on the Protein and Energy Digestibility of a Manufactured .Abalone Diet Six diets were fomiulated in total, including the basal diet (Table 1). Four of the diets consisted of the basal diet with 5% of the semolina substituted for one of the following nonnutritive fill- ers: kaolin, bentonite. diatomaceous earth, and beach sand. The fifth diet contained 5% of pre-gelatinized waxy maize starch (Wades BOllC, Goodman Fielder Mills. NSW. Australia). The beach sand was obtained from West Beach. South Australia and was autoclaved at 121°C for 20 min before inclusion in the diet. The size of the sand grains ranged from around 192-346 fjim. Each dietary treatment was randomly allocated to one of 18 digestibility tanks to provide three replicate fecal samples per diet. Experiment 2 — The Effect of Kaolin Inclusion Level on the Protein and Energy Digeslihilily of a Manufactured Abalone Diet Six diets were formulated in total including the basal diet and the basal diet with 5% pre-gelatinized waxy maize as in experi- ment 1 (Table 2). The other four diets contained increasing levels of kaolin (5%. 107r. 15%. and 20%) again at the expense of semolina. The six diets were randomly allocated to one of 18 digestibility tanks to provide three replicate fecal samples per diet. Abalone and Feeding Juvenile greenlip abalone (shell length 40-60 mm) were used in the experiments. The abalone had been obtained from a com- mercial hatchery and raised on manufactured abalone feed. The abalone were preconditioned for one week on the test diet assigned to their tank. During both the pre-conditioning and experiment periods the animals were fed to excess every day at approximately 1700 h. 793 794 Vandepef.r et al. TABLE 1. Composition, proximate analysis and gross energy content (M,I/kg) of experimental diets from experiment 1 (g/kg, air dry basis). Basal SC, 5<7r 5% 5% 5% Diet Starch Bentonite D. Earth* Sand Kaolin Basal diel 995.0 945.0 945.0 945.0 945.0 945.0 Starch 0 50.0 0 0 0 0 Bentonite 0 0 50.0 0 0 0 Diat(.)maceous earth 0 0 0 50.0 0 0 Sand 0 0 0 0 50.0 0 Kaolin 0 0 0 0 0 50.0 Chromic oxide 5.0 5.0 5.0 5.0 5.0 5.0 Gross energy (MJ/kg) 17.79 17.77 17.03 16.93 16.98 16.90 Crude protein (N x 6.25) 311.9 311.9 312.5 312.8 3 1 1 .9 311.3 Dry matter 919.2 916.4 923,4 927. S 926.3 922.4 * diatomaceoiis earth. Tanks and Collection System Conical shaped digestibility tanks were used. Abalone were housed in 20-L buckets (approximately 80-100 per bucket) that fitted inside the tanks. All the buckets were fitted with plastic mesh bottoms (1.3 cm x 1.3 cm mesh) allowing containment of the abalone while permitting feces to drop into the collection tube at the base of the tank. Three 25 cm lengths of PVC pipe (8 cm in diameter) were placed in the buckets as shelters for the abalone. Attached to the bottom of each digestibility tank was a screw-on collection tube (1 i cm long. 1.5 mm diameter). Tanks were on a flow-through water system at a rate of about 2 L/min. The seawater was filtered to 30 jj-rn by primary sand filters, then to 10 p.m by secondary composite sand filters before entering the tanks. Aera- tion was supplied at 0.5 L/min to each tank at all times by an air stone. Water temperature and lighting were controlled during the experiment with temperature maintained at 18.0°C ± 1.0 and a light regimen of 12 h light; 12 h dark. Salinity was 35-36%f throughout the experiment. Fecal Collection Feces were collected by settlement every day until 5-6 g of feces (dry weight) was collected for each replicate sample. This took approximately 2 wk for each replicate. On each day of fecal collection the buckets containing the abalone were removed and the digestibility tanks were drained of water and all fittings were cleaned of feces and uneaten feed. Following cleaning, the tanks were refilled and the buckets replaced. Collection tubes were fitted by 0900 h. A small foam container was placed underneath each tube and filled with ice to keep the collecting feces cold and thus reduce its degradation by microbes. The feces were collected from the lubes at about 1630 h by gently pouring the contents onto a 1 mm diameter mesh. The mesh was then placed into a petri dish and frozen at -30°C. The following day the frozen fecal sample was scraped off the mesh, pooled into a composite sample, and stored in the freezer until required for analysis. Prior to analysis the samples were freeze-dried and ground with a mortar and pestle. Chemical Analyses Dry matter was determined by drying samples at IOO°C over- night until a constant weight was achieved. Gross energy was determined using a Parr 1281 bomb calorimeter. Crude protein was determined bv the combustion method using a LECO® CN-2000 Carbon and Nitrogen Analyser (Royal Australian Chemical Insti- tute 1999). Chromic oxide was determined using atomic absorp- tion spectroscopy based on a modification of the methods de- scribed by Hillebrand et al. (1953). The modified methodology involved preliminary ignition of the sample at 500"C to remove organic material and the dissolution of the sample in hydrochloric acid instead of sulphuric acid (M. Frith, personal communication. University of Tastnania, Launceston, Australia). Digestibility Determination The apparent digestibilities of nutrients in the diets were cal- culated using the following formula (Hardy 1997): Apparent digestibility : C/y,.^.,,, X Nutrient j,^., where Cr is chromium content and Nutrient is nutrient or energy content of the diet. Statistical Analysis The data were analyzed by use of general linear model and the treatment means were compared by least significant difference. Prior to analysis, data were analyzed using a univariate procedure and normal plots to establish that the data were in fact normally distributed, which was the case. The presence of outliers was as- sessed using the RANK procedure in SAS (SAS Institute Inc.. 1988) and normal scores computed from the ranks following Blom (1958). As no outliers were detected using the RANK procedure, all data were used in the calculation of digestibility estimates for all parameters, respectively. RESULTS Experiment 1—Tlie Effect of Different Nonnutritive Fillers on the Protein and Energy Digestibility of a Manufactured Abalone Diet Although very close, no significant difference was found among the six diets in their apparent protein digestibility for aba- lone at the 0.05 significance level (F^,,,, = 2.76: P = 0.08()4i (Table 3). However, the diets were significantly different at the 0.10 significances level. This was entirely due to the 57r bentonite diet having significantly lower apparent protein digestibility than all the other diets. A significant difference was observed in the apparent dry matter digestibility (F5 ,o = 6.93; P = 0.0048) and The Effect of Fillers in Abalone Diets 795 TABLE 2. Composition, proximate analysis and gross energy content (MJ/kg) of experimental diets from experiment 2 (g/kg, air dry basis) Basal Diet 5% Starch 5% Kaolin 10% Kaolin 15% Kaolin 20% Kaolin Basal diet Starch Kaolin Chromic oxide Gross energy (MJ/kg) Crude protein (N x 6.25) Dry matter 995.0 945.0 945.0 895.0 845.0 695.0 0 50.0 0 0 0 0 0 0 50.0 100.0 150.0 200.0 5.0 5.0 5.0 5.0 5.0 5.0 17.65 17.74 16.87 15.97 15.11 14.21 .^15..^ 308. 1 310.3 302.2 297.2 291.3 914.7 920.6 924.9 928.0 9.16.7 940.7 apparent gross energy digestibility of the diets (F^ m = 3.29. P = 0.0516) (Table 3). The diet containing 5% starch had significantly higher apparent dry matter digestibihty than all the other diets (Table 3). No significant differences in dry matter digestibility occurred among the diets with fillers and the control diet (Table 3). The diet with 5% starch had significantly higher apparent gross energy digestibility than all the other diets, excluding the kaolin diet (Table 3). There was no significant difference in apparent gross energy digestibility among the control, bentonite. diatoma- ceous earth and sand diets. Similarly, there was no significant difference in apparent gross energy digestibility among the kaolin, bentonite. diatomaceous earth and sand diets, but the kaolin diet did have significantly higher apparent gross energy digestibility than the control diet (Table 3). Experiment 2 — The Effect of Kaolin Inclusion Level on the Protein and Energy Digestibility of a Manufactured Abalone Diet The digestibility coefficients for the control diet and that with 5% starch were comparable with those from experiment 1 (Table 3 and Table 4). No significant difference in protein digestibility occurred among the six diets in experiment 2 (F, ,„ = 1.41. P = 0.3017) but there were significant differences among the diets in apparent gross energy (Fs j,, = 18.99. P = 0.0001 ) and dry matter digestibility (F5 8.56. P = 0.0022) (Table 4). The diet with 5% starch had significantly higher apparent dry matter digestibility than all the other diets (Table 4). The diet with 10% kaolin had significantly higher dry matter digestibility than the control diet, but not the other kaolin diets. Apparent gross energy digestibility significantly increased with increasing level of kaolin in the diet (Table 4). The diet with 20% kaolin had significantly higher gross energy digestibility than all other diets (Table 4). DISCUSSION Substitution of semolina with kaolin in a manufactured diet for juvenile greenlip abalone increased the apparent digestibility of its gross energy. The gross energy digestibility of the manufactured diet significantly increased with increasing inclusion level of ka- olin (Table 4). Unlike kaolin, the addition of other inert fillers (sand, bentonite and diatomaceous earth) did not significantly im- prove abalone's apparent gross energy digestibility of the diet when included at 5% (Table 3). It is possible that these fillers may improve the apparent gross energy digestibility if included in diets at higher levels. The improved digestibility/growth rates observed in other ani- mals fed diets supplemented with inert fillers such as kaolin, may have resulted, singly or in combination, from their assisting with the breakdown of diets through an abrasive/grinding effect, in- creasing gut transit time and through supplementation of minerals that may be deficient. Any of these mechanisms could also explain why kaolin significantly improved the gross energy digestibility of a commercial diet for juvenile abalone. Two other possible mecha- TABLE 3. Apparent fecal digestibility coefTicients of protein, gross energy and dry matter of the 6 diets from experiment 1. Statistics Basal Diet 5% Starch 5<7f Bentonite 5% D. Eartht 5% Sand 5% Kaolin SEM 0.708 ±0.01069 0.665 ±0.01287 0.699 ± 0.00543 0.703+11.00742 0,713 ±0.01 144 Prolein 0.717 ±0 01303 Gross energy 0.490^ ± 0.00589 0.560" ± 0.00841 0.5091^ ± 0.02258 OSOI*^ ± 0.00945 0.515'^ ±0.00665 0.540"" ± 0.00901 DMD 0.362" ± 0.00840 0.434" ± 0.03165 0.331' ± 0.fW935 0.327-' ± 0.01 303 0.34S' ± 0,01041 NS 0.370" ±0.00614 0.01137 0.01435 0.0452 0.01479 0.0466 * P = 0.05. **P< 0.001. t diatomaceous earth. NS, nol significant (P > 0.05). SEM, standard error of the mean. DMD, dry matter digestibility. L.SD. least significant difference. Diets in a row with different superscripts differ significantly (t,, 05, ,, Data are mean ± SE, n = 3. 2.23). 796 Vandepeer et al. TABLE 4. Apparent fecal digestibility coefficients of protein, gross energy and dry matter of the 6 diets from experiment 2. Basal Ditl 5% Stercli Protein 0.687 ± 0.00542 0.687 ± 0.00846 Gross energy 0.455'' ± 0.00897 0.549'^ ± 0.007484 DMD (I..U7' ±0.0I.'!20 0.426° ±0.005 15 5% Kaolin 10% Kaolin ISri Kaolin 20 "^f Kaolin SEM LSD 0.685 ±0.00612 0.505' ± 0.02084 0.362'"-± 0.01828 0.696 ± 0.00495 0.553'' ±0.01 155 0..376''± 11,01 148 0.688 ±0.0071.? 0.55 1 "±0.00788 O.UI'" ±0.01225 0.707 ± 0.00532 0.605^ ±0.01 1 12 11.358'"' ±0,01002 NS 0.00692 0.01164 0.0367 0111169 0,0369 **/> 0.05). SEM. standard error of the mean. DMD. dry matter digestibility, LSD. least significant difference. Diets in a row with different superscripts differ significantly (Im,,,! Data are mean ± SE. 2.23). nisms are its reducing digesta viscosity and osmotic pressure in the lumen. Rediulioii in Osmotic Pressure in the Lumen The increase in apparent gross energy digestibility of diets with the increased level of kaolin could be related to the unusual oli- gosaccharides in soyflour, the second largest component of the diet in this study. Pedersen (1989) suggested that the post-weaning diarrhea found in piglets fed soy-based diets might not be due solely to an allergic reaction elicited by soy antigens, but to the soy's oligosaccharides. The accumulation of these components in the intestine not only provides a substrate for the growth of unde- sirable bacteria, but also could lead to an "osmotic" diarrhea that occurs when body fluid is drawn into the lumen of the gut to counter a raised osmotic pressure (Pedersen 19S9). Soyflour may possibly produce the same response in the gut of abalone. The positive effect kaolin had might be related to its ability to coun- teract this osmotic diarrhea caused by soyflour and thus increase the digestibility of dietary nutrients. Evidence for this is that the addition of zeolites (alumino-silicates) to piglet diets has been found to reduce diarrhea. Inclusion of zeolite to the diet of piglets severely affected with scours (prolonged dianhea in livestock) markedly reversed the progress of this disease within a few days (Morita 1967). Similarly, the incidence of scours in young swine fed a diet supplemented with 5% clinoptilolite was significantly reduced (England 1975). Zeolites are characterized by their ability to lose and gain water reversibly and by a very effective ion exchange capacity (Mumpton & Fishman 1977. White & Ohlrogge 1974). It is these properties of zeolites (ion exchange and revers- ible binding of water) that could have reduced diarrhea in piglets when added to their diet. Kaolin, also an alumino-silicate, could have had the same effect in the gut of abalone resulting in the increased gross energy digestibility of the diet. Abrasive or Grinding Effect Kaolin may perform a siinikir function to sand and grit that abalone ingest while feeding on their natural diet, assisting in grinding up and breakdown of food. Insoluble grit given to chick- ens remains in their gi/zard and assists in grinding, crushing and breaking up food particles (Bruce 1976). Sill is believed to have a similar effect in the style sac of mussels (Murken 1976). Sand and grit, which may have a grinding effect in the gut. is taken in by wild abalone when they feed on their natural diet algae. In the wild, abalone consume algae by either catching it as it drifts past or by grazing it off rocks. When they graze rocks, they also take in sand and other sediments. The fine sand and grit can be seen in their intestine when dissected. A study on the composition tif food in the stomachs of wild H. laevigata. H. rubra and H. nwi (Shep- herd 1973) revealed algae, and other browsed matter, including sand grains, small gastropods, bryozoa and detrital matter. The stomachs of H. laevigata and H. scalaris also contained quartz sand (Shepherd & Cannon 1988). Kaolin contains approximately 46.3% silica (Reis 1908). Like sand, silica may have an abrasive/ grinding action in the gut of abalone. However, the mechanism that kaolin improves digestion in abalone through its grinding effect in the abalone's gizzard implies that the addition of sand, bentonite and diatoinaceous earth would be expected to increase their digestibility. Therefore, it does not seem likely that a grinding effect of kaolin caused the increase in digestibility observed in this present study. Supplementation of Minerals It is possible kaolin is not nonnutritive but is actually supplying essential minerals that abalone require but that are deficient in manufactured diets. Deshimaru and Kuroki (1974), using a semi- purified diet, found that mineral rich diets (as high as 19.5% ash) produced the best growth of Penaeiis japoniciis. Similarly, Wheeler and Oltjen (1977) reported faster daily weight gains, feed conversion efficiency and carcass quality in finishing steers fed a corn and hay ration cttntaining 8% protein and 3.5% cement kiln dust to replace trace minerals, than in control animals receiving an otherwise similar diet containing 1 2% protein plus trace minerals. They speculated that the cement kiln dust may have provided hitherto unknown but necessary trace minerals. Also, increasing levels of bentonite in a high roughage ration for steers reduced the retention of dietary calcium but increased the retention of phos- phorous (Martin et al. 1969). Thus kaolin may have provided minerals essential for certain enzyme systems and biochemical functions involved in digestion in abalone but lacking in the diet used in this study. Alteration of Gut Transport Time It is possible that kaolin increases digestion in abalone by al- tering gut transport lime. Increased gut transport time can increase digestibility, because the food is exposed for a longer time to digestive enzymes. The improved caloric efficiency by the addi- The Effect of Fillers in Abalone Diets 797 tion of kaolin to poultry diets as observed by Osterhaut (1967), was due possibly to a slowing down of the rate of feed passage through the intestinal tract (Quinsberry 1968). Kurniek and Reid (1960), in their study of the effect of the addition of bentonite to 3 diets differing in energy levels for Leghorn cockerels, observed a sig- nificant increase in growth rate with the feeding of 2.5% bentonite to the low energy level diet. For each diet, the feeding of bentonite in the diet slightly delayed the passage of feed through the diges- tive tract. Similarly, by testing the effect of inclusion of silica, cellulose and chabamin at 10% and 20% in diets for European seabass juveniles Dias et al. (1998) observed that in fish fed the control diet, the totality of the feces was expelled after 31 h. whereas in those fed with 20% bulk incorporated diets, fecal eges- tion continued over 35 h. By contrast, however. Grove et al. ( 1978) reported that the gastric evacuation time in rainbow trout was reduced from 15 h to 10 h when the energy content of the feed pellet was reduced by 50% by dilution with kaolin. In the wild, herbivores, such as abalone, eat macrophytes, which are low in digestibility and possess high levels of indigest- ible materials that pass rapidly thi'ough the gut (Wee 1992). Their relatively long guts increase retention time and allow enzymes to work on the ingested material to optimize nutrient extraction and absorption. Wild abalone take appro.ximately 24 h to digest pre- ferred species of algae and species less digestible may remain identifiable in abalone guts for more than 48 h (Foale & Day 1992. Day & Cook 1995). The addition of indigestible kaolin to the diet may have affected transit time in a manner similar to that of poorly digestible algal species that remain in the gut for a longer period of time, thereby increasing exposure to digestive enzymes. The di- gesta transit times of the diets in this study were not measured and thus cannot be compared, however, an increase in transit time caused by the addition of kaolin can not be ruled out as a possible mechanism for the increase in energy digestibility of the diet. Reduction in Diet Viscosity Kaolin may have increased the gross energy digestibility of the commercial diet by reducing its viscosity and hence increasing access to it by digestive enzymes. The basal diet used in this study consisted mainly of semolina and soyflour. It is well known, par- ticularly for poultry, that soluble polysaccharide (fiber), such as arabinoxylans in wheat (semolina) and (J-glucans in barley and oats elicit negative effects on digestion through increasing intes- tinal viscosity (Annison 1990. Bedford et al. 1991, Choct & An- nison 1992, Annison 1993, Choct et al. 1996, Dusel et al. 1997). High gut viscosity decreases the rate of diffusion of substrates and digestive enzymes and hinders their effective interaction at the mucosal surface (Choct 1997). The viscous polysaccharides prob- ably complex directly with digestive enzymes and reduce their activity (Ikeda & Kusano 1983). As with poultry, non-starch polysaccharides in wheat may also elicit negative effects on di- gestion in abalone through increasing intestinal viscosity. There- fore, the significant improvement in digestion by the addition of kaolin may be because it decreases intestinal viscosity. If kaolin did increase the digestibility in abalone by decreasing the diet's viscosity, the fact that other fillers used (bentonite. di- atomaceous earth and sand) had no effect on its digestibility may be due to their physical/chemical properties. The contradictory results of the effects of nonnutritive fillers incorporation on feed evacuation time in fish are. in some cases, related to the different physico-chemical properties of the various bulk agents tested (Dias et al. 1998). The properties, such as ion binding or water holding capacity, have a strong influence on solubility, gelling and viscos- ity of food during its passage through the intestinal tract. The phenomenon of gelation caused by polysaccharides is because they are hydrophylic molecules and consequently have the ability to hold water. Kaolin is hydrophilic. disperses in water readily, is non-expanding, has low viscosity, low sorptivity, and a low sur- face charge (Murray 1993). Thus, it may reduce viscosity caused by soluble polysaccharides in diets by binding with water and reducing the polysaccharide's gelation ability. The same did not occur with the other fillers and this may be due to their different chemical properties. For example, bentonite, albeit hydrophilic, has high viscosity, high sorptivity, is expanding, and has a high surface charge (Murray 1993). The effect of nonnutritive fillers seems related to their physico-chemical properties and properties of other ingredients in the diet. Thus, the positive effect of kaolin on digestibility may have been related to the basal ingredients in the diets and it may not have a positive effect on diets with dif- ferent basal ingredients. ACKNOWLEDGMENTS The authors are grateful to Murray Frith for chromic oxide analysis and Debra Partington for help with .statistical analysis. This research was supported by financial grants from the Fisheries Research and Development Corporation. Annison. G. 1990. 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Anti-nutritive effect of wheat pentosans in broiler chickens: roles of viscosity and gut microflora. British Poultry Sci. 33:821-834. Choct, M., R. J. Hughes. J. Wang, M. R. Bedford. A. J. Morgan & G. Annison. 1996. Increased small intestinal fermentation is partly respon- sible for the anti-nutritive activity of non-starch polysaccharides in chickens. British Poultry Sci. 37:609-621. Day. R. & P. A. Cook. 1995. Bias towards brown algae in determining diet and food preferences: the South African abalone Haliotis mulue. Mu- rine and Freshwater Research. 46(3):623-627. Deshimaru O. & K. Kuroki. 1974. Studies on a purified diet for prawn-l. Basal composition of diet. Nippon Suisan Gakkaishi 40:413^19. Deshimaru. O. & K. Kuroki. 1974. Studies on a purified diet for prawn-I. Basal composition of diet. Nippon Suisan Gakkaishi 40:41.^^19. Dias. J.. C. Huelvan. M. T. Dinis & R. Metailler. 1998. 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THE DIGESTIBILITY OF WHOLE AND DEHULLED LUPINS (LUPINUS ANGUSTIFOUUS) FED TO JUVENILE GREENLIP ABALONE, HAUOTIS LAEVIGATA MEEGAN E. VANDEPEER,' PATRICK W. HONE," JON N. HAVENHAND,^ AND ROBERT J. VAN BARNEVELD^ ^South Australian Research and Development Institute. PO Box 120. Henley Beach. South Australia 5022: -Fisheries Research and Development Corporati(m. PO Box 222. Deakiii West. Australian Capital Territoiy 2600: -Flinders University. GPO Box 2100. Adelaide. South Australia 5001: ^Barneveld Nutrition Ptv. Ltd.. 19-27 Coonaii Road. South Maclean. Queensland. Australia 4280 ABSTRACT The apparent digestihility of protein, amino acids, and gross energy from whole and dehulled lupms {l.iipimis an^iis- lifoliiis) and defatted soyflour was investigated for greenhp ahalone, Haliotis laevificiia Donovan. Protein digestibility of all three feedstuffs was high with a coefficient of 0.91 obtained for soytloiir and whole lupins and a coefficient of 0.92 obtained for dehulled lupins. Gross energy digestibility was much more variable with the energy from soyflour being significantly more digestible (coef- ficient of 0.87) than that from dehulled lupins (coefficient of 0.83). which was significantly more digestible than the energy from whole lupins (coefficient of 0.50). The significantly lower energy digestibility of the whole lupins compared with the dehulled lupins could possibly be due to abalone's poor ability to digest cellulose, which is the major component of the hull of L aiif;iislifi>liiis. KEY WORDS: ahalone, digestibility, lupins. Luphiiis antiit.sufnliiis. Halioiis liicvigiilii INTRODUCTION hull. To investigate this the digestibility of both whole and de- hulled L aiif^iistifoliiis for abalone was determined. In Australia, lupins, in particular the species Lupinus angusti- foliiis. are used as a source of dietary protein and energy for both pigs and poultry. L. angustifdlius has protein levels ranging from 27 1 .9 to 372.3 g/kg and a gross energy content of 1 7.9-1 8.6 MJ/kg (Petterson et al. 1997). Lupins are an attractive feedstuff for use in animal diets, as selective breeding programs have produced vari- eties of lupins with low concentrations of anti-nutritive factors such as alkaloids (Annison et al. 1996). Successful results have been obtained on incorporation of L. angustifolius in manufactured diets for a range of aquaculture spe- cies including juvenile prawns, silver perch, snapper, carp, gilt- head seabream and rainbow trout (Jenkins et al. 1994. Viola 1988. Robaina et al. 1995, Gomes et al. 1995, Smith et al. 1998, Allan et al. 1998). Jenkins et al. ( 1994) found no significant differences in growth rates of snapper when fed balanced diets containing either 20% soybean meal, 15% L. angustifolius seed meal plus 13% soybean or 28% L. angustifolius seed meal. Similarly, excellent results with L. angustifolius have been found for carp. Growth of carp fed a diet containing 30% lupins exceeded that of a control diet by 25% while carp fed a diet containing 45% lupins grew at the same rate as those on the control diet (Viola et al. 1988). Robaina et al. (1995) found no significant difference in weight gain, protein efficiency ratio and feed efficiency between gilthead seabream (Sparus aurata) fed a fishmeal based control diet and a control diet with 30% of the fishmeal protein replaced by L an- gustifolius as a source of protein. Positive results have also been reported for prawns with no significant differences in daily growth rate, feed intake, feed conversion rate, protein conversion effi- ciency or apparent protein utilization of P. monodon when fed diets containing either dehulled L. angustifolius or deffated soy- bean meal as the main source of protein (Sudaryono et al. 1999). This study was undertaken to determine the apparent digest- ibility of protein, amino acids and gross energy from the lupin, Lupinus angustifolius, for greenlip abalone, Haliotis laevigata. A potential problem that could affect abalone's ability to digest en- ergy from L. angustifolius is the large proportion of cellulose in its MATERIALS AND METHODS Diets Two lupin diets, one containing whole, and the other containing dehulled L. angustifolius. were formulated (Table 1). A diet con- taining defatted soyflour was formulated as a control diet for com- parative purposes ("Bakers Nutrisoy" brand; Archer Daniels Mid- land Company, Decatur, Illinois). Each feedstuff was the sole source of protein in the diet and each diet was formulated to have a crude protein content of 160 g/kg. The lupins were ground in a hammer mill and then in a ball mill before inclusion in the diets. Equal amounts of vitamins and ininerals. as described by Uki et al. (1985). Jack Mackerel oil (Triabunna Fish Oils, Triabunna, Tas- mania), and pre-gelatinized maize starch were added to each diet. Kaolin was included as filler and chromic oxide was included as an indigestible marker (0.5%) for use in digestibility calculations. Shipton and Britz (2001 ) found that chrotnic oxide was a suitable marker in protein digestibility studies on Haliotis midae as it was inert, was not absorbed, did not interfere with the digestive pro- cess, and moved through the intestine at a similar rate to the protein. Proximate analyses of the experimental diets and of each feedstuff were determined (Table 2 and Table 3). Diet Allocations The three diets were randomly allocated to 4 of 12 digestibility tanks to provide four replicate fecal samples per diet based on a completely randomized design. .Xhalnne Feeding and Feeal Collection Abalone and Feeding Juvenile greenlip abalone (shell length 40-60 mm, 70 g wet weight) were used in the experiment. The abalone had been ob- tained from a commercial hatchery and raised on manufactured abalone feed. The abalone were preconditioned for one week on 799 800 Vandepeer et al. TABLE L Composition of experimental diets (g/kg, air dry basis). TABLE 2. Proximate analysis, total amino acid content (g/k;;. air dry basis), and gross energy (M.j/kg, air dry basis! of experimental diets. Whole Dehulled Lupin Lupin 500.0 421.1 100.0 100.0 369.4 448.4 Soyflour .Soytlour' 33.S.4 Lupin (whole)" Lupin (dehulled)- Pre-gelatinised starch' 100.0 Kaolm .';34.0 Diet Soyflour Diet Whole Lupin Diet Dehulled Lupin Diet Crude protein (N x 6.25) Gross energy (MJ/kg) Dry matter Ash 167.5 8.28 968.1 539.4 169.8 9.88 950.0 373.8 155.0 10.45 974.6 462.2 Jack Mackerel oil 20.0 Mineral premix"* 2.0 Vitamin preniix'' 3.0 Ascorbic acid 0.5 DL- -tocopheryl acetate 0. 1 20.0 20.0 2.0 2.0 3.0 3.0 0.5 0.5 0.1 0,1 Fiber extract Crude Acid detergent Neutral detergent Ether extract (bp. 40-60°C) 25.0 427.6 719.3 12.4 34.3 270.0 46').0 38.1 55.3 387.8 618.6 38.6 Chromic oxide 5.0 5.0 5(1 Anuno acids Aspartic acid Glutamic acid Serine Glycine 11.60 26.10 7.70 6.75 11.50 32.50 8.50 7.00 9.30 23.40 6.60 6.05 ' Defatted soyflour (Baker's Nutrisoy; ADM). " Liipiiuis cingiistifolius cv. Gungunu. ^ Pre-gelatinized waxy maize starch (Wades BOl IC, Inpak Foods. South Australia). "* Vitamin and mineral preniixcs as described by Uki el al. ( 1985). Histidine Arginine 3.95 9.60 4.00 16,0(1 4.15 13,00 Threonine 6.50 6,00 4.95 the test diet assigned to their tank. During both the preconditioning Alanine 7.05 5.50 5.10 and experimental periods the animals were fed to excess every day at approximately 1700 h. Proline Tyrosine Valine 14.0 5.70 8.30 7.50 6.50 5.50 10.55 5.70 5.65 Tanks and Collection System Methionine Cystine 1.25 3.0(1 2.50 2.00 1.05 1,65 Conical shaped digestibility tanks were used. Abalone were Isoleucine 7.75 6.50 5.85 housed in 20-L buckets (approximately 80-100 per bucket) that fitted inside the tanks. All the buckets were fitted with plastic mesh bottoms ( L3 X 1.5 cm mesh) allowinc containment of the abalone Leucine Phcnylanaline Lysine 13.45 8.85 8.20 12.00 6.50 7.50 9.80 6.05 4.80 while permitting feces to drop into the collection tube at the base of the tank. Three 25-cm lengths of PVC pipe (8 cm in diameter) were placed in the buckets as shelters for the abalone. Attached to the bottom of each digestibility tank was a screw-on collection tube ( 1 1 cm long, 15 mm dianieter). Tanks were on a flow-through water system at a rate of about 2 L/min. The seawater was tillered to 30 p.m by primary sand filters, then to 10 jjim by secondary composite sand filters before entering the tanks. Aeration was supplied at 0.5 L/min to each tank at all times by an air stone. Water temperature and lighting were controlled during the experi- ment with temperature maintained at I8.0°C ± 1.0 and a light regimen of 12 h light: 12 h dark regimen. Salinity was 35~36%f throughout the experiment Fecal Collection Fecal collection occurred every day until 6-10 g (dry weight) of feces was collected for each replicate sample. This took 22 days in total. On each day of fecal collection the buckets containing the abalone were removed and the digestibility tanks were drained of water and all fittings were cleaned of feces and uneaten feed. Abalone were out of water for about 1 to 2 min. Following clean- ing, the tanks were refilled and the buckets replaced. Collection tubes were fitted by 0900 h A small foam container was placed underneath each tube and filled with ice to keep the collecting feces cold and thus reduce degradation by microbial action. The feces were collected from the tubes at about 1630 h by gently pouring the contents onto a 1 mm mesh. The mesh was then placed into a petri dish and frozen at -30°C. The following day the frozen fecal sample was scraped off the mesh, pooled into a composite sample, and replaced into the freezer until required for analysis. Prior to analysis the samples were freeze-dried and ground with a mortar and pestle. Chemical Analyses Chemical analysis for proximates (i.e.. dry matter, ash. ether extract, neutral-detergent fiber, acid-detergent fiber, and crude fi- ber) were undertaken using the methods of the Association of Official Analytical Chemists (1984). Gross energy was determined using a Parr 1 28 1 bomb calorimeter. Proteins were analyzed by the combustion method using a LECO® CN-2000 Carbon and Nitro- gen Analyser (Royal Australian Chemical Institute 1999). All amino acids were determined by the Water's PICOTAG amino acid analysis method (Sarwaret al. 1988) using pre-column phenylisothiocyanate (PITC) derivilization and liquid chromatog- raphy was used for the determination of all amino acids. Protein sources were hydrolyzed for 22 h in duplicate with 6 N HCL at 1 10°C for the determination of all amino acids, except for methi- onine and cystine. Hydrolysates for the determination of methio- nine as methionine sulfone and cystine as cysteic acid were pre- pared by performic acid oxidation of the protein prior to hydrolysis using 6 N HCL. Alpha-aminobutyric acid was used as an internal standard. Chromic oxide was determined using atomic absorption spec- troscopy based on a modification of Hillebrand et al.'s (1953) method. The modified method involved preliminary ignition of the sample at 500°C to remove organic material and the dissolution of the sample in hydrochloric acid instead of sulphuric acid (M. Frith, The Digestibility of Lupins for Abalone 801 TABLE 3. Proximate analysis and total amino acid content (};/kg. air dry basis), and gross energy content (MJ/kg, air-dry basis) of soytlour, whole and dehulled lupins used in experimental diets. Feedstuff Soyflour Whole Lupin Dehulled Lupin Crude prolein (N x 6.25) 454.0 338.6 380.0 Gross energy (MJ/kg) 17.33 17.74 18.28 Dry matter 876.5 894.5 891.9 Ash 58.8 25.0 25.3 Fibre extract Crude 30.5 76.9 93.9 Acid detergent 79.9 132.8 108.6 Neutral detergent 94.6 186.3 157.3 Ether extract (bp. 40-60°C) 7.5 63.5 64.4 Amino acids Asparticacid 61.40 32.85 41.00 Glutamic acid 88.10 75.85 82.15 Serine 26.15 18.00 18.45 Glycine 20.80 14.85 14.95 Histidine 12.70 7.60 9.85 Arginlne 35.65 37.65 37.60 Threonine 18.(15 12.15 13.05 Alamne 19.10 11.60 11.65 Proline 20.65 21.15 23.95 Tyrosine 17.95 13.55 12.35 Valine 24.45 12.50 13.45 Methionine 6.10 2.35 2.00 Cystme 6.70 3.50 2.00 Isoleucine 23.10 13.20 14.30 Leucine 36.90 21.40 22.75 Phenylanaline 24.95 13.45 14.05 Lysine 30.85 14.60 15.35 personal communication. University of Tasmania. Launceston, Australia). Digestibility Determination The apparent digestibilities of nutrients in the diets were cal- culated using the following formula (Hardy 1997): Apparent digestibility = 1 Cr.iu'i ^ l^'i"''eM/„c. Cr,, 'jeces ^ Nutrient j,^,,/ where O" is chniniium content and Nutrient is nutrient or energy content of the diet. The digestibility of gross energy for each ingredient was cal- culated by subtracting the amount of digestible energy contributed from the oil, sodium alginate and pre-gelatini/ed starch in each diet. Statistical A nalysis The data were analyzed by a general linear model and the treatment means were cotnpared by least significant difference. Prior to analysis, data were analyzed using a univariate procedure and normal plots to establish that the data were in fact normally distributed, as was the case. The presence of outliers was assessed using the RANK procedure in SAS (SAS Instittite Inc.. 1988) and normal scores computed from the ranks following Blom (1958). As no outliers were detected using the RANK procedure, all data were used in the calculation of digestibility estimates for all pa- rameters, respectively. RESULTS A significant difference was found among the lupins and soy- llour in abalone's digestibility of protein (F-,,, = 17.22, P = 0.0033). gross energy (F^^, = 1340.96, P = 0.0001), arginine (F, 7.24. P = 0.0251). proline (F,„ = 10.87, P = O.OIOI). methionine (F, , 9.16. P = 0.0150) and isoleucine (F,, 18.94, P = 0.0026) (Table 4). Dehulled lupins had significantly higher apparent gross energy digestibility for abalone than whole lupins, but had significantly lower gross energy digestibility for abalone than soyflour (Table 4). The protein fnmi dehulled lupins was significantly more di- gestible than that from either whole lupins or soyflour (Table 4). DISCUSSION Dehulling significantly improves abalone's digestibility of en- ergy from L. anf^Ksrifolius. The low gross energy digestibility of whole L. in}i;iislitoliiis seeds and the significant increase in gross energy digestibility of dehulled compared with whole L. angiisti- folius has also been observed for other aquacultured species. For example, the gross energy digestibility increased from 45-74% for juvenile P. monodon (Smith et al. 1998) and from 59.4-74% for silver perch {Bidxanus Indyamis) (Allan et al. 1998) by use of dehulled as against whole L. angustifoUus lupins. The abalone's significantly lower digestibility of energy from whole compared with dehulled L. angustifoUus indicates it has a poor capacity to digest the cellulose in lupins. Cellulose constitutes approximately 57.3-58.4% of the hull of L angustifoUus (Evans & Cheung, 1993) and thus is likely to be cause of abalone's poor gross energy digestibility of whole L. angustifoUus. Indeed, Uki et al. (1985) demonstrated that the growth rate of Haliotis discus hannai decreased as the cellulose content of the diet increased from 0-20% and concluded that abalone must have a poor capacity for digesting it. Some may expect that abalone should be able to digest cellu- lose, given that it forms the structural basis of many algae, their natural diet (McCandless 1985) and cellulase activity has been documented for a number of Haliotis spp. (Gianfreda et al. 1979, Elyakova et al. 1981, Boyen et al. 1990, Gomez-Pinchetti & Gar- cia-Reina 1993). A complication in interpretation of those results is that despite many studies, the origin of the cellulose activity in abalone is inconclusive. This is because most studies have not identified whether the cellulase was of microbial origin. Erasmus ( 1997) was the first to demonstrate that abalone possess a cellulase by detecting the presence of carboxymethylcellulase in gnotobiotic H. midae. Erasmus (1997) suggested that this cellulase was most likely to have been poly-fi-glucanase, which hydrolyzes car- boxymethylcellulose. Whether abalone pos.sess the other two cel- lulases required to hydrolyze cellulose remains to be confirmed. The substrate carboxymethylcellulose does not require a true cel- lulase for hydrolysis. Erasmus et al. ( 1997) hypothesized that bac- teria possibly secrete a true cellulase to completely degrade cellu- lose and abalone only partially hydrolyze the substrate. This hy- pothesis was supported by Enriquez et al. (2000) who examined the in vitro digestion of pure cellulose (alphacel) using stomach extracts from Haliotis fulgens and reported that bacteria play an important role in cellulose digestion in abalone, as stomach ex- 802 Vandepeer et al. TABLE 4. Apparent faecal digestibility coefficients of protein, gross energy and amino acids in soyflour, whole and dehulled L. anumtifoliiis lupins fed to juvenile greenlip abalone I//. laevif;ala). Data are mean ± SK, ii = 4. Statistics Whole Lupin Dehulled Lupin Feedstuff Soytlour P SEM LSD Protein 0.904' ±0.00151 0.909" ± 0.00267 0.919" ±0.00222 ** 0.00199 0.0069 Gross energy 0.870'' ± 0.00293 0.499*" ± 0.00502 0.83 r± 0.00838 *** 0.00556 0.0192 Amino acids Aspartic acid 0.980 ±0.00153 0.949 ± 0.03647 0.978 ±0.00130 NS 0.02078 Glutamic acid 0.981 ±0.00146 0.982 ±0.00168 0.979 ±0.00124 NS 0.00157 Serine 0.938 ± 0.00233 0.935 ± 0.00229 0.938 ± 0.00335 NS 0.00228 Glycine 0.919 ±0.00573 0.924 ± 0.00306 0.923 ± 0.00924 NS 0.00591 Histidine 0.914 ±0.00702 0.918 ±0.00448 0.929 ± 0.00607 NS 0.00655 Arginine 0.947" ± 0.00425 0.962" ±0.00142 0.962" ± 0.00230 * 0.00306 0.0106 Threonine 0.880 ±0.00716 0.895 ± 0.00360 0.884 ± 0.00666 NS 0.00472 Alanine 0.902 + 0.00561 0.889 ± 0.00250 0.893 ± 0.00363 NS 0.00361 Proline 0.947" ± 0.00549 0.9 16" ±0.00927 0.945" ± 0.00450 * 0.00525 0.0182 Tyrosine 0.885 ± 0.00397 0.908 ±0.00153 0.907 ±0.01080 NS 0.00723 Valine 0.873 ±0.00170 0.857 ± 0.00367 0.869 ± 0.00808 NS 0.00447 Methionine 0.815" ±0.01077 0.907" ± 0.00474 0.828" ± 0.02372 * 0.01647 0.0150 Cystine 0.972 ±0.01379 0.975 ± 0.00688 0.969 ±0.01379 NS 0.00344 Isoleucine 0.693" ± 0.00664 0.660" ± 0.00225 0.596" ± 0.00664 ** 0.01 130 0.039 1 Leucine 0.881 ±0.00716 0.888 + 0.00174 0.887 ±0.00716 NS 0.00455 Phenylanaline 0.870 ±0.01009 0.875 ± 0.00206 0.870 ±0.01010 NS 0.00577 Lysine 0.927 ± 0.00895 0.920 ± 0.00634 0.913 + 0.00895 NS 0.00624 NS, not .significant. * P < 0.05 ** P < 0.01 ***/>< 0.001 SEM. standard error of the mean LSD, least significant difference Values in a row with different superscripts differ significantly (t,)( 2.45). tracts showed significantly decreased cellulolytic activity in the presence of antibiotics. A factor to consider when evaluating abalone's ability to digest cellulose based on previous studies done to determine cellulase activity, is that many have estimated cellulase activity by exam- ining the production of glucose from abalone stomach homoge- nates incubated with a-cellulose. This type of cellulose is a powder prepared by treating de waxed cotton (ethanol extracted) for 8 h with 1% boiling sodium hydroxide solution (Whistler & Smart 1953). Thus, it is probably much easier for the abalone's own endogenous enzymes and gut bacterial enzymes to digest then cellulose present naturally in algae or in land based plants. Evi- dence for this is that cellulose that has been treated with alkali to increase the proportion of amorphous cellulose is more rapidly decoiTiposed than untreated cellulose (Whistler & Smart 1953). In addition bacterial attack of cellulose is inhibited by the presence of lignin (also a component of lupin hulls). Thus bacteria that readily hydrolyze isolated cellulose may have little, if any. effect on wood (Whistler & Smart 1953). In addition, algae contain only low levels of cellulose. In divisions Phaeophyta. Rhodiiphyta and much of the Chlorophyta. cellulose is a minor coinponent of the struc- tural polysaccharides. In fact, in Phaeophytes and Rhodophytes. the amount of a-cellulose ranges from 1 to 8% of thallus dry weight whereas the proportion is often 30% in terrestrial plants (Kloareg & Quatrano 1988). Thus it is possible that although aba- lone possess cellulase it does not necessarily mean that they are able to effectively hydrolyze the cellulose present in terrestrial plants, thereby resulting in the lower gross energy digestibility of the whole compared with dehulled lupins in this study. Abalone must be able to digest the major energy reserves in the cotyledon of lupins (lipids and cell wall non-starch polysaccha- rides (Evans 1994) relatively well, as indicated by the significant increase in gross energy digestibility of the lupins after dehulling. The lipid content of the cotyledon is relatively low and comprises approximately 85 g/kg by dry weight (Evans & Cheung 1993). The predominant monosaccharides in the cotyledon non-starch polysaccharides are galactose (67%), arabinose (12%) and uronic acid ( 10%) residues (Evans & Cheung 1993), and are the constitu- ent sugars of the reserve pectic substances. Pectin is also one of the principal polysaccharides in cell walls of algae (Chapman & Chap- man 1973). and so it would not be surprising if abalone were able to digest it. The finding of comparable digestibility of lupins and soyflour indicates that there is potential lor use of lupins in manufactured feeds for abalone. Since lupins are considerably cheaper than de- fatted soyflour. the replacement of soyflour with lupins in diets would result in a reduction in the cost of manufactured feeds. ACKNOWLEDGMENTS The authors thank Murray Frith for chromic oxide analysis, Tom Lam for amino acid analysis and Debra Pailington for help with statistical analysis. This research was supported by financial grants from the Fisheries Research and Development Corporation. The Digestibility of Lupins for Abalone 803 LITERATURE CITED Allan, G. L.. V. P. Gleeson. A. J. Evans & D. A. J. Stone. 1998. Replace- ment of fish meal in diets of silver perch: digestibility of Australian lupins. In: Fishmeal replacement in aquaculture feeds for silver perch. Project 93/120-03. Final report to the Fisheries Research and Devel- opment Corporation, Canberra, Australia. 233 pp. Association of Official Analytical Chemists. 1984. Official Methods of Analysis of the Association of Official Analytical Chemists. 14th edi- tion. 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Fisheries and Culture, University of Capetown, February 6-1 1 2000. 18 pp. Erasmus, J. H., P. A. Cook & V. E. Coyne. 1997. The role of bacteria in the digestion of seaweed by the abalone Hcilii>ti.\ midae. Aquaculture 155:377-386. Evans. A. J. 1994. The carbohydrates of lupins, composition and uses. In: Proceedings of the first Australian Lupin Technical Symposium. Perth. Western Australia. October 17-21. 1994. Evans. A. J. & P. C-K Cheung. 1993. The carbohydrate composition of cotyledons and hulls of cultivars of Lupiiius imi>ustifolius from Western Australia. J. Sci. Food. Agric. 61:189-194. Gianfreda, L., A. Imperato, R. Palescandolo & V. Scardi. 1979. Distribu- tion of P-1.4-glucanase and P-glucosidase activities among marine molluscs with different feeding habit. Comp. Biochem. Physiol. 63B: 345-348. Gomes. E. F.. P. Rema & S. J. Kaushik. 1995. Replacement of fish meal by plant proteins in the diet of rainbow trout iOucorhynchus inyki.ss): digestibility and growth pertbrmance. 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Unusual feedstuffs (tapioca and lupin) as ingredients for carp and tilapia feeds in intensive culture. Isr. J. Aquacult. Bamidgeh 49:29-34. Whistler. R. L. & C. L. Smart. 1953. Polysaccharide Chemistry. New York: Academic Press Inc. pp. 66-1 1 1 . Journal ofShellthh Research. Vol. 21, No. 2. 805-809. 2002. LARVAL ESCAPE THROUGH ABALONE CULTURE EFFLUENT SYSTEMS: AN ANALYSIS OF THE RISK C. D. HAWKINS'* AND J. B. JONES" ^ Depuriment of AiiricuUitre. Western Australia. PO Box 16. Moora, WA 6510, Australia: 'Department of Fisheries Western Australia. Research Division. P.O. Bo.\ 20, North Beach. WA 6020. Australia ABSTRACT Expansion of atialone culture in Western Australia has resulted in the acquisition of juvenile stock (Haliotis laevigala) from interstate, panicularly South Australia. This movement has raised concerns about the possibility of compromising the genetic integrity of domestic wild stocks of abalone with e.xotic genetic material. Such a compromise could arise from the escape of larvae from a production system in the event of a spawning event during the growing period. The use of a fine mesh filter may reduce this risk but is considered impractical because of frequent blockage. A risk model was constructed, which interfaces with an abalone growth model, to predict spawning events and the likelihood of viable larvae surviving in the open sea until they mature and spawn again. The model predicts that the probability of a spawning event leading to larvae escaping, maturing and spawning again in the wild is in the order of 3.7 x 10"'' per production cycle (about 3 y), and if such an event occurred, around 10 abalone would reach maturity, or about 1 7 in the absence of a settling pond for effluent water before discharge. Sensitivity of the model to input variables and implications for further research are discussed. KEY WORDS: abalone culture, larval escape, risk analysis INTRODUCTION Abalone {Haliotis spp.) are highly prized edible marine gastro- pod mollusks. Their culture is increasing around the coastline of Australia with national production anticipated in the order of 485 tons in 2000-2001 (Hone & Fleming 1997). Increasing world prices for abalone have increased the viability of abalone aquacul- ture. with the majority of Australian aquaculture production being in South Australia. Victoria, and Tasmania. The industry is com- parable to most intensive animal production systems, with a degree of specialization occurring. In particular, the potential exists for the production of large quantities of juvenile abalone for sale to grower units enabling growers to focus on a single aspect of pro- duction by purchasing stock from a specialist producer. Production occurs in both sea- and land-based systems, with a trend toward land-based systems. Land-based systems tend to be operated close to the ocean, with sea-water supplied from the ocean, and effluent returned some distance from the inlet system. Australian abalone farms vary in size and layout. The profitability of such farms with either a 100 or 200 ton annual production was modeled by Weston et al. (2001 ). Development of the industry in Western Australia is expanding, although only a small number of hatcheries have been established to date. To facilitate industry development, there has been pressure on Government to permit the introduction of juvenile stock from other states in Australia. Movement of any aquaculture stock into Western Australia is subject to an environmental assessment pro- cess. When assessing the movement of juvenile green-lip abalone (Haliotis laevigata) from South Australia to Western Australia, a concern was expressed that the genetic integrity of the Western Australian stocks could be compromised. During the growing pe- riod of some three years, it was hypothesized that a spawning event could occur in the growing tanks, resulting in discharge of viable juvenile abalone into the wild. Although the species of abalone in Western Australia is the same as that around other parts of the coastline, biotypes may vary (Brown 1991. Elliot et al. 2001 ) and an exotic or hybrid biotype may establish in the wild following a spawning event (Gaffney et al. 1996). *Corresponding author. E-mail; chawkins@agric.wa.gov.au This proposed risk to the genetic integrity of wild abalone has been the subject of unpublished consultancies, and there is evi- dence that in greenlip abalone. stocks are genetically differenti- ated, that differences are detectable over small distances, and that interaction between different stocks is limited. More recent data published by Hancock (2000) show only small differences among Haliotis roei populations in Western Australia and, on this basis, genetic zones should no longer be applied for broodstock collec- tion with this species. For black lip abalone (H. rubra), genetic differences seem to be even smaller than for green lip. with data on other species being inadequate for reliable conclusions to be made (Westaway & Norriss 1997). However, the potential for introduc- ing new genetic material into existing stocks, and the possibility of reducing genetic diversity remain of concern to environmentalists and the abalone aquaculture industry, and management of this perceived risk is appropriate. Recommended risk management strategies include the use of progeny derived from a sufficiently large number of parent stock to ensure adequate genetic diversity; obtaining broodstock from local populations; and locating the sea culture facilities at a prescribed distance from wild populations of abalone. In addition, circumstances may require that effluent tank water be discharged over sand or filtered to minimize the ability of escaped larvae to settle on a suitable substrate. CuiTcnt policy in this state dictates the use of a settlement pond or sediment filter for land-based farms. Other risk reduction measures have been suggested, including the culture of polyploid (specifically triploidi organisms and har- vesting before sexual maturity is reached. However, these options are not viable using present technology, because polyploidy is difficult to guarantee, and sexual maturity may occur some con- siderable time before harvestable size is reached in some species. One management strategy to minimize the risk of larval escape from abalone culture ventures is the use of an effluent filter to trap larvae should a spawning event occur. In practice, given the vol- ume of water involved, the use of a fine-mesh (100 |jim) filter on the discharge outlet from abalone farms poses significant difficul- ties because of rapid clogging with particulate matter and restric- tion of water flow. It is the intent of this risk assessment to quantify the probability of escape of larvae from a land-based abalone culture system using 805 806 Hawkins and Jones flow-through seawater in the absence of an effluent filter. The assessment relates to the risks of a spawning event, which is af- fected by the volume of water used, but independent of the number of abalone in the farm and their density. METHOD A quantitative risk model (Vose 2000) was constructed using Microsoft® Excel, with the Palisade® @Risk add-in. However, because of the changing risks as.sociated with the growth and de- velopment of abalone during the growing period, relevant parts of the growth and production system were also modeled. The model tracks abalone growth and associated aquaculture events daily. Abalone production is based on a grow-out raceway fed by seawater, the flow rate of which increases as the abalone grow. Initially, appro.ximately 0.2 megaliters (ML) of water per day is used, increasing to 40 ML per day for the 2 mo before harvest. A linear relationship between size and water flow is assumed: Flow Rate = Age (days) * 0.04 ML/day to day 1010, then 40 ML thereafter. Indications from Purdom ( 1 980) are that growth in fish is linear under optimal conditions, restrained by food, temperature, and sexual maturity. Social interaction has an effect in free swimming fish, but it is anticipated that such interaction is minimal in aba- lone. Hahn (1989) also showed that growth in length is relatively linear for abalone over a wide size range. Sexual maturity in green- lip abalone is generally after reaching 90 mm, so the constraints of feed and temperature remain the major ones, although precocious maturation occurs in abalone farms. Under farmed conditions, feed should be supplied ad libitum and temperatures maintained at or around optimum. Thus, a linear growth rate between 0.2 and 70 mm, over about 1,000 days is used. The growth expression is; Size (mm) = Age (days) * 0.07. Considerable attrition is noted in farmed abalone, with survival to maturity being very low. Large losses before settlement have been noted. Shepherd et al. (2000) observed .settlement rates be- tween 0.02-1.2% for H. rubra, and 0.1-6.5% for H. laevigata in experimental stock enhancement trials in South Australia. At the request of the Department of Fisheries, two linear death rates were used. In this situation, mortalities reach 93% by the time abalone are 5 mm (approximately day 1 27 of the model ) and a further 50% mortality by the time abalone reach 88 mm. Simple linear regres- sion lines were used for this purpose. Abalone are harvested at approximately 70 mm in length, some 20 mm short of the anticipated size for sexual maturity. However, abalone have been reported to spawn when only 25 mm in length. Synchronous spawning may occur in abalone, although Hahn (1989) reports that spawning is poorly synchronized in some spe- cies. Egg production is from 0.5 x 10'' to .3 x 10" per abalone, with larger shellfish producing more eggs. The relationship between size and spawn production is considered linear. Throughout this model, assumptions are made that favor the production of larvae and their escape, so as to avoid underestimating the risk. Thus, abalone as small as 15 mm are permitted to spawn in the model. The quantity of spawn (eggs) produced is calculated as: Spawn (millions) = length in mm * 0.023809524 + 0.142857143. Discharge water from the grow-out tanks is fed into a settling pond, which is believed to remove up to 80% of solid material. Although more accurate figures would be desirable, this percent- age is an upper limit, with the minimum and means probably being 30 and 65%. There is a negative correlation between the flow rate of seawater through the farm and the efficiency of the settling pond: Proportion settling in the pond is: flow (ML) * (-0.0125628) + 0.8. The effectiveness of the settling pond was evaluated by running the model with zero settling. From the settling pond, effluent is discharged through a pipe- line to a subsurface outlet in the sea. This outlet is located at the maximum practical distance from the seawater inlet for the facility. Discharge is required to be over sand, to minimize the chance of any abalone larvae that escape of finding a suitable (rocky) habitat on which to settle. However, the effect of discharge over sand is not directly modeled, rather a probability that such larvae will survive and settle is used. Water from the subsea discharge pipe is expected to plume rather than disperse evenly into the sea. thereby maintaining a higher level of suspended particles for some distance from the outlet. The model uses a random number generator to determine if a spawning event occurs during a growing cycle. It is assumed that spawning is an unexpected event that may occur once before har- vest. The likelihood of spawning is set as a probability, using a beta-pert distribution. This type of distribution has been chosen, because it offers the capacity to specify minimum, maximum, and most likely values, and the curve is such that the extremes are not favored, and the bulk of the values generated cluster around the most likely value. The shape of the beta-pert curve is one that can be grasped intuitively, appearing as a bell-shaped curve, with or without a skew. The values for the distribution are: Minimum: 0.001; Most Likely 0.005; Maximum 0.0095. That is, there is a 1 in 200 chance of a spawning event occurring, but this may be as low as 1 in 1,000, or as high as (approximately) 1 in 100. These data have been provided from production experience. Although spawning is most likely to occur with larger abalone in the third year of the growing period, the model allows spawning on any day once abalone reach 15 mm. (This may seem very low, but gonad maturation occurs very early for some species; e.g., Haliutis roei) The day on which the spawning event occurs is randomly set between the time abalone reach 15 mm, to the end of the growing period. When a spawning event occurs, a proportion of abalone present in the grow-out tank is able to spawn as females. This is also represented as a beta-pert distribution, with the following values: Minimum 0.05; Most Likely 0.1; Maximum 0.3. Proportions cal- culated in this manner were subsequently used to determine the number of individuals reaching the next stage, as a binomial prob- ability. Although a number of environmental variables interact at the time of spawning to influence fertilization, and the window of opportunity for fertilization may be short, is assumed that all spawn are fertilized — the worst case scenario. (In practice, any lag between males and females spawning in high flow rate tanks will impede fertilization.) There is always a concern that "something drastic could go wrong" — the unexpected, rare event that throws the whole system into disarray, such as a meteor crashing directly into the abalone farm. In this model, the possibility of a rare event is incorporated in the form of a total washout of all particulate matter from the grow-out tanks; that is, whatever fertilized spawn is present gets washed straight out to sea without the benefit of a settling tank. The likelihood of such a rare event is a beta-pert distribution with the following parameters: Minimum 0.0000001; most likely Abalone Larval Escape from Culture: A Risk Analysis 807 0.000001; Maximum 0.000005. That is. the event is most hkely to occur with a 1 in a milhon probabihty. but could be as high as 5 in a million, or as low as 1 in ten million. This parameter is included simply to allow for the occurrence of the most extreme adverse event. Any fertilized eggs (larvae) that escape the system are subject to several events: settlement, survival through the first year, and survival to sexual maturity. Although biologic events, these are treated as probability distributions as follows. Survival to settle- ment (Shepherd et ai. 2000) as a beta-pert distribution: Minimum 0.0002; Most likely value 0.02; Maximum 0.08. Survival for the first year — a uniform distribution between 8 and 16%. Survival from the first year to spawning as a beta-pert distribution: Mini- mum 0.1; most likely value 0.2; Maximum 0.5. (It should be noted that Shepherd et al. (2000) modeled survival of larvae released directly onto a reef. They also found that survival depended on the density of abalone already on the reeL Our model assumes that any larva escaping from the farm will be carried to a reef area as if it was being released for reseeding. Clearly, this is a worst case scenario.) An abalone reaching maturity and then spawning is considered the end point of the model. Outputs from the model are: • total fertilized spawn (larvae) discharged: • number of abalone surviving in the wild to spawn; and • probability of a spawning event in aquaculture. and subsequent survival to spawn in the wild. That IS. if a spawning event occurs, and there is no filter, how many larvae escape, how many survive to spawn again, and what probability can be associated with such events occurring? The model was run for 1.000 iterations, each iteration repre- senting a full grow-out cycle (approximately 3 y). A random num- ber seed is generated internally by (S'Risk, and this is allowed to vary between runs of the model. Consequently, results vary in detail between runs, but not in order of magnitude. Computation- ally, the model is a matrix of 17 variables calculated each day for 1,100 days (i.e., 18,700 individual variables determined on each iteration of the model), of which some 9,000 are generated as ©Risk statistical distributions. This matrix is treated as an Excel lookup table, and outputs are computed by accumulating looked up values over the total number of iterations. Hence, outputs are not single values, but aggregations of data that are presented as means with upper and lower 95% confidence intervals, where appropri- ate. Limitations imposed on the model by capacity of ©Risk to handle large numbers restricts maximum sizes used in some cal- culations. This was managed by performing such calculations on a per megaliter of water basis. For example, the use of binomial sampling is limited to population sizes of less than or equal to 32,767 — a number far smaller than the expected number of spawn produced by abalone. For the calculation of outputs, the total vol- ume of water is taken into account. Where appropriate, sensitivity of output variables to the inputs was analyzed using the facility available in @Risk. Suitable input variables are day of spawning, proportion of abalone spawning, and probability of a spawning event, the main drivers of the model. Other variables are not suited to this process, because they are calculated independently each day of the growing cycle (about 1,000 days), and consequently, each daily probability contributes to a very small component of the final output. The modeled abalone farm is stocked with five million juvenile abalone, of two millimeter (2 mm) length. TABLE I. Outputs from risk model, with a settling pond. Output Mean Value Lower 5% Confidence Interval Upper 95% Confidence Interval Total spawn discharged Abalone surviving to 13,184 10 3.920 0 26.80(1 40 spawn again Probability of spawning event and survival to spawn in the wild 3.7 X 10"" 6.1 X I0-' 9.5 X 10-" RESULTS Details of Output Variables Are Presented in Table I. The number of spawn discharged is variable. However, the mean value is under 20,000, with an upper confidence limit of the order of 30,000^0,000. Of these, very few survive, with around 10, but fewer than 100, expected to reach sexual maturity and subsequently spawn again. The likelihood that there will be a spawning event in the grow-out tank, and larvae will escape, de- veloping to sexual maturity, is very low. in the order of 4 in a million, but may be as high as 1 in 100,000. These results include the possibility of a rare, but catastrophic event, as described pre- viously. The effect of the settling tank, although detectable, was small. The effect of removing the settling tank from the model is given in Table 2. Output graphs for the data in Table 2 follow (Figs. 1-3). The impact of a "rare event" is small. Because the rare event operates in the same manner as removing the settling pond, the occurrence of a rare event, if it happened at the time of spawning, would be to increase the number of larvae escaping and reaching maturity. Because of the very low probability of a rare event, it does not affect the very low probability that larvae would escape and establish in the wild. Sensitivity Analysis As indicated in Tables I and 2. the model demonstrates that the presence of a settling pond reduces the quantity of larvae escaping if a spawning event occurs. However, because the effectiveness of the settling pond is not perfect, and its effectiveness decreases in efficiency as flow rates increase, the settling pond does not greatly affect the probability of larval escape. TABLE 2. Outputs from risk model, without a settling pond. Output Mean Value Lower 5% Confidence Interval Upper 95% Confidence Interval Total spawn discharged Abalone surviving to 20,796 17 8,382 0 38,520 80 spawn again Probability of spawning event and survival to spawn in the wild 4.0 X 10-" 6.5 X lO"' 9.1 X 10-" 808 Hawkins and Jones 5- CD CO > 4,500^ 4.000 3.500-- 3,000 2.500 2 000 1.500-- 1.000 0 500 0.000 leojp- 140- riitean=:3 972:.f.9E-0e 17,5 35 52 5 Spawn discharged (thousands) 5% 5% : 8 38 38 52 Figure 1. Distribution I frequency! histogram for total spawn dis- charged. Horizontal bar indicates percentiles. The built-in facility of @Risk to detenniiie the correlation of input variables to output variables indicates a correlation between total spawn discharged, and the input variables day of spawning (r = 0.8) and proportion of abalone spawning (r = 0.41). Survival of escaped larvae to spawn again subsequently is correlated to day of spawning (r = 0.2) and the proportion of abalone in the grow- ing tank spawning (r = 0.16). Probability of a spawning event in the growing tank, and subsequent survival to spawn again in the wild, is also correlated to the day of spawning (r = 0.40) and probability of a spawning event (r = 0.,^l. CONCLUSIONS The model constructed and described here is a hybrid of risk and biologic models. Biologic components (growth of abalone. deaths, water flows) drive the development of abalone in culture, and risk components drive the events of interest; that is. spawning. settling, escaping, and maturing. Where available, published data have been used to determine probabilities. However, the relative newness of the abalone culture industry is such that much data is either lacking, or dependent on limited production experience. In regard to the initial question of the importance of a filter to prevent the escape of larvae should a spawning event occur, the incorporation of a filter would seem to be a low utility procedure. The likelihood of a spawning event leading to establishment of abalone in the wild is very small (in the order of 10" ''-10*'^). The consequences of a spawning event would seem to be likewise 0 060-'^ 17 [ 0.050- >, 0 040- u c 1 0,030- 0) r«te3r, ■-MAP.7 1 -? ' 0 020- 0.010- 0.000- lj:2 r, — 1 — 1 Number surviving 120 5% : 80 Figure 2. Distribution (frequency) histogram of abalone surviving to spawn in the wild. Horizontal bar indicates percentiles. V C Is TO > 120- 1 00- 60 40- Pt otaabilitv ot spawning and survival to spawn again Values in 1 0"-6 I 5% Figure .1. Distribution (frequency) histogram of the probability of a spawning event and survival of abalone to spawn in the wild. Hori- zontal bar indicates percentiles. small, with very few abalone surviving to spawn again. This result is in accord with practical experience. Most attempts to outplant hatchery-reared juvenile abalone on reefs have failed (Burton & Tegner 2000). It should be remembered that the data used in this model are considered pessimistic; that is, overestimates of the input values, and the outputs can likewise be considered pessimis- tic. It will require additional research to quantify the variables of concern better. It may also be of benefit to investigate improving the efficiency of settling ponds, to further reduce the numbers of abalone escaping should spawning occur. The nature of the model, using a large matrix as a lookup table tends to mask the effects of input variables. This occurs because an input variable may be refen-ed to once in a chain of "events" over a large i-ange (in this case, 3 y, or about 1,000 day-events). The model itself has some 9,000 input variables, which refer back either directly or by way of another variable, to the biologic and stochastic variables that provide the basic inputs for the models. It is difficult to determine relationships between inputs and outputs under these circumstances. However, for the primary driving vari- ables, day of spawning, probability of a spawning event, and the proportion of abalone spawning, positive correlations with output variables were observed. In view of the nature of the model, such correlations strongly suggest close relationships between the out- puts and the input parameters, despite traditional statistical think- ing on the nature of correlations. These correlations suggest that it would be beneficial to collate observations associated with spawning events in cultured abalone and to determine what range of triggers influence such an event, with the goal of manipulating these triggers to control or prevent spawning in culture. Based on the results of this modeling exercise, the source of broodstock for land-based farms is of little importance in terms of genetic impact on wild populations. However, the model does not address disease risks and fine filtration (100 micron absolute) has been applied to systems in Western Australia to reduce disease risks associated with use of stock from interstate. Similariy. settlement ponds did not greatly influence the risk of larval escape, but this is not their primary purpose. Settlement ponds are used primarily to reduce nutrient release into the pristine coastal areas usually selected as abalone farming intake locations. ACKNOWLEDGMENTS The authors acknowledge the helpful technical advice provided either directly by Dr. Greg Maguire of Fisheries Western Australia or through him from his numerous industry contacts. Abalone Larval Escape from Culture: A Risk Analysis 809 LITERATURE CITED Brown. L. D. 1991. Genetic variation and population structure in the black- lip abalone Haliotis rubra. Austral J. Mar. Freshwater Res. 42:77-90. Burton, R. S. & M. J. Tegner. 2000. Enhancement of red abalone Haliotis rufescens stocks at San Miguel Island: reassessing a success story. Mar. Ecol. Prog. Series. 202:303-308. Gaffney. P. M., V. P. Rubin. D. Hedgecock. D. A. Powers. G. Morris & L. Hereford. 1996. Genetic effects of artificial propagation: signals from wild and hatchery populations of red abalone in California, .-^i/uacul- ture. 143:257-266. Elliot, N., N. Conod. G. Maguire. B. Evans & J. Bartlett. 2001 . Preliniuiary genetic comparison of Western Australian and Tasmanian greenlip aba- lone. Proceedings .Annual Abalone Aquaculture Workshop July:200l . Hahn. K. O.. editor. 1989. Handbook of culture of abalone and other marine gastropods. Boca Raton. FL: CRC Press Inc. 348 pp. Hancock. B. 2000. Genetic subdivision of Roe's abalone. Haliotis roei Grey (MoUusca: Gastropoda), in South Western .Australia. Mar. Fresh- water Res. 51:679-687. Hone. P. & A. Fleming. 1997. Abalone. In: K. W. Hyde, editor. The new rural industries. A handbook for farmers and investors. Canberra: Rural Industries Research and Development Corporation, pp. 83-90. Purdom, C. E. 1980. Growth in fishes. In: T. L. J. Lawrence, editor. Growth in animals. London: Butterworths. pp. 273-285. Shepherd. S. A.. P. A. Preece & R. W. G. White. 2000. Tired nature's sweet restorer? Ecology of abalone [Haliotis spp.) stock enhancement in Australia. In; A. Campbell, editor. Workshop on rebuildmg abalone stocks in British Columbia. Ed. A. Campbell. Canadian. Special Pub- lication. Fisheries Aquatic Science 130:84-97. Vose. D. 2000. Risk analysis. A quantitative guide. 2nd ed. Chichester: John Wiley & Sons Ltd. Westaway, C. & J. Norriss. 1997. Abalone aquaculture In Western Aus- tralia: dscussion paper and draft policy guidelines. Fisheries Manage- ment Paper No. 109, Fisheries Western Australia. 24 pp. (See also the subsequent 1999 version: Abalone Aquaculture in Western Australia: Policy Guideline. Fisheries Management Paper 133, Fisheries Western Australia, Perth. 10 pp.) Weston. L.. S. Hardcastle & L. Davies. 2001. Profitability of selected aquaculture species. Chapter 3. Abalone. Australian Bureau of Agri- cultural and Resource Economics (ABARE) GPO Box 1563. Canberra, ACT, Australia. 2601. research report 01.3, pp. 16-27. Journal of Shellfish Research. Vol. 21, No. 2. 81 1-815. 2002. ISOLATION AND CHARACTERIZATION OF MICROSATELLITE LOCI IN THE PACIFIC ABALONE, HALIOTIS DISCUS HANNAI QI LI,' CHOULJI PARK/ AND AKIHIRO KIJIMA^* ' Fisheries College. Ocean University of Qingduo. Qiugdao 266003. China: 'Education and Research Center of Marine Bio-resources. Graduate School of Agricultural Science. Tohoku University. Onagawa. Oshika 986-2242. Japan ABSTRACT Four microsatellite loci, designated HdhLUl. Hdh7S. Hdhl76I, and Hdhl457. were isolated from the Pacific abalone. Haliotis discus hannai, using an enrichment method based on magnetic/biotin capture of microsatellite sequences from a size-selected genomic library. Primers designed to amplify via polymerase chain reaction the microsatellite loci were used to screen 30 individuals from a natural Pacific abalone population in Onagawa Bay. Miyagi Prefecture. Japan. The four microsatellite loci were all polymorphic, with an average of 14.3 alleles per locus (range 7-20). The mean ob.served and expected heterozygosities were 0.48 (range 0.30-0.97) and 0.79 (range 0.60-0.92). respectively. Significant deviations from Hardy-Weinberg expectations were observed at three loci as a result of homozygote excess. The expected heterozygosity values were considerably higher than those previously found for allozymes (range 0.101-0.125), suggesting that these microsatellite loci should provide useful markers for studies of trait mapping, kinship, and population genetics. KEY WORDS: microsatellite loci. Pacific abalone, Haliotis discus hannai, genetic vanahility INTRODUCTION The Pacific abalone. Haliotis discus hannai, is distributed along the coastal waters of East Asia, where it is one of the most valuable and popular fisheries resources. Although cultured seeds of the Pacific abalone were produced 20 years ago. the develop- ment of abalone culture has long been hampered by the problems of low growth rate and mass mortality during seed production (Hara & Sekino 2001). To resolve these problems and improve animal breeds for aquaculture production, many genetic studies on growth-related traits and temperature tolerance have been per- formed in the Pacific abalone (Wilkins et al. 1980. Okumura et al. 1981. Hara 1990. Kobayashi et al. 1991. 1992, Kobayashi & Fujio 1994, 1996, Furutono et al. 1995, Kijima et al. 1995, Kawahara et al. 1997. 1999). Allozyme analysis suggested the presence of ho- mozygote excess and inbreeding depression (Kijima et al. 2002); however, genetic control of the target traits in H. discus hannai remains unclear. To tlnd genetic markers associated with loci that control economically important traits to assist in selective breeding programs, the development of molecular markers is needed. For this purpose, allozymes are not appropriate because there are too few of them and they are not sufficiently variable (Kijima et al. 1992). Microsatellites are tandemly repeated arrays of short nucleotide motifs found in all prokaryotic and eukaryotic genomes analyzed to date (Zane et al. 2002). Because they are evenly dispersed throughout genomes, usually characterized by high length poly- morphism, and generally inherited in a Mendelian fashion, micro- satellite markers have been widely used for genomic mapping, linkage analysis, pedigree analysis, and population genetics of bio- logic resources (Schlotterer et al. 1991. Knapik et al. 1998. Hol- land 2001). The traditional approach to obtain microsatellites is to create a size-selected genomic library in a plasmid or phage vector and then screen clones using oligonucleotide probes containing differ- ent repeat motifs. For microsatellite repeats that are less abundant in the genome, it is difficult to isolate them using the method. *Corresponding author. E-mail: a-kijima@mail. cc.lohoku.ac.jp Recently, different approaches have been developed to enrich for microsatellites (Zane et al. 2002). One method is based on selec- tive hybridization, a protocol that is commonly used in enrichment procedures (Ostranderet al. 1992, Kandpal et al. 1994, Kijas et al. 1994, Refseth et al. 1997, Zane et al. 2002). In the present study, we report the first isolation of microsatellite loci in H. discus hannai by magnetic bead hybridization selection and assess poly- morphism at microsatellite loci in individuals from a natural popu- lation. MATERIALS AND METHODS DNA Extraction from Abalone For constructing a genomic DNA library, high-molecular weight DNA was extracted from a live Pacific abalone from Ona- gawa Bay, Miyagi Prefecture, Japan. The foot muscle was re- moved from the abalone and ground with dry ice in a mortar. About 100 mg of the tissue was digested overnight at 37''C in 0.7 niL of lysis buffer (6 M urea. 10 mM Tris-HCl. 125 mM NaCI, 1% SDS, 10 mM EDTA, pH 7.5) and 35 |xL of proteinase K (20 mg/mL). The reaction mixture was extracted with phenol xhloro- form (1:1). precipitated with isopropanol, and dissolved in TE ( 1 0 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was treated with DNase-free RNase (20 p.g/mL) at 37°C for 1 h, and purified again using a phenol/chloroform extraction. Digestion of Genomic DNA, Size Fractionation, and Ligation of Adapters Extracted DNA was digested with HaelU, Dral. and Hindi using 100 U each for 50 p-g of abalone DNA. The digested DNA (5 (xg) was electrophoresed on a 2.5% NuSieve GTG agarose gel (EMC Bioproducts), and fragments of 400-800 bp were excised and purified using a Qiagen column (QIAquick Gel Extraction Kit. QIAGEN). The fragments ( 1 p.g) were ligated with 200 pmol of an EcoR\-Notl-BamHl adapter (TaKaRa) using a DNA Ligation Kit (TaKaRa), then collected by ethanol precipitation, and resus- pended in 20 p.L of H^O. 811 812 Ll ET AL. Magnetic Isolation of Tarin't Sequences and Adapter Polymerase Chain Reaction iPCR) One 0.6-niL tube of the Streptavidin MagneSphere® Paramag- netic particles (Promega) was washed according to the manufac- turer's instructions, resuspended in 300 p,L of 5x SSC ( 1 x SSC = 150 mM NaCl. 15 inM sodium citrate), and mixed with 300 pmol of biotinylated oligoprobe: 5'-(CA)|,GCTTGA-biotin. The six- base noncomplementary region at the 3' end was used to prevent the probe, copurified with target DNA during magnetic isolation, from acting as primers in subsequent adapter PCR (Gardner et al. 1999). The beads and probe were incubated for 15 minutes at room temperature, then washed three times in 5x SSC and resuspended in 100 |jiL of hybridization solution (0.5 M NaCl. 47f polyethylene glycol 8000) at 56°C (Gardner et al.l999). The fractioned DNA (20 |jlL) was mixed with 80 |j.L of the hybridization solution, denatured by incubating at 95°C for 5 min, added quickly to the beads, and incubated at 56°C for 20 min. The beads were then washed four times at room temperature in 200 ixL 2x SSC fol- lowed by four times at 30°C in 200 piL 1 x SSC. Immobilized DNA fragments were eluted from the beads in 50 |xL 0.15 M NaOH at room temperature for 20 min. The beads were then removed, and the supernatant was neutralized by the addition of 5.5 |a,L lOx TE, 3.25 JJ.L 1.25 M acetic acid. DNA was purified by a Qiagen col- umn (QIAquick PCR purification kit. QIAGEN) and eluted in 50 |jiL of TE buffer. PCR amplification was performed in 50 |jlL volumes containing 5 jxL of the released DNA, 1 .25 U of Ampli- Taq Gold (Perkin Elmer), 5 |JiL of GeneAmp lOx PCR buffer (Perkin Elmer), 0.2 mM dNTP mix, 1.5 niM MgCK. and 0.6 jxM of the adaptor sequence primer, 5'-CGGCGGCCGCGGATCC-3'. Reactions were denatured at 95°C for 1 1 min before 35 cycles of 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min, followed by a 5 min, 72°C final extension. PCR products were purified using a QIAquick PCR purification kit (QIAGEN). Cloning of the PCR-Amplified DNA Fragments The purified PCR products were digested with Null, inserted into the yVofl site of the pBIuescript II SK(-t-) vector (Stratagene), and the recombinant plasmid vector was transformed into XLl- Blue MRF' supercompetent cells (Stratagene) following the manu- facturer's protocol. Recombinant clones were selected by plating on LB media containing ampicillin (100p.g/mL), IPTGdOO (jlL of 10 mM stock solution) and X-Gal ( 100 |jlL of 27c stock solution). PCR Screening of Microsatellite-Containing Clones A small portion of a white colony was transferred to 10 (xL of 10 mM Tris-HCI (pH 8.5) with a toothpick, incubated at 95°C for 10 min, and then used as template (1 p-L) in the PCR with two vector primers (T3 and T7) and the nonbiotin-labeled (CA),, primer (Gardner et al. 1999). Each 10-[xL reaction mixture con- tained 0.25 U of AmpliTaq Gold (Perkin Elmer), Ix the supplied buffer (Perkin Elmer), 0.2 mM dNTP mix, 1.5 mM MgCK, and 0.2 (i-M of each pnmer. Screening amplifications were pert'ormed as follows: 1 1 min at 95''C followed by 35 cycles of I min at 94"C. 1 min at 57°C, and 1 min at 72=C, followed by 5 min at 72°C. PCR products were electrophoresed on 1 .5% agarose gels. Inserts pro- ducing two or more bands were considered likely to contain a microsatellite locus. Positive plasmid DNAs were purified using Qiaprep spin columns (QIAGEN), and then double sequenced on a Shimadzu DSQ-2000L DNA sequencer (Shimadzu) using Ther- moSequenase cycle sequencing kit (Amersham) in combination with the universal forward (5'-CGCCAGGGTTTTCCCAGT- CACGAC-3') and reverse (5'-GAGCGGATAACAATTTCACA- CAGG-3') primers, respectively. PCR primers for each microsat- ellite locus were designed using the OLIGO software package (National Biosciences Inc.). Assessment of Polymorphism in Microsatellite Loci Primer pairs were tested on a random sample of 30 Pacific abalones from Onagawa Bay. Abalone DNA was extracted by the method as described above. PCRs were performed in lO-p-L vol- umes containing 0.25 U of AmpliTaq Gold (Perkin Elmer), Ix PCR buffer, 0.2 niM dNTP mix. 1.5 mM MgCK, 1 piM forward FITC-labeled primer and reverse primer, and about 100 ng tem- plate DNA. The PCR conditions for all loci were 1 1 min at 95°C followed by 35 cycles of 1 min at 94 C, 1 min at the annealing temperature listed in Table 1, and 1 min at 72°C, with a final extension of 5 min at 72-'C. Amplification products were resolved via 6% denaturing polyacrylamide gel on a Shimadzu DSQ-2000L DNA sequencer equipped with DSQ-SA program (Shimadzu), and a pBIuescript II SK(-(-) sequencing reaction was co-electrophoresed as a size marker. Statistical Analysis Number of alleles per locus, expected and observed heterozy- gosities, and x^ tests of deviations from Hardy-Weinberg expec- TABLE I. Characterization of four polymorphic microsatellite loci in the Pacific abalone, Haliotis discus hannai. Locus Repeat Motif Primers (5'-3') .\nnealing Size No. of GenBank Temperature IC) (bp) Alleles H,, H^ Ace, No. HdbLU! (CGCA)j(CA)|s TTCTGAGATGAGACGCACCAC TTGGCAGCAGGCGTCGTGT Hdh7R (CACCD^CACTTlCACCT), GTCGAAACTAGCACAAACATCT GATCACCGGTCACATCATAC Hdhl76l (CA),TA(CA),. ..(CCACA),3 GTCCAACACAACCAACTCCTC CCTGAGCATGTTTGTGGATAAC Hdhl457 (CGCCA),,(CTCCA)„. .. CACCTGTGTTTCGTACCCAC (CTCCA),5. . .(CTCCA),, GGGACAATTCCAAGTAGATGC H„, observed heterozygosity; H^, expected heterozygosity. *P<0.01. 62 272-,^62 20 0.97 0.92 AB084076 54 177-332 7 0.33* 0.60 AB084077 64 405-596 18 0.30* 0.92 AB084()78 62 481-601 12 0.33* 0.71 AB084079 Isolation of Microsatellites in H. discus hannai 813 tations (HWE) were calculated using GENEPOP 3.1 software (Raymond & Rousset 1995). RESULTS Isolation of Microsalellites in H. discus hannai An enriched library of 350 white colonies was screened using the PCR-based technique, and 84 clones were identified by the presence of two or more bands on the agarose gel. Sequencing the 84 clones gave 46 loci containing microsatellites arrays with a minimum of five repeats, primarily (CA)„, but some in combina- tion with other tetra- or pentanucleotide array motifs. Primer pairs were designed for 10 of the 46 sequences with long, uninterrupted repeats and adequate unique regions flanking the microsatellite array. Of the 10 primer sets developed, four loci were successfully amplified with polymorphisms (Table I). Genetic variability of Microsatellites in H. discus hannai Figure I shows the alleles of microsatellite loci Hdhl32l. Hdh78. Hdhl76L and HdhI457 identified in six Pacific abalone individuals. The presence of minor peaks below the major ampli- fication product ("stutter") was observed. At locus Hdhl761. the peaks of longer alleles tended to be smaller than those of shorter alleles. Primer sequences, repeat motif, annealing temperature, number of alleles, amplified product size range, the observed (Hq) and expected (H,.) heterozygosities for the four microsatellite loci 252 262 272 282 292 302 312 322 332 342 352 362 372 382 392 A]kles (bp) 162 172 182 192 202 212 222 232 242 252 262 272 282 292 302 312 322 332 342 352 Alleles (bp) 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 Alleles (bp) 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600 610 620 Alleles (bp) Figure I, Alleles of microsatellite loci Hdhlill. Hdli78. Hdhl76l. and HdhI457 identiPied in six Pacific abalone individuals from a natural population in .Miyagi Prefecture. are summarized in Table I . The four microsatellite loci were all highly polymorphic, whereas the degree of variability was differ- ent at each locus. Hdhl321 had the highest number of alleles (20). whereas number of alleles at Hdh78. Hdhl761. and Hdhl457 was 7. 18, and 12, respectively. The expected heterozygosity ranged from 0.60 at Hdh78 to 0.92 at both Hdhl.Ul and Hdhl761. Sig- nificant deviations from HWE for the observed heterozygosities were observed in the Hdh78. Hdhl76l. and Hdhl457 (P < 0.01). Allele frequencies of the four microsatellites for samples from Miyagi Prefecture are shown in Table 2. At loci Hdh78 and HdhI457. the frequencies of major alleles 267 and 563 were 0.617 and 0.500. respectively. At loci HdhlJ21 and Hdhl76l. allele frequencies were overall low. with the most common alleles being 0.150 and 0.167. respectively. DISCUSSION The enrichment efficiency seen here (13.1%) is similar al- though lower than that reported by Gardner et al. (21%, 1999). By the colony hybridization method, the percentage of positive clones containing microsatellite repeats was 0.52-0.66'7f in the European flat oyster (Naciri et al. 1995). 0.1% in the quagga mussel (Wilson et al. 1999), and an average of 1.96% in molluscs (Zane et al. 2002). Compared with traditional methodologies, the enrichment procedure using the magnetic bead hybridization selection is more efficient. The presence of the microsatellite stutter bands seen at all four microsatellite loci is a typical artifact of PCR amplication of mi- crosatellite loci (Johansson et al. 1992). This phenomenon is due to slipped strand mispairing during PCR (Weber 1990). In this study, although the variability observed in the microsatellite loci (average 14.3 allele per locus) is possibly underestimated because of small sample size (30 individuals), it was still much higher than that of allozymes in the Pacific abalone population (Kijima et al. 1992). They surveyed 18 allozyme loci using 445 Pacific abalone indi- viduals in six groups from coastal waters of Japan, and found the average number of alleles per locus in allozymes was 2.01 (range 1.89-2.17). The average expected heterozygosity in total popula- tions was 0.116 (range 0.101-0.125). The high level of length variation found here is similar to that found in H. discus discus and other abalone species (Huang & Hanna 1998. Kirby et al. 1998. Miller et al. 2001. Sekino & Hara 2001). Significant deviations from HWE occurred for the observed genotype frequencies at loci Hdh78. Hdhl761. and Hdhl457 be- cause of homozygote excess. The departure from HWE with an excess of homozygotes may be the result of one or more of the following reasons. (1) Large allele "dropout" artifacts in the PCR amplification process: At locus Hdhl76I. we observed that large allele bands tended to be less intense than small alleles. In hetero- zygous individuals, preferential amplification of a smaller allele over a larger allele would result in the mis-scoring of heterozy- gotes for homozygotes even though larger alleles may indeed exist. The problem of large allele dropout during PCR has been well documented for humans (Day et al. 1996), Minke whales (Van Pijien et al. 1995), and chinook salmon (Banks et al. 1999). (2) Small sample size: As microsatellite DNA has a rapid mutation rate, resulting large number of alleles, a large sample size is needed for accurate reflection of genotypic frequencies (Ruzzante 1998). This sample size was suggested to be at least 50 individuals per population for microsatellite loci studies. (3) Presence of null alleles: Null alleles of microsatellite regions, which occasionally 814 Li et al. TABLE 2. Allele frequencies of four microsatellite loci of Pacific abalone, H. discus hiiiinai, from Miyagi Prefecture. Allele Hdhl32l Allele Hdh78 Allele Hdh 1 76] Allele Hdhl457 272 0.017 177 0, 1 33 405 0.067 4X1 0.033 290 0.150 182 0.017 408 0.017 493 0.017 292 0.033 187 0.067 418 0.167 509 0.017 294 0.033 202 0.033 426 0.050 518 0.033 300 0.117 262 0.083 433 0.067 548 0.017 304 0.150 267 0.617 442 0.033 563 0.500 306 0.017 332 0.050 448 0.167 568 0.033 310 O.I 00 450 0.017 578 0.167 312 0.017 478 0.017 586 0.117 316 0.067 483 0.017 591 0.017 320 0.017 526 0.067 596 0.017 322 0.017 537 0.033 601 0.033 324 0.05O 544 0.067 328 0.10(1 553 0.033 336 0.033 570 0.033 340 0.017 576 0.033 346 0.017 591 0.083 348 0.017 596 0.033 356 0.017 362 0,017 fail to yield an amplification product. Jiiay arise tlirough mutations such as point mutations in the primer annealing site (Callen et al. 1993. Pemberton et al. 1995). Kijima et al. (1997) have found null alleles at allozyme loci in the Pacific abalone by mating experi- ment. If null alleles are present but not accounted for. the resulting .scoring error of heterozygotes for homozygotes can create an ap- parent excess of homozygotes in population studies (Jones et al. 1998). (4) Inbreeding effects: Extensive heterozygote deficiency has also been reported at allozyme loci in natural populations of the Pacific abalone (Fujino 1978, Fujio et al. 1986). Hara and Kikuchi (1992) showed an excess of homozygotes in all natural populations of H. discus harmed collected from nine different sam- pling sites, suggesting extensive inbreeding. The excessive ho- mozygotes for microsatellite loci were also described for H. rubra. H. discus discus, and H. kamtschatkana populations (Huang et al. 2000. Miller et al. 2001. Sekino & Hara 2001). (5) Presence of size homoplasy: Size homoplasy is the co-occurrence of alleles that are identical in state (PCR products of the same size) without being identical by descent (Ardien et al. 1999). Mistaking homoplasy for homology can lead to underestimation of the genetic divergence within and among population (Taylor et al. 1999). Microsatellite allele size homoplasy has been confirmed in various animal spe- cies (Estoup et al. 1995. Viard et al. 1998. Ardren et al. 1999. Taylor et al. 1999); however, was not reported for abalones. Fur- ther studies of natural populations and controlled crosses of H. discus hannai will help to clarify this deviation from HWE. The high variability of the microsatellite markers identified in this study will make them excellent tools for paternity testing, population studies, and the linkage analysis of genes related to traits with economic significance in the Pacific abalone. ACKNOWLEDGMENTS The study was supported by grants from the Japan Society for the Promotion of Science (No. P00335). Agriculture. Forestry and Fisheries Research Council of Japan, and National Natural Science Foundation of China (No. 30170735). LITERATURE CITED Ardren. W. 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Evol. 12: 459-472. Viard, F.. P. Franck. M. Dubois. A. Estoup & P. Jame. 1998. Variation of microsatellite size homoplasy across electromorphs, loci, and popula- tions in three invertebrate species. / Mol. Evol. 47:42-51. Weber. J. L. 1990. Informativeness of human (dC-dA)n.(dG-dT)n poly- morphism. Genomics 7:524-530. Wilkins. N. P.. K. Fujino & K. Sasaki. 1980. Genetic studies on the Pacific abalone. IV. Thermostability difference among phosphoglucomutase variants. Nippon Suisan Gakkaishi 46:549-553. Wilson. A. B.. E. G. Boulding & K. A. Naish. 1999. Charactenzation of tri- and tetranucleotide microsatellite loci in the invasive mollusc Dreis- sena bugensis. Mol. Ecol. 8:692-693. Zane. L.. L. Bargelloni & T. Patarnello. 2002. Strategies for microsatellites isolation: a review. Mol. Ecol. 11:1-16. Journal of Shellfish Re-iearch. Vol. 21. No. 2, XI 7-824. 2002. TRANSMISSION OF WITHERING SYNDROME IN BLACK ABALONE, HALIOTIS CRACHERODII LEACH CAROLYN S. FRIEDMAN.' - * WENDY BIGGS.' JEFFREY D. SHIELDS,' AND RONALD P. HEDRICK- Califonua Department of Fish and Game and 'Department of Medicine and Epidemiology, University' of California. Bodega Marine Laboratoiy. P.O. Bo.x 247. Bodega Bay. California 94923: Virginia Institute of Marine Science. The College of William and Maiy. Gloucester Point. Virginia 23U62 ABSTRACT Withering syndrome (WS) has been associated with catastrophic declines in black abalone populations in southern and central California. In an effon to identify the etiological agent of WS and to characterize the progression of this disease, we initiated a transmission study in which abalone from Ano Nuevo Island, a location free of WS. shared aquaria with animals from Vandenberg Airforce Base, a location where WS is epizootic. The mean incubation period of WS (time to develop overt signs of the disease) was 245 days with a mean time to death after development of clinical signs of 42 days. Median time to death was 41 wk in the experimentally exposed Ano Nuevo Island abalone and 16 wk in the positive control Vandenberg abalone. Cumulative mortality was significantly different between the negative control (unexposed) Ano Nuevo Island abalone (25% mortality) and both the exposed Ano Nuevo Island abalone (85% mortality; P = 0.0001) and the positive control Vandenberg abalone (100% mortality; P = 0.0001). In addition, significant differences in prevalences of a recently described Rickettsiales-like procaryote (RLP). "Candidatus Xenohaliotis califomiensis." were observed between negative control animals (no RLPs) and those with WS (both the experimentally exposed Ano Nuevo Island and Vandenberg abalone were infected with RLPs: P < 0.001). All abalone infected with the RLP had signs of WS, including decreased condition indices, foot muscle atrophy, and digestive gland degeneration {P < 0.05). No correlation between intensity of RLP infection and degree of WS was observed (P > 0.05), suggesting a complex relationship between the RLP and clinical disease in black abalone. Despite this, these data in conjunction with a lack of observation of any other significant pathogens in the abalone provides evidence that the RLP infecting abalone {"Cundidtitus Xenohaliotis califomiensis") is the etiological agent of WS. KEY WORDS: withering syndrome, black abalone, Huliulis. rickettsiales, "CcJ/d/n/ii/i/.v .Xenohaliotis califomiensis" INTRODUCTION Withering syndrome (WS) has been associated with cata- strophic declines in black abalone populations in southern and central California (Haakeret al. 1992; Steinbeck et al. 1992, Fried- man and Haaker unpublished data). Initial studies identified a pre- viously undescribed coccidian parasite. Margolisiella ( = Pseudok- lossia) haVwtis (Friedman 1991; Friedman et al. 1995, Desser & Bower 1997). in black abalone with WS that was subsequently determined to be nonpathogenic as evidenced by field and labo- ratory studies (Friedman et al. 1993. 1997). VanBlaricom et al. (1993) documented WS on San Nicolas Island in April of 1992. These researchers a observed Rickettsiales-like procaryote (RLP) that was recently described as a new taxon and has been given the provisional status of "Candidatus Xenohaliotis califomiensis" (Friedman et al. 2000). Two of six abalone with clinical WS har- bored RLPs. whereas apparently healthy animals were devoid of the RLP. The authors indicated that Rickettsiales-like bacteria were commonly observed in marine invertebrates and that the pathogenicity of these organisms was unknown. Gardner et al. (1995) also observed RLPs in association with WS in black aba- lone from San Nicolas and San Clemente Islands in southern Cali- fornia. Healthy abalone from Ano Nuevo Island in central Cali- fornia were not infected with RLPs. suggesting an association between the RLP and WS. Friedman et al. (1997) examined the association between the RLP. degeneration of the digestive gland, and mortality in a laboratory study. No clear associations between intensity of RLP infection and either condition of the digestive gland or time to mortality were observed. Recently. Moore et al. *Corresponding author. School of Aquatic and Fishery Sciences. University of Washington, Seattle, WA 98195. E-mail: carolynf@ u. Washington. edu (2000) observed a significant relationship betv\een the intensity of RLP infection and degree of WS in cultured red abalone. These conflicting data indicate further examination of the role of the RLP in WS is warranted. This study was designed to examine the trans- missibility of WS and to determine the relationship between RLP infection and WS in black abalone. MATERIALS AND METHODS Animals Healthy black abalone were collected on March 28, 1995 from Ano Nuevo Island, where WS had never been observed. Black abalone with WS were collected from Vandenberg Airt'orce Base (Vandenberg) and Cayucos on April 24. 1995. Abalone were trans- ported to the Pathology Quarantine Facility at the Bodega Marine Laboratory, where they were placed in an 88-L aquaria and re- ceived ambient (8-10°C). flow-through, full-strength seawater. Macrocystis pyrifera was collected from Bodega Bay and was surface sterilized by soaking in a tamed iodine solution (Prepo- dyne: Westagro. Kansas City, MO) for 15 min followed by a freshwater rinse. Animals were fed M. pyrifera twice per week. All abalone were lagged and the following data were collected: maxi- mum length, foot length and total volume (TV), and total weight (TW). Animals were bled from the pallial sinus with a tuberculin syringe and a 26-gauge. 0.5-inch needle and the density, cell-type, and condition of circulating hemocytes was determined using a hemocytometer. Visual condition of the abalone was assessed ac- cording to the following scale: (3): healthy abalone with a foot and viscera that fills the entire shell volume; (2): visible mantle retrac- tion and moderate atrophy of the foot muscle; and ( 1 ); severe atrophy of the foot muscle. 817 Friedman et al. Histology Selected tissues were placed in Invertebrate Davidson's solu- tion (Shaw & Battle 1957) tor 24 h and processed for routine paraffin histology. Deparaffini/ed 5-|j.iti sections were stained with hematoxylin and eosin (Luna 1968) and viewed by light microscopy. The intensity of RLP infection was quantified using the following logarithmic scale at 200x magnification: (0): no bacterial foci; (1): 1-10 foci per field; (2): I 1-100 foci per field; and (3): >100 foci per field (Friedinan et al. 1997). Infection in- tensity was quantified in both the postesophagus (PE) and diges- tive gland (DG). and an overall infection intensity was calculated by summing the intensity in the PE and DG (range of 0-6 possible) (Moore et al. 2000). Intensities were examined according to tissue type to determine whether the location of infection was correlated with animal health. Unless otherwise specified, the term RLP in- fection refers to overall infection intensity. Condition of the di- gestive gland and foot muscle were assessed using the ( 1 )-( 3 ) scales of Friedman et al. (1997), in which normal was scored as (3), moderate (up to 30%) alteration from normal was scored as (2), and tissue that was severely (>30%) altered was scored as ( 1 ). Three specific morphologic changes that characterized observed alterations in digestive gland architecture were individually scored according to the following (1H3) scale: (1) normal architecture; (2) moderate (up to 25%) degeneration (characterized by an in- crease in connective tissue between digestive tubules, the primary tissues responsible for secretion of digestive enzymes and nutrient absorption in abalone) (Voltzow 1994). transport duct metaplasia, or inflammation; and (3) abundant (>25%) transport duct metapla- sia, an increase in connective tissues between degenerating tu- bules, or inflammation. Transmission Experiment Groups of 12 abalone from Ano Nuevo Island were randomly placed in each of two negative control (NC) and two experimental aquaria (EA). Groups of 12 abalone with WS (EWS) were ran- domly added to each of the two experimental aquaria and to each of the two positive control aquaria (PC) (Fig. 1). Animals were maintained on ambient seawater for the first 3 mo of the s£udy. During this time temperatures ranged between 8-10°C (X = 9.53X) for the first 4 wk. 11-15^ (X = 12.89"C) for the fol- lowing 4 wk and 10.5-15''C (X = I2.47°C) for the third month. After this time, the animals were acclimated over a 2-wk period to 18 ± rC, the temperature at which the abalone were maintained for the remaining 34 wk of the 46-wk study. Physical measure- ments and hemocyte counts were assessed approximately every 8 wk over the course of the experiment. All moribund abalone or mortalities were sampled as above, including shell weight (SW) and shell volume (SV), and selected tissues (foot, digestive gland. PE, and kidneys) were processed for histology. The intensity of RLP infection and condition of the digestive gland and foot muscle were quantified as described above. The condition of the abalone was also assessed upon death using the body weight condition index of Friedman et al. (1997) = [(TW - SWi/TW]. In addition, the percentage of live tissue volume relative to the entire volume of the animal was determined = [(TV - SV)/TV]. Statistical Analysis The Fisher's exact test was used to test the independence of exposure to WS and mortality; abalone were grouped as exposed or unexposed and as alive or dead. Chi square contingency table analysis (X") was used to test independence between exposure to WS and measured health parameters. Abalone were grouped as exposed (laboratory or field exposed) and unexposed. The follow- ing health parameters or responses were grouped as normal (scores of 3 for animal condition and that of the digestive gland and foot and 0 for RLP presence), whereas those with signs of WS and RLP infection were grouped as abnormal. Observed versus expected frequencies in each category were compared using 2x2 contin- gency table analyses. The Fisher's Exact test was used when fewer than five observations were observed in any cells. These analyses Experimental Design n=12 n=12 O O I n=12 n=12 n=12 11=12 n=12 O O 1 Replicate 1 Replicate 2 n=12 (VAFB)' (EWS & EA)- (Ano Nuevo Is.)^ } Animals Positive Experimental Negative } Treatment Control Control Figure 1 Experimental Design. The dark circles represent black abaU.ne «ith WS collected from the field (Vandenberg Airforce Base or Cayucos (VBCS)). The open circles represent healthy, naive (no exposure to WS before study) black abalone from Ano Nuevo Island. Animals with WS collected from Vandenberg Airforce Base and Cavucos in the positive control treatments. -Animals from VBCS (EWS) or Ano Nuevo Island (EA) with and without WS. respectively, in experimental treatments. 'Animals in the negative control treatments without WS that were collected from Ano Nuevo Island. Withering S\'ndrome in Black Abalone 819 were also used to test the independence of RLP infection and WS. Animals were grouped as infected and uninfected and as above for survival and health parameters. In a separate analysis to further assess the independence of specific lesions and RLP infection intensity animals were grouped as low overall infection levels (0-3) and high overall infection levels (4-6) and the response (e.g., metaplasia) was grouped as present or absent. Spearman rank cor- relation coefficients were calculated and tested for a (linear) rela- tionship between intensity of RLP infection and condition of the digestive gland and foot, condition indices, density of circulating hemocytes, cumulative mortality, and time of exposure. Stepwise forward and backward regression models were used to predict the intensity of RLP infection in exposed abalone from the following variables: condition of the digestive gland and foot, weight con- dition index, visual condition assessment, and duration of expo- sure. Multiple logistic regression analysis was used to predict pres- ence of RLP infection by using combinations of the five indepen- dent variables listed above. RESULTS All abalone from the PC treatment and all except three animals in the experimental treatment (EWS and EA) that died in this study had visible signs of WS, including weakness, weight loss, and visible atrophy of the foot muscle (Fig. 2). The two EA abalone in the experimental aquaria that died during the first week of the study lacked visual and histopathological signs of WS. RLP in- fections, or visible injuries. A third abalone from the experimental treatment that died during the 21st week of the study was too decomposed for gross or histologic examination. The six NC aba- lone that died during the experiment and 1 8 NC survivors sampled upon termination of the study did not have visible or microscopic signs of WS (Figs. 3 and 4). Both Vandenberg/Cayucos (PC and EWS) and Ano Nuevo Island (EA) abalone with clinical WS had histopathological and hematological signs of this disease, includ- ing degeneration and inflammation of and/or metaplastic changes in the digestive gland, depletion of muscle bundles in the foot, (Figs. 3-5), and the presence of necrotic cells, cellular debris, and small hemocytes (-4.5 p,m) with a large nucleus to cytoplasmic ratio within the hemolymph. In addition, all PC and EWS animals and all except the two EA abalone that died during the first week of the study were infected with the RLP. whereas none of the NC animals were infected. Other than the nonpathogenic renal coccid- ian, Margolisiella { =PseudokIos.sia) luiliotis. no other parasites were observed in any of the abalone examined in this study. In this study, the incubation time for clinical WS is detuied as the dura- tion between initiation of the study and development of gross clinical signs such as mantle retraction or visible atrophy of the foot muscle. The mean incubation period for the EA abalone was 245 days (;; = 21) with a range of 154-301 days. The duration between onset of visible signs of WS and mortality averaged 42 days (n = 21) with a range of 6-1 13 days. The two abalone that died during the initial week of the study from handling stress and the single animal that died at 21 wk did not show signs of WS or were too decomposed for assessment of WS, respectively, and were not included in these calculations. Cumulative mortality ap- proached 100% in the PC aquaria, 85% of the EA animals in the experimental aquaria, and 25% in the NC aquaria (Fig. 6). A significantly higher proportion of abalone died upon exposure to WS (22/24) relative to unexposed animals (6/24; P < 0.0001, Fish- er's exact test). Median time to mortality was significantly differ- ent between the exposed EA (41 wk) and PC ( 16 wk) abalone {P < 0.0001, Mann-Whitney test). As only a few NC abalone died during the study, median time to death was not calculated for this group. A significantly higher proportion of abalone exposed to WS (EA and PC) had reduced condition indices, morphologic changes, and RLP infections than did unexposed animals (NC). Reduced condition indices were observed in 18/24 EA and 19/24 PC aba- lone. whereas only 3/24 NC animals lost condition {P = 0.002 and P < 0.001. respectively. X' test). Morphologic changes were ob- served in the digestive gland of 20/21 EA and 17/24 PC abalone, whereas only 1/24 NC abalone had an abnormal digestive gland architecture (P < 0.001. X' test). Of these, degeneration was ob- served in 14/21 EA and 6/10 PC animals, metaplastic changes in 9/21 EA and 4/10 PC abalone, and inflammation in 6/21 EA and l/IO PC abalone, whereas 1/24 of the unexposed animals only had mild digestive gland degeneration. Pedal atrophy was observed in 15/21 EA, 17/24 PC, and only 1/24 NC abalone (P = 0.001 and P < 0.001. respectively, X' test). Infections with "Candidatus Xe- nohaliotis califomiensis" were observed only in EA (22/21) and PC (24/24) treatments (P < O.OOI, X- test and P < 0.001, Fisher's exact test). As above, significantly higher proportions of animals with RLP infections died and had clinical signs of WS than did unexposed abalone (/* < 0.001, A"" test). Spearman rank correlation coefficients for relationships be- tween intensity of RLP infection of individuals in each WS- exposed group (EA, EWS, and PC) versus visual condition, con- dition indices, condition of the foot and digestive gland, and den- sity of circulating hemocytes were low and ranged between -0.275 and 0.486 for the Ano Nuevo Island animals and -0.0175 and 0.0567 for Vandenberg animals. Except for metaplasia and overall Figure 2. Black abalone with and without WS. \. Healthy animals from .\no Nuevo Island from a negative control treatment. B, \n (EA) abalone from Ano Nuevo Island that contracted WS from infected black abalone in an experimental treatment. 820 Friedman et al. <^^-: ■ ^■^^■L^yj' ■ ^) -■■' - mm.' ' l:-A B ■^.■.- >' •-. .-^^- . . > . i'.? 'v:S>*v=-'..>^-^ •:, * "^ . i i't^ :^<^ ?.^/V- -^■.. ?';'"-■ r ■ - -^■*. . ■ ■■ ^- - „ - V - 1 ■ *^- »y4S^^-, ^,,.ff tf ■' rf r"':!^- ^^' * ** ^^ . • ,,-:-jf", ^ .. w^^^r"- '^ •-■, 'C* .-" i ...t _-->. ■*'■, ^ . ' ; . • •,-;-■. ^.' . - •!" \ • -"■^"' *;■•• * «-'-' • ■'.•■•-!'•■ ?'^' --—•.- ' - . ,, ■.■*■* '^ . ' - " "y^i^:^^^^^" ■ y ■ ■■ . . ■ ;"x.';fv->i'v ■^; « "'V'i. - ■ ff' '^ - - " , . - . . V 'v ^ - -•* - 4 - '.----'■ .-■'■^^.^ -"■^.- » '• '-c.'. ■' ■• ■ -' "' *■ .'''^"'ri^^, ■■■ : . . "" ' ■^' • • ■ ? ^ . ' . ■- V* ^"■^.J••. v^ ,' . . • - •;v::;:y^^vvr>;r- ■ v.--/' ..;/ ^"':,-:- "1*' ' . '. * y ' .'■■■'■ ' ,'*. ■"'■">' ' , -■ ^"^" ". . -y" . ' '. " -■*•'_■*■ ■''■-. ' ■ ' * ' '- " ■— • wS^-'•i^^6^''-• '' ^-".- •: .' ■:.'.,■■'". TT?' Figure 3. Microscopic anatomy of the foot muscle of black abalone with and without \V S. The toot muscle of an uninfected animals are illustrated in A ( Vandenberg abalone) and B ( Ano Nuevo Island abalone). Note thai dense bundles of muscle libers comprise most the loot. The pedal muscle atrophy of an abalone that contracted \VS in the field (C) is also observed in those that acquired W'S in this laboratory study (D). Note the severe reduction in muscle fibers and increase in visible connective tissue in affected individuals. Hematoxylin and eosin, bar = 15(1 pm. RLP burden in the EA abalone {P < 0.03), all coefficients were nonsignificant (P > 0.05). This relationship was also mirrored in X' analysis in which a higher proportion (8/9) of EA abalone with high overall RLP burdens (scores of 4-6) had metaplastic changes as the sole or partial response to RLP infections than did those with low (scores of 0-3) infections ( 1/7. P < 0.01 ). Several of the PC abalone were too necrotic to assess specific lesions in the digestive gland, a tissue that degrades more quickly than other tissues (Friedman, personal observation), and resulted in small samples sizes for this specific analysis. Intensity of RLP infection in labo- ratory EA abalone was predicted from the duration of e.xposure (time) with weight condition index, visual condition, condition of the foot and digestive gland, and time as independent variables in the model (P = 0.0156. Forward and Backward stepwise regres- sions). No prediction of presence of RLP infection could be made using Multiple logisitic regression analyses using all possible com- binations of the five independent variables used in this study (P > 0.500). We did observe a significant correlation between hemocyte numbers and weight condition index of the EA abalone (P = 0.0469), PC abalone (P = 0.0016) and NC abalone (P = 0.0015). Correlation coefficients, however, were low to moderate and ranged between 0.2668-0.5612. DISCUSSION The present study describes the transmission of WS from black abalone with WS to previously healthy black abalone held in the same aquaria. The similarity in physical, histopathological, and hematological characteristics of WS between black abalone ex- posed to WS in the laboratory and field, combined with a lack of these signs in the negative control animals, confirmed that the experimental abalone contracted WS in this stud\ (Haaker et al. 1992. VanBlaricom et al. 1993, Gardner et al. 1995. Shields et al. 1996, Friedman et al. 1997). These data also suggest that WS is directly transmissible between sympatric abalone by cohabitation. WS is a chronic, slow-progressing malady in which clinical signs appear in the final stages of the disease. The presence of advanced microscopic morphologic changes throughout the pedal muscle and digestive gland of affected abalone supports this con- clusion (Figs. 2-5). Our data also suggests a long incubation pe- riod for WS (-35 wk) followed rapidly by mortality (-42 days) under the conditions used in this study. As shown in Figure 6, once the animals developed clinical WS, the slopes of the mortality curves from the experimentally (EA) and field-exposed (PC) ani- mals were very similar. However, median survival times between Withering Syndrome in Black Abalone 821 Figure 4. Microscopic anatomy of tlie digestive gland of black abalone with and without WS. The digestive gland of unexposed abalone from both Vandenberg (Al and Ano Nuevo Island (D) is composed primarily of terminal tubules and little transport/absorptive duct epithelia. whereas those of abalone that contracted VVS in the field (B, Cl and laboratory (E, F) show a reduction in numbers of terminal tubules and an increase in connective tissue and transport/absorptive duct epithelia. The digestive gland of some abalone with WS is characterized by an atrophy and loss of terminal tubules (B, E), while other individuals respond to RLP infection (arrow heads) with a transport/absorptive duct metaplasia (arrows; C, F). Hematoxylin and eosin, bar = 150 fim. these two groups were quite different (41 wk for Ano Nuevo and 16 wk for Vandenberg/Cayucos animals) and may be due to a variety of factors. The Ano Nuevo Island EA abalone was unin- fected before initiation of the study, whereas the Vandenberg and Cayucos PC abalone were in varying stages of WS. In addition, differences in susceptibility may e.xist between abalone from these geographically distant locations. In an earlier study in which asymptomatic but previously exposed black abalone were col- 822 Friedman et al. ai CO +j to Q> u -a -a c o O LU w + 1 O o > pcch't\ ycssoensis. with comments on their significance. J. Fish Dis. 9:6^)-71. Friedman, C. S., T. McDowell, J. M. Groff, J. T. Hollibaugh, D. Manzer & R. P. Hedrick. 1989. Pre.sence of Bonamia ostreae among populations of the European flat oyster, Ostrea edulis Linne, in California, USA. J. Shellfish Res. 8:133-137. Friedman, C. S. 1991. Coccidiosis of California Abalone. Hciliatis spp. J. Slieilfish Res. 10:236. Friedman, C. S.. W. Roberts, G. J. Kismohandaka & R. P. Hedrick. 1993. Transmissibility of a coccidian parasite of abalone. Haliotis spp. / Shellfish Res. 12:519-525. Friedman, C. S., G. R. Gardner, R. P. Hedrick. M. Stephenson, R. J. Cawthom & S. J. Upton. 1995. Pseudoklossia haliotis sp. n. (Apicom- plexa) from the kidney of California abalone. Halimis spp. (Mollusca). / Inverlebr. Pathol. 66:33-3S. Friedman, C. S.. M. Thomson, C, Chun. P. L. Haaker & R. P. Hedrick. 1997. Withering syndrome of the black abalone, Haliotis cracherodii (Leach): water temperature, food availability, and parasites as possible causes. J. Shellfish Res. 16:403^11. Friedman, C. S. & T. W.-M. Fan. 1998. Withering Syndrome of black abalone: causes and physiological alterations. In: California Sea Grant Reports of Completed Projects 1994-97. La Jolla. CA: Sea Grant Col- lege System, pp. 101-106. Friedman, C. S.. K. B. Andree, K. A. Beauchamp, J. D. Moore. T. T. Robbins, J. D. Shields & R. P. Hedrick. 2000. "Camlidattis Xenohali- otis califomiensis", a newly described pathogen of abalone, Haliotis spp., along the west coast of North America. Frielier, P. F., J. K. Loy & R. Kruppenbach. 1993. Transmission of nec- rotizing hepatopancreatitis in Penaeits vaivuimci. ./. Invcrlchr. Puihnl. 61:44-48. Gardner. G. R., J. C. Harshbarger, J. L. Lake, T. K. Sawyer. K. L. Price, M. D. Stephenson, P. L. Haaker & H. A. Togstad. 1995. Association of prokaryotes with symptomatic appearance of withering syndrome in black abalone Haliotis ermherodii. J. Invertehr. Pathol. 66:111-120. Gulka. G. & P. W. Chang. 1984a. Pathogenicity and infectivity of a rick- ettsia-like organism in the sea scallop, Placopecten inagellanicus. J. Fish Dis. 8:309-318. Gulka. G. & P. W. Chang. 1984b. Host response to rickettsial infection in blue mussel. Mylilus edulis L. / Fish Dis. 8:319-323. Haaker. P. L., D. V. Richards, C. S. Friedman, G. E. Davis, D. O. Parker & H. Togstad. 1992. Mass mortality and withering syndrome in black abalone Haliotis cniehemdii in California. In: S. A. Shephard, M. J. Tegner & S. A. Guzman del Proo. editors. Abalone of the world. Oxford: Blackwell Scientific, pp. 214-224. Le Gall, G., D. Chagot. E. Mialhe & H. Grizel. 1988. Branchial nckettsi- ales-like infection associated with a mass mortality of sea scallop Pecten ma.xinuis. Dis. Aqiiat. Org. 4:229-232. Le Gall. G.. E. Mialhe. D. Chagot & H. Grizel. 1991. Epizootiological study of rickettsiosis of the Saint-Jacques scallop Pecten maximus. Dis. Ai/iiat. Org. 10:139-146 Luna, L. G., editor. 1968. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw-Hill, pp. 38-39. Min, K.-T. & S. Benzer. 1997. Wolbachia. normally a symbiont of D;v).ot- phila. can be virulent, causing degeneration and early death. Proe. Natl. Acad. Sci. USA 94:10792-10796. Moore. J. D., T. T. Robbins & C. S. Friedman. 2(J00. Withenng syndrome in fanned red abalone, Haliotis nifescens: thermal induction and asso- ciation with a gastrointestinal Rickettsiales-WVc prokaryote. / Aquut .Antuial Health. Shaw, B. L. & H. L Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster. Cras.wstrea virginica (Gmelin). Can. J. Zool. 35:325-347. Shields, J. D., F. O. Perkins & C. S. Friedman. 1996. Hematological pathology of wasting syndrome in black abalone. J. Shellfish Res. 15:498. Steinbeck, J. R., J. M. Groff C. S. Friedman, T. McDowell & R. P. Hedrick. 1992. Investigations into a coccidian-like protozoan from the California black abalone, Haliotis cracherodii. In: S. A. Shephard. M. J. Tegner & S. A. Guzman del Proo, editors. Abalone of the world. Oxford: Blackwell Scientific, pp. 201-213. VanBlaricom. G. R., J. L. Ruediger. C. S. Friedman. D. D. Woodard & R. P. Hedrick. 1993. Discovery of withering syndrome among black abalone Haliotis cracherodii Leach, 1814 populations at San Nicolas island. California. J. Shellfish Res. 12:185-188. Voltzow, J. 1994. Gastropoda: prospbranchia. In: I. Mollusca, F. W. Har- rison & A. J. Kohn, editors. Microscopic anatomy of invertebrates, vol. 5. New York: Wiley-Liss. pp. 1 1 1-252. Joiinml of Shellfish Research. Vol. 21. No. 2. 825-S30. 2002. HISTOPATHOLOGICAL EVALUATION OF THE YELLOW ABALONE HALIOTIS CORRUGATA AND THE BLUE ABALONE HALIOTIS FULGENS FROM BAJA CALIFORNIA, MEXICO MA. DEL CARMEN ALVAREZ TINAJERO,' JORGE CACERES-MARTINEZ,'* AND JOSE GUADALUPE GONZALES AVILES' ^ Laboratorio de Patologia de Mohtscos del Departamento de Aaiiciilliini. Ceiitro de liiveslii;acidn Cientifica y de Ediicacidii Superior de Ensenada, Apdo. Postal 2732. 22S00 Ensenuda. Bcija Ccdifornia. Mexico: 'Sociedad Cooperativa de Produccidn Pesquera. Pescadores Nucionales de Ahuldn. S. C. de R. L. Av. Ryerson 1 17. 22820 Ensenada. Baja California. Mexico ABSTRACT The yellow abalone Halioris corrugala and the blue abalone Haliotis ful,^eiu are caught in Baja California. Emerging diseases are affecting the fishery of abalone in several countries around the world. To determine the health status of the yellow and the blue abalone in Isia de Cedros and Islas San Benito in Baja California. Mexico, a histopathological survey of commercial stocks of these species was conducted. The results showed the presence of bacterial foci in epithelial cells of the digestive tract resembling to the intracellular bacterium "CiuuliJulus Xenohaliotis califoniiensis." considered to be the etiological agent of the Withering Syndrome (WS). The prevalence was higher in the blue abalone than in the yellow abalone. 100% and 63'7r. respectively; and their presence was not correlated with the external signs of WS. These bacteria were found in WS symptomatic and nonsymptomatic abalone. Protozoans and copepods were found between the branchial filaments, with a maximum prevalence of 79% and 37%. respectively; gregarines were also found with a prevalence of 29%. With the exception of the intracellular bacterium, the other organisms seem to he innocuous for the studied abalone. KEY WORDS: abalone. Hulions fiili-ens. Halialis corrugatci. histopathology. Rickettsia, withering syndrome INTRODUCTION Abalone production in Baja California (Mexico) was 365 met- ric tons in 1998. reaching a value of about 36 million US dollars. The abalone fishery has been one of the main sources of economic resources in the Peninsula of Baja California. Cunently. there are more than 1.300 direct employees involved in this activity (Ponce et al. 1998). Two species comprise 97% of this fishery, the blue abalone Haliotis fulgens and the yellow abalone Haliotis corrii- gata. Other abalone species included in this production are the black abalone Haliotis cniclieiodii. the red abalone Haliotis nife- scens. and the white abalone Haliotis soreiiscni (Ramade et al. 1998). During the last few years, an important decrease in the production has been recorded, which has been related to overfish- ing, inefficient application of management regulations, and envi- ronmental fiuctuatii)ns that have favored the development of some diseases (Haaker et al. 1992. Vanblaricom et al. 1993, Olivas- Valdez & Caceres-Marti'nez 2002). In 1984, dramatic mortalities of California black abalone were recorded after the occurrence of El Nifio. Lately, it has been suggested that the agent responsible for those mortalities was an intracellular bacteria from the order Rickettsiales (Gardner et al. 1995), tentatively named "Candidatus Xenohaliotis califomiensis" (Friedman et al. 2000, Moore et al. 2000, Friedman et al. 2002). External signs in the abalone include reduction of the foot muscle in relation to shell size, loss of ad- herence capacity, and death. Because of the foot muscle appear- ance, these symptoms are called withering syndrome (WS) (Haaker et al. 1992). It has been suggested that very high tem- peratures may increase the mortality of abalone affected by the WS (Friedman et al. 1997). Other parasites that have been recorded in abalone species are the protozoan Margolisiella ( = Pseudoklns- sia) haliotis, which infect the kidney and was first associated with the WS (Friedman et al. 1995). and the eukaryotic protist Laby- *Corresponding author. E-mail: jcaceres(a'cicese.mx riiitliiiloides haliotidis. which is pathogenic to juvenile abalone of H. kamtschatkana and H. rufescens (Bower 1987). Metazoan para- sites such as the nematode Echinocephalus pseudoimcinatus have been detected in H. cnrnigata. H. fulgens. and H. cracherodii (Milleman 1963). Damage to the host by these parasites varies and some of them cause severe mortalities (Bower et al. 1994). Studies of the parasites and symbionts in abalone from Mexico are scarce. These studies revealed a trematode belonging to the family Opecolidae in H. fulgens (Romero 1996), a boring clam in H. fulgens and H. corrugata (Alvarez-Tinajero et al. 2001 ). and the parasitic load of H. rufescens from a culture facility (Caceres- Marti'nez & Tinoco-Orta 2001). The present study was conducted as part of a health evaluation of a commercial catch of H. fulgens and H. corrugata from Isla de Cedros and Islas San Benito. Baja California. MATERIALS AND METHODS In the first sampling (November 1997), 19 individuals of H. corrugata and 16 individuals of H. fulgens were obtained from Punta Norte, north of Isla de Cedros; in March 1 998. 26 H. fulgens were studied from Islas San Benito; finally, in June 1998. 21 H. corrugata and 14 H. fulgens were obtained from San Agusti'n. south of Isla de Cedros (Fig. 1). Abalone species were obtained from commercial catches (mean size of 145 mm ± 1.73 SD) during commercial season when abalones are not in the reproductive stage. The external appearance of the abalone was recorded from a visual examination using a modification of the WS scale of Friedman et al. (1997) as: (0) no external symptoms, (1) from I to lOVr reduction of the foot muscle in relation to the shell, (2) from II to 20% reduction of the foot muscle. (3) from 21 to 30% reduction, and finally (4) from 31 to 50% reduction of the foot muscle. The visceral mass of the abalone was fixed whole in the David- . son's fixative (Shaw & Battle 1957) for at least 24 h. Seven trans- verse sections that contained portions of the digestive tract (pos- 825 826 Carmen Alvarez Tinajero et al. San Agustin RESULTS Figure 1. Map showing the sampling sites in Isla de Cedros and Islas San Benito, Baja California, Mexico. terior esophagus included), kidney, gonad, muscle, epipode, and gills were processed for histology. Sections of 5 |xm were stained with iron hematoxylin and eosin (Gray 1934). The rickettsia-like prokaryotes (RLPs) infection intensity was estimated considering the number of bacterial foci in each histologic preparation at 200x magnification, which were enumerated using a modification ot the scale of Friedman et al. (1997): (0+) no RLP. (1 + ) 1 to 10 RLP. (2+) 11 to 100 RLP. (3+) 101 to 1000 RLP. and (4+) >1000. Moreover, following the criteria of Friedman et al. (1997). the appearance of the digestive gland was marked as ( 1) normal tissues (see Antonio et al. 2000). (2) moderate tissue degeneration, and (3) severe tissue degeneration. Protozoa in the digestive tract and gill branchiae were counted in each histologic preparation. Prevalence of the other organisms was estimated as (number of infested aba- lone/number of abalone examined) x 100. The Mann-Whitney U Test was used to compare differences in the intensity of organisms per abalone species and the Spearman rank order coirelation test was used to determine the relationship between degeneration of the digestive gland, intensity of RLP. and external signs of WS (Zar 1984). RLPs The RLPs were found in H. fidgens and H. cornigata in the three study sites. These bacteria infected the epithelial cells of the digestive tract, including the posterior esophagus, stomach, diges- tive diverticula, and intestine (Fig. 2a and b). The RLPs formed colonies inside a large vacuole in the cytoplasm of the infected cells. These colonies varied in shape from round to elongated and their size ranged from 17.5 x 10.8 [xm to 24.3 x 10.2 |xm. The host cell nucleus was displaced to the limit of the cytoplasm, the host cell became hypertrophied. and in a heavy infection there was a metaplasia of the epithelium within the digestive gland. Some host cells were disrupted and bacterial colonies were expelled to the lumen of the digestive tract (Fig. 2a). Table 1 shows the RLP intensity of infection in H. fulgens and H. cornigala per studied site. The yellow abalone from Punta Norte showed a RLP prevalence of 63.2%; of these, 52.6% had a severe infection (3-i- to 4+). including a moderate degeneration (2) of the digestive gland in 41% of the animals, whereas the remain- ing 52.6% of the abalone did not show signs of degeneration of the digestive gland. Blue abalone from the same area showed a RLP prevalence of 43.7%. with severe infection (3+ to 4+) in 31.3% of the animals. Only 12.4% of the infected abalone showed a mod- erate degeneration of the digestive gland (2). The results showed a higher grade of infection in yellow abalone than blue abalone. but this was not significant statically (U Mann-Whitney. P = 0.33). There was no significant correlation between the degree of degen- eration in the digestive gland and the intensity of infection by RLP in the yellow and blue abalone species (Spearman rank order cor- relation ;■ = -0.19. P = 0.93 and r = 0.34. P = 0.18. respec- tively). This correlation was neither significant between the exter- nal signs and degeneration degree of the digestive gland (Spear- man rank order correlation r = 0.36. P = 0.12 and /• = -0.23, P = 0.37). The blue abalone from Islas San Benito showed a RLP preva- lence of 96.2%. and the infection was severe (3-i- to 4+) in 92.4% of infected abalone. In 56.2% of these abalone the digestive gland was normal whereas the remaining 43.8% showed a moderate Figure 2. (a) RLP in the intestinal epithelium. An RLP-infected area is shown where the epithelium of the post-esophagus has lost its structure by hypertrophy of infected cells and rupture (lA). The epithelium in front is in normal omdilion (HEl. There is mucus or cellular debris (M) in the intestinal lumen. Scale bar = 40 fim. (bl RLP in digestive diverticula. The hypertrophy of infected cells is compressing the neighbor cells. Scale bar = 20 pm. Evaluation of Haliotis corrugata and H. fulgens 121 TABLE 1. Intensity and pre>alente of RLP in H. corrugata and H. fulgens from different localities studied. TABLE 2. External signs scale of the WS in H. corrugata and H. fulgens. Punta Norte Islas San San Agusti'n Punta Norte San Agusti'n H H H H H. H. Islas San H. H. Scale corrugata fulgens Benito H. corrugata fulgens corrugata fulgens (<7r) Benito H. fulgens ( % ) corrugata (%) fulgens VVS (%) {Vc) fulgens ( % ) (%) {%) Scale 0 1 89.4 5.3 75.0 18.8 88.5 0 47,6 4.8 71 6 0 36.8 56.3 3.8 38.1 0 7,1 1 5.3 6.2 0 4.8 21.4 -) 5.3 6.2 7.7 28.6 0 2 5.3 6.2 3.8 42.8 21.4 3 0 0 3.8 19.0 14.2 3 42.1 18.8 46.2 14.3 35.8 4 0 0 0 0 7.1 4 10.5 63.2 12.5 43.7 46.2 96.2 0 61.9 21.4 100 /; 19 16 26 21 14 Prevalence // 19 16 26 21 14 degeneration (2). There was no significant correlation between the presence of RLP and degeneration of the digestive gland (Spear- man rank order correlation /• = 0.34, P = 0.08) or between WS external signs and the degeneration degree of the digestive gland (Spearman rank order correlation r = 0.33. P = 0.08). The yellow abalone from San Agusti'n showed a RLP preva- lence of 61.9% and the infection intensity was medium (2+) in 42.8% of the organisms. In relation to the degeneration of the digestive gland. 57.1% of the infected organisms had a moderate degeneration (2), 14.28% had a severe degeneration (3), and 28.6% had a normal digestive gland. There was no significant correlation between the RLP intensity and degeneration degree of the digestive gland (Spearman rank order correlation r = -0.03. P = 0.86). However, a sign correlation was observed between the external signs of WS and the degree of digestive gland degenera- tion (Spearman rank order correlation r = 0.75. P < 0.001). The blue abalone from the same area showed a RLP prevalence of 100%, with severe infections (3-i- to 4-i-) in 57.2% of these abalone. Of the severely infected abalone, 28.5% showed a moderate de- generation of the digestive gland, 14.28% showed severe degen- eration, and 57.14% showed a normal appearance of the digestive gland. In both species, the correlation between the RLP intensity and degeneration of the digestive gland was not significant (Spear- man rank order correlation ;■ = 0.08. P = 0.75), but it was sig- nificant between the WS external signs and the degeneration of the digestive gland (Spearman rank order correlation /■ = 58.36, P < O.OOI). The infection prevalence from San Agusti'n was signifi- cantly higher in the blue abalone (U Mann-Whitney P < 0.01 ) hut not from Punta Norte (Table I). Table 2 shows the percentage of both abalone .species with external signs of the WS. At the microscopic level, all these or- ganisms showed some tissue disorders, such as an increase in connective tissue between digestive tubules; lack of gonadal matu- ration and in a few occasions duct metaplasia or intlammation. The foot muscle showed a reduction in muscle fibers and loss of muscle bundle orientation, increase in connective tissue and infil- tration by hemocytes. There was no correlation between the WS external signs and the presence of RLP in tissues (Table 3); in some individuals with WS external signs, infection by RLP was not detected (Fig. 3). Ciliated Protozoan A ciliated protozoan (Fig. 4a) was found in the mantle cavity and gill filaments of the yellow and blue abalone. The unidentified ciliates measured 26.5 |jim x 12.7 \x.m. No histologic disorders or lesions were associated with these organisms. Prevalences were low in both species with 10.57f of the yellow and 6.3% of the blue abalones from Punta Norte (Table 4. Mann Whitney U, P = 0.66). Prevalences were higher at the other two locations where over 40% of the blue abalone from Islas San Benito and over 70% of both yellow and blue abalones from San Agusti'n were infested (Table 4. Mann Whitney U. P = 0.25). Copepods Copepods were found among the filaments of the gill where some compression and infiltration of hemocytes in the tissue was detected (Fig. 4b). The mean size of the copepods was 200 (xm x 50 |j,m. Copepod prevalence in the yellow abalone from Punta Norte was 36.5%, there were no copepods in blue abalone from this locality. Copepod prevalence in blue abalone from Islas San Benito was 34.6%. The yellow abalone from San Agusti'n showed a prevalence of 19.0%, in the blue abalone only one copepod was recorded. There were no significant differences of copepod inten- sity between both abalone species (pooled data, U Mann-Whitney P = 0.34). Gregarines Low prevalences of trophozoites of a gregarine protozoan (Fig. 4c) were observed in the branchial epithelium, esophagus epithe- lium and kidney of the blue abalone (Table 4). The organisms measured 22 jxm x 14(jLm and no host reaction against this proto- zoan was observed. Prevalences ranged between sites from 0 to a high of 28.7% (Table 4). The limited number of data precluded the use of a statistical test for comparison. TABLE 3. Spearman rank correlation between the WS external scale and the abundance of RLP in H. corrugata and H. fulgens. Punta Norte Islas San Benito H. fulgens San Agusti'n H. H. corrugata fulgens H. H. corrugata fulgens R P value 0.05 0.06 0.82 0.79 0.18 0.37 -0.09 -0.008 0.68 0.97 828 Carmen Alvarez Tinajero et al. O Abalone without WS ■ Abalone without WS and with RLP ■ Abalone with WS □ Abalone with WS and RLP H. commala H. fulaens _H. I'uluens ii. corru^ata H. fulgens Punta Norte Islas San Benito San Agustin Figure 3. Percentage of the WS symptomatic and nonsymptomatic abalone and the intensity of RI>P infection lor Haliotis corriigata and Haliotis fulgeiis. « '-* G Figure 4. (al filiated protozoan (I'l bet»een branchial gill lilamenls. Scale bar = 20 Mm. (bl Copepod between gill lllaments (arrow). Note the intlltration of hemocytes in lilamenls surrounding the copepod (IN). Scale bar = 40 urn. (c) (Jregarinc protozoan ((i) in the right kidney of H. fulgens. Scale bar = 15 jim. Evaluation of Haliotis corrugata and H. fulgens 829 TABLE 4. Other organisms in abalone H. corrugata y H. fulgens per locality. Locality Ciliated proto/oan Measured: 26.5 x 12.7 |xni Location: Mantle cavity and gil Punta Norte filaments Islas San Benito San Agusti'n Copepods Measured; 200 x 50 jjim Location: Filaments of gill Punta Norte Islas San Benito San Agusti'n Gregarines Measured: 22 x 14 \km Location: Branchial, esophagus, and kidney Punta Norte Islas San Benito San Agusti'n Abalone Species H. corrugata H. fulgens H. fulgens H. corrugata H. fulgens H. corrugata H. fulgens H. fulgens H. corrugata H. fulgens Mean H, corrugata O.I H. fulgens 0.2 H. fulgens 4.6 H. corrugata 62.7 H. fulgens 11.1 1.0 0.5 0.3 0.1 0.06 (1.07 0.4 Prevalence 10.5 42.3 71.4 78.5 36.5 34.6 19.0 7.1 6.2 3.8 28.5 Max. Intensity 2 4 28 594 346 DISCUSSION Caceres-Marti'nez and Tinoco-Oila (2001) and Culver and Ri- chards (1992) mentioned that the WS was observed in the black (Haliotis cracherodii) green (H. fulgens) and red {H. rufescens) abalone from Baja California. Mexico; however, this is the first record of RLP infecting the tissues of H. fulgens and H. corrugata. The histologic appearance, shape, size, and targeted cells are simi- lar to that of the intracellular bacterium "Catulidatus Xenohaliotis califomiensis" causing the WS in H. cracherodii and H. rufescens in California. (Friedman et al. 2000). Further characterization is needed to confirm this hypothesis, therefore molecular methods for RLP detection are necessary (Andree et al. 2000. Antonio et al. 2000). The absence of a relationship between the intensity of RLP infection with the external signs of WS and the degeneration de- gree of the digestive gland in both abalone species was previously observed in studies on WS in black abalone (Friedman et al. 1997, Friedman et al. 2002). Moreover, Caceres-Marti'nez and Tinoco- Orta (2001) found changes of the digestive gland (metaplasia) in red abalone infected by RLP but also in noninfected organisms. Only in San Agusti'n was the relationship between the WS external signs and the degeneration of the digestive gland significant. Fried- man et al. (1997) and Friedman et al. (2002) showed that the WS disease agent requires a long incubation period before the externa! symptoms become apparent. This could help us to explain this contradictory result. Another possibility is the existence of sub- species or strains of RLP, some pathogenic and other nonpatho- genic. Moreover, the external signs of the WS are not exclusive of this disease, other diseases or starvation conditions may result as external symptoms similar to those of the WS (Milleman 1963, Dixon et al. 1991); also, differences in susceptibility between aba- lone species and individuals are possible. Friedman et al. (2002) noted "studies that examine the interaction between host gastroin- testinal cells and the RLP may provide insight into the cellular physiology of the host and the physiology and disease mechanisms of the bacterium." Vanblaricom et al. (1993) also found a suctorian protozoan in gill squashes from the black abalone H. cracherodii. both in healthy specimens and in organisms affected by the WS. Ciliated protozoa are common in marine invertebrates, and most of them are considered as commensals (Lauckner 1983). Our histopatho- logical results did not show any host response to the protozoa, and the low numbers suggested a lack of potential for a health problem. To our knowledge, this is the first record of a copepod associ- ated with abalone species. Copepoda and mollusk associations have been widely studied, Mytilicola intestiiudis and P.^eiidomyi- cola spinosus are two of the most studied copepods associated with bivalve mollusks. These copepods inay produce severe damage to the epithelial cells of the digestive tract, they may produce encap- sulations in the connective tissue of the digestive gland, and they may produce obstruction and rupture of reproductive follicles (Sin- dermann 1990. Olivas-Valdez & Caceres-Marti'nez 2002). In this study, no evidence of copepods inside of the abalone digestive tract was found, but some inflammation in the gills of abalone was observed in some cases. Their prevalence suggests a symbiotic association but more studies are needed for determining a kind of interaction between the copepods and the host. In the blue abalone, we recorded the presence of gregarines in the renal tissue and no histologic evidence of reaction of the host against the protozoan was observed. Some authors (Vanblaricom et al. 1993) found similar protozoa in the black abalone. Haliotis cracherodii from California and from Baja California, Mexico. In 830 Carmen Alvarez Tinajero et al. both studies, no evidence of pathogenic activity was found. A similar gregarine infection has been observed in mussels, oysters and other marine bivalves, in some cases the infection has been associated with a focal, benign inflammatory response, without significant health effects (Bower et al. 1994). AKNOWLEDGMENTS The authors thank Rafael Muiio/ Gallardo and Amado Castro from the Cooperative Pescadores Nacionales de Abulon for an agreement with CICESE for the financial support of this work. WORKS CITED del C. Alvarez-Tinajero, M., J. Caceres-Mati'ne/. & J. G. Gonzalez-Aviles. 2001. Shell boring clams in the blue abalone Htilioti.s fiilgens and the yellow abalone Haliotis cornigaia from Baja California Mexico. J. Shellfish Res. 20:889-894. Andree. K. B., C. S. Friedman. J. D. Moore & R. P. Hcdrick. 2000. A polymerase chain reaction assay for the detection of genomic DNA of a Rickettsialesdike prokaryote associated with withering syndrome in black abalone Htiliolis cnicheriHlli (Leach). ,/. Shfllll\h Res. 19:21.5- 218. Antonio. B. D.. K. B. Andree. J. D. Moore. C. S. Friedman & R. P. Hedrick. 2000. Detection of Rickettsiales-like Prokaryotes by in situ hybridization in black abalone, Haliotis cracherodii. with Withering Syndrome. / Imertehr. Pathol. 75:180-182. Bower, S. M. 1987. Labyriiithuloides haliotis n. sp. (Protozoa: Labyrintho- morpha), a pathogenic parasite of small juvenile abalone in a British Columbia mariculture facility. Can. J. Zool 65:1996-2007. Bower, S. M., S. E. McGladddery & I. M. Price. 1994. Synopsis of infec- tious diseases and parasites of commercially exploited shellfish. Ann. Rev. Fish D/j 4:1-199. Caceres-Martinez. J. & G. D. Tinoco-Orta. 2001. Symbionts of cultured red abalone Haliotis nifescens from Baja California. Mexico. J. Shell- fish Res 20:875-881. Culver, C. S. & J. B. Richards. 1992. Black abalone mortality: Establishing a research agenda. Report No. T-CSGCP-024. LIniversity of California: California Sea Grant College. Dixon. M. G.. T. Hecht & C. R. Bryt. 1991. Identification and treatment of Clostridium and Vibrio infection in South African abalone. Haliotis midae L. J. Fish Dis 14:693-695. Friedman, C. S., G. R. Gadnerd, R. P. Hyrick, M. Stephenson, R. J. Caw- thorn & S. J. Upton. 1995. Pseudoklossia haliotis sp. n. ( Apicomplexa) from the kidney of California abalone. Haliotis spp. (Mollusca). / bivertehr. Pathol. 66:33-38. Friedman. C. S., M. Thomson, C. Chun, P. L. Haaker & R. P. Hedrick. 1997. Withering syndrome of black abalone. 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EFFECT OF EL NINO 1997-98 ON THE SNAIL ASTRAEA UNDOSA (WOOD) POPULATION ALONG THE BAJA CALIFORNIA WESTERN COAST MARIA GEORGINA GLUYAS MILLAN,*' CASIMIRO QUINONEZ VELAZQUEZ," AND JESUS TALAVERA MAYA' 'in.stituta Nacional de La Pesca-SAGARPA, Centra Regional de Investigacion Pesquera-La Paz, Carretera a Pichilingue Km 1 s/n. C.P. 23020, La Paz. B.C.S., Mexico: 'Centra Interdisciplinario de, Ciencias Marinas-bistitiito Politecnico Nacional. Apdo, Postal 592. C.P. 23000, La Paz, B.C.S.. Mexico. ABSTRACT The effect of the 1997-98 El Niiio on the snail Astraea undosa population along the Baja California western coast on biomass changes in June 1997 and July 1998 was evaluated. In 1998. dead snails >40 mm basal diameter were collected and their basal diameter measured. Individual weight and density (snail,s/10 nr) decreased significantly in 1998 from those registered in 1997, with an average 47% decrease in biomass. We discuss the possible causes of those changes as a function of the disappearance of the Macrocystis pyrifera forest and of an e.xtended period of positive anomalies of the sea surface temperature that were present previous to July 1998. KEY WORDS: El Nifio 1997-98, snail, Aslraea undosa. biomass, Baja California. INTRODUCTION The 1997-98 El Nifio has been considered the most intense of the twentieth century (Kerr 1998, McPhaden 1999). It was de- tected along the Baja California western coast through positive anomalies of the SST >3°C. In July 1998. we observed dying as well as dead snails Aslraea undosa (Wood) in the imertidal zone and in fishing banks. No kelp forests M. pyrifera (Agardh) were found in Bahia Tortugas from October 1997 to April 1998 (Ladah et al. 1999). This species together with Eisenia arborea (Aresch) are the food source and habitat of A. undosa. A. turlxviica (Dall), and other species of herbivores of commercial interest such as the abalone Haliotis fulgens (Philippi) and H. ci>rrus>atu (Wood) (Guzman del Proo et al. 1991 ). In July 1998 we monitored the A. undosa populations located on the Baja California peninsula western coast to evaluate the effect of the 1997-98 El Niiio. This region carries the highest abundance of A. undosa. where >80% of its catch is obtained (Gluyas-Millan et al. 2000). The size structure of live and dead snails, average size and weight, density and biomass of July 1998. were contrasted with those obtained from the same fishing banks in June 1997. We assumed that the sampled snails in July 1998 reflect the effect of the 1997-98 El Nino. MATERIALS AND METHODS The study area is located between Punta Eugenia (27°5rN- 115'04'W) and Bahi'a Asuncion (27°08'N-1 14'^I7'W) (Fig. 1). The area was divided into three zones and three fishing banks were selected out of each one (Table 1 ). We believe the selected geo- graphical area was adequate because it is the main area of abun- dance of A. undosa in its distribution range along the Baja Cali- fornia western coast, and also because of the accessibility of the sampling places, and because this is where the main snail fishing banks are located. Monitoring was done in June 1997 and in July 1998 in the same places. As an environmental indicator of the 1997 to 1998 El Nino conditions we used the SST monthly anomalies of 1996 to 1998 from the deviations of the monthly average of the ten-year period before 1997 for the Bahi'a Tortugas area. The SST values were extracted from NOAA COADS records (Comprehen- sive Ocean and Atmospheric Data Set) for the Bahi'a Tortugas area. The comers of the polygons that comprise the area fishing banks were estimated using a GPS receiver (Gamiin model 45XL) (Table 1 ). The monitoring in June 1997 and July 1998 consisted of conducting up to 10 dives at each fishing bank (a, b, c), using a Hooka diving equipment at depths between 5 and 20 m. In each dive, all snails inside a 10-m"-sample unit (SU) were taken and the basal diameter (BD) of each snail was measured, grouping them in intervals of 5 mm to obtain the size frequency distribution. A subsample was obtained by randomly selecting up to five snails of each size interval and measurements were made of the BD of the shell (±1 mm) and the weight (±1 g) of the adductor muscle to estimate the biomass. The area of each fishing bank was transformed to SUs. Snail density for each fishing bank was stratified according to the snail number frequency distribution in each SU (Cochran 1978). The number of density intervals (DI) was determined with the Sturges rule (Daniel 1984). An estimate of the population total abundance of snail was obtained according to the estimators of the random stratified sam- pling technique (Schaeffer et al. 1987, Conquest et al. 1996): Average population estimate was A^ E ^. y, Total abundance population estimate was Nv, S ^. ■^' Where N = total number of SU, N, = SU number in the fishing bank,, L = number of density intervals, / = density interval av- erage. From the total population abundance estimation, the biomass was calculated by size interval with the following relationship: BIT ={aJ^)* ^N,y, * r *Corresponding author. E-mail: gmillan@balandra.uabcs.m,\ where BIT = size interval biomass. a and p = weight-size rela- tionship parameters, (Xf= i N, y,) = biomass in snail numbers, 0, 8.31 832 Gluyas Millan et al. Punta Eugenia \ r ^ ,.\- ^ \ \ A \__^ Bahia"~- ^ ^\^^ )\K^ "27°43' ^ "Tortugas ^ '-J ~-T~ \^^ •v^— -^ ^ Ic ^\ \ \\\% c lla ^ ^_^^ Bahia San\ ] \ "" ^^^ristobal "^^''^^^ | lib N. \ \ PACIFIC lie. Isia San^ OCEAN L iii^ • -^ Roque 27°13' lllbSN/ ^ lllc Bahia 114°5r 1 114°28' Asuncion 1 Figure I. Baja Calitornia Hestern coasC and liicalion of sampling sites. The names of tlie sampling locations are in Table I. = relative frequency accumuluted by size interval from total snails measured. The total biomass for all the size intervals was estimated by: Bt ^BIT To determine the empty shells of snails from individuals that died in 1998, and that did not come from the commercial catch of that year or earlier, observations of presence-absence of fauna and flora epibiotics were made inside the shell. Additionally, we know thai the snails are not killed in the fishing banks, and furthermore the snail-fishing season was suspended in 1998. except in Bahia Tor- tugas and Isla Natividad. The latter is 7.5 km NW off Punta Eu- genia (Fig. 1). RESULTS In July 1998 we observed that A. undosa snails at depths of 5 to 20 ni were dying. In addition empty shells were found in the intertidal zone, and few M. pyrifera stalks between Punta Eugenia and Bahia Asuncion. July 1998 was preceded by a period of in- tense warming, the SST rose to 26°C (Fig. 2A). SST positive anomalies >3°C persisted from September 1997 to January 1998 (Fig. 2B) and the M. pyrifera forests disappeared in Bahia Tortu- gas. The June 1997 monitoring was not preceded by conditions of intense warming (Fig. 2A. B). and the snails showed normal con- ditions, the appearance of the adductor muscle was not tlabby as in July 1998; dead snails were not observed, and the M. pyrifeni forests were present. Dead snails in the inlcilida! area in July 1998 showed remains of viscera, a flabby adductor muscle, the opercula fixed to the muscle, and absent epibiotic fauna and flora in the shell internal surface. This indicates that these snails were not the product of fishing and that they had recently died. In each sampling zone in July 1998. the range and the size frequency distribution of the living as well as of the dead snails were similar (P > 0.05. KS-test) (Fig. 3): indicating the 1 997-98 El Nifio affected proportionally those snails whose BD was >40 mm. This result is reinforced when contrasting the size frequency dis- TABLE I. Astraea undosa fishing hanks by zone on the Baja California western coast. See Figure 1 for geographic location. References above the Fishing Fishing Banks Zone Banks Location Dimensions (nr) 1 a La Banderita-Punta Quebrada .118.(10(1 1 b Punta Priela-La Cantina 318.000 Ic Los Morros-Clam Bay 318.000 lla Cabo Pruneda-Cerro del Calvillo 104.000 II b El Muerto-Punta Prieta-Salado 124.000 lie San Pablo 136000 III a Punta .San Pablo-Puerto San Pablo 51.000 lllh Vuelta del Cerro-Reef-Crucecita 63.000 IIU Isla .San Roque 75,000 tribulion of 1997 and 1998 (Fig. 3). which showed no significant differences (P > 0.05. KS-test 1. This suggests that the effect of El Nino 1997 to 1998 was not selective for snails >40 mm BD. In 1997 and 1998 the mean BD of the live snails by zone was not significantly different (P > 0.05. r-test) (Fig. 4). This confirms the previous results that indicate an effect on the population with- out size selectivity. The mean individual weight, density, and bio- mass were significantly greater in 1997 than in 1998 (Fig. 4) {P < 0.05. /-test). This shows a negative effect on individual weight and density of the snail and in consequence the 1998 biomass de- creased. The magnitude of the biomass was variable among the zones because of the different area sizes (Table 1 ). The biomass decreased 47% on average in 1998 as compared with that regis- tered in 1997. Because of this decrease, the commercial catch season was suspended in 1998 in most of the study area. Observations made in June 1998 of the A. undosa stomach contents indicated important proportions of silt and crustose cor- _ 27 O a; 23 3 re E H 15 o OS E o c m Q) re c re ^ ■d O c re ^ O c re Q. < O 0) Q. E 4 n ft^^^^^^ B 2 - 0- 2- -3 A^^ ,a ^ ^.-A 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ' —5 Q. 3 < -^ o ^ O. 3 U < ^ o ^ re a. 3 t3 O 1998 1996 1997 Figure 2. i.\) Monthly sea surface temperature (SSI), and (B) SST anomalies from 1946 to I^VS. Data obtained from NOAA COADS records of the Bahia Tortugas area. El Nino Effect on the Astraea undosa Population 833 35 30 25 □ live snail 1998 -^dead snail 1998 ■i live snail 1997 Zone I 40 50 60 70 80 90 100 110 120 130 140 Basal diameter (mm) Figure 3. Size frequency distribution of the snails Astruea undosa from the Baja Cahfornia western coast in June 1997 and July 1998. alline algae (Lithothamnium or Lithophylwn). articulated coralline algae (Bossiella orbigniana [(Manza) Johans], and Coratlina offi- cianalis [L.]). They were the predominant flora then and are of temperate and subtropical affinity (Guzman del Pioo et al. 1991 ). These algae covered a great extension of the rocky surface at all the levels of the sampling depths. In June 1997 the diet compo- nents were varied, and we observed in the snail stomachs an im- portant proportion of M. pyrifem. which was still present in the area (Raiil Reyes Tisnado. pers. com.). It was not possible, how- ever, to quantify the stomach contents. DISCUSSION Though the 1997-98 El Nifio was detected from summer 1997 in the northern Pacific Ocean (Liu et al. 1998. Connolly & Rough- garden 1999). its impact on the A. undosa population along the Baja California western coast was not evident until the summer of 1998, when it became obvious that the biomass of snails had decreased and dead snails were seen. These events were also re- ported at La Bocana. 70 km south of Isla .San Roque (Daniel Aguilar Osuna. pers. com.). The results and the observations suggest that the observed mor- ^1201 • 1997 g diameter 00 o o o " 1 CD I/) CD ^ 60 J P=0 21 1 o 1998 t P = 0 45 P=0 32 80 ro 60 - CT 40 ^ 20 0 cT 50 E o 40 I 30 ^ 20 « 10 CD D P = 0 02 P=0 03 I 1 0 P<0 01 P=0 01 P<0 01 i \ \ P<0 01 40 n ■Z: 30 m 20H CD E o CO 10 \ P<0 01 30 20 10 0 4 \ 12n P<0 01 P<0 01 { Zone Zone Zone Figure 4. Average and standard deviation of the basal diameter (mm I. individual weight (g), density (snails/l(l nr), and biomass III by sam- pling zone of the snail Aslraea undosa from the Baja California west- ern coast in June 1997 and July I99S. tality and the diminished biomass that occurred in 1998 were caused by weakness and starvation because of the absence of M. pyrifem, and a possible long-warming period temperature effect that contributed to the disappearance of kelp forest (Ladah et al. 1999). Nearly all snails >40-mm BD are mature individuals, be- cause the minimum reproductive size is attained at 58-mm BD (Belmar-Perez et al. 1991). Those snails would be >2-years-old according to the BD-age relationship reported by Gluyas-Millan et al. (2000). The individuals <40 mm-BD are in the cryptic phase (Gluyas-Millan et al. 1999, Gluyas-Millan et al. 2000) and they were not observed in the study period. The biomass decrease of the A. undosa population from 1997 to 1998 was the result of both an increased natural mortality and 834 GLUYAS MlLLAN ET AL. decreased individual vveiL'hl and can be attributed to two factors: 1 ) The prolonged disappearance of the forests of M. pyrifeni from October 1997 to April I99S (Ladah et al. 1 999 ) affected the habitat and the food availability of A. imdosa. This alga is one of the main food sources of /\. iindosa (Guzman del Proo et al. 1991) and also provides protection against predators; and 2) The physical factors associated with 1997-1998 El Nifio had a density-independent influence because their potential effect in each member population >40-mm BD is the same regardless of population size (Sissenwine 1984). Therefore, the increase of SST and the change of the pre- dominant diet coinponents could have affected the mollusk physi- ology and consequently its growth efficiency. The decrease of the 1998 snail biomass resulted in an important economic loss for the fishing sector. Because of the low yields and the poor quality of the adductor muscle fishing ceased at most of the fishing banks. These fishing yields decreased from 166 t in 1997 to44 t in 1998. El Nino negative effects have been documented in the eastern Pacific Ocean on the productivity, abundance and distribution of invertebrates, the recruitment failure of some fish, and the devas- tation of the M. pyrifeni forests (Lenarz et al. 1995, Teagner & Dayton 1987, Ladah et al. 1999). There are also positive effects in the recruitment of the balanus Balanus glaruhihi and Chthamahis spp. along the California coast attributed to an increase of the transport toward the coast (Connolly & Roughgarden 1999). For A. iindosa along the Baja California western coast, the 1997-1998 El Nino negatively affected its survival. The effects of this on the structure and function of the M. pyrifera forest ecosys- tem where A. imdosa is an important species, cannot be ignored, even when the con.sequences of these impacts on the structure and function of the ecosystem are difficult to quantify (Gislason et al. 2000). Moving from population to the ecosystem level increa.ses complexity and we lack the general rules to help us predict changes in the abundance of the interacting species (Lawton 1999). However, the annual evaluations of the A. imdosa biomass from the National Institute of Fishing (INP-Mexico) will allow the evaluation of the recovery of .4. imdosa associated with the M. pyrifera forests. ACKNOWLEDGMENTS The authors thank Ramon Hernandez, from CRIP-La Paz. for his collaboration during the monitoring. We also thank the tech- nicians and members of the following fishing cooperatives for their support in the collection of the biologic material: SCPP •'Bahi'a Tortugas", SCPP "Emancipacion" and SCPP "California San Ignacio". CQV was supported by COFAA-IPN and CONA- CYT-Mexico. Thanks to Dr. Ellis Glazier who edited the English- language text, and two anonymous reviewers for advice and edi- torial help. LITERATI) Belmar-Perez, J.. S. Guzman del Proo & I. Martinez-Morales. 1991. 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Symposium overview: incorporating ecosystem objectives within fisheries manage- ment. ICES J. Mar Sci. 57:468-475. Gluyas-Millan. M. G., C. Quiiionez-Velazquez, J. A. MasstV-Rojas & F. N. Melo-Barrera. 1999. Diferencias en la relacion talla-edad del cara- col panocha Aslraea imdosa entre dos localidades de Bahia Tortugas. Baja California Sur, Mexico. Cienc. Mar. 25:91-106. Gluyas-Millan, M. G.. C. Quiiionez-Veldzquez & J. Talavera-Maya. 2000. Parametros poblacionales del caracol Astraea undosa (WOOD 1828) de la costa occidental de la Peninsula de Baja California. Cienc. Mar 26:643-658. Guzman del Proo, S., S. Mille-Pagaza, R. Guadarrama-Granados. S. de La RE CITED Campa, J. Carrillo-Laguna. A. Pereira-Corona, J. Belmar-Perez, M. J. Parra-Alcocer & A. Luque-Guerrero. 1991. La comunidad bentonica de los bancos de abulon (Haliotis spp. mollusca:gaster6poda) en Bahia Tortugas. Baja California Sur. Mexico. An. Esc. Nac. Cienc. Biol. Mexico 36:27-59. Kerr. R. A. 1998. Models win hig in forecasting El Niiio. Science 280: 522-523. Ladah, B. L., J. A. Zertuche-Gonzalez & G. Hernandez-Carmona. 1999. Giant kelp [Macrosislys pyrifera. Phaeophyceae) recruitment near its southern limit in Baja California after mass disappearance during ENSO 1997-1998. / Phycol. 35:1106-1112. Lawton, J. H. 1999. Are there laws in ecology? Oikos 84:177-192. Lenarz, W. H., D. Ventresca, W. M. Graham, F. B. Schwing & F. Chavez. 1995. Explorations of the El Nifio events and associated biological population dynamics off Central California. Cal.COFI Rep. 36:106- 118. Liu, W. T.. W. Tang & H. Hu. 1998. Spaceborne sensors observe El Nino's effects on ocean and atmosphere in north Pacific. EOS 79:249-252. McPhaden, M. J. 1999. Climate oscillations-Genesis and evolution of the 1997-98 El Niiio. Science 283:950-954. Schaeffer, R. L. W. Mendenhall & 1. On. 1987. Elementos de muestreo. Iberoamerica. Mexico D.F.: Grupo Editorial 321 pp. Sissenwine, M. P. 1484. Why do fish population vary' In: R. Ed & M. May, editors. Exploitation of marine communities. Dahlem konteren- zen Bedin: Spring-Veriag. pp. 59-94. Teagner, M. J. & P. K. Dayton. 1987. El Niiio effects on southern Cali- fornia kelp forest communities. Adv. Ecol. Res. 17:243-279. Journal of Shellfish Research. Vol. 21, No. 2, 835-841. 2002. REPRODUCTION OF THE SPOTTED PINK SHRIMP, FARFANTEPENAEUS BRASILIENSIS (DECAPODA: PENAEIDAE) MARTHA E. SANDOVAL-QLINTERO' AND ADOLFO GRACIA" ^ Centra Regional de Inve.stii;aci(in Pesquera de Puerto Morelos. Instititto Nucional de la Pesca. Apartado Postal 580. Ccinciin. Quintana Roo. 77501. Me.xico. Fa.x: 99H 871 0076. insandovaI@ww2.net.in.\ 'Institiito de Cieneitis del Mar y Linmologia: UNAM. Apartado Postal 70-305. Me.xico. D. F. 045 JO. Me.xico. Fa.x: 55 5616 2745. grucia@inar.icmyl.whim.mx ABSTRACT Based on the information from catches in Contoy. QR, Mexico (Fehruary-Decemher 1993). the reproductive cycle of the spotted pink shrimp. Farjanlepenaeiis hrasiliensis. is described. Population si/e structure shows that fishery exploitation affects small sizes. Females were more numerous than males, particularly during months of greatest reproductive activity. The period for ripe females period occurs from February to August, peaking in March and April. Main recruitment offshore follows from this spawning population. The estimated size at first reproduction averaged 148 mm. According to this estimation. 65-80% of the females have not been able to reproduce at least once before being caught. We observed a significant relationship (Pearson P = 0.05) between the larger size shrimps and a proportionally larger number of ripe females with depth from February to August. Based on catch-per-unit-effort indices (CPUE) of ripe females, we propose the region located between 21''40' to 22"10'N and 86°30' to 86°S0'W. at depths of approximately 25 to 40 fathoms (33 to 59 m) as the main spawning area for the spotted pink shrimp. F. hrasiliensis in the northeast region of Contoy. QR. Mexico. KEY WORDS: spotted pink shrimp, reproductive dynamics, maturity, sex ratio INTRODUCTION The spotted pink shrimp, FarfanlepenaeiLS hrasiliensis La- treille 1817, is a commercially important species in Mexico where the main fishery is located in the Mexican Caribbean along the coast of the state of Quintana Roo. This impotlant commercially area is near the noilhem end of the species geographic distribution which extends from Florida. USA. to the Rio Grande do Norte in Brazil (Perez-Farfante 1969. 1988). In the Mexican Caribbean, F. hrasiliensis is the target of a trawl fishery. Landings also consist of rock shrimp, Sicyonia breviros- tris, which is of lesser commercial importance and represents ap- proximately 55'7r of the total catch. Recorded production of spot- ted pink shrimp is low compared with the penaeid shrimps har- vested in the Gulf of Mexico (F. aztecus. F. duorarum and Litopenaeus setiferus). However, fishery exploitation of the spot- ted pink shrimp represents an important regional activity account- ing for more than 45% of the crustacean production of the state (Anonymous 1995). The annual landings of spotted pink shrimp have declined since 1984—1987. when maximal production was attained (309-474 Metric tons), falling to less than 200 tons during recent years. Similar to other tropical penaeid stocks (Graci'a et al. 1997). the spotted pink shrimp, P. hrasiliensis. has reached its maximal sus- tainable exploitation levels, or even surpassed them, posing serious problems for the resource. Despite its regional importance, the spotted pink shrimp fishery has not been described adequately, and the available literature about its biology and ecology is scarce. There have been some previous studies about growth (Arreguin Sanchez 1981a), gonad development (Sandoval-Quintero and Gra- cia 1998), general distribution (Porras-Ruiz et al. in prep.) and some fishery aspects (Arreguin Sanchez 1981b. Soto-Aguirre in prep.), but information about main population processes is still lacking. Basic information on the reproductive biology of this species is needed to establish adequate management policies to sustain this resource. This study presents data on the temporal pattern of re- productive activity and distribution of females of F. hrasiliensis in different stages of gonadal maturation in relation to depth. STUDY AREA The F. hrasiliensis fishery in Quintana Roo. Mexico operates for most of the year, except for the closed season which occurs around August and September since 1994. The main fishing loca- tion is on the continental shelf of the Yucatan Peninsula, northeast of Contoy Island, between 21=27' to 22°I8'N and 86°34' to 87°0I 'W (Porras-Ruiz et al. in prep.). The marine bottom is sandy, with large coralline formations that limit the operation of trawlers to regions locally called "blanquizales" which are free from rocks and corals. It is reported that F. hrasiliensis is also distributed over coralline bottoms, hence part of the population is not subjected to fishing gears (Soto-Aguirre in prep.). MATERIAL AND METHODS Spotted pink shrimp were collected from the fishing area of Contoy from February to December 1993 onboard shrimp trawlers. The monthly information obtained consisted of sampling coordi- nates, depth and duration of each of the tows, total length of the shrimp (TL. measured from the tip of the rostrum to the lip of the telson. at a 0.5 mm precision), sex, and gonadal stage of females by visual inspection according to a gonad color key based on a dissecting analysis (Silva Neto et al. 1982, Sandoval-Quintero and Gracia 1998). The calculated appropiate minimum size of the monthly sample taken from the commercial catch was 90 individu- als (Daniel, 1987). at a confidence level of 95% {P = 0.05). Sex ratio for each month was analyzed through a x"^ independence test (Mendez et al. 1984). Periods of greatest reproductive activity during the year as demonstrated from the commercial catch were determined from an analysis of gonadal development of females. Females were grouped according to maturation stage as non-ripe (I and II) or ripe (III and IV) to avoid misclassification of gonadal stage determi- nation. The last two stages indicate proximity of the spawning 835 836 Sandoval-Quintero and Gracia period, and can only be distinguished precisely by histologic sec- tioning of the gonads (Sandoval-Quintoro and Gracia 1998). We used catch-per-unit effort (CPUE) as an index of abun- dance of these stages to analyze variations in the proportion of non-ripe and ripe females, estimating it monthly from each tow in the following manner: [CPUE, ^1 = In /t) {%I.M), where n = number of females in the sample, t = duration of trawl (hr) and %I.M = percentage of non-ripe (1) or ripe (M) females, expressed in terms of total number of females per hour towed. Size at first maturity was defined as mean total length of fe- males when 50'7f had reached maturity. The relationship between size distribution and depth was analyzed through a linear regres- sion analysis (Zar 1974, Bhattacharya & Johnson 1977). We cal- culated the correlation coefficient (r) to determine whether there was a statistical association between the gonad stage and depth for those months in which the variables were not independent (Everitt 1977). Localities were classified arbitrarily according to whether they yielded >50'7<- non-ripe or ripe females. RESULTS Population Size Structure Total length for entire study ranged from 35 to 205 mm both sexes, with females typically reaching greater maximum lengths than males (Fig. I). Both sexes size distribution was unimodal during most of the months. When two modes were found they conesponded to large females and small males groups. Male and female size distribution varied along the period stud- ied (Table 1). Usually female modal sizes were greater than 140 mm TL. Female average total length varied from 124 mm (inma- ture females) to 166 m (ripe females). Male average lengths varied from 116 to 133 mm TL. Modal frequencies were close to the average in most of the months. May and September modes suggest the presence of two cohorts (Fig. 1 ). Sex Ratio Sex ratio varied in the months sampled (Table 2). Females prevailed in the months of March to May and in July. Males prevailed in February and August and from October to December. The X" test for independence revealed significant differences (X'ob. = 276.8; x",ah- ^-^^ = 18.307) in the sex proportions during the year. Size of First Reproduction The size at which 5(y/r of the females are mature was estimated as 148 mm TL (Fig. 2). However, mature females with a minimum size of 95 mm were found in February and March, while a mini- mum size of 105 mm was observed in April. In the other months, minimum sizes of mature females ranged from 1 10 to 125 mm TL. Distribution of Females on the Fishing Ground A significant correlation (Pearson P < 0.05 ) between increasing mean size of spotted pink shrimp females and increasing depth of capture (Table 2, Fig. 3) was found from February to August. In most cases the relationship was positive, except in August when large females were found at shallower depths. The degree of ripeness of the females and the depth showed a significant linear relationship (Pearson P ■£ 0.05 ) from February to May and during July and August. April. May. July, and February FEMALES NON RIPE (NR) ^ RIPE (R) Februao' '"^'^=-^08 R=171 100 50 0 rriTfTfflwTKTWI < Q Z SO 40 i 0 1 100 50 0 - 100 50 0 100 50 0 May 345 '"rrrfliiTlML. NR=223 R=137 June n = 345^^^ NR=106 R=239 July n = 454 August n = 370 R=180 =274 100 50 0 100 - 50 . 0 - 200 100 0 - 80 I 40 4 0 L 100 50 0 100 50 0 MALES February^ March n = 7 April May n = 30. June n = 356 July n = 274 JL NR=I80 JL R=190 100 August „ L Cri 30 15 0 — September n=187 60 30 0 October n = 274 NR=89 R=98 NR=193 30 . September JdM^^^lfh- R=81 30 November 15 n=110 0 — -n NR=37 R=73 December NR=92 30 - 15 . n= 184 35 70 105 140 175 Total length (mm) 30 15 0 30 15 0 November n=147 'IdMlL December ca Hk ^ 35 70 105 140 175 Total length (mm) Figure I. Size structure of the spotted pink shrimp, F. brasiliensis, stock from Februar\ to December 1993. yielded positive correlations between the degree of ripeness and depth. The inverse condition was observed in March and August. During all months, except March, at least one area was found in which more than 50'/f of the females were ripe. From February to June ripe females were more widely distributed. In March, no area had high percentages of ripe females; however, the CPUEs in some of the areas were among the highest ( 1 7. 20, 28 ripe females/ hr towed) along with those observed in April ( 1 3-20 ripe females/ hr towed). Sites with larger CPUE for non-ripe females occurred in Feb- ruary (8-15 and 15-20 non-ripe females/hr towed) and March (35, 45. 52 non-ripe females/hr towed). During other months, these values ranged from 1-13 non-ripe females/hr towed. One main area which contained ripe females (more than 50%) dunng almost all months was delimited (Fig. 4). Based on this we propose that the main spawning area within the general fishing ground is located at 2r'4()' to 22°10'N and 86 '30' to 86^50' W. In subarea B. determined only for April, with depths of 25 to 40 fathoms (approximately 42-67 m). the CPUE was estimated at 13 Reproduction of F. brasiliensis 837 TABLE 1. Female and male size distribution. Average Size Maximum Size Minimum Size Mode Size Range 95% Observations Immalure females 129-157 195 35 110-160 S5-195 Ripe females 146-166 205 95 130-170 115-180 Males 116-133 200 35 110-145 80-170 Both sex 124-147 205 35 120-160 80-185 February anJ Mareh mean length 130 and 136 mm (recruits) May and September, two modes July. August and October, two modes to 20 ripe females/hour towetd. Subarea C located between 20 to 35 fathoms depth (approximately 33-59 m) was also present for most of the year with a CPUE of 2 to 11 ripe females/hr towed. Reproductive Periods Ripe females were present throughout the whole year, indicat- ing that reproduction may be continuous. The highest percentages were found in the months of April (72.3%), June (69.3'7r). and November (66.4%) (Fig. 5a). Female percentages registered in May and July to October were lower (less than 50%) than the previous months. These differences are also related to the catch of this species within the year, which reaches a maximum in April (Soto-Aguirre in prep.). Catches yield a relatively high number of females in April, suggesting that the greatest reproductive activity occurs during this month. If we analyze the reproductive activity using CPUE as an index of abundance (ripe females/hr towed), the pattern obtained differs from that of percentages (Fig. 5a and 5b). mainly on the last part of the year. Ripe females are better represented in March and April, with a CPUE of 8.3 ripe females/hr towed, indicating that these months are most important in terms of reproduction. June, also considered as an important month for reproductive activity in terms of percentage of mature females, had lower CPUE values of 5.6 females/hr towed. February, May, July, and August, varied from 4.6 to 4.8 ripe females/hr towed. November registered as important in terms of percentage of ripe females had one of the lowest values based of CPUE. From September to December the number of females decreased from 3.2 to 1 .6 ripe females/hr towed (Fig. 5c). TABLE 2. Coefficient correlations (r) of female pink spotted shrimp (n) size and depth, and monthlv sex ratio (calculated on the basis of females! in the Contoj fishing ground. Month Sex Ratio (.Male/Female) February 0.246* 479 1.17 March 0.414* 1118 0.66 April 0.471 988 0.48 May 0.220* 360 0.83 June 0.185* 345 1.03 July 0.351* 454 0.60 August -0.197* 370 1.12 September 0.060 187 0.95 October 0.096 274 1.35 November 0.047 110 1.33 December -0.073 184 1,38 DISCUSSION Ninety-five percent of the spotted pink shrimp catches con- sisted of organisms that ranged from SO to 185 mm TL, which suggests that shrimp are recruited at small sizes about 80 mm TL. This agrees with the emigrating size (80-100 mm TL) of juveniles of other penaeid shrimps from the nursery areas in the Gulf of Mexico (Bielsa et al, 1983, Gracia 1989a, Gracia & Soto 1990. Gracia 1995). Juveniles off. brasiliensis seem to leave the nursery areas at 67-84 mm TL according to data recorded in coastal la- goons near the study area (May 1999). Sex Ratio In general, the months with the greatest ratio of males/females are within the main reproductive period (February to August). In contrast, the months of October to December, which are not con- sidered as part of the main reproductive period, had a higher pro- portion of males (1.33-1.38 per female). Guitarl and Hondares ( 1980) also found a difference in the female/male ratio of F. duo- rarwn and F. azwciis in the Cainpeche Bank. Female Spatial Distribution Pattern Some relationships between size of females and gonad stage were found; however, no consistent pattern was observed. Several authors have reported that mature female shrimp migrate toward deeper regions during the spawning time (Bielsa et al. 1983. Brusheret al. 1972. Garcia and Le Reste 1986, Gracia 1992). This may be the case for F. brasiliensis. since the reproduction period established in this paper (February to August) coincides with a significant relationship of size and gonad stage with depth. The best correlation between size of females and depth of catches was observed during March and April that are the months of greatest reproductive activity. If we also consider that female size can be related to maturity, one could expect a similar behavior ' Significant at 5% level. C/3 s 35 65 95 125 155 185 Total length (mm) Figure 2. Size of first maturity of F. brasiliensis with the criterion of 50%. 838 Sandoval-Quintero and Gracia 200 150 t/D w 100 J < 50 s tu 0 tlH tlH o 100 75 e^ w 50 CQ ?5 S D 0 Z • Non Ripe 15 20 25 30 35 40 45 Depth ( fathoms ) Figure 3. Number of ripe and non-ripe females of F. depth. brasiliensis vs for the relation^hip between ripeness and depth. This was found for April but did not occur in March, when no significative correlation was observed. This correlates well with the fact that March is a month of marked recruitment to the fishing ground (Porras-Ruiz et al. in prep.) which can mask this relationship. In August, when smaller organisms were found at greater depths, the relationship of size and depth depicts a negative slope. This finding is related with the second recruitment period of the population towards the fishing grounds (Soto-Aguirre in prep.) during summer. In general terms, the results suggest that there may be a relationship of size and female gonad stage with depth of the fishing ground, especially during reproductiiin periods. Nonetheless, monthly variations of non-ripe and ripe females ocurrence in the fishing ground suggest an area of recruitment with small shrimp troughout the year (21°30' to 2I°50'N and 86^30' and 86"40'W) and a spawning area (21°40' to 22°10'N and 86°30' to 86''50'W) at depths of 20 to 40 fathoms (approx. 33-67 m) which can be useful as reference areas for the fishery. This study suggests that the reproductive period for pink spot- ted shrimp occurs throughout the whole year, but the highest re- productive activity in terms of number of ripe females occurs from February to August, peaking in March and April. According to Garcia and Le Reste (1986). variation in reproductive activity of shrimps is typically linked to seasons of the year, with two repro- ductive peaks. Other authors (Crocos and Kerr 1983. Crocos 1987a and b, Garcia 1988) have also observed a bimodal pattern in egg B STW 86°40' 86°20' 87°00' 86°40' 86°20' 135-1^ iio-iiiJ Q. Rol) March 'i CPUE L (fem/t-h) i 7 1 R A B c D E F 42 10-15 15-22 7-14 35 52 20 3-10 2-4 2-4 17 28 il .22''10' 21 "50' 21°30' 87°00' 86°40' 86°20' 87°00' 86°40' 86°20' August CPLiH22°10' ^fem/t-hj 2r50' .21°30' 87°00' 86°40' 86°20' r, October U CPUE I (fem/l-h)J 7 1 K A B C 12 M 4-7 K 3 1 ^-155 L i 1 L21°50' 2r30' 87°00' 86°40' 86f20' CPUE L22°10' (fem/t-h) 2-3 L2I°S0' L21°30' 2I°30' 21°50' 21°30' Figure 4. Montlily main distribution spawning area of tiie spotted pinit shrimp F brasiliensis. Reproduction of F. brasiuensis 839 ary, and hcpatopancreas of Parapenaeus longirostris females at different stages of ovarian de>elopnient ((mly the quantitatively most important fatly acids are represented). Muscle Ovary Hcpatopancreas Fatty ,\cids Stage 1 Stage 2 Stage 3 Stage I Stage 2 Stage 3 Stage 1 Stage 2 Stage 3 14:0 0.2 + ().()■' 0.2 ± 0.0" 0.3 ±0.1" 2.6 + 0.9" 3.2 ± 1 .3" 4.0 ±2,0'" 6.4 ± 1.2' 8.3 ±1.5'' 8.7 ± 1.9" 16:0 4.6 ± c.g-" 4.7 ± 0.7" 4.6± 1,1" 24.3 ± 4.2" 32.7 ± 3.7' 38.7 ±5.1' 54.7 ± 4.9'' 61.7 ±6.4"' 73.9 ± 7.9' 18:0 1.7 + 0.5" 1.7 ±0.4" 1.4 + 0.8" 5.4 ±1..^" 9.0 ±1.7' 9.5 ±1.9' 9.2 ±2.1' 10.2 ±2.7' 11.6 ±3.0' X saturated 7.1 ±1.3" 7.2 ±1.5" 6.9 ±1.2" 34.3 ± 5.9" 47.5 + 6.8' 57.4 ± 7.9'' 764 ±10.-^' 87.4 ±12.1" 98.2 ± 9.9s 16:l(n-7) 1.5 ±0.8-' 1.6 ±0.9" 1.5 ±0.6" 8.5 ± 2.2" 11.0 ±4.5" 19.5 ±3.6' 28.6 ± 8.9'' 33.3 ± 7.6"' 37.6 ± 6.3' 18:l(n-9) 4.3 ±1.6-' 3.6 ± 0.9" 3.6 ±1.2" 44.3 ±8.2" 61.1 ± 10.9' 74.3 ±12.4"' 83.1 ±12.5'' 107.8 ±18.1"' 1 16.8 ± 20.6' 18:l(n-7) 1.1+ 0.5" 1.1 ±0.6" 1.3 ±0.4" 12.1 ±3.2" 13.5 ±2.8" 16.0 ±3.3"' 29.0 ± 7.5' 28.4 ±6.0' 26.0 ± 5.4' 20:l(n-9) 0.7 ± 0.3" 0.2 ±0.0" 0.2 ±0.1 '■ 8.3 ± 1.5^ 10.3 ±2.3' 18.0 ±2.7'' 16.9 ±4.2"' 20.1 ±3.4" 21.5 ±5.0" 20:l(n-7) 0.1+0.0" 0.1 ±0.0" 0.1 ±0.0" 1 .5 ± 0.5" 2.2 ± 0.8" 7.7 ± 3.6' 5.1 ± 1.3' 6.1±2.r 6.6 ±2.4' 1 monounsaturated 8.0 ± 1 .9" 6.8 ±1.6" 7.0 ± O.S" 83.0 ±15.6" 109,0 ±25.3"' 140.7 ± 19.8' 169.0 ±23.5'" 204.0 ±18.7"' 224.6 ± 26.6' 18:2(n-6) 0.3 ±0.1' 0.3 ± 0.2" 0.2 ±0.1" 1.5 ±0.3" 1,3 ±0.5" 2.5 ± 0.8"' 3.2 ± 0.9' 4.2 ±2.1' 3.3 ± 1.3' 20:4(n-6) 1.5 ±0.8" 1.5 ±0.6" 1.8 ±0.9" 3.4 ± 0.8" 4.9 ± 1.7" 7.7 ± 3.4' 11.1 ±3.2'^ 12.9 ±2.2" |4,y±4.4" 20:5(n-3) 3.8+1.2" 3.8 ±1.5" 4.0 ±1.0" 1 1 .3 ± 2.8" 14.2 ±4.4"' 16.5 ± 3.0' 23.2 ±4.2" 27.2 ±4.0"' 30. 1 ± 3.9' 22:6(n-3) 4.4 ±1.7" 5.4±2.1" 5.7 ± 1 .9" 24.0 ± 5.8" 30.3 ±7.1' 19.7 ±6.3" 24.5 ± 3.3" 29.5 ± 4.8' 3 1.4 ±5.2' 2. polyunsaturated 10.9 ±2.5" 1 1 .8 + 0.9" 12.2 ±0.9" 46.1 ±15.6" 58.5 ± 12.4"' 51.4± 12.0" 72.9 ±11.7' 87.0 ± 14.5" 90.0 ± 12.6" Values are the means ± SD of three pooled samples. Different superscript letters within rows represent significant differences (P < 0.05). Biochemistry and Reproduction of Rose Shrimp 847 storage in the adult, egg. and prefeeding larva (Xu et al. 1994. Nates & Mckenney 2000), comprising primarily 16:0 and omega-9 family fatty acids (Teshima et al. 1988). PL. DAG. and sterols are the other main lipid classes found in marine shrimps and are also associated with the maturation of oocytes (Teshima 1997. Ravid et al. 1999. Wouters et al. 2001). The HP is the major lipid storage and processing organ for postembryonic stages (Voght et al. 1985). but during maturation the ovary becomes an additional center for lipid metabolism, in- cluding lipogenesis — TAG synthesis (Teshima et al. 1988). In the present study, and like some previous studies in decapods (Castille & Lawrence 1989. Cavalli et al. 2001). the increase in ovarian lipids is not accompanied by a decrease in HP lipids. Under these circumstances, the lipid requirements of the developing ovary seem to be more dependent on the ingestion of dietary lipids than on HP reserves. The profile of fatty acids in the ovaries of P. longirostris is a reflection of the fatty acid requirement of this tissue or of what is required for transfer to the developing embryos after fertilization. Alava et al. (1993). Cahu et al. (1994). and Cahu et al. (1995) demonstrated the benefits of high levels of highly unsaturated fatty acids in the diet on reproductive parameters and on offspring qual- ity of penaeid shnmps. In fact, the long-chain fatty acids are nec- essary for vitellogenesis of crustaceans (Middleditch et al. 1980. Millamena & Pascual 1990) and ARA and EPA are precursors of eicosanoids in marine animals (Lawrence et al. 1979. Sargent. 1995). The high values of MUFA in the ovary and HP are con- sistent with previous findings of Clarke et al. (1990) and Roustaian et al.( 1999), which indicated that these compounds are the major sources of energy during embryonic and early larval development. In the present study, the protein content of the ovary and HP did not vary significantly. On the other hand, the seasonal (intra- annual) variation of protein content of the muscle may be linked with changes in feeding activity. Muscle protein loss during star- vation has been observed in other deep-sea decapod species (Dall 1981). Because cholesterol is a precursor of steroid hormones (Kanazawa & Teshima 1971), the increase of ovarian cholesterol levels with maturation in the P. longirostris was not surprising. This increase can also be related to the role of cholesterol as precursor of ecdysteroids. as these compounds are known to in- crease during gonadal maturation (Wilder et al. 1991). Similar trends were observed in HP cholesterol, which differs from the results obtained by other authors (Adiyodi & Adiyodi 1970, Lau- tier & Lagarrigue 1988), where the decrease in HP cholesterol during vitellogenesis suggests that mobilization of HP cholesterol stores may contribute to the build-up of ovarian cholesterol. The explanation of our findings could be found in Teshima et al. (1988). which indicated that cholesterol is sequestered to the ova- ries from the muscle stores. In fact, in the present study, the muscle cholesterol content decreased significantly from SI to S3, and the seasonal variation of the cholesterol content also seems to confirm the conclusions of Teshima et al. (1988). On the other hand, be- cause cholesterol stores within the HP and gonads are derived from the diet (Middleditch et al. 1980). because of the incapacity of de novo synthesis of the steroid ring, if the steroid resources are mobilized from the HP to the gonad, then the HP resources seem to be compensated by those gained from feeding. Glycogen did not show significant variations throughout the maturation process, in contrast to what was stated by Kulkami and Nagabhushanam (1979). Moreover, because carbohydrates have specific roles in the production of nucleic acids, are precursors of metabolic intermediates in the production of energy and nones- sential amino acids, and as a component in ovarian pigments (Har- rison 1990). they have to be especially important for the matura- tion process and for embryogenesis. Among the different tissues analyzed in this study, the glycogen is mainly stored in the HP and to a lesser extent in the muscle, but according to the studies by Hagerman et al. (1990) and Baden et al. (1994). on the decapod Nephiops nonegkus (Linnaeus 1758). the occurrence of glycogen depletion in the muscles, following hypoxia and starvation, sug- gests that the muscle contains a particularly important store of glycogen, because it is more readily accessible when there is a shift to anaerobic metabolism or when there is decrease in the feeding activity dunng winter. This can explain the seasonal variation of glycogen content in the muscle of P. longirostris. because the lowest values were obtained in the winter. Although there is no evidence of a decreasing feeding activity during this period of the year, the diet composition of P. longirostris should vary signifi- cantly between seasons like the other deep-sea crustaceans species (Cartes & Sarda 1989, Labropoulou & Kostikas 1999), because these changes correspond basically to the period of abundance of the different dietary group in the deep-sea environment (Cartes 1994). In summary, it is evident that reproductive cycle has profound effects upon the biochemistry of P. longirostris. Reproduction and gonadal maturation have large associated energy costs attributable to the increase in biosynthetic work, which will support the leci- thotrophic strategy (reliance on egg yolk nutrition) of the embryos and prefeeding larval stages. Moreover, these processes seem to be influenced or even synchronized with seasonal feeding activity or food availability. Despite not being mentioned or discussed in this study, moult cycle can also have an important effect in the bio- chemistry and physiology of decapod Crustacea, manifested in changes in their physiological ecology and behavior. Lack of data on this matter in P. longirostris indicates that further work will be necessary to understand better the biochemistry and physiology of this species. 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P.O. Sm 5667. St. John's. Nfld.. Canada. AlC 5X1 ABSTRACT A new approach to study growth performance in squid is described, based on modeling the ratio of body weight to mantle length (BW/ML) as a function of age (squid "condition"). This approach has several advantages above traditional size-at-age modeling, including the concurrent use of two size indices, the fact that it is not constrained by theoretical considerations regarding the form of growth in absolute size, and that it is also less severely affected by sampling bias. It was tested on three species of the genus lllex using raw data sets for two species as well as BW/ML values calculated from sizes predicted by models available in the literature for all three species. Several experimental models were explored, but the specific logistic model was found to be the most suitable, especially when most of the life cycle was sampled. This model was successfully applied to all species and case studies, thereby elucidating common features of growth performance throughout the genus llle.x. It provided similar or superior model fits when compared with corresponding models of length-at-age or weight-at-age. Comparisons within and among species indicated some effects that have also been suggested from independent length-at-age studies, including a positive effect of temperature on growth perfor- mance. This new approach also indicates a relationship between growth performance and sexual maturation. KEY WORDS: squid, lllex. growth performance, age INTRODUCTION Three species of short-finned squids of the genus llle.\: (Teuthoidea. Ommastrephidae) are commercially exploited (Roper et al. 19981; lllex illecehrosiis (Lesueur 1821 ). /. ari>entinus (Cas- tellanos 1960), and /. coindetii (Verany 1839). The fisheries they support have recently gained increasing attention because of con- cerns regarding potential for overexploitation (Haimovici et al. 1998, O'Dor & Dawe 1998, Sanchez et al. 1998). Effective squid fisheries management, however, is hampered by uncertainties concerning the life cycle. As is typical of most squid, llle.x species show great and unpredictable variability in growth, maturation, and spawning patterns over very short time periods (Mangold 1987, Forsythe 1993, O'Dor & Lipinski 1998). Such great variability in life history parameters largely accounts for the great variability evident in size-at-size (e.g., weight-at- length) and size-at-age relationships observed in lllex sp. and the difficulty in applying length-frequency methods (Caddy 1991, Jereb & Ragonese 1995) conventionally used for estimating growth rates in other fisheries resources (Pauly & Morgan 1987). Recent direct ageing techniques based on squid statolith micro- structure (Jereb et al. 1991; Jackson 1994), despite the inevitable existence of ageing bias (Pauly 1998, Gonzalez et al. 2000), indi- cate very high growth rates, even higher than those observed in fast growing pelagic fish such as the scomberoids (Longhurst & Pauly 1987, Jarre et al. 1991). Despite the many studies of the past decade, there is no con- sensus on which model is the most suitable to describe squid growth. Among those used for various species and portions of the life cycle, the simple linear, power, log linear, piece-wise, expo- nential, seasonally oscillating von Bertalanffy, double exponential (or Gompertz), and logistic (Jackson 1994) have most frequently been applied. To date, five models have been used to describe *Corresponding author. E-mail: ragonese(3irma.pa.cnr.it absolute growth in either length or weight within the genus lllex (Table 1). Until now, size-at-size and size-at-age relationships have been analyzed separately, despite the strong correlation existing be- tween body mass and mantle length. Here, these relationships are analyzed jointly by relating body mass to mantle length and in- vestigating trends in this ratio with age. The main purpose of our study was to combine two indices of size (both important factors in ecology and evolution studies; Peters 1983, La Barbera 1989) into a new index, which may better indicate variation in growth performance than either length or weight alone and thereby facili- tate broad-scale comparison within and among species. Potential advantages of this approach include that variability surrounding models of weight-at-length on age may be lower than for more familiar models of size on age. Weight-at-length on age models may be simpler, more consistent, and provide better fits than single size variable on age inodels. They may also better reflect suitabil- ity of the biotic environment than growth in either length or weight alone. The models we developed were applied to three congeneric squid species {lllex spp.) to test the utility of this approach for comparison of growth performance patterns. MATERIALS AND METHODS The weight-at-length index (WaL) is defined as the ratio of whole body weight (BW; g) to dorsal mantle length (ML; mm), i.e.. WaL = BW/ML; it is, therefore, analogous to other more conventional condition indices, derived from the classic Huxley's allometric formula y = ax*" (La Barbera 1989) and widely used in aquaculture and fisheries science (Dawe 1988, Bolger & Connolly 1989, Scott Cone 1989). Two kinds of size-at-age databases were used (Tables 1 and 2). The first one consisted of original individual estimates of size (ML and BW) and age. Such data sets were available for two micro- cohorts of /. coindetii from the Strait of Sicily (Arkhipkin et al. 851 852 Ragonese et al. 3 a. o £ BiD [ij at J 1 sa * ^ o ■o Oi 2 O = Q. .- < J ^ a, -n rt ON ^ n; J -1.) x: D- -I 0^ o; >. - I — — c 3 ^ J CQ ■ c " ■ •- 3 c ?= P O c?5 < O 02 Q c« n: S*"5q 0^ O OO CL o I sC' r- 30 oc Tj- o zc X ~ — r- CiO - Y Cl Cl. C- I j< >< >< D U U ^ >^ >* >. PS >. >*>%>.>.>. >.>~.>^>.>^>.>-. >>^^ c = c c r E >3 >; nil Oij Qlj DJj CO DJj =ij DJ o c o fN) ^ -^ ,^ ^- cc r-; in — ri 00 w-i — On ON 00 ■ I ^rJ - ^ p X X X X f 00 i~^i o -T ^ Y =r "/". r^i ir^i 1 p o ^^ * 9 vD T ? "T x: X X X ^ n ? 1 a. 1 X X X X D. Cl Cl 4^ ■s- X X * 00 r^ X X X X ■«■ X ON r- * * •^r- 1 * * * ir. ON oJ U V, r- iri U^j o. Q. T -rt ON U-, ^ r- + + + r*-. r^i ri y^ ^ X * X r-_ p ri d .^ d o r- o O "^ d + * ■Sr" + + + + ri -t + + + O + NO ri O; S + + rj ^ oo ri + oc 4 o ^ ri + On r*~\ o 00 Tf o W". oc U-; d r*-i — ! >-. >-, >* >. >-> >^ >* >* >> >. >. >-, >~, >^ >-. >-. >. >. >% >. o _ tn Q O o O o «n o O in O m O o o c o o 00 r*~, ON r- i£ f*-, r- in ON «n o m, in r*-) ~~ ri ri ~ NO nD ri -^ ri — -^ ri — ■^ "^ -^ r*"i "^ r*^ tN r*"i rj ri rj n ri rj ri ri r<^ r*~i 1/1 O m, o _ '/". w"- '/^. O — OO' — O-"! — — r^, OCOC ^' -J ^ b Cm b a. c — U -t -.'i fli _3 flj r3 Q i; Q S C/p S I<5 -O T3 ii ii t t>5 o " t 1/5 SOSO<0< Q- S-C« 5 C/5 S S Cl ^ "^ C- ^ — < S S < S 5 — rir*-, ^ — rir^. -t— ri— 'rir;-, -t_,~|r^, -j-i/-, sO — Growth Performance Across the Squid Genus Illex 853 TABLE 2. Ranges of ape. mantle length (ML), and weight-at-length ratios (WaLl for data sets based on individual observations and on predicted size values for Illex species and populations. For cases A and E, the predicted values were calculated only for the age ranges sampled (i.e., without extrapolation). Original 1 individual 1 Observations Theoretical (mean) Observations Age Range MI. Range WaL (g/mm) ■Age Range ML Range WaL ( g/nim ) Species-Area Case Sex Group Idaysl (niml Imin) (max) N (days) (mm) (min) (max) N A cvlmlcni Al f Oct.-Dec. 75-240 41-202 0.073 1.284 189 70-230 33-186 0.054 0.891 17 Central Mediterranean A2 f May-July 74-181 52-175 0.102 1.027 144 70-180 31-156 0.089 0.687 12 A.I ni Oct.-Dcc. 76-230 45-171 0.078 1.1.54 218 80-230 50-143 0.105 0.866 16 A4 m May-July 81-191 56-141) 0 1 17 0.857 98 100-180 89-129 0,251 0.720 9 /, coindelii Bl r Oct-March 220-475 115-204 0.407 1.206 IS W Mediterranean B2 r Apr.-Sept. 160-460 92-205 0.266 1.218 21 83 ni Oct. -March 160-430 68-165 0,191 1.127 19 84 m Apr.-Sept. 160-370 69-158 0.198 1 .042 15 /. coindt'tii CI f pooled animals 90-450 37-tOl 0.099 4.160 25 Galician (Atlantic) C2 m pooled animals 90-390 53-248 0.053 2.773 21 L coindetii Dl f Sierra Leone 120-255 113-192 0.418 0.757 10 NW African Coasts D2 f W. Sahara 90-300 84-294 0,282 2.522 15 D3 m Sierra Leone 105-210 84-135 0,206 0.627 8 D4 ni W. Sahara 90-255 80-192 0,187 1.823 12 /. illecebrosus El r March 116-247 L54-299 0.383 1.753 32 115-250 166-261 0,488 1.070 10 NW Atlantic E2 f April 108-228 159-286 0.396 1.7.34 32 100-235 150-291 0,366 1 ,574 10 E3 f May 112-195 175-281 0.491 1.697 12 120-210 181-297 0.535 1.869 7 E4 m March 116-204 152-227 0.409 1.128 48 115-220 169-225 0.488 0.922 8 E5 m April 115-216 152-246 0.357 1.447 35 115-220 174-237 0.463 1,189 8 E6 m May 141-200 217-265 0.917 1 620 17 135-210 212-268 0,812 1.749 6 /. araentinus Fl r June 180-360 149-311 0,377 2.206 13 Palagonian Shelf F2 m June 1 80-360 172-298 0.589 2.567 13 1998, Arkhipkin et al. 2000; Case A: Tables 1 antJ 2) and three micro-cohorts of /. illecebrosus from the Northwest Atlantic (Dawe & Beck 1997; Case E; Tables 1 and 2). /. coindetii samples were collected during an experimental depth-stratified random bot- tom trawl survey (stretched mesh size in the cod-end of 31 mm), whereas /. illecebrosus samples were collected from the New- foundland inshore jig fishery during July to November 1990. The second kind of database consisted of size values (ML and BW) at age predicted by the specific size-at-age models reported by sex for different hatching groups or populations (pooled over hatching groups) of Illex spp. in the literature (Tables I and 2). When only ML-at-age models were available (Case B), the corre- sponding BW-at-age data were roughly approximated from pub- lished BW-ML relationships obtained for the same season. There- 00 -0.5 O 1 O A A l^^^^^o ^ -1.0 A^ ^0 J^ AOO o -1.5 A CP^^^I 1)8 o O A1-F A A3-M -2.0 - Al - F com - - - A3 - M com -0 9, — 1 70 90 110 170 190 210 130 150 Age (days) Figure I. Individual log,, of body weight (BVV) at mantle length (ML) ratio vs. age and computed (com) specified logistic model for Al and Xi cases, Illex coindetii females (F) and males (M) of the Central Mediterranean (see Table 1 for case specifications). 854 Ragonese et al. CD (U o> o 0.0 -0.5 -10 -15 -2.0 -2.5 > Aa Q .^^ °0 A Ad A^°A A _A O O A2-F A A4-M A2 - F com - - - A4 - M com r r 70 90 110 130 150 Age (days) 170 190 210 Figure 2. Individual !»(;,. of hody weigiit (BW) al maiUk- lengtii (Mlj ratio vs. age and computed (com) .specified logistic model for A2 and A4 cases, lllex coindelii females (F) and males (M) of tlie Central Mediterranean (see Table 1 for case specifications). fore, they have to be considered only as representing the form of growth. To explore the most suitable models to apply. WaL-at-age scat- ter plots were analyzed by points interpolation with a model-fitting procedure (Systat 1992). A variety of available models were ap- plied to the untransformed WaL and age data, as well as to data sets with the dependent variable only and with both variables transformed (using natural, base e, logarithms). This exploratory analysis indicated that the relationship of WaL on age (t) was best described by the power function in some cases (WaL = at*") and by asymptotic models in other cases. Among the asymptotic mod- els, the sigmoidal logistic model was preferred (Ratkowsky 1983). Therefore, the following two models (applied to log trans- formed data to stabilize the variance and normalize the residuals) were tested: the allometric model, hereafter referred to as the log-log model, which represents the linear transformation of the power model: TABLE 3. Estimated coefficients of the log-log and specific logistic models by using individual vveight-at-length (WaL I at-age observations: log = natural (log,) logarithm; r' = coefficient of determination; MSE = mean squared error; N = sample size. Case Sex Group Log-log Model (i) Specific Logistic Model (ii) Species-Area log^a b r" MSE A B C r" MSE N /. coiinli'lii Al f Oct.-Dec. -13.552 2.549 0.859 0.059 0.1335 0.0314 159.4 0.950 0.054 189 Central Mediterranean s.e. 0.378 0.076 s.e. 0.0856 0.0021 6.2 A2 f May-July s.e. -11.754 0.493 2.26(1 0.101 0.777 0.055 s.e. -0.2708 0.0499 0.0508 0.0040 114.4 2.6 0.953 0.041 144 A3 m Oct.-Dec. s.e. -13.134 0.298 2.479 0.060 0.887 0.050 s.e. -0.0373 0.0515 0.0360 0.0017 143.0 3.5 0.968 0.041 218 A4 m May-July s.e. -13.118 0.547 2.552 0. 1 1 3 0.840 0.057 s.e. -0.2046 0.0602 0.0527 0.0045 114.4 3.2 0.961 0.041 98 /. illecebrosus El f March -9.9.34 1 .883 0.685 0.040 Model failure 25 NW Atlantic s.e. 1.359 0.266 E2 f April s.e. -14.893 1 .542 2.877 0.302 0.777 0.039 s.e. 0.7126 0.2702 0.0335 0.0082 175.3 17.3 0.839 0.037 28 E3 f May -13.280 2.618 0.827 0.013 Model failure 11 s.e. 2.052 0.400 E4 m March -6.288 1.164 0.5,36 0.024 Model failure 47 s.e. 0.814 0.162 E5 m April -7.827 1.488 0.416 0.090 Model failure 35 s.e. 1 .549 0.307 E6 ni May -9.L54 1.819 0.590 0.019 Model failure 15 s.e. 2.168 0.420 Growth Performance Across the Squid Genus Illex 855 log^WaL = log^.a + blog^t + e and; the specific logistic (Weatherley 1972): log^WaL = A - log,( 1 + e-'"'^'^') + e' where log^.a and b are the intercept and the slope parameters re- spectively, A denotes the asymptote (log^WaL-,), B is the slope, C is the inflection point (days), t is the age (days), considered as the independent variable, and e and e' are the error terms. A possible disadvantage of the specific logistic model is the apparent lack of a direct biologic interpretation for the asymptotic parameter (A). However, a direct biologic meaning may be found for the parameter C, which, when modeling growth in length, represents the inflection point at the end of the initial exponential growth phase and the beginning of the logarithmic growth phase. We investigate the possible relationship of the age at inflection point (i.e.. the C parameter) with the approximate mean age of all mature animals of a specific group, as derived from the literature. Model coefficients were estimated by the ordinary (Type I) simple least squares method, using both linear (Model i) and non- linear (Model ii; "Quasi Newton" and "Simplex") iterative proce- dures. The coefficient of determination (r) and the mean square errors (MSE) were used as approximate indices of goodness of tit. The former statistic, in particular, was used to compare results derived by modeling WaL with those derived by modeling ML and BW separately (original size models. Table 1 ). Probability levels for acceptance of significance of the regression coefficients and the overall regressions were fixed at P < 0.05* (significant) and P < 0.01** (highly significant). Statistical analysis, model fitting, and computations were performed using the package Systat (1992). RESULTS Models for Data Sets from Individuals I. coindetii The log-log transformation (Model i) was not successful in achieving linearity between WaL and age for /. coindetii from the Strait of Sicily, with the exception of females of the October to December group (Al). However, the scatter-plots of log^. trans- formed WaL values vs. age according to Model ii (Figs. 1 and 2), showed an acceptable homogeneity of variance. Male and female curves overlapped, but the two seasonal groups did not. October to December specimens (A1-A3) showed lower log^.WaL values for a given age than did their May to July counterparts (A2-A4), suggesting a seasonal effect with higher growth performance of spring-summer group. Considering cases A and Model i (Table 3). all regressions were significant, but the regression parameters were rather impre- cise, as reflected by high standard errors. The Model i. therefore, did not properly fit the data sets, as already indicated by failure to achieve linearity. Improvement in both r~ and MSE was achieved by fitting Model ii (Table 3). For both hatching groups and sexes convergence was rapid, and various starting values led to very similar final estimates of regression parameters, regardless of which non-linear fitting procedure was used. The precision of the regression parameters was acceptable. Model ii parameter values were similar between sexes of the May-July group (A2 and A4: Table 3, Fig. 2), whereas some sex effect was detected in the October to December group (Al and A3, Fig. 1). In that fall to winter group, females exhibited higher asymptotic values than males and a small difference in the slope parameter and inflection points (Table 3, Fig. 1 ). Overall, /. coindetii specimens hatched in May to July showed a higher growth performance than their Oc- tober to December counterparts. /. illecebrosus For this species (E cases), parameter estimation was achieved only using Model i (Table 3), except for the E2 case. The precision in the estimation of parameter A (Model ii) was very low in that case, however, as reflected by the ratio of that parameter to its standard error (0.27/0.71, i.e.. 38% of the variation). The Model i (log-log regression) coefficients (Table 3) indicate that the slopes were higher for May and April than for March, and higher for females than males. Models for Data Sets of Predicted Values Results obtained from fitting both models to the data sets of predicted values (all cases) are presented in Table 4. The consis- tently higher model fits (reflected by higher r and lower MSE) than for models fitted to data sets of individuals (Table 3), reflects the much lower variance in the data sets of values predicted by other (single size-at-age) models than in raw empirical data sets. A comparison of specific logistic models among cases is shown for females (Fig. 3) and males (Fig. 4). It is interesting to initially compare the A and E cases for which models were also fitted to the data sets based on individuals. Case A: /. coindetii The log^WaL-at-log^.-age plots (Model i) were nearly linear for the October to December group (cases Al and A3) and more strongly curvilinear, with an asymptotic trend, for May to July hatched squid (cases A2 and A4). The log^.WaL-at-age plots (Model ii) showed a curvilinear trend in October to December specimens and a clear asymptote in May to July animals. Application of Model (i) resulted in clear differences in models between seasonal groups and sexes. Model fits were better (higher r and lower MSE) and slopes were higher for October to Decem- ber than for May to July models and also for female than for male iTiodels (Table 4). The application of Model ii (Table 4) resulted in rapid conver- gence and good agreement between empirical and predicted val- ues. The asymptotic (A) and inflexion (C) parameters were higher in the October to December group; with the exception of the A parameter (which showed great variability), males and females within the each group showed very similar inflection and steepness parameters (Table 4). Case E: /. illecebrosus Linearity was not fully achieved by the log-log transformation and the application of Model i resulted in different parameter values among groups (cases El to E6; Table 4); however, the fit was good (high r and low MSE) with only slight differences between sexes, except for E3 and E6 cases (higher slopes for females than males in the May-hatched group). The log^WaL-at-age plots (Model ii) resulted in very similar (virtually coincident) curves for both sexes of the March (El, E4) and May (E3. E6) groups while larger values of log^,WaL-at-age were observed in females than in males of the April group. (E2, E5); the slopes consistently increased from March to May for both sexes, suggesting increase in growth rate throughout the spring. 856 Ragonese et al. TABLE 4. Estimated coefficients of the log-log and specific logistic models bv using predicted weight and mantle length to derive weight-at-length (WaL)-at-age observations: log = natural (log,! logarithm; r" = coefficient of determination: MSE = mean squared error; N = sample size. Case Sex Group Log-log Model (i( Specific L jgistic Model (iil Species-Area log,.a b /■■ MSE A B C r' MSE N /. cnindetii Al f Oct.-Dec. -14.029 2.628 0.964 0.037 0.002 0.038 148.7 0.997 0.004 17 Central Mediterranean A2 f May-Julv -11.912 2.288 0.918 0.048 -0.301 0.054 108.5 0.996 0.003 12 A3 m Oct.-Dec. -11.551 2.152 0.953 0.026 -0.065 0.036 140.0 0.998 0.00 1 16 A4 m May-Julv -8.650 1.639 0.799 0.03 1 -0.315 0.078 108.4 0.999 0.000 9 /. cointletii Bl f Oct.-March -8.569 1.418 0.999 0.0001 0.7747 0.0065 443.0 0.999 0.0000 18 W Mediterranean B2 f Apr.-Sept. -8.729 1 .452 0.999 0.0003 0.5215 0.0083 360.0 0.999 0.0002 21 B? ni Oct.-March -10,S25 1.803 1.000 0,0001 0 8'-)73 0.0085 443,0 0.997 O.OOIO 19 B4 m Apr.-Sept. -11.680 1.982 1 .000 0.00(.)0 0.4756 0.0119 323.1 0.999 0.0002 15 /. coindetii CI r pooled animals -13.337 2.405 0.997 0.0041 1 .5600 0.0140 355.9 0.997 0.0037 25 Galician (Atlantic) c: m pooled animals -15.041 2.689 1.000 0.0000 1 .0460 0.0181 297.0 0.995 0.0083 21 /. coindetii Dl f Sierra Leone -4.258 0.727 0.941 0.0024 -0.2693 0.0251 110.8 0.994 0.0003 10 NW African Coasts D2 f W. Sahara -9.506 1.845 0.995 0.0020 1.0167 0.0196 198.5 0.999 0.0007 15 D3 m Sierra Leone -8.214 1.473 0.889 0.0185 -0.4887 0.0482 118.3 0.994 0.0012 8 D4 m W. Sahara -10.972 2.126 0.961 0.0229 0.5799 0.0327 152.9 0.996 0.0026 12 /. ilU'cehrosus El f March -5.947 1.010 0.999 0.0001 0,3350 0.0131 161.8 0.999 0.0000 10 NW Atlantic E2 f April -8.848 1.705 1.000 OOOOO 0.8022 0.0180 1 89.3 0.999 0.0002 10 E3 f May -11.324 2.235 1.000 0.0000 1.2347 0.0205 202.0 0.999 0.0001 7 E4 ni March -5.364 0.981 0.999 0.0000 0.2224 0.0139 146.6 0.999 0.0000 8 E5 ni April -7.662 1 .454 1.000 0.0000 0.5779 0.0164 178.5 0.999 0.0000 8 E6 m Mav -8.728 1.737 1.000 0.0000 1.1411 0.0171 196.4 0.999 0.0000 6 /. an^eutinus Fl f June -14.200 2.548 1 ,000 0.0000 1.2977 0.0140 333.2 0.999 0.0002 13 Patagonian Shelf F2 ni June - 1 1 .606 2.130 1,000 0.0001 1.6207 0.0113 358.9 0.999 0.0000 13 -3.50 60 90 120 150 240 270 300 330 360 -0-A1-IC -•-A2-IC -^B1-IC -^ B2-IC -»^C1-IC -ChDI-IC -m- D2-IC -^E1-ll -♦^ E2-II E3-II H-FI-IA 180 210 Age (days) Figure 3. Log„ of body v»eight (BW) at mantle length (ML) ratio (based on predicted values, symbols) vs. age and computed specified logistic models (lines) for Illex females (see Table 1 for case specifications). Growth Performance Across the Squid Genus Illex 857 1.50 -3.50 60 90 120 150 270 300 330 360 A3-IC A4-IC B3-IC -k- B4-IC -^ C2-IC -O- D3-IC -^- D4-IC ^ E4-II -*- E5-II E6-II -^ F2-IA 180 210 240 Age (days) Figure 4. Log^ of body weight (BW) at mantle length (ML) ratio (based on predicted values, symbols) vs. age and computed specified logistic models (linesl for Illex males (see Table 1 for case specifications). Application of Model ii resulted in rapid convergence and sat- isfactory agreement between predicted and empirical values (Table 4). Both the slopes (B) and asymptote (A) increased with month of hatching in each sex. Differences in these parameter values be- tween sexes were slight for all hatching groups. Case B, C, D, and F: Illex spp. For these cases, for which no raw data were available, both models performed quite well (Table 4). but Model i did not achieve complete linearity in D cases. There were some strong differences in model parameters between the sexes but none that were con- sistent among groups and between Models i and ii. All cases for all three species are compared using Model ii for females (Fig. 3) and males (Fig. 4). Two patterns emerged: relationships with a slight curvature (Cases B1-B4; C1-C2; F1-F2) and relationships with a pronounced curvature and asymptotic trend (all D cases). Clear differences in model slopes between sexes were evident in /. coindetii off Sierra Leone (Dl, D3) and in /. argentinus (Fl , F2), but in the former the slope was higher for females than males, whereas the opposite was true in the latter. A comparison among and within species indicates that B cases (/. coindetii from the Catalonian Sea; Figs. 3 and 4) are unique. They are consistently different from the other curves, suggesting relatively slow and near linear growth, regardless which model is considered. The Inflection Point and Maturity The relationship between age-at-the-inflection point (i.e.. C pa- rameter; Table 4) and the mean age of mature animals of a specific group (as directly computed or roughly estimated from the litera- ture) was examined (Fig. 5). Despite the rough approximation, a positive and significant linear trend between the two parameters was evident, suggesting that C values reflect to a certain extent ages at maturity. The estimated slope ( 1 .7). however, is higher than the value expected in case of direct proportionality (close to 1 ). The inflection parameter tended to be relatively lower than (or equal to) the age a! maturity for groups where almost all the life stages were represented in the samples (i.e., A1-A4 and D1-D4). It tended to be relatively higher in cases in which mature speci- mens were underrepresented in the samples, i.e.. mean age at ma- turity was probably underestimated (i.e.. C1-C2 and Fl). DISCUSSION The most appropriate model for describing growth in cephalo- pods is currently a subject of strong debate. One argument is that squid are fundamentally different from fish and so any of the several available empirical growth models may be appropriate and applied (Jackson 1994). The alternative argujiient is that most principles of fish population biology do apply to squid: therefore the most appropriate growth function to use is the von Bertalanffy Model (Pauly 1985. Longhurst & Pauly 1987. Pauly 1998). There is no need of entering into this matter here because an index of growth performance was iflodeled. not growth in absolute size itself. Therefore we were justified in embracing, the view of Ricker ( 1979). that "the only criteria for choosing a growth curve that have proven valid are goodness of fit and convenience." It is, however, of considerable interest to compare trends in growth performance obtained here with trends in growth from studies of absolute size-at-age. 858 Ragonese et al. 260 300 140 180 220 Mean age (days) of mature specimens Figure 5. Relationship between age at inflexion point (C parameter of llie specific logistic model) and mean age of mature Illex females (see Table 1 for case specifications). Tlie fitted straight line is significant Ir" = (1.835: df = 9). Trends in Growth Performance A single curve has seldom been suitable to describe the whole life cycle when modeling size-at-uge (Ricker 1979, Jackson 1994); therefore, a single model is more often a compromise that deals with different growth phases, each of which could be better de- scribed independently. In the case of lUe.x spp. at least three phases of growth can be identified: an early (larval) exponential phase, a "juvenile"' (power or log linear) phase and a "mature and post mature" phase. Be- cause of this, the exponential and the power models are often defined as "early growth curves" (Peters 1983). Linear modeling of length-at-age data fits satisfactorily some restricted portions of the life cycle (Rodhouse & Hatfield 1990. Gonzalez et al. 1996, Dawe & Beck 1997), but clearly does not apply to the entire cycle (Dawe & Beck 1997. Gonzalez et al. 1998). A logistic model was already used quite satisfactorily to rep- resent growth in length and weight of A1-A4 squids (Arkhipkin et al. 2000). However, several different models can fit the same set of data (Arkhipkin et al. 1998) and in some situations both linear and curvilinear models could perform quite well, as was recently re- ported for length-at-age of/, argentinus (Uozumi & Shiba 1993). In our study of growth performance, the logistic model was quite suitable for cases in which most of the ontogenetic develop- ment was represented in the samples (i.e.. individual and predicted data for cases A and values based on predicted sizes for cases D). Nevertheless, some imprecision in the estimate of the A (asymp- totic) parameter was evident, likely as a consequence of the poor representation in the samples of spent males and females, i.e., a poor representation of the last portion of the life cycle. Besides the problem related to sampling the whole life cycle, poor model fits obtained with raw data may be caused by several different biases that are related to the different aspects of the methodology applied, starting with the partitioning of specimens into groups or "microcohorts" based on periods of hatching (e.g.. month). Statolith increment counts in fact, can be affected by systematic and measurement errors due to preparation techniques and reader's interpretation (Jackson 1994. Dawe & Beck 1997. Arkhipkin et al. 1998. Gonzalez et al. 1998, Gonzalez et al. 2000). Thus, the hatching month to which any individual is assigned may be largely a function of ageing error (Gonzalez et al. 2000). The use of mean values, despite statistical problems in fitting models, reduces the incidence of these errors thus reflecting in a better model fit. Comparisons of absolute growth can also be biased by differ- ences in the sampling gear used. Although little is known about the catching capacity of gear such as trawls, traps and jigs, it is most likely that their selectivity by size do differ, so that none of them individually would provide a truly representative sample. Our ap- proach facilitates comparisons when such sampling biases are present, since different gears are likely to be much more highly selective for absolute size than for physical condition. Evaluation of Models One of the immediate questions to answer, i.e.. "Is there any advantage to modeling WaL above modeling ML and BW sepa- rately'?" requires comparisons to be answered. Such comparisons were possible only for individual /. coindetii (A1-A4 cases) and /. Growth Performance Across the Squid Genus Illex 859 illecebrosiis {E1-E6) data sets, based on the logistic models re- ported in Arkhipkin et al. (2000) and the simple linear and log-log models presented in Dawe and Beck ( 1997). The r^ for A cases ranged between 0.87-0.91. 0.77-0.89, and 0.95-0.97 for ML-at-age, BW-at-age, and log^WaL-at-age, respec- tively. Considering the E cases, the r values ranged between 0.37-0.84, 0.37-0.90, and 0.35-0.90 for ML-at-age, log^.BW-at- log^age, and log^.WaL-at-age, respectively. Results indicate that similar (E1-E6) or even better (A1-A4) model tits were obtained by using WaL instead of ML and BW separately, with the ad- vantage of using only one model to take into consideration two size indices. A possible explanation for that is that while length will increase with age under any conditions, weight may increase or decrease, but does not account for concurrent increases in length. However, weight-at-length does account for concurrent length increase, and so it is a better descriptor of growth perfor- mance. Another very interesting point is the comparison of results ob- tained by applying the same models to the same data sets and using alternatively raw data from individuals and values computed from predicted lengths and weights. While both Models i and ii (i.e., the log-log and the specific logistic) do fit consistently well for both /. coincletii and /. illecebrosiis datasets when using data based on predicted sizes, when raw individual data were used. Model ii fitted well /. coindetii datasets (Cases A), but did not fit /. illece- brosiis datasets. Such differences in Model ii fits are likely the consequence of the "quality" of the available datasets. Raw indi- vidual age data in fact, are almost neither "balanced" (i.e.. the same number of aged specimens for each age level) nor "optimal" (i.e., number of counts proportional to the variance of the esti- mates) because statolith processing is very time consuming and age classes are numerous. In cases of such high variability, few points could have a great influence on the estimates and the model may not fit properly. If the samples are larger, however, or if repeated blind counts are available (see, for example, Arkhipkin et al. 1998), then a statistical "weight" can be assigned to each point and a weighed regression performed to improve the fitting quality (Neter et al. 1985). Comparisons Within and Among Species WaL modeling showed some common patterns among species and groups, with the exception of the /. coindetii population of the Catalonian Sea. Regardless of the model applied to those excep- tional cases, the parameters generated were quite different from those of all the other cases. To a certain extent, such differences may be related to the indirect method used in the present work to compute body weight-at-age, but it is also likely that age was originally overestimated for that group, as a consequence of the ageing method used (Arkhipkin personal communication: Dawe & Beck 1997, Gonzalez et al. 2000). Results obtained for the other groups showed several common features of growth performance within the genus Illex, which are consistent with results of other studies (e.g., Dawe & Beck 1997, Haimo\ici et al. 1998, Arkhipkin et al, 2000). These features in- clude superior growth performance of /. coindetii and /. illecebro- siis specimens hatched in warm conditions relative to those hatched in colder conditions. Also consistent with previous stud- ies, a superior growth performance in populations inhabiting warm waters (Strait of Sicily in the Central Mediterranean Sea. West Sahara, and Sierra Leone in the Eastern Atlantic) relative to that of con-specitlc or con-generic populations living in colder waters resulted. Last but not least, the previous conclusion that the Gali- cian /. coindetii population appears to be more similar to the South Patagonian stock of /. argentinus than to any other Illex popula- tions (Gonzalez et al. 1996) was confirmed. One of the most interesting results of our study is the direct relationship found between the intlexion parameter C (from appli- cation of the specific logistic model) and the age-at-maturity. Size- at-maturity in Illex squid has rarely been objectively defined (e.g., as the size at which 50% of the specimens were classified as mature; cf Jereb & Ragonese 1995). Usually, only ranges or mean size values of mature animals have been reported, although the onset of sexual maturation is considered to be a critical point in cephalopod life cycles (Mangold et al. 1993). The C parameter may represent a useful maturity objective index for future studies of changes or trends in sexual maturation. For example, Pauly (1998) argued that principles of fish dynamics are also applicable to cephalopods. If this were true, then tempera- ture would be directly related to growth rate and inversely related \.o maximum size- and age-at-maturity. Consequently, when com- paring among populations, a shift of the inflection parameter to younger ages would be expected from colder to wanner habitats, related to an improvement in growth performance. This is consis- tent with results obtained in the present work. Obviously further comparative studies are required especially within populations. Independent estimates of size- and age-at- maturity would be very useful in evaluating if a true relationship exists between the C parameter from the specific logistic model and the age at the onset of maturity. LITERATURE CITED Arkhipkin. A. 1. 1996. Geographical variation in growth and maturation of the squid Illex coindetii (Oegopsida, Ommastrephidae) off the North- west African coasts. J. Mar. Biol. Ass. U.K. 76:1091-1 1()6. Arkhipkin, A. I., P. Jereb & S. Ragonese. 1998. Age determination of Illex coindetii from the Strait of Sicily by statolith increment analysis. Pages 47-58. In: A. I. L. Payne, M. R. Lipinski, M. R. Clarke & A. C. Roeleveld, editors. Cephalopod biodiversity, ecology and evolution. S. Afr J. Mar. Sci. 20. Arkhipkin. A. I., P. Jereb & S. Ragonese. 2000. 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COMPARISON OF NUTRIENT COMPOSITION OF GONADS AND COELOMIC FLUID OF GREEN SEA URCHIN STRONGYLOCENTROTUS DROEBACHIENSIS CHANDRIKA LIYANA-PATHIRANA,' FEREIDOON SHAHIDI,' " * AND ALAN WHITTICK' ^Department of Biology, -Department of Biochemistry. Memorial University of Newfoundland. St. John's, Newfoundland, AlB 3X9, Canada ABSTRACT The compositional characteristics of sea urchin gonads and coeloniic lluid from Stnmgylocemnitus droehuchiensis harvested in the coasts of Newfoundland and thereafter reared in an aquaculture facility and fed on a Laminaria diet for a 3-week period, were assessed. Evaluations were performed on the basis of proximate composition, lipid class distribution, fatty acid compo- sition, total and free amino acid composition, and contents of nucleic acids and carotenoids. Noticeable changes existed between proximate composition of sea urchin gonads and coelomic fluid. Moisture content was 74.7 + 0.04 and 96.5 ± 0.03% in gonads and coelomic fluid, respectively. Gonads contained very high levels of lipids, proteins, and carbohydrates; whereas, these were present at very low levels in the coelomic fluid. Major nonpolar lipid classes were triacylglycerols (TAG), free fatty acids (FFA). and sterols (ST) while dommant polar lipid classes were phosphatidylcholine (PC), phosphatidylethanolamine (PE). sphingomyelin/ lysophosphatidyl- choline (SM/LPC). and phosphatidylserine / phosphatidylinositol (PS/PlI in both the gonads and the coelomic fluid. Major saturated fatty acids (SPA) were 14;0 and 16:0; whereas. 20:ln-15 was the main monounsaturated fatty acid (MUFA) present. Furthermore, 20:5n-3 (eicosapentaenoic acid, EPA) was the dominant polyunsaturated fatty acid (PUFA) in the gonads and the coelomic fluid. The total amino acid (TAA) and free amino acid (FAA) profiles were dominated by glycine. The total FAA content was much higher in the gonads than in the coelomic fluid. In addition, the total carotenoid content of sea urchin gonads was approximately 6.4 times greater than that of coelomic fluid. Hence, most of the carotenoids were concentrated in the gonadal tissue. Echininone and fucoxanthin were the dominant carotenoids in the gonads and the coelomic fluid, respectively. The content of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) was much higher in the gonad than in the coelomic fluid, thus indicating greater biomass and protein synthetic activity in the former tissue. The present study demonstrates that sea urchin gonads have much in common with sea urchin coelomic fluid on a qualitative basis. However, there were marked quantital;ve differences between the two tissues. KEY WORDS: amino acid composition, carotenoids. fatty acid composition, lipid class distribution, nucleic acids, Slmngyhcen- trolus droehachiensis INTRODUCTION Sea urchins belong to the marine invertebrate phylum Echino- dermata or spiny-skinned animals. These relatively small echino- derms have spherical bodies enclosed in a hard shell or "test" completely covered with numerous sharp spines. Sea urchins are omnivorous animals that live on the ocean floor, feeding on small crustaceans, fish offal, but mainly seaweed (Smith 198(5). Thus, the eating quality of sea urchin gonads is dictated, to a certain degree, by the quality of kelp consumed. Laminaria kelps are the preferred source of feed for sea urchins. Kramer and Nordin ( 1979) reported that the green sea urchin Strongyloceiitniliis droebachien- sis produces high-quality gonads when the availability of fresh kelp is adequate. The edible green sea urchin S. droehachiensis is abundantly distributed in the North Atlantic, Arctic, and North Pacific Oceans, but this species is currently exploited to a much lesser extent in the Northwest Atlantic and in the Northeast Pacific, and Northeast Atlantic (Walker & Lesser 1998). Furthermore, S. droehachiensis is a target species for the development of commer- cial echiniculture (Hagen 1996). The edible portions of the sea urchin body are its reproductive organs; ovaries, and testes. Gonad yield from sea urchin may vary with the time and the site of harvest and generally ranges from 8-20% of the total body mass. When sea urchins are processed for gonads, the initial step is to break the shell and open it so that the five gonad sacs are exposed. The cracked shells are then allowed to drain for several minutes to dispose of coelomic fluid. Thus, during extraction of sea urchin gonads, large amounts of coelomic fluid are obtained. So far, there are no effective means of using sea urchin coelomic fluid in a useful manner. Furthennore, no infor- mation is available on the nutrient composition of sea urchin coe- lomic fluid. In fact, knowledge of nutrient composition may be useful to determine whether sea urchin coelomic fluid could serve as a potential source of a flavoring in fabricated seafood. The objective of this study was to assess the nutrient compo- sition of sea urchin coelomic fluid as compared with that of the gonads. Thus, proximate composition, lipid class distribution, fatty acid composition, amino acid composition, and contents of carot- enoids and nucleic acids of gonads and coelomic fluid were de- termined. This may lead to potential commercial utilization of the processing byproducts from sea urchins, which would otherwise be discarded. MATERIALS AND METHODS Materials *Corresponding author. Tel.: (709) 737-8552; Fax: (709) 737-4000; E-mail: fshahidi@mun.ca One hundred twenty-five sea urchins were procured from the Sea Urchin Research Facility (SURF) at Bonavista Bay, New- foundland and subsequently transported in aquarium coolers to our laboratory at Memorial University of Newfoundland. Urchins were captured from the wild (June 2000) and raised in raceways feeding on a Laminaria diet. Urchins were harvested for analysis after three weeks of feeding on a purely algal diet. Live urchins were stored at 4°C before the extraction of tissues. The gonads and coelomic fluid of sea urchins were separated after breaking the shell, using a specially devised sea urchin cracking tool. After extraction, sea urchin gonads were homogenized for 2 min using a cooled Waring blender (Dynamics Corporation, New Hartford, CT), and coelomic fluid was used for analysis as it is. In this study. 861 862 Liyana-Pathirana et al. sea urchin male and female gonads were pooled together for analy- sis. The tissues (both gonads and coelomic fluid) were flushed with liquid nitrogen and stored at -20°C until used for further analyses. All chemicals used were obtained from either Fisher Scientific (Fair Lawn. NJ) or Sigma Chemical Co. (St. Louis. MO). The solvents were of ACS-, pesticide-, or HPLC-grade. Determination of Proximate Composition Moisture and ash contents of sea urchin tissues were deter- mined according to the standard AOAC ( 1990) procedures. Crude protein content was obtained by Kjeldhal method (AOAC 1990). and total lipids were extracted and quantified by the Bligh and Dyer ( 1959) procedure. Carbohydrate content of each sample was determined by difference. Analysis of Lipid Classes by latrnscan Instrumentation The crude lipids obtained from Bligh and Dyer ( 1959) extrac- tion were chromatographed on silica gel-coated Chromarods - S III and then analyzed using an latroscan MK-5 (latroscan Laborato- ries Inc., Tokyo) analyzer equipped with a flame ionization detec- tor (FID) connected to a computer loaded with TSCAN software (Scientific Products and Equipment. Concord. ON) for data han- dling. A hydrogen flow rate of 160 niL per min and an airflow rate of 2.000 mL per min were used in operating the FID. The scanning speed of rods was 30 sec per rod. Preparation of Chromarods The Chromarods were soaked in concentrated nitric acid over- night followed by thorough washing with distilled water and ac- etone. The Chromarods were then impregnated by dipping in a 3% (w/v) boric acid solution for five minutes to improve separation. Finally, the cleaned Chromarods were scanned twice to burn any remaining impurities. Standards and Calibration A stock solution of each of the nonpolar lipids; namely, free fatty acid (FFA; oleic acid), cholesterol ester (CE). cholesterol (CHOL), monoacylglycerol (MAG; monoolein). diacylglycerol (DAG; diolein). and triacylglycerol (TAG; triolein), and the polar lipids; namely, phosphatidylcholine (PC), phosphatidylethanol- amine (PE). phosphatidylinositol (PI), phosphatidylserine (PS), lysophosphatidylcholine (LPC). lysophosphatidylethanolamine (LPE). cardiolipin (CL). and sphingomyelni (SM) was prepared by dissolving each in a chloroform/methanol (2:1. v/v) solution and stored at -20°C. A range of dilutions of the stock solution, from 0.1 to 10 |j.g per (xL. was prepared for use as working standards. Each compound was developed individually and run on the latro- scan-FID to determine its purity and Rf value. For each compound peak, area was plotted against a series of known ct)ncentrations to obtain the calibration curve. latroscan (TLC-FID) Analysis of Sea Urchin Lipids The total lipids extracted were dissolved in chloroform/ methanol (2:1, v/v) to obtain a concentration of 1 p.g lipid per mL. A I ji,L aliquot of sample was spotted on silica gel-coated Chro- marods - S III and conditioned in a humidity chamber containing saturated CaCl, for 20 min. The Chromarods were then developed in two solvent systems. The solvent system hexane/diethyl ether/ acetic acid (80:20:2. v/v/v) was u.sed for separation of nonpolar lipids (Christie 1982). Following their development, Chromarods were dried at 1 10°C for three minutes and scanned completely to reveal nonpolar lipids. For polar lipids, following the same proce- dure and drying, the Chromarods were scanned partially to a point just beyond the MAG peak to bum the nonpolar lipids. These partially scanned Chromarods were developed in a second solvent system of chloroform/methanol/water (80:35:2, v/v/v) for the sepa- ration of polar lipid classes (Christie 1982) followed by drying at 110°C for three minutes. Finally, the Chromarods were scanned completely to reveal polar lipids; the identity of each peak was determined by comparison with a chromatogram of standards ac- quired concurrently with the samples. The determination of weight percentages of individual lipid classes was achieved using the standard curves obtained for each authentic standard. Analysis of Fatty Acid Composition of Lipids Fatty acid composition of lipids was determined using gas chromatography (GC) as described by Wanasundara and Shahidi (1997). Fatty acid methyl esters (FAMEs) of total lipids of sea urchin gonads and coelomic fluid were prepared by transmethyl- ating approximately 10 to 20 mg of each lipid sample in 2 mL of freshly prepared transmethylating reagent [6% (v/v) sulfuric acid in 99.9 moK/f HPLC-grade methanol containing 15 mg of t- butylhydroquinone (TBHQ)] at 65°C for 15 h in a 6 mL Teflon- lined screw-capped conical vials. After incubation, the mixture was cooled, and 1 mL of distilled water was added to it. This was followed by extracting the FAMEs three times with 1.5 mL pes- ticide-grade hexane. A few crystals of TBHQ were added to each sample before extraction with hexane. The hexane layers were removed and combined in a clean test tube followed by washing twice with 1.5 mL of distilled water by vortexing. The aqueous layer was discarded after the first wash, while the hexane layer was removed and placed in a GC vial following the second wash. Hexane was evaporated under a stream of nitrogen in a fume hood. The dried FAMEs were then dissolved in 1 mL of carbon disulfide and used for GC analysis. FAMEs were separated using a gas chromatograph (Hewlett-Packard 5890 Series II, Hewlett-Packard. Mississuaga, ON) equipped with a fused silica capillary column (SUPELCOWAX-IO. 0.25-mm diameter. 30-m length. 0.25-p,m film thickness; Supeico Canada Ltd.. Oakville, ON). The sample was injected into the GC analyzer using a Hewlett-Packard 7673 autoinjector (Hewlett-Packard. Toronto. ON). The temperature of the oven was programmed at 220°C for 10.25 min followed by ramping to 240°C at 20°C per min. where it was held for nine minutes. Helium at a flow rate of 2 mL per min was used as the carrier gas. The FAMEs were identified by comparing their reten- tion times with those of authentic standard mixtures (GLC - 461. Nu-Check-Prep) and literature values (Takagi et al. 1980. Takagi et al. 1986). The relative content of fatty acids in the sample was determined using the peak areas of fatty acids. Carotenoid Pigments Extraction and Determination of Total and Individual Carotenoids Carotenoids from each tissue were extracted three times with a total of 50 mL of acetone for two minutes. The homogenized samples were centrifuged (lEC Centra MP4 Centrifuge. Interna- tional Equipment Co.. Needham Heights. MA) at 4000 x g for five minutes. The supernatant was subsequently filtered through a Comparison of Nutrient Composition of Sea Urchins 863 Whatman No. 1 filter paper. Carotenoid pigments in acetone were then transferred to 40 niL of n-hexane in a 250-mL separatory funnel. One hundred niilhliters of a 0.57c sodium chloride solution were added to the mixture to maximize the transfer of carotenoids. The hexane layer was then transferred into a 50-mL volumetric flask and made up to volume. The absorption spectrum was then recorded (400-600 nm) using a Spectronic spectrophotometer (Spectronic Genesis. Toronto. ON I. The total and individual ca- rotenoid contents were determined by the method of McBeth (1972). The total content of carotenoids present per 100 g of tissue was calculated using the following equation. mg Carotenoid per 100 g tissue = (A x V x 10')/e x W) where, A = absorbance at \„„,; V = total volume of the sample (mL); e = molar extinction coefficient, and W = weight of the tissue (g). Because the crude extracts usually contained a variety of carotenoids an average coefficient of 2,500 was used in the cal- culations. The total pigment extracted was separated into individual ca- rotenoids by means of thin-layer chromatography (TLC). The crude carotenoids were separated by preparative TLC on silica gel G (20 X 20 cm, 230 (jim, Aldrich Chemical Co., Inc., Milwaukee, WI) using acetone/n-hexane (3:7, v/v) as the developing solvent. Characterization of Fractions Cochromatography on TLC provided the ultimate test for iden- tification when authentic samples were available for comparison with unknown pigments. The unknown fraction and the authentic sample were spotted on either side in an equally proportionated mixture of the two pigments on silica gel G plates (20 x 20 cm. 250 fjLm, Aldrich Chemical Co., Inc., Milwaukee, WI): unknown frac- tions were considered to be identical to the authentic sample if the two did not separate upon subsequent development of the plate. When authentic samples were unavailable, the type of carotenoid in each fraction was tentatively identified according to its absorp- tion maximum in n-hexane, ethanol, and chloroform (Goodwin 1955, Krinsky & Goldsmith 1960, Fox & Hopkins 1966, Britton 1995). Determination of Total Amino Acids The amino acid composition of sea urchin gonads and coelomic fluid was determined according to the procedure described by Blackburn (1968). Samples were lyophili/.ed and then hydrolyzed for 24 h at 1 10°C with 6M HCl. Hydrochloric acid in the hydro- lyzate was removed under vacuum, and the dried sample was reconstituted with a lithium citrate buffer (0.2 M, pH 2.2) for analysis. The amino acids in the hydrolyzate were separated, iden- tified and quantified using a Beckman 121 MB amino acid analyzer (Beckman Instruments Inc., Palo Alto, CA). Sulfur- containing amino acids were determined by oxidizing the samples with pertbrmic acid before their hydrolysis in a 6M HCI solution (Blackburn 1968). Cysteine and methionine were measured as cys- teic acid and methionine sulphone, respectively. To determine tryptophan, samples were hydrolyzed in 3M mercaptoethane- sulfonic acid at 1 10°C for 22 h under nitrogen and then neutralized with lithium hydroxide and adjusted to pH 2.2 (Penke et al. 1974). Determination of Free Amino Acids Samples (10 g) were extracted with 20 mL of a 6% (v/v) perchloric acid (PCA) solution by homogenization using a Poly- tron homogenizer (Brinkmann Instruments, Rexdale, ON) at 10,000 rpm for two minutes in an ice bath. The homogenized samples were then incubated in an ice bath for 30 min. This was followed by centrifugation (lEC Centra MP4 Centrifuge, Interna- tional Equipment Co., Needham Heights, MA) at 2,000 x g for 15 min. The residue was re-extracted with another 20 mL of 6% PCA. The supematants were combined and filtered through a Whatman No. 4 filter paper. The pH of the filtrate was adjusted to 7.0 using a 33% KOH (w/v) solution. Precipitates of potassium perchlorate were removed by centrifugation at 2000 x g for 10 min. The supernatant was then acidified to pH 2.2 using a 10 M HCI solu- tion, and the volume of the extract was brought to 30 mL with distilled water. Three milliliters of lithium citrate buffer (pH 2.2, 0.3M) were added to I mL of the extract, and the resultant solution was analyzed using a Beckman 121 MB amino acid analyzer (Beckman Instruments, Inc., Palo Alto, CA) for individual amino acids. Determination of Nucleic Acids The DNA and RNA contstituents of gonads and coelomic fluid of sea urchins were extracted according to the method of Schmidt and Thannhauser ( 1945) as modified by Munro and Fleck (1969). Five grams of each sample were homogenized in 80 mL ice-cold deionized water using a Polytron homogenizer (Brinkman Instru- ments, Rexdale, ON) at 10.000 rpm. Five milliliters of the homo- genate were allowed to stand for ten minutes in ice and then centrifuged (lEC Centra MP4 Centrifuge, International Equipment Co., Needham Heights, MA) at 2000 x g for ten minutes. The residue was subsequently washed with 2.5 mL of ice-cold 0.2 M PCA and centrifuged at 2,000 x g for ten minutes followed by digestion of the residue in 4 mL of a 0.3 M KOH for one hour at 37°C in a water bath. The resultant solution was cooled in ice and mixed with 2.5 mL of 1.2 M PCA and allowed to stand for ten minutes, which finally resulted in the coagulation of proteins. The mixture was centrifuged at 2,000 x g for ten minutes, and the supernatant was recovered (Extract No.l). The precipitate was then washed twice with 2.5 mL of a 0.2 M PCA solution and centrifuged at 2,000 x g for five minutes. The supernatant was combined with extract No. 1 and 10 niL of a 0.6 M PCA were added to the mixture. This was used for RNA determination after diluting it up to 100 niL with distilled water. The residue was dissolved in 17 mL of a 0.3 M KOH solution at 37 C and diluted to the 50 mL mark in a volumetric flask with distilled water. The content of DNA in the samples was estimated by determining the deoxyribose content in the extract using the indole procedure of Ceriotti (1952), while RNA was determined by recording the ab- sorbance of the nucleotide extracts at 260 nm using a Hewlett- Packard diode array spectrophotometer (Hewlett-Packard, Model 8452A, Hewlett-Packard [Canada] Ltd., Mississauga, ON). Protein interference at this wavelength was eliminated by employing a correction factor of 0.001 absorbance unit for each I (xg per mL protein concentration in the extracts. The protein concentration ot the extracts was measured using the Folin-phenol procedure ot Lowry et al. (1951). Bovine serum albumin (BSA) was used as a standard. Calf thymus DNA (containing 82% single stranded DNA) and calf liver RNA (96% purity) were used as the standards for DNA and RNA determinations, respectively. Statistical Analysis Each experiment was replicated three times and mean values ± standard deviations reported for each sample. For statistical analy- 864 Liyana-Pathirana et al. ses, mean values of the experimental data were subjected to one way analysis of variance (ANOVA) using GraphPAD Instat Ver- sion 1.0. Significance was determined at 5% probability level. RESULTS I'roximale Coiiiposilidii Proximate composition of sea urchin gonads and coelomic fluid is shown in Table I . The moisture and ash contents of sea urchin coelomic fluid were much higher than those of the gonads. On the other hand, the levels of protein, lipid, and carbohydrate in the coelomic fluid were much lower than those in the gonads on a fresh weight basis. Lipid Class Distrihiilion The nonpolar and polar lipid classes of gonads and coelomic fluid of sea urchin S. droebachiensis are shown in Table 2. Major nonpolar lipid classes were TAG. FFA. and ST; whereas, main polar lipids classes were PC. PE. SM/LPC. and PS/PI in both gonads and coelomic fluid. Triucylglycerols constituted the main energy reserve in both tissues, contributing more than 63% to the total nonpolar lipids. On the other hand, PC was the dominant polar lipid, accounting for more than 60% in both gonads and coelomic fluid. The polar lipid classes SM and LPC as well as PS and PI did not show a clear chromatographic separation from each other during latroscan analysis. Fatty Acid Composition Fatty acid composition of sea urchin gonads and coelomic fluid is presented in Table 3. Qualitatively, the fatty acid compositions were the same in both tissues, while there were significant (P < 0.05) quantitative variations. In both tissues. 14:0 and 16:0 were the main saturated fatty acids. In addition. 18:0 and 20:0 were present in considerably high levels. The fatty acid 20: In- 1 5 was the dominant MUFA in both gonadal and coelomic fluid lipids. Furthermore, 16:ln-7. l6:ln-9. 18:ln-7, 20:ln-7, 20;ln-9, and 22: In- II were detected in noticeable amounts. Among PUFA, 20: 5n-3 contributed the highest proportion to the total fatty acid con- tent in both gonadal and coelomic fluid lipids, Carotenoid Pigments The total carotenoid content, on a dry weight basis, of sea urchin gonads and coelomic fluid was 23.2 ± 0.04 and 3.7 ± 0. 1 mg per g tissue, respectively. Crude pigments from gonads and coe- TABLE 1. Proximate cumposition of sea urchin gonads and coelomic fluid after feeding urchins on a Laminaria diet for three weeks. Constituent Gonads Coelomic Fluid Moisture 74.7 (0.04) 96.5(0.031 Ash 2.2 (0.2) 3.0(0.02) Protein 7.4 (0.2) 0.1 (0.02) Lipid 4.7(0.1) 0.1 (0.03) Carbohydrate' 10,6 (0.2) 0.4(0.1) TABLE 2. Quantification of non-polar and polar lipids (weight % ) of sea urchin gonads and coeliiniic fluid alter feeding the urchins on Laminaria diet for three weeks. Lipid Cla.sses Gonad Coelomic Fluid Non polar lipids TAG 66.7(0.8) 56.5 (0.7) FFA 22.7(1.)) 37.4(1.0) •ST 10.6(1.2) 6.1 (0.8) MAG tr tr DAG tr tr Polar lipids PC 65-7(1.4) 68.9 (0.8) PE 17.3(0.2) 27.3 (0.9) SM/LPC 8.4 (0.7) 2.6 (0.2) PS/PI 8.5 (0.6) 1.1 (0.5) Results are mean values of three replicates (standard deviation). Values in each row with the same superscript are not different (f > 0.05) from one another. ' Determined by difference. Results are mean values of three replicates (standard deviation). Values in each row with the same superscript are not different (P > 0.05) from one another. Abbreviations: TAG. triacylglycerol; FFA, free fatty acid; ST, sterol; MAG, monoacylglycerol; DAG, diacylglycerol: PC, phosphatidyl- choline; PE, phosphalidylelhanolaniine; SM, sphingomyelin; LPC. lysi- phosphatidylcholine; PS, phosphatidylserme and PI, phosphatidylinositol; and tr. irace. lomic fluid were separated by TLC into eight and seven fractions, respectively. Crude pigments of both tissues, upon TLC separa- tion, exhibited two major bands. In gonads, fractions I (Rf = 0.96) and II (Rf = 0.88) ran close to the solvent front, but they were adequately separated. For coelomic fluid, fraction I (Rf = 0.94) ran almost close to the solvent front; whereas, fraction IV (Rf = 0.42) ran well behind. The carotenoid fraction I of both gonads and coelomic fluid of sea urchin S. droehachiensis was confirmed to be 3-carotene using an authentic ^-carotene sample as established by cochromatography on silica gel TLC plates. Similarly, gonadal fractions III. IV. VI. and VII contained astaxanthin ester (Rf = 0.57), zeaxanthin (Rf = 0.51), canthaxanthin (Rf = 0.22), and free astaxanthin (Rf = 0,1), respectively. Furthermore, fraction II of gonadal crude pigments corresponded to echininone by means of absorption maxima in hexane. chloroform, and ethanol (Good- win 1955. Krinsky & Goldsmith I960. Fox & Hopkins 1966. Britton 1995). Thus, the observed X^nax values of echininone were 484/460. 466. and 475 nm in hexane. ethanol. and chloroform, respectively. In coelomic fluid, the pigment in fraction IV corre- sponded with fucoxanthin based on absorption maxima of 424/ 447/474, 423/446/472, and 454/488 nm in hexane. ethanol, and chloroform, respectively. Other minor carotenoids in the coelomic fiuid were astaxanthin etser (Rf = 0.58), canthaxanthin (Rf = 0,23), and free astaxanthin (Rf = 0.1), which coiresponded to fractions III, V, and VI, respectively. The other minor carotenoids were not analyzed because of their insufficient concentration to obtain absorption maxima and also lack of authentic .samples. Amino Acid Composition The total and free amino acid compositions of sea urchin go- nads and coelomic fluid are shown in Tables 4 and 5, respectively. Results so obtained did not show any clear variation on a quali- tative basis for tissues examined. Thus, the spectrum of amino acids, both total and free, present was nearly the same for both gonads and coelomic fluid. Total amino acid profile indicated the Comparison of Nutrient Composition of Sea Urchins 865 TABLE 3. Fatly acid composilimi (Ht'i(;lit '7f I of total lipids sea urchin gonads and coeloniic fluid after feeding the urchins with iMininaria diet for three weeks. TABLE 4. Total amino acid content Img/g protein! of sea urchin gonads and coelomic fluid after feeding urchins on a Laminaria diet for three weeks. Fatty Acid 14:(1 15:0 KrO IS:() 20:0 14:ln-7 l6:ln-9 l6:ln-7 16:1 11-5 lS:lii-4 lS;ln-7 lS:ln-5 20; In- 15 20:ln-9 20:ln-7 22: In- 11 22:ln-9 16:2n-fi Hi:4n-h l(r4n-3 l.S:2n-'-) lS:2n-6 lS:3n-6 lS:3n-3 lS:4n-.^ 20:2A5. 1 1 20:2A5. 13 20:2n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:5n-6 22:5n-3 22:6n-3 Gonads Coelomic Fluid Amino Acid Gonads Coelomic Fluid 9.4(0.1)' 0.4(0.02)'' 11.1 (O.!)-* 2.2(0.04)'' 2.9(0.1)-' 0.8 (0.03)" 4.8(0.1)"'= 1.5(0.1)" 0.3 (0.03)° 1.8(0.1)" 3.6(0.1)'' 0.5 (0.02)" 7.5 (0.2)" 4.0(0.03)" 2.2(0.1)" 2.9 (0.04)" 0.4(0.03)" 0.5 (0.03)" 1.9(0.1)" ND ND 1.1 (0.1)" 1.4(0.03)" 1.3(0.04)" 3.8(0.03)" 1.8(0.1)" 0.9(0.1)" 1.7(0.1)" 7.0(0.1)° 1.7(0.2)" 1.2(0.2)" 16.3(0.1)" 0.2(0.1)" 0.6(0.1)" 1,4(0.1)" 8.4(0.1)" 0.7 (0.02)" 17.6(0.3)" 2.3(0.1)" 1.2(0.1)" 0.3(0.03)" 1.6(0.1)" 3.4(0.2)" 0.6(0.04)" 2.2(0.1)" 4.2(0.1)" 3.4(0.2)" 5.6(0.1)" 1.2(0.03)" 1.9(0.1)" 1.2(0.1)" 1.1(0.1)" 0.2(0.02)" ND 2.8(0.1)" 0.5 (0.02)" 0.9(0.1)" 1.3(0.1)" 0.5 (0.04)" 2.2 (0.03)" 1.7(0.1)" 0.7(0,03)" 1.7(0.1)" 9.9(0.1)" 0.9 (0.04)" 0.3(0.02)" 16.5(0.5)" 0.3 (0.03)" 0.7 (0.04)" 0.6(0.1)" ResuUs are mean values of three replicates (standard deviation). Values in each row with the same superscript are not significantly different {P > 0.05) from one another. ND, not detected. dominatice of glycine in both tissues analyzed. However, on a dry weight basis, the content of glycine in sea urchin coelomic fluid was significantly (P < 0.05) higher than that of gonads. Almost all the essential amino acids were present in both gonads and coelo- mic fluid of S, droebachiensis. With respect to FAA content of sea urchin tissues, the total FAA content was much higher in sea urchin gonads than that in the coelomic fluid. However, in both tissues glycine was the dominant FAA contributing 57.1 and 56.3% to the total amount in the gonads and coelomic fluid, re- spectively. Content of Nucleic Acids The content of nucleic acids in sea urchin gonads and coelo)nic fluid, on a dry weight basis, was different. The content of DNA of sea urchin gonads and coelomic fluid was 3.93 ± 0.1 and 1.02 ± 0.07 jjLg per g tissue, respectively, whereas, corresponding values for the content of RNA were 2.63 ± 0.06 and 0.49 ± 0.03 p,g per g tissue, respectively. Thus, the content of DNA was higher than Alanine Arginine Cysteine Glutamic acid Glycine Histidine Hydro\yproline Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Total (mg/g protein) 42.5 (0.4)" 80.7(0.5)" 7.2(0.1)" 87.2(0.2)" 118.0(0.7)" 35.5 (0.3)" 3.3 (0,2)" 77.1 (0,1)" 65,9(1.0)" 81.1 (0.9)" 1.2(0.3)" 53.2 (0.9)" 40.2(0.5)" 47.1 (0.1)" 47.8 (0.4)" 1.4(0.1)" 36.3 (0,8)" 78,3 (0,4)" 989,4(2,2) 63.1 (0.5)" 74.2 (0,6)" 5,7 (0,2)" 90.6 (0.6)" 143.6(0.7)" 27.0 (0.8)" 2.6 (0.2)" 60.9 (0.9)" 62.6 (0.3)" 77.1 (0,9)" 0,9(0,1)" 42.1 (0,6)" 48.4(0,1)" 46.5 (0,7)" 47.2 (0,5)" 1,1 (0,2)" 38.6 (0,4)" 75.1 (1.0)" 985.4(1.3) Results are mean values of three replicates (standard deviation). Values in each row with the same superscript are not different (P > 0.05) from one another. that of RNA in both tissues analyzed. Furthermore, the ratio of RNA/DNA was 0.7 ± 0. 1 and 0,5 ± 0,03 for sea urchin gonads and coelomic fluid, respectively, DISCUSSION Reproductive Stale of Sea Urchins For feeding experiments, sea urchins were obtained from the wild in the month of June, representing the spring season. In gen- eral, gonad development in sea urchins may include five different stages of resting, growing, premature, mature, and spawning (de Jong-Westman et al, 1996). In resting, gonad size is at a minimuiT), which usually occurs after spawning. S. droi'lnwhiensis has an annual reproductive cycle with major spawning period in the late winter or early spring (Keats et al. 1984). Therefore, at this stage, uichins were presuniably in the state of resting; hence, they have undergone a large drop in gonad size following spawning. In general, gonadal yield is strongly affected by the seasonal reproductive cycle of sea urchins. During spawning, a high pro- portion of the gonad mass is released as gametes (Thompson 1984). Once spawning occurred, this may exert a significant effect on the biochemical composition of gonads. Because urchins were fed on a Laminaria diet after harvesting and consequently subject to intense feeding, this may have a significant effect on the nutrient composition of sea urchin tissues. Generally, both food quality and quantity affect sea urchin growth (Lawrence & Lane 1982), thus excessive feeding resulting in the accumulation of nutrients in the tissues. Proximate Composition The major nutrients of sea urchin S, ilroehachiensis gonads were polysaccharides, proteins, and lipids, siniilar to that reported 866 Liyana-Pathirana et al. TABLE S. Free amino acid conttnl in/ft. dry weight) of sea urchin gonads and coelomic fluid after feeding urchins on Laminaria diet for three weeks. characteristics of sea urchins' coelomic tliiid. In our study, when urchins were fed on u Lainimiria diet, gonads contained 74.7 ± 0.04% moisture, which was significantly (f < 0.05) lower than that of the coelomic tluid. Gonads Coelomic Fluid Amino Acid Mg/g % Mg/g % Alanine 2.872 (77 )■■ 14 899(17)" 7.5 a-aminoadipic acid 68 IS)-* 0.3 Arginine 180 (3I'' 0.9 507(19)" 4.2 Asparagine 13(1)-' 0.1 Aspartic acid 73 (Sr 0.4 80(3)" 0.7 Cystathionine 115(2)" 0.6 76 (.^9)" 0,6 Cysteine 214(12f 1.0 90(7)" 0,7 Glutamic acid 874(12)" 4.2 478(14)" 4,0 Glulamine 647 (34)" 3.1 184(8)" 1,5 Glycine 11.751 (223)-' 57.1 6,771 (4.30)" 56,3 Histidine 100(6)- 0.5 111 (10)" 0,9 Hydro.xyproline 124(11)- 0.6 88(6)" 0,7 Isoleucine 313(11)-' 1.5 175(6)" 1,5 Leucine 370 (23)" 1.8 293(7)" 2.4 Lysine 356 (7)" 1.7 312(11)" 2.6 Methionine 68 (4)" 0.3 134(8)" 1,1 Phenylalanine 164(2)" 0.8 164(12)" 1.4 Proline 140(10)" 0.7 4.909(147)" 2.7 Sarcosine 332(14) 1.6 Serine 316(5)" 1.5 163(10)" 1.4 Threonine 521 (27)" 2.5 189(12)" 1,6 Tryptophan 264(11)" 1.3 140(3)" 1,2 Tyrosine 217(16)" 1.1 212(5)" 1.8 Valine 273(16)" 1.3 272(9)" 2.3 Total ( mg/g ) 21 (1,0)" 12.0(0.8) Results are mean values of three replicates (standard deviation). Values in each row with the same superscript are not different (P > 0.05) from one another. by Fernandez et al. (1995). However, coelomic fluid contained 96.5 ± 0.03% moisture; thus, its contents of lipid, protein, and polysaccharide were extremely low. On the other hand, the ash content of sea urchin coelomic tluid was much higher than that of the gonads. In general, sea urchin gonads are known to contain high levels of protein. They also have considerably high lipid levels; whereas, carbohydrate levels are low (McClintock & Pearse 1987). However, in our study, the carbohydrate content was high and accounted for approximately 10% of the total amount, on a fresh weight basis. Furthermore, in coelomic fluid, although the relative proportion of carbohydrate was only 0.4 ± 0.1%. this was four times more than that of its protein and lipid contents. From this study, it is apparent that content of lipid, protein and carbo- hydrate in the coelomic fluid was much less than that of the go- nads. The urchins in this work were given a diet purely comprised of Lantinaiia kelp to resemble the urchin's prefened natural diet. In general, the diet plays a very important role in the compositional characteristics of these animals (Nishikiori 1989, Fernandez et al. 1995, Agatsuma 1998) and almost all studies have only determined compositional characteristics of the gonads. Thus. Agatsuma (1998) showed that a diet of fishmeal increased moisture levels, and Nishikiori (1989) observed that moi.sture content in the gonads of 5. niiJus was below 70% when the urchins were fed Laminaria japonica to satiation. None of these studies reported compositional Lipid Class Composition The lipid composition of marine invertebrates is influenced by several factors, including pattern of feeding, gametogenesis, and possibly environmental conditions (Jezierska et al. 1982). Wax esters have been reported to constitute energy reserves in various marine invertebrates (Sargent 1976). but this was not the case for sea urchin 5, draelniciucnsis. TAG formed the main energy reserve in these animals, and their gonads and coelomic fluid were quali- tatively composed of similar lipid classes. The same nonpolar lipid class distribution has been observed in S. droelyuchiensis gonads and coelomic fluid collected form Nova Scotia (Takagi et al. 1980). The nonpolar lipids of gonads and coelomic tluid consisted mainly of TAG, FFA, and ST. Triacylglycerols are usually con- sidered to serve as storage lipids in eukaryotic cells (Sul et al. 2000). Thus, sea urchin lipids contained much larger amounts of storage lipids, principally TAG, which constituted more than 60% of the total nonpolar lipids of gonads and coelomic tluid. Although, qualitative composition of nonpolar lipids of gonads and coelomic fluid of .S'. droehachiensis was similar, relative con- tent of individual classes differed. Hence, both sea urchin gonads and coelomic fluid were composed of the same major lipid classes, both nonpolar and polar, but their relative contents were markedly different. Thus, relative content of TAG in sea urchin gonads was much higher than that of coelomic fluid; whereas, that of FFA in gonads was much less than that of coelomic fluid. It was apparent in the preliminary experiments that both gonads and coelomic fluid contained high levels of FFA. Therefore, it was thought that partial hydrolysis of TAG may lead to an underestimation of TAG con- tent. However, the impact of this on a nutritional value of the fatty acids involved is inconsequential. To verify the above fact, fatty acid content was determined in a set of freshly harvested sea urchins. The gonads were extracted as quickly as possible at 0°C immediately after homogenization. It was assumed that hydrolysis of lipids because of the activity of endogenous enzymes is mini- mized under these conditions. The FFA content was 15.5 ± 1.7% of the total nonpolar lipids upon latroscan analysis. Hence, the high levels of FFA observed for stored sea urchin tissues following homogenization could be attributed to the hydrolysis of TAG dur- ing storage of samples at -20°C. In general, the energy supplied to the animal by the breakdown of lipid reserves comes primarily from oxidation of fatty acids. Farkas (1979) has shown that the production of FFA can be in- duced by stress. Thus, the environmental temperature and diet can be specified as factors exerting a major impact on the content and metabolism of fatty acids in animals (Farkas et al. 1978). There were noticeable differences in the relative content of sterols in sea urchin tissues. In general, cholesterol level may depend on dietary level and stage of sexual development (Love 1970), In fact, diet and nutritional status are known to be the main factors that influence cholesterol levels (Dave et al. 1975). On the other hand, during gametogenesis, a redistribution of cholesterol takes place that may lead to high levels of cholesterol in the gonads (Idler & Tsuyuki 1985). Therefore, all relevant factors must be considered to explain the content of cholesterol in different tissues. Comparison of Nutrient Composition of Sea Urchins 867 III the present study, the relative content of ST in the gonads was significantly (P < 0.03) higher than that in the coelomic fluid. Vaskovsky and Kostetsky (1969) have performed TLC on polar lipids of sea urchin 5. iiiuhis and S. intennediiis. The polar lipid fraction was separated into five components of which PC, PE, and SM constituted the major polar lipid classes present. Furthermore, lipid extracts of different organs of the same animal had a similar qualitative polar lipid composition (Vaskovsky & Kostetsky 1969). In this study, both gonads and coelomic fluid showed quali- tative similarities in their polar lipid fractions. Thus, PC, PE, SM/ LPC, and PS/PI constituted the polar lipids of S. droebachiensis gonads and coelomic fluid, and PC was dominant in both tissues, with a contribution of more than 65'^/c to the total content of polar lipids. Similarly, Floreto et al. ( 1996) demonstrated that sea urchin Tiipneiistes gratila fed on a seaweed diet had PC and PE as the major lipid constituents, and PC contributed a greater proportion than PE. Fatty Acid Composition The fatty acids of total lipids of sea urchin gonads and coelomic fluid were typically similar to those of other marine species with a dominance of 16:0 and 20:5n-3 (Wanasundara 1996). Although. 22;6n-3 is a typical fatty acid in marine lipids, it contributed only 1 .4 ± 0. 1 and 0.6 ± 0. 1 % to the total fatty acids in the lipids of sea urchin gonads and coelomic fluid, respectively. Holland (1978) reported that the predominance of 20:5n-3 and 22:6n-3 in typical marine fatty acids is a result of low- temperature adaptation. This helps in the maintenance of cell membrane fluidity in organisms living in the cold environments. Considerable data are available on the fatty acid composition of sea urchins (Takagi et al. 1980. Kaneniwa and Takagi 1986). The fatty acid 16:0 was the major SFA in the sea urchin 5. droe- bachiensis harvested from Herring Cove, Nova Scotia (Takagi et al. 1980). Fujino et al. (1970) analyzed fatty acid composition of sea urchins Anthocidaris crassispina. S. piilclwninuis. S. fraii- ciscanus. S. intermedins, and Echinus esculentus. In all these samples, 16:0 was the prominent SFA followed by 14:0. The fatty acid 18:0 was found to occur in considerable amounts. Similarly, in the present study, the predominant SFA were 16:0 and 14:0 in the lipids of both gonads and coelomic fluid of S. droebachiensis. Among MUFA 20: In- 1 5 was present up to 11% in the total fatty acids of urchins (Takagi et al. 1980). Ackman and Hooper (1973) reported that such marine animals as periwinkle {Littorine littorea). moon snail (Liinata triseriata). and sand shrimp (Crangon septemspinosu.s) contain 20:ln-l5, but at much lower levels of up to 0.2% of the total fatty acids. However, this has not been commonly reported as being typical of marine lipids. In our study, 20:ln-15 was also the major MUFA in both tissues ana- lyzed. On the other hand, seaweeds, the natural diet of sea urchins, have not been reported to contain 20:ln-15 (Ackman & McLachlan 1977); hence, the formation of 20:ln-l5 in sea urchin tissues may be biosynthetic in origin, because this was not depen- dent on the diet. The occurrence of such unusual ."i-olefinic fatty acids as 18:ln-13, 20:ln-15, 20:2A5,11, 20:2A5,13, 20:3A5,1 1,14. and 20:3A5, 11,14,17 has been noticeable in lipids of sea urchins ac- counting for as much as 6-2 1 % of the fatty acids of total lipids (Takagi et al. 1980. Kaneniwa & Takagi 1986). In this study, the amount of 5-olefinic acids found in the lipids of both gonads and coelomic fluid was in the range of 7-10%. The presence of 5-ole- finic fatty acids has been reported in 12 species of Echinoidea collected in Japan (Takagi et al.l986); thus, they serve a common and characteristic feature of sea urchin lipids. The amount of eicosapenlaenoic acid (20:5n-3) was quite high in sea urchin lipids (Takagi et al. 1980). Pohl and Zurheide (1979) reported that urchins that consumed Laniinaria had a high content of 16:4n-3, 18:4n-3, 20:4n-6. and 20:3n-3. Similariy. sea urchin S. droebachiensis in our study consumed Laniinaria for only a three week period, and their gonadal and coelomic fluid lipids contained quite high levels of these fatty acids. Thus, the fatty acid profiles of sea urchin tissues somewhat reflect that of their diets as was also observed by Floreto et al. (1996). However, certain fatty acids, such as 16:4n-3, 20:4n-6, 20:5n-3, and 20:ln-l I, which constitute the major fatty acids of sea urchin tissues, were not detected in their diets; therefore, suggesting that sea urchins are capable of synthesizing them from lower fatty acid precursors. Similarly, in the present study 16:4n-3, 20:ln-l 1, 20:4n-6, and 20:5n-3, among others, may have been formed by chain elongation of precursors. In general, the sea urchin fatty acids; namely, 16:4n-3, 20:4n-6, and 20:5n-3, may possibly confer some structural function and, hence, are purposely synthesized by the animal (Floreto et al. 1996). Carotenoid Pigments In the sea urchin .S'. dnfcbacliiensis. carotenoids were mainly concentrated in the gonadal tissue. Hence, the total content of carotenoids in the gonads was about 6.3 times more than that of the coelomic fluid. However, the content of carotenoids in different tissues may vary with the reproductive stage of urchins. Hence, during gametogenesis most of the carotenoids in other tissues may be transferred into gonads, consequently increasing their carot- enoid content (Griffiths & Perrott 1976). Echininone and fucoxanthin were characterized as the major carotenoids present in the gonads and the coelomic fluid, respec- tively. In addition, (J-carotene was identified in both tissues. Echininone was found to be the main pigment with a lesser amount of P-carotene in the gonads of S. piirpiiraliis (Griffiths 1966), S. droebachiensis (Griffiths & PeiTOtt 1976) and Tripnenstes gratila (Shina et al. 1978). Tsushima et al. (1995) found that (J-echininone and p-carotene were the major carotenoids in the gonads of 1 9 out of 20 sea urchin species examined. Meanwhile, the major carot- enoids of brown algae, the natural preferred diet of sea urchins, consist of p-carotene, violaxanthin, and fucoxanthin (Matsuno & Hirao 1989). Furthermore, there is bioconversion of p-carotene to P-echininone via P-isocryptoxanthin in sea urchins; which takes place mainly in the gut wall, and the resultant p-echininone is incorporated into the gonads (Tsushima et al. 1993). Kawakami et al. ( 1998) showed that fucoxanthin, the major carotenoid in brown algae, did not accumulate in the gonads. In fact, in the present study on 5. droebachien.sis. fucoxanthin did not occur in the go- nads. On the contrary, coelomic fluid had fucoxanthin as its major carotenoid. Amino Acid Composition Although marine invertebrates characteristically contain a high intracellular concentration of FAA, the composition of the FAA pool may vary among species (Gilles 1979). In the present study, glycine was the dominant amino acid in both TAA and FAA 868 Liyana-Pathirana et al. profiles in both sea urchin gonads and cocloniic fluid. Komata el al. (1962) reported that glycine was dominant ni the gonads ot" sea urchin S. piilclwnimiis. and its content ranged from 35—41% of total FAA. Lee and Haard ( 1982) reported that glycine constituted 18-60% of the FAA in the gonads of sea urchin S. droebachiensis. The gonads and coelomic fluid of sea urchin .S'. droebachiensis in this study contained 1 1.9-14.6% glycine in the TAA profile, re- spectively. However, glycine was not the dominant amino acid in the gonads of the sea urchin Paracentrotus lividiis. although it contributed a considerable amount to the TAA pool (Cruz-Garcia et al. 2000). Other than glycine, alanine, arginine. glutamic acid, lysine, and methionine are considered important for taste, even though some of them were present in small quantities (Lee & Haard 1982). These amino acids were present in considerable amounts in both gonads and coelomic fluid of sea urchins in this study. It has been found that different combinations of taste-active components (substances that influence the taste of any food) as well as their relative amounts are of paramount importance in producing the characteristic flavor of each seafood (Puke 1994). In general, glutamine and glycine, which were present in higher amounts in the gonads than coelomic fluid, are known to be taste- active in sea urchins and other seafoods, regardless of their quan- tity. Sea urchin gonads seemed to be sweeter when little or no glutamine was present, and alanine was found in considerably high levels. Alanine is a taste-active component in sea urchin tissues, contributing noticeably to both TAA and FAA contents. Further- more, valine and methionine are known to be taste-active only in sea urchins; whereas, arginine was also taste-active in sea urchins because of its high content (Fuke 1994). Both methionine and arginine were present at a higher proportion in the coelomic fluid than in the gonads. Similarly, the contents of aspartic acid, histi- dine. and especially proline were much higher in the coelomic fluid than those in the gonads. Thus, amino acids play a major role in the taste of sea urchin gonads. In our study, various amino acids contributed differently to both the TAA and FAA of sea urchin gonads and coelonnc fluid. Contents of Nucleic Acids In general, quantitative analysis of nucleic acid provides a rela- tively simple means of estimating recent growth rate of sea ur- chins. The processes of cellular growth and division require the synthesis of nucleic acids and proteins. The fact that RNA is a precursor to protein synthesis led to its use as an indicator of growth rate (Church & Robertson 1966). The primary function of RNA involves protein synthesis; whereas. DNA is the primary carrier of genetic information. Because the majority of cellular DNA is chromosomal, the quantity of DNA per cell is quasicon- stant in soinatic tissues; the tissue DNA concentration reflects cell numbers (Sulkin et al. 1975; Bulow 1987). Therefore. DNA con- tent has usually been used as an index of cell numbers or biomass (Regnault & Luquet 1974). In this study, the DNA content in the gonads was approximately four times higher than that in the coe- lomic fluid. Although the gonad is a tissue with a higher biomass as compared with coelomic tluid, the latter contains mostly coe- lomic fluid with a lower biomass. On the other hand, the RNA/ DNA ratio has been used as an estimate of growth for a variety of invertebrates (Sulkin et al. 1975). Thus, the RNA/DNA ratio is an index of protein synthetic activity per cell and reflects the protein synthesizing capacity for estimating recent //; situ protein increa,se (Bulow 1987, Hovenkamp & Witte 1991 ). In fact, correlation be- tween RNA concentration or RNA/DNA ratio and growth rate has been observed for a wide variety of organisms (Sutcliffe 1970). Furthermore, the gonadal RNA content was about 5.4 times higher than that in the coelomic tluid, thus demonstrating higher protein synthetic activity in the gonads. In general, gonad is the site of gametogenesis, which involves much protein synthesis. Further- more, the RNA/DNA ratio was much lower in coelomic tluid than that in gonads, indicating greater protein synthetic activity per cell in the gonads. This is an indication that gonad is a tissue with greater //; sitit protein growth as compared with coelomic fluid. CONCLUSIONS The pi'esent study demonstrated that sea urchin gonadal and coelomic tluid tissues had many common compositional charac- teristics. Most of the parameters analyzed did not show qualitative diffeiences; whereas, there were quantitative differences. In fact, gonads of sea urchins are a site of nutrient storage in addition to being the reproductive organs. The accumulation of nutrient re- serves contributes to the growth and development of the commer- cially important sea urchin gonads. Although .sea urchin coelomic fluid has not yet been exploited commercially, evaluation of its composition may lead to its potential use as a flavoring source. ACKNOWLEDGMENTS The author (C. L.-P.) gratefully acknowledges the assistance of the Canadian International Development Agency (CIDA) through a Mai'ine Science Scholarship. Thanks are also extended to Mr. Keith Collins at the Sea Urchin Research Facility (SURF) at Bonavista Bay, Newfoundland for providing sea uichin samples for the study. Acknian. R. G. & S. N. HcmpL-i, 1973. Non-methylene interrupted fatty acids in lipids of shallow water marine invertebrates: a comparison of two mollusks {Littorina liltorea and Limatia triseriata) with the sand shrimp {Cnmgitn .scpli'iiispiiin^ii.s). Copiii. Bim hem PItysiol. 41:4 x lO'' formalin:seawater appeared to prevent hypno- spore enlargement, with no hypnospores recovered from these con- centrations. The extremely weak formalin (1:4 x 10" formalin: seawater) appeared to have little effect on trophozoite viability. with treated tissues exhibiting a similar enlarged hypnospore abun- dance to that of the control. Similarly, colchicine concentrations of 10""" M or 10"" M had little effect on parasite viability. Colchicine has been demonstrated to be a potent microtubule inhibitor at such concentrations (Wiest et al. 1993). thus preventing cell division. The fact that parasites treated with colchicine remained viable and underwent division once out of the treatment is consistent with microscopic observations that no cell division occurs during tro- phozoite differentiation to hypnospores and cell enlargement. Swelling of Perkinsus cells in FTM had been suggested to occur as a result of the direct uptake of the media (Ray 1932). although the mechanisms of this process are not yet described. As nonviable trophozoites failed to enlarge in FTM. and thus failed to become iodinophilic, we tested whether hypnospore stages could be killed and still retain their iodinophilic priiperties. All treatments tested killed hypnospores. The sensitivity of hyp- nospores to low temperature is in accordance with Chu and Greene (1989). who ob.served that hypnospores of P. marimis exhibited 1009f mortality at 0°C for 24 h. Goggin et al. (1990) observed motile zoospores from cultured tissues previously chilled at 4"C. 0°C and frozen at -20°C. showing that trophozoites are more tolerant to low temperatures than hypnospores. In all cases, nonviable hypnospores were iodinophilic. Thus, such tissue could be transported risk-free to processuig plants to serve as a positive reference for sample comparison. Although this is not a control for FTM development, it still supplies users with a positive control that is safe, noncontagious, and simple to use. ACKNOWLEDGMENTS Mr. Tavis Anderson. Department of Microbiology and Parasi- tology. University of Queensland, assisted with experimentation and viability testing, and Dr. David Hunter, Department of Chem- istry, University of Queensland, guided the in'adiation experi- ments. Financial support from the Fisheries Research and Devel- opment Corporation (2000/151) is gratefully acknowledged. Chu, F. E. & K. H. Greene. 1989. Effect of temperature and salinity on in vino culture of the oyster pathogen Perl3 y) remained problematic, and the cause of mortality in these animals could not be determined. While two of the diets tested are potentially useful for supplemental feeding of adult unionids to increase glycogen levels during quarantine, or during short-term captive maintenance in the laboratory, none can be recommended without reservation for long-term maintenance because of the lack of survival after three years during this study. KEY WORDS: unionidae diets INTRODUCTION Nearly 70% of the freshwater mussels (Unionacea) in North America are currently facing extinction (Williams et al. 1993). Conservation efforts have focused on relocation of endangered populations, and aquaculture of recently transformed larvae. Cap- tive maintenance of endangered animals is a common technique used to enhance and preserve species-at-risk. however unionids have proved difficult to maintain in captivity or to relocate into new habitats (Cope & Waller 1995). Most aquaculture efforts have concentrated on developing live algal diets that will support the growth and survival of larvae ( 1 3 mo (Table 3 and Table 4). None of the unionids showed any shell growth while on these diets. Percent survival and growth did occur on the last commercial feed TR#1. Individuals of all species sur- vived at least 15 mo and two species, P. grandis and L. fragilis. showed shell deposition during the first four months, but not af- terwards. Four out of 10 adult P. grandis and 3 out of 10 adult L fragilis grew 1-2 mm over the first four months on these dried green algae, but shell growth then ceased. Mortality rates of 100% occurred within 13 mo. Four of the five experimental diets (TR #7-10) were successful in supporting initial limited growth and survival, but long-term survival (>3.5 y) was still problematic (Table 2 and Table 4). The exception was the encapsulated feed, TR#6. This encapsulated feed was ingested by the unionids, but within 12 h of feeding all the animals were gaping and non-responsive to touch on soft body pans. We dropped this feed from the diet tests after 30 days and 60% mortality in all species. The best diet for supporting survival, growth and reproduction of adults of most species, at least up to year 3. was the high-protein egg chow (TR#7). The exception was L fasciola, all of who died regardless of diet treatment. At the beginning of year 3. the sur- vival rates of the adult unionids feeding on TR#7 were; A plicata 81%. C. mberculata 80%, L. ventricosa 72%, L. siliquoidea 65%, Leptodea fragilis 64%, P. grandis 71%, and Q. qiiadnila 69%. TABLE 2. Test response of adult and subadult unionids to treatment diets. Survived Showed Injjested but at least Survived Survived Survived shell Initiated Ingested caused stress 6 months ~\ year -2 years -3 years growth glochidia TR#1. Dried Chlorellu + + -f + TR#2. Marine algal paste + + (1) TR#3. Hatchfry encapsulon + -y + TR#4. Fish Flake Food + + + TR#5. Yeast -I- + TR#6. Encapsulated feed + -Kl) TR#7. Egg chow -1- + + + -H + + (5) TR#8. Bacterial slurry #A + -K2) + + (4) + TR#9. Bacterial slurry #B + + -h (4) (4) + TR#10. Bacterial slurry #C + + -H (3) (3) -1- -1- ( 1 ). Stopped testing after one month (2). Kills Pyganodon grandis almost immediately and Leptodea fragilis within a couple a weeks (3). Tested only for one year (4). Tested only two years. (5). 20% of females died after glochidial relea,se 878 Nichols and Garling TABLE 3. Percent survival of adult unionid at 12 months on treatment diets. N = 10 for each species, each diet, except for C. luberculata and L. fasciola where N = 5/diet tested. TR#I. TR#3. TR#8. TR#9. TR#1(I. Dried Hatchfrv TR#4. TR#5. TR#7. Bacterial Bacterial Bacterial Unfed Chlorella Encapsulon Fish flake food Yeast Egg show. slurry #A slurry #B slurry #A A. plicala 45% 100% 51% 25% 100% 100% 100% 100% 100% C. tuhenulata * * * * * 100% * 100%> 100% L fascinla * * * * * 0% * 0% 0%. L venlricosa 21% 100% 14% 5% 2% 100% 3% 100% 100% L siliqouidea 15% 100% 11% 10% 1% 100% 4% 100% 100% L fragilis 12% 100% 6% 0%. 0% 100% 0%> 0% 100% P. grandis 32% 100%c 37% 0% 0%. 100% 0% 0% 100%. Q. qiuidruki 0% 100% 54% 0% 79% 100% 100% 100% 100% Indicates diet not fed to that species Twenty-one percent of the P. grandis and 15% of the L fragilis feiTiales formed glochidia during year 2, on this diet. However, during the third year adult unionids began to die. and by the beginning of year four all had perished. The body weight and glycogen levels (discussed below) of these animals were high. indicating that starvation was not a factor. At times soft tissue growth was so rapid that the mussels could not completely close their shells. Autopsies showed no signs of parasitism or other disease factor, but all of these animals had greatly enlarged kid- neys. The proximate analysis indicates that TR#7 is a high protein (~65%)/low carbohydrate (22%)/high lipid (13%) feed that is natu- rally high in cholesterol (78%- of total lipid), but contains no phy- tosterols. The series of bacterial/ciliate slurries. TR#s 8-10, differed in their ability to support adult unionid growth and survival. Treat- ment #8 proved an acceptable feed for species such as A. plicala and Q. qiiadnila. but killed all of the P. grandis within a day or two of the initial feeding and 99%* of the Linnpsilis species within a few weeks. Most of the other species died within the 13-month period. On the other hand, Amblema plicata and Q. quadrnia adults survived and grew well on this diet during the two years i.i'i testing. The greatest increase in shell growth was seen in A. pli- cata. The average increase was 6 mm; maximum was 1 1 mm and minimum 3 mm over the 48-nio period. The amount of growth in Q. (jiiadrnla was about half that seen in the A. plicara adults. No reproductive effort was seen in any species (Table 4). TR#9 did not improve survival and growth when compared with TR#8. Neither adult P. grandis nor L. fragilis could tolerate this feed, but once again, 82% of the A. plicata and 77% of the Q. qiiadmla survived for 2 y. Amblema plicata grew more than Q. qiiadnda, averaging 9 mm. with a maximum of 17 mm. and a minimum 4 mm over the 48-mo period. The amount of growth in Q. quadrnia averaged 9 mm, with a maximum of 12, and a mini- mum of 2. No reproductive effort was seen in any species (Table 4). Treatment #10, which combined the basic bacterial/ciliate slurry, dried algae, with various micronutrients and lipids proved acceptable to adults of all species. Pyganodcm grandis and L. fragilis did well on this diet, as did all the other unionid species tested. Survival of the adults of all species was 100% after one year, with the exception of L. fasciola. which we were not suc- cessful at handling regardless of what they were fed or handled. Note that this diet was only tested for a one-year period and long- term data is not available. TR#IO is a low protein (-8%), low lipid (5%), high carbohydrate (87%) feed. Cholesterol comprises 75% of total lipid, algal sterols at 20% and miscellaneous lipids at 5%. Pyganodon grandis fed TR#7 and TR #10 showed a consistent increase in glycogen levels from January 1998 to December 1998. The glycogen levels of animals on TR#IO rose from an average of 7.2 ± 1.9 mg/g in January 1998. to an average of 9.7 ± 2.4 mg/g by December 1998 but this increase was not significantly different (f-test. n = 10. P = 0.059). During the same period, animals on the TR#7 showed a statistically significant increase {P < 0.05) in glycogen from an average of 7.6 ± 1.1 mg/g to 1 1.2 ± 0.5 mg/g (/-test. H = 10. P = 0.036). Glycogen concentrations of P. ^ra«rf(.5 that had been feeding on TR#7 for at least 36 mo (36-45 mo) were significantly higher than P. grandis that had been on TR#7 for only 12 mo, averaging 14.1 ± 2.6 mg/g as compared with the 1 1.2 ± 0.5 mg/g (?-test. n = 20. P = 0.042). Initial glycogen concentrations are not available for the animals kept on TR#7 for the 36-mo period. Siihadiills The growth rates of subadult uniomds led on TR#7 and onTR#IO differed more by species than by diet. Pyganadon gran- dis and L. fragilis grew significantly faster on TR#7 and on the TR#IO than did the other species over a 350-day period, with P. grandis showing an average increase in shell length of 8.7 mm and L. fragilis. 8.1 mm. There was no significant difference in growth rates between these species on either diet. The thick-shelled spe- cies {A. plicata. C. tuherculata. and Q. quadrnia) grew signifi- cantly less, averaging 6 mm. Lxnnpsilis siliqnoidea and L. ventri- cosa grew even less, averaging only 3 mm during the test period. Survival during the test period was 100% for all species except L. fa.uiola. Mortality rates for L fasciola were 100%. The growth equations, r", and P values are presented in Figure I . Significant differences in growth rates are as follows, with species sharing a line not significantly different (Arcsine transformed data. AN- COVA. P < 0.05): A = A. plicata. C = C. tuherculata. L = L. fragilis. P = P. grandis. Q = Q. quadrula. E = TR#7, B = TR#10. Within diet LB PB AB CB QB LE PE AE CE QE Between diets AB AE CB CE LB LE PB PE QB QE Unionid Diets 879 TABLE 4. Adult unionid % survival at 24 montlis on various diets. N = 10 for each species, eacli diet, except for C. luberailata and L. fasciola where N = 5/diet tested. The bacterial slurry #C was not tested for longer than one year. TR#1. TR#3. TR#8. TR#9. Dried Hatchfrv TR#4. TR#5. TR#7. Bacterial Bacterial Unfed CMorella Encapsulon Fish flake food Yeast Egg chow slurry #A slurry #B A. plicala 0% 0% 0% 0% 0% 87% 78% 82% C. tuberculata * * * * * 89% * 0% L. fasciola * * * * * 0% * 0% L. ventricosa 0% 0% 0% 0% 0% 78% 0% 0% L siliqouidea 0% 0% 0% 0% 0% 71% 0% 0% L. fragilis 0% 0% 0% 0% 0% 70% 0% 0% P. grandis 0% 0% 0% 0% 0% 72% 0% 0% Q. quadnila 0% 0% 0% 0% 0% 75% 80% 77% * indicates diet not fed to that species. Water Quality Water quality was difficult to maintain in the test chambers, particularly when feeding the bacterial slurries. There were 22 episodes of water quality problems during the four years of tests. During these events, dissolved oxygen levels would plummet to <1 ppm and ammonia levels rise to >3 ppm often in less than 12 h. Mortality was seen in adult mussels of all ten species being fed TR#2 and TR#6 during these events (25% and 60% respectively within 24 h). None of the other test animals died during these events, but gaping and lack of response to touch on soft tissue body parts were noted. No other diet treatments lead to mortality events affecting all ten species of unionids within 1 2 h of feeding. Rapid mortality seen in TR#8 and 9 were limited to two species (P. grandis and L. fragilis). not all ten. DISCUSSION One problem in evaluating these diets is that none of the union- ids survived for longer than 3.5 y. although a couple of feeds supported growth and short-term survival (<3.5 y). Our data in- dicate that unionids are capable of feeding on a wide variety of materials, and can survive and grow for months on non-live algae diets. Long-term survival as would be needed for broodstock inain- tenance remains problematic. Other than survival, the criteria we selected for measuring diet success, such as growth, reproduction TR#7-Egg Chow ■« — Pyganodon Leptodea — — Amblema --K- Quadrula « — Cyclonaias TO T2 T4 T6 T8 TIO Time Figure 1. Comparison of growth equations of various unionid subadults fed the diet TR#7 for 350 days. Ambelma plicata y = 3.33677Ln(x) - 0.7147 R- = 0.9095; Cyclonais tuberculata y = 3.8l23I.n(x) - 0.6746 R" = 0.935: Leptodea fragilis y = 5.7222Ln(x) grandis y = 5.5916Ln(x) - 0.8214 R" = 0.9602: Quadrula quadrula y = 3.1152Ln(.\) - 0.6959 R" = 0.9112. 1.0147 R- = 0.941; Pygaiidoii 880 Nichols and Garlfng and glycogen concentrutinn were not capuble ol predicting the gradual die-off of ail test animals after 36 mo. Additional criteria relating to physiologic mcchanisins and underlying digestive ca- pabilities are needed so that changes in diet and environmental conditions can be implemented before mortality occurs. The two diets that were most effective in our tests. TR#7 and TR#1(), differed substantially in protein, lipid, carbohydrate, and phytosterol composition, but did not differ in their ability to sup- port subadult unionid growth or adult survival. TR#10 is a low protein (-8%), low lipid (5%), high carbohydrate (87%) feed with added algal sterols. In contrast, TR#7 is a high protein (-65%), low carbohydrate (22% I. higher lipid ( 13%) feed that is naturally high in cholesterol, but contains no phytosterols. Our hypothesis is that TR#10 is the closest in nutritive content and physical structure to the food resources used by wild unionids in shallow temperate rivers (see Nichols & Garling 2000). This diet proved acceptable to all species tested in our study, supported significantly higher growth rates in subadults and kept 100% of the test animals alive for the entire year of the study. However, while promising results were obtained, the problem in whole-heartedly recommending this diet is that it was tested for only one year. It is possible that this diet, like TR#7 cannot support long-term (>3 y) survival. Up until year three of our study, we thought TR#7 was a successful diet formula. It was easy to make in the laboratory, was readily ingested by all species, and supported growth, survival, glycogen storage, and limited reproduction, at least until year three. The question remains unanswered as to why long-term sur- vival was not supported. One problem might relate to protein levels. This TR#7 is a high protein feed (-65%- protein), and high protein may not be a dietary requirement of the age class of mus- sels used in this study. Subadults grew as well on the low protein (8%) levels found in TR#IO as they did on the high protein TR#7 (Fig. I and Fig. 2). Our work on a unionid community in a small temperate river indicate that they preferentially use a lower protein food (the <28 jx fine particulate organic matter.-6.7% protein) as their main food source (Nichols & Garling 2000). Feeding a high-protein feed such as the TR#7 as a sole diet at the amount used in this study may have overfed the animals. Glycogen levels in animals fed on egg chow were far higher than those recorded by Naimo et al. (1998), Naimo and Monroe ( 1999), and Patterson et al. (1999) for newly caught unionids. However, food quantity may not have been the only problem. Feeding ex- cessive dietary protein to vertebrates that normally use low protein feeds will support rapid growth and reproduction initially, but can cause high mortality due to excess nitrogen excretion causing kid- ney failure after a few years and alter mineral tnetabolism. This supposition is not a direct cause-and-effect relationship in union- ids. While all the dying unionids on this feed did show greatly enlarged kidneys, unionid kidneys do not excrete excess nitrogen. as do vertebrate kidneys; the gills perform that function. However, the primary function of unionid kidneys is to control ionic balance, including mineral balance, of bodily fluids. Theoretically, excess protein can affect ionic balance due to alteration of blood pH through excess amine production and stress the metabolic balance of the unionid; we could not determine cause and effect from our study. We cannot recommend the long-term use of high protein feeds for unionids. However, short-term or supplemental feeding at a lower ration than used in this study might be acceptable to prevent glycogen loss during quarantine as reported by Patterson et al. (1999). TR#10-Bacterial Slurry C 12 1 Time Figure 2. Comparison of growth equations of various unionid subadults fed diet TRSKI over 350 days. Amhelma plicala y = 3.9346I,n(\l - 0.4899 R= = 0.9649: Cychnais liiherculala y = 4.l877I.n(\( - 0.3063 R" = 0.9909; l.iplodea fragilis y = 5.5873Ln)x) - 0.7751 R" = 0.9608: Pygandon grandis y = 5.77851>n(x) - 0.903 R" = 0.9426: Qiiadnihi qiHidnila \ = 3.l988I,n(\l - 0.5076 R" = 0.9533. Unionid Diets U There is certainly the possibility that unionid mortality after three years in captivity relates more to en\ ironment than nutrition. Even aquaculture efforts that feed their unionids live algae report incidents of poor water quality that at times does not lead to immediate mortality (Gatenby et al. 1994, Gatenby et al. 1996). However, such events may produce sublethal stress that over a period of time and after a number of incidents may eventually kill the adult unionids. One of the greatest problems in using the types of feeds we tested is that of maintaining water quality, especially with bacterial/ciliate slurries (Tr#s 8-10). There are a nuinber of other environmental factors that may produce sublethal stress, including altered tlow. light, and tem- perature regimes. Such environmental factors need further study before we can establish captive management protocols for the various unionid species, as there will be variability in environmen- tal tolerances. Survival was to some degree species-specific. L fasciola could not be kept alive under any type of environmental condition, even though we were able to keep other Lampsilis spe- cies alive for several years. Unionids can survive, grow, and even reproduce for about 3-3.5 y on non-live algal diets, but long-term survival remains problematic. Health monitoring criteria using glycogen concentra- tions, shell growth rates, reproduction, and survival did not provide enough warning to prevent mortality. Additional criteria to judge success or failure of captive management protocols, other than death, need to be developed. These types of non-live algal diets may function for supplemental feeding, but at this time, maintaining adult unionid populations in captivity will require access to natural foods and water supplies from water systems that support native unionid fauna to increase the likelihood of long-term survival. LITERATURE CITED American Public Health Association (APHA). 1989. Standard Methods for the Examination of Water and Wastewater. In: M. Franson. editor. Washington DC: APHA. Association of Official Analytical Chemists (AOAC). 1995. Official meth- ods of analysis. 16th ed. Arlington. Virginia: AO.-^C. Buchal. M. & C. Langdon. 1995. Lipid spray beads for the delivery of water-solable materials to marine bivalves. Annual meeting of the Na- tional Shellfisheries Association. Pacific Coast Section and Pacific Coast Oyster Growers Assoc. 14:227. Cope. W. & D. Waller. 1995. 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Absorbed ration, respiratory costs and resultant scope for growth in the mussel Aiilaeomya ater (Molina) fed on a diet of kelp detritus of different ages. Mar. Biol. Lett. 3:289-306. Williams, J., M. Warren. Jr., K. Cummings, J. Harris & R. Neves. 1993. Conservation status of freshwater mussels of the United States and Canada. A«i. Fish. Soc. 18:6-22. Joimwl of Shellfish Research. Vol. 21. No. 2. 883. 2002. ABSTRACTS OF TECHNICAL PAPERS Presented at The 4th Annual INTERNATIONAL CONFERENCE ON SHELLFISH RESTORATION Hilton Head Island. South Carolina November 15-18. 2002 883 Shellfish, Restoration. Hilton Head Island. SC Abslracls, December 2002 885 CONTENTS S. A. Allen. Jr. Brood stock sources for hatchery-based stock enhancement of oyster reefs: Essential questions and recommendations. . 889 T. D. Alphiii and M. H. Posey Ecological function of oysters in southeastern North Carolina 889 D. Bishop Simplification of shellfish restoration methods 889 M. Bowen, K. Groves, C. Heinig, and A. Frick Expanding and sustaining shellfisheries of Casco Bay 889 M. F. Frana, E. A. Venso, K. Brohawn. W. Beatty, M. Ellwanger, R. McKay, B. Evans, and M. Phipps-Dickerson DNA fingerprinting of nonpoint source Escherichia coli contamination in a Chesapeake Bay watershed 890 D. W. Brown A national strategy for coastal habitat restoration 890 R. J. Bruckner and R. L. Takacs Beyond the project: Values of community-based habitat restoration 890 R. I). Brunbaugh. W. J. Goldsborough. L. A. Sorabella, and J. A. Wesson Oyster bloodstock enhancement in Virginia and application of a new monitoring technique 891 D. Bushek, R. F. Dame. D. M. Allen, A. J. Lewitus, E. T. Koepfler. and D. Edwards Nutrient cycling in intertidal creeks along the southeast US: Are oysters in control? 891 G. W. Calvo. L. M. Ragone Calvo, and E. M. Burreson Disease resistance in a selectively bred Cntssostrea virginica strain 891 D. Cheney. R. Elston. B. MacDonald, A. Kinnan, A. Suhrbier, G. Cherr. C. Friedman, F. Griffin, A. Hamdoun, J. Mitchell, L. Righetti. and L. Burnett Summer mortality of the Pacific oyster. Crassostrea gigas: Influences of culture methods, site conditions, and stock selection °"'- F.-L. E. Chu. V. G. Encomia, S. Stickler. S. Allen, and J. La Peyre Physiological condition and defense-related activities among easleni oyster populations 892 P. Comar. L. Kracker, P. Bauersfeld, and M. Meaburn A unified information system for shellfish restoration 892 M. Davidson. L. Hoist. H. Bokuniewicz, C. Smith, and K. Tetrault Effects of pearl net stocking density on survival, growth, and gonadal maturation of bay scallops 893 R. A. Elston, R. M. Estes, A. Gee. R. P. Herwig. K. Kinnan, and S. Rensel Probiotic approach to enhance health of hatchery produced shellfish seed 893 E. Fahy Management by size limit of the Whelk Biicciinim wuhuwn Fishery in the south west Irish Sea 893 L. D. Coen and A. Fischer Managing the future of South Carolina's oysters: An experimental approach evaluating current harvesting practices and boat wake impacts 894 P. M. Gaffney. K. S. Reece, and J. C. Pierce Genomic approaches to marker development and mapping in the Eastern oyster. Crassostrea virginica 894 W. J. Goldsborough. R. D. Brumbaugh. D. W. Meritt, and J. A. Wesson Community-based oyster restoration: Case studies from Chesapeake Bay 894 M. Gomez-Chiarri and P. Munoz Molecular immune responses of the Eastern oyster to the parasite Perkinsus marimts 895 G. C. Cherr, C. S. Friedman. F. J. Griffin. A. Hamdoun. J. Mitchell. L. Righetti. D. P. Cheney. R. A. Elston, and B. McDonald Factors affecting the stress response in oysters on the west coast: Implications for summer mortality 895 N. H. Hadley and L. D. Coen Community-based oyster habitat restoration and enhancement in South Carolina 895 J. M. Harding and R. Mann Essential or just opportunistic fish habitat' Utilization of restored complex shellfish habitat by fish species 896 E. E. Hofmann, J. M. Klinck, E. N. Powell, S. E. Ford. S. Jordan, and E. Burreson Effects of climate variability on the prevalence and intensity of Dermo and MSX diseases in Eastern oyster populations 896 886 Abstracts, December 2002 Shelllish. Restoration. Hilton Head Island, SC C. Smith, S. Dumais. L. K. Hoist, and M. Davidson Field trial of a Bay scallop [Argopecten inadians) spawner sanctuary 896 A. C. I be and P. O. Abohweyere Restoring critical habitats in the Niger Delta for shellfish production 896 M. Janowicz Stakeholder and concensus - How do we make these elements work for effective action? 897 S. J. Jordan, K. N. Greenhawk, C. B. McCollough, and M. L. Homer Oyster biomass and abundance in niirthern Chesapeake Bay: Trends in relationship lo harvest, recruitment, parasitic diseases, and environmental variation 897 C. J. Judy and E. Campbell Public and private oyster restoration in Maryland's Chesapeake Bay 897 P. Kamerinans and A. C. Sinaal Mussel culture and cockle fisheries in The Netherlands: Finding a balance between economy and ecology 898 R. J. Diigas, W. R. Keithly, M. Bourgeois, P. Meier, D. Lavergne, and A. Diagne An economic analysis of public ground oyster reef restoration in central Louisiana damaged by hurricane Andrew 898 A'. E. Landrum, K. M. St. Pe, B. Ache, and F. Kopfler The rerouting of stormwater discharges for wetlands enhancement, levee protection, and oyster habitat protection and restoration 899 K. E. Landrum The shellfish challenge initiative: A cooperative success story in the Barataria-Terrebonne National Estuary 899 T. Landry, M. Ouellette, and P. Cormier Oyster population restoration in Caraquet, N.B.; Phase 1. population assessment 899 A'. L. LeBlanc Community-based initiatives for improving water quality in Southwestern New Brunswick. Canada — an update on success 899 A. T. Leggett, R. Brumbaugh, W. Goldsborough, and A. McDonald Application of commercial-scale oyster aquaculture to reef restoration 899 M. Luckenbach, F. O'Beirn, P. Ross, J. Nestlerode, and L. Sorabella Dollars and sense of oyster restoration: An examination of nitrogen removal by a restored oyster reef 900 R. B. Luftig and W. Pelon Reduction in the Vibrio vulnificus load of oysters by a novel short-term combination biodepuration treatment 900 R. Mann and J. M. Harding Coming soon to a restoration site near you: The invading predatory oriental gastropod Rapana venosa 900 R. Mann Up close and personal: A suggested quantitative approach to broodstock enhancement on shellfish restoration sites .... 901 D. W. Webster and D. W. Meritt Linking public and private partners for restoration aquaculture in Maryland's seaside bays 901 D. W. Meritt and S. Tobash Production of disease-free oyster seed using shallow water nurseries in the Mid-Chesapeake Bay 901 K. A. Nelson and G. L Scott A multifactorial approach for describing the relationship between the classification of shellfish harvesting waters and adjacent land use in Murrells and North Inlet, South Carolina 901 J. A. Nestlerode, M. W. Luckenbach, P. G. Ross, and F. X. O'Beirn Influence of oyster reef structure on fish assemblages: Does the placement of artificial substrate enhance fish populations? 902 M. Norman Shellfish restoration in Ireland: The need for novel partnerships 902 M. Ouellette, M. Hardy, T. Landry, N. G. MacNair, and A. Boglien Reproductive biology of the northern quahaug. Mercenaria mercciuiiia. in Prince Edward Island, Canada 902 K. T. Paynter, Jr. Considerations for oyster restoration in Maryland: Disease genetics, density, reproduction, and habitat creation 903 Shellfish. Restoration. Hilton Head Island, SC Abstracls. December 2002 887 P. J. Pazo Culture techniques applied to wild bivalve beds in Galicia. NW Spain 903 A. L Pereira. F. A. Ruano. L. Chicharo. and D. Matias The influence of environmental factors in Japanese oysters health condition cultivated in the South of Portugal 903 P. Perina and D. Perina The "EEEOHM" (Easttlelds" Environment Enhancing Oyster Holding Module) 903 E. A. Bochenek, E. N. Powell, J. M. Klinck, and E. E. Hojmann A biochemically-based model of the growth and development of Crassostrea gigas larvae 904 J. M. Klinck, E. N. Powell. J. N. Kraeuter, and S. E. Ford A fisheries model for managing the oyster fishery during times of disease 904 J. Power and D. B. Walker Shellfish data management and reporting system (SDMRS) 904 S. P. Powers, C. H. Peterson, and J. H. Grabowski Restoring oyster reefs for fish: Estimating enhanced secondary production of restored oyster reefs 905 B. RaLonde Restoring the littleneck clam resource for Native American subsistence use in the Prince William Sound, Alaska 905 A. M. Reece, W. L. Ribeiro, K. L. Hudson, and S. A. Allen, Jr. Development of Crassostrea virginica microsatellite markers for a genetic linkage map and genetic monitoring of restoration projects 905 D. B. Rouse, R. A. Wallace, and F. S. Rikard Oyster reef restoration research in Mobile Bay. Alabama 905 D. C. Sherwood Transplanting broodstock oysters, Crassostrea virginica. onto reconstructed oyster reefs to increase spat recruitment in the Piankatank River 906 S. E. Shumway Harmful algal blooms and shellfish restoration: Can they co-exist? 906 L. A. Sorabella, M. W. Luckenbach, and F. X. O 'Beirn A comparison of two oyster {Crassostrea virginica) strains for productivity and suitability for use in oyster reef restoration efforts 906 S. M. Tobasfi and D. W. Meritt Oyster restoration and the University of Maryland: Interactions between research, industry, and the public 907 A. A. Volety, M. Savarese, and S. G. Tolley Effects of watershed alterations on oyster populations in Southwest Florida estuaries: An ecological approach 907 J. A. Wesson and L. B. McKay The Virginia Oyster Heritage Program 907 J. J. Whitten Enhancing and sustaining North Shore shellfishing through aquaculture 908 A. Willner Community-based oyster restoration in an urban estuary: Developing an oyster culture and reef restoration program in the Hudson-Raritan Estuary 908 Shellfish, Restoration. Hilton Head Island. SC Abstracls. December 2002 889 BROOD STOCK SOURCES FOR HATCHERY-BASED STOCK ENHANCEMENT OF OYSTER REEFS: ESSEN- TIAL QUESTIONS AND RECOMMENDATIONS. S. K. Allen, Jr.. Aquaculture Genetics and Breeding Technology Cen- ter, Virginia Institute of Marine Science, College of William And Mary, Gloucester Point, VA 23062. Oyster populations are subdivided into genetically distinct units with major divisions occurring over large geographic scale be- cause of larval dispersal. Populations resist local adaptation be- cause of population mixing through migration. However, over the course of the last 50 years, populations of Chesapeake Bay oysters may have lost alleles for disease resistance from the combination of disease mortality followed by heavy harvesting of survivors. Artificial breeding can increase the frequency of disease-resistant alleles, and several varieties of disease-resistant oysters are avail- able as brood stock today. Some of these strains have been used to populate reefs and are likely to survive, grow, and breed on the reefs. Reproduction of the disease-resistant strains will produce disease-resistant spat over surrounding areas. There may also be natural stocks of oysters that resist disease, such as those from the Gulf of Mexico, where Dermo historically occurs. However, using "artificial" or genetically distmct oysters from the hatchery for reef restoration could also entail some risk to natural genetic diversity. For example, artificially selected populations have (by definition) reduced genetic variation over wild stocks. Interbreeding of the two may alter wild populations. At present, the risk (or benefit) of using alternative stocks is unknown. The results and recommen- dations from a workshop on genetic considerations for hatchery- based reef restoration will be presented. ECOLOGICAL FUNCTION OF OYSTERS IN SOUTH- EASTERN NORTH CAROLINA. T. D. Alphin and M. H. Po- sey, Center For Marine Science, UNC-Wilmington, 1 Marvin Moss Lane, Wilmington, NC 28409. Oysters serve a variety of functions within the estuarine sys- tems of Southeastern North Carolina. A number of juvenile tlsh and decapods use oyster habitat for refuge/forage during some portion of their lives. Here, we present results from several studies evaluating the use of oyster habitat by juvenile fish and decapods compared with alternate habitats. An abundance of fish and deca- pods were examined in isolated and mixed oyster habitats using Breder traps in small tidal creek estuarine systems whereas net sampling was used to compare faunal abundances in oyster reefs along larger spatial scales in the presence and absence of sea grass beds. In both cases, abundance in oyster reefs were compared with vegetated or unvegetated marsh edge habitats. On the larger scale, use of oyster reefs was also compared with abundance patterns within seagrass beds. The mixed results of these studies indicate that the importance of oyster reefs as a refuge/forage habitat varies seasonally within a given system as well as among small estuarine systems based on the presence of alternate habitats. We also present preliminary information on more indirect effects of oysters as modifiers of water quality using transplant/removal studies. SIMPLIFICATION OF SHELLFISH RESTORATION METHODS. D. Bishop, Fukui North America, P.O. Box 119, 523 Island View Drive, Golden Lake, Ontario, Canada. As the shellfish aquaculture industry continues to grow, meth- ods of husbandry to reduce labor, increase yield, and produce higher quality products are in a constant evolution. Based on the simple fact that smart people learn from their mistakes and really smart people learn from other's mistakes, there is a lot that can be learned from the aquaculture industry to transfer to restoration projects. Although all the answers for husbandry are not in place, many dynamics and protocols positively affect restoration projects by moving them forward at a faster pace. Attitudes towards labor efficiency using different equipment ideas and management tech- niques will be discussed. This will give attendees references that, used as-is or with slight modification, could benefit their efforts significantly. An interactive audiovisual presentation with ex- amples of methods from around the world used today will enhance the presentation. EXPANDING AND SUSTAINING SHELLFISHERIES OF CASCO BAY. M. Bowen,' K. Groves,- C. Heinig,' and A. Frick,'' 'Normandeau Associates, Normandeau Associates Inc., 251 Main Street. Yarmouth. ME 04096; "Casco Bay Estuary Proj- ect, University of Southern Maine, Law School Building, Portland. ME 04104; 'Mer Assessment Corporation, 14 Industrial Parkway, Brunswick. ME 04011; and ■*Albert Frick Associates. Inc.. 95a County Road, Gorham ME 04038. One of the missions of the Casco Bay Estuary Project is to ensure communities around Casco Bay in Maine have a healthily shellfish harvest that sustains commercial and recreational shell- fishing for generations to come. A "clam team" of stakeholders, including the US Environmental Protection Agency, the Friends of Casco Bay, Maine Department of Marine Resources, individual cities and towns, and the Maine Department of Environmental Protection was formed to find the most productive shellfish areas currently closed to harvest, determine sources of contamination, and find ways to remediate. A field review of the 57 clam flats — 800 acres — of soft-shell clam habitat that are currently closed to harvest targeted 22 of these, totaling 370 acres of highly produc- tive clam flats. Review of water quality data pinpointed sources of contamination. Many of the flats are closed simply because of the presence of an overboard discharge system that treats household waste. The project is currently supporting an intensive effort to design and construct replacement systems, a collaborative effort between the towns, state, and individual homeowners. Additional water sampling efforts are in progress to determine other nonpoint sources of contamination, including farm runoff, leaking septic systems, and wildlife. A third element of the project is investigat- 890 Abstracts. December 2002 Shellfish, Restoration, Hilton Head Island, SC ing the sustainability aspect, investigating the effectiveness of regulatory options, including licensing, harvest limits and tech- niques, and conservation closures. DNA FINGERPRINTING OF NONPOINT SOURCE ES- CHERICHIA COLl CONTAMINATION IN A CHESA- PEAKE BAY WATERSHED. M. F. Frana.' E. A. Venso,' K. Brohawn/ W. Beatty,' M. Elluanger,' R. McKay/ B. Evans,"' and M. Phipps-Dickerson.'' 'Department of Biologi- cal Sciences, Salisbury State University, 1101 Camden Avenue. Salisbury, MD 21801; "Environmental Health Science, Salisbury State University, 1101 Camden Avenue, Salisbury. MD 21801; 'Maryland Department of the Environment, Technical & Regula- tory Services Administration, 2500 Broening Highway, Baltimore. MD 21224; and ""Wicomico County Environmental Health Depart- ment. Seth H. Hurdle Health Center. 108 East Main Street, Salis- bury, MD 21801. Fecal coliform contamination has closed shellfish harvesting areas and public beaches and threatened recreational areas in the Chesapeake Bay watershed. Bacteriological water quality testing currently performed in these watersheds does not identify the sources of contamination. Therefore, no pollution control or miti- gation efforts have been undertaken, despite the large economic impact for this area of the Midatlantic. Possible sources include runoff from crop fields, wildlife, discharge from boats, and runoff from >1..^00 animal production farms on the Eastern Shore of the Chesapeake Bay. Although municipal waste water plant effluent and on-site waste water treatment (septic) systems could contrib- ute, shoreline surveys conducted by the Shellfish Sanitation Pro- gram of the Maryland Department of the Environment indicate that nonpoint sources are responsible for the elevated levels of coliform bacteria in this watershed. It is understood that these sources would contribute not only bacteria but also excess nutrients and possibly other water contaminants that can negatively impact public health as well as the sensitive plant and animal species that dwell in the watershed. The methodologies used to determine the specific sources oi Escherichia colt contamination are described, including choice of sample locations and environmental variables, sampling techniques. DNA analysis of strain-specific E. cc^li. and interpre- tation of the data. Preliminary data are presented, including se- lected DNA fingerprints and relationships among and between total conforms and E. coli MPNs and six environmental and water chemistry variables. Ultimately. Geographic Information Systems mapping will be used for spatial analysis as a key to the under- standing needed for pollution control and mitigation. A NATIONAL STRATEGY FOR COASTAL HABITAT RESTORATION. D. W. Brown, National Marine Fisheries Ser- vice. 1313 East West Hwy. Ssmc#.3. Room 15221. Silver Spring. MD 20910-3282. Shellfish habitats make up a significant portion of the important aquatic habitats in our coastal waters that provide the living space for marine and estuarine fish and shellfish. Unfortunately, in many areas along our coastline, many habitats, including shellfish habi- tats, are being destroyed and the natural systems they support are failing. The National Oceanic and Atmospheric Administration recently joined with Restore America's Estuaries and the Pew Charitable Trusts to launch a major partnership initiative to restore important habitats in our coastal estuaries. A major element of this initiative is to develop a national strategy for coastal habitat res- toration, including important shellfish habitats. The purpose of the strategy is to identify specific habitat problems in each coastal region and to determine the most viable restoration approaches to address degraded areas for these regions. The National Strategy will ( 1 ) actively promote the increased protection of existing habi- tats; (2) establish specific regional and national restoration goals and objectives; (3) provide a framework for setting restoration priorities; (4) identify and integrate the science and new technolo- gies needed for effective restoration; and (5) energize cooperative partnerships among private and public stakeholders. This presen- tation will review ongoing and planned actions by the NGO com- munity, federal agencies, and the private sector to develop a na- tional strategy for coastal habitat restoration, including shellfish habitats, by the fall of 2001. BEYOND THE PROJECT: VALUES OF COMMUNITY- BASED HABITAT RESTORATION. R. J. Bruckner' and R. L. Takacs,- 'NCAA Restoration Center, 1315 East West High- way, Silver Spring, MD 20910 and "NCAA Chesapeake Bay Of- fice, 410 Severn Avenue, Annapolis, MD 21403. The NCAA Community-based Restoration Program (CRP) be- gan in 1996 to inspire local efforts to conduct meaningful, on-Ihe- ground restoration of marine, estuarine. and riparian habitat. The CRP is a systematic effort to catalyze partnerships at the national and local level to contribute funding, technical assistance, land, volunteer support, or other in-kind services to help citizens imple- ment restoration projects that promote stewardship and a conser- vation ethic for living marine resources. The CRP links funding and technical expertise to citizen-driven restoration projects and emphasizes collaborative strategies built around improving NCAA trust resources and the quality of the communities they sustain. Oyster restoration projects, although not all explicitly off-limits to harvesting, have emphasized the habitat benefits of reef restora- tion, from three-dimensional habitat conducive to spat settlement, to the benthic organisms that make up the ecological diversity of oyster reefs themselves, to the fish and openwater communities that aggregate around hard-bottom reef habitat. In addition to implementing projects, this innovative funding/partnership source has provided the mechanism to "field test" new restoration strat- egies, such as reef design and construction, unique management approaches like sanctuaries, reserves, and satellite bars, and often has served as the springboard for larger-scale, river-wide restora- tion efforts. The availability of technical expertise and matching Shellfish. Restoration. Hilton Head Island. SC Abstracts. December 2002 891 funds and the positive results achieved by community-based shell- fish restoration efforts have catalyzed other federal, state, and local entities to participate, effectively broadening the partnering and stewardship opportunities, increasing the areas available for shell- fish restoration, and leveraging the amount of funds available for habitat restoration efforts. OYSTER BROODSTOCK ENHANCEMENT IN VIRGINIA AND APPLICATION OF A NEW MONITORING TECH- NIQUE. R. D. Brumbaugh.' W. J. Goldsborough,' L. A. Sora- bella,' and J. A. Wesson," 'Chesapeake Bay Foundation, 142 W. York Street. Suite 318. Norfolk. VA 23510 and -Virginia Marine Resources Commission. P.O. Box 756. 2600 Washington Ave.. Newport News, VA 23607. The transplanting of both wild and hatchery-produced oysters onto oyster sanctuary reefs is increasingly frequent as a component of oyster restoration efforts in the Chesapeake Bay. Since 1996. oysters have been added to more than a dozen state or privately managed sanctuary reefs in Virginia in an effort to enhance local- ized oyster spawning success. Wild oysters, purchased with both state and private funds, have accounted for approximately 70% of the total number of oysters added to reefs. Increasingly, however, the oysters added to reefs are hatchery produced, grown by citizens and students volunteering through programs such as the Chesa- peake Bay Foundation's Oyster Corps. To date, more than 800.000 oysters grown by volunteers have been added to Virginia's system of reefs. Although definitive data are scarce, there appears to be good empirical evidence that these stocking efforts have enhanced spat settlement rates on and around sanctuary reefs. Dive surveys and patent-tong data show substantial increases in localized spat settlement in tributaries where oysters have been added to reefs in recent years. To better understand spat settlement dynamics around the reefs, spat cages, small cages filled with a known volume of shell, are now being used to monitor oyster settlement around selected reefs. A strong correlation exists between spat cage data and diver-surveys on nearby reefs (r = 0.95, P < 0.01). suggesting that spat cages may be a low-cost means of both involving the public in restoration and of evaluating results of broodstock en- hancement and reef restoration projects. NUTRIENT CYCLING IN INTERTIDAL CREEKS ALONG THE SOUTHEAST US: ARE OYSTERS IN CONTROL? D. Bushek,' R. F. Dame," D. M. Allen," A. J. Lewitus.' E. T. Koepfler,- and D. Edwards,' 'Baruch Marine Field Laboratory. Baruch Institute for Marine Biology and Coastal Research. Uni- versity of South Carolina. Georgetown. SC 29442 and "Depart- ment of Marine Science, Coastal Carolina University. Conway, SC 29528. Ecologically, oyster reefs provide habitat, filter water, and fa- cilitate nutrient cycling. We experimentally removed oyster reefs to examine their role in the structure and function of intertidal creek ecosystems. Surprisingly, the removal of oyster reefs did not significantly alter nutrient concentrations, nekton usage, or phy- toplankton production. Our calculations show that oysters do not produce enough ammonium to satisfy phytoplankton productivity, but nekton, water column remineralization, and sediments more than account for the deficit. These observations were interpreted as an indication of functional redundancy in the system. Flagellates, which are preferred over diatoms as food by the oysters, dominated the phytoplankton during summer when ammonium concentra- tions were high. Diatoms dominated during the colder months. Shifts in phytoplankton dominance corresponded to the seasonal arrival and departure of nekton in the creeks. Because nekton comprised more than double the biomass of oysters during sum- mer, fishes and macrocrustaceans may play a greater role in nu- trient remineralization than has been previously considered. At the meso-scale ecosystem level, the loss of nutrient remineralization activities attributable to the removal of oyster reefs was compen- sated by other components within the system, but phytoplankton communities changed, apparently in response to changes in graz- ing. Oysters clearly play important roles, but defining the impor- tance of shellfish restoration in the management of coastal eco- systems requires an understanding of the ecosystem science, a consideration of scale, and the realization that tidal creek systems exhibit complex responses. DISEASE RESISTANCE IN A SELECTIVELY BRED CRAS- SOSTREA VIRGINICA STRAIN. G. W. Calvo, L. M. Ragone Calvo. and E. M. Burreson, Virginia Institute of Marine Science (VIMS). College of William and Mary, Gloucester Point, VA 23062. During 1997 to 1999, DEBY oysters, a VIMS stock that was selectively bred for four generations at a disease endemic site in the lower York River, Virginia, were evaluated for survival, growth, and disease susceptibility in comparison with progeny from wild Mobjack Bay (MB) and Tangier Sound (TS) brood stocks. MB and TS stocks are relevant to rehabilitation of Chesa- peake Bay oysters because the former have been routinely used for aquaculture and the latter have been recently used for reef resto- ration because of their putative disease resistance. Oysters (n = 1500 of each group, mean shell height = 15-17 mm) were de- ployed in floating mesh cages at a low salinity (<15 ppt) site and a moderate salinity (15-25 ppt) site in the lower Chesapeake Bay, and at a high salinity (>25 ppt) site on the Atlantic Coast of Virginia. Twenty-eight months after deployment, cumulative mor- tality in MB and TS was 84-100%. In contrast, cumulative mor- tality in DEBY at low, moderate, and high salinity sites was, respectively, 21, 51. and 36%. By November 1999. mean shell height in MB and TS at low-, moderate-, and high-salinity sites was. respectively. 77. 88-90. and 57-59 mm. In comparison, mean shell height in DEBY was 92. 101. and 72 mm. Although similar low levels of MSX were observed in all groups, P. marimis infec- 892 Ahsmicis. December 2002 Shelltish, Restoration. Hilton Head Island, SC tions in MB and TS were more intense than in DEBY throughout the study. This promising oyster strain has potential to facilitate commercial aquaculture and reef restoration efforts in Chesapeake Bay. SUMMER MORTALITY OF THE PACIFIC OYSTER. CRASSOSTREA GIGAS: INFLUENCES OF CULTURE METHODS, SITE CONDITIONS, AND STOCK SELEC- TION. D. Cheney.' R. Elston.' B. MacDonald.' K. Rinnan,' A. Suhrbier.' G. Cherr." C. Friedman." F. {Jriffin.- A. Ham- doun,- J. Mitchell,- L. Righetti," and L. Burnett,' 'Pacific Shell fish Institute, 120 State Ave NE #142. Olympia, WA 98501 ; "Uni- versity Of California, Davis. Bodega Marine Laboratory. P.O. Bo,\ 247, Bodega Bay, CA 94923; and 'Grice Marine Laboratory, 20.5 Fort Johnson, Charleston, SC 29412. During the late summer to early fall period. Pacific oy.sters cultured on the west coast of the United States and elsewhere may experience high levels of mortality. In the 1960s to 1980s, this condition was subject to intensive investigation focusing on broad areas of disease pathology, genetics, physiology, and the environ- ment. Results of these studies were largely inconclusive or pointed to a poorly defined etiology. Recent studies in Puget Sound. Wash- ington and Tomales Bay, California, center on the influence of multiple stressors and their affects on oyster survival, physiology, and pathology. The goal of this research is to identify possible modifications in culture practices, brood stock selection, or grow- out location to increase survival of Pacific oysters. Field observa- tions indicate oysters are subject to extreme variations in a number of parameters during intertidal cycles. An increased rate of oyster mortality and modified physiological response appear to be strongly correlated with both elevated temperatures and extended periods of depressed DO. The DO reductions are sometimes coupled with heavy macroalgae blooms and high phytoplankton densities. This and other works indicate oyster summer mortality rates are also strongly influenced by ploidy and broodstock origin/ stock selection. The.se observations have renewed interest in test- ing stocks selected for reduced rates of summer mortality and which retain desirable characteristics of good growth and meat yield. This research was supported by grant numbers NA86RG0015 and NA96RG0488 from the National Sea Grant College Oyster Disease Research Program and matching contribu- tions from West Coast shellfish farmers. PHYSIOLOGICAL CONDITION AND DEFENSE- RELATED ACTIVITIES AMONG EASTERN OYSTER POPULATIONS. F.-L. E. Chu.' V. G. Encomio,' S. Stickler.' S. Allen,' and .1. La Peyre." 'Virginia Institute of Marine Science. College of William and Mary, Gloucester Point. VA 2.^062 and "Louisiana State University, Baton Rouge, LA 70803-6002. The goal of our study is to identify oyster slocks that are re- sistant/tolerant to the disease caused by the parasite, Perkinsus iiuiniiKs (Dermo). We are comparing the physiological condition and defense factors of putative "Dermo resistant" and "nonresis- tant" oysters (Crasssostrea virgiiiica) deployed in the fall of 1999 at two sites in the Chesapeake Bay (Port Kinsale. Yeocomico River; Regent Point. Rappahannock River), where Dermo disease is known to occur, but not MSX (disease caused by Haplospo- ridiiim nelsoni). These oysters are Fl progenies from presumably genetically distinct oyster populations (three Gulf of Mexico and three Chesapeake Bay populations, and one hatchery strain) and represent geographical disparity. Oysters have been sampled monthly since May 2000. Initial analysis showed that all the stocks have grown significantly since deployment and the Rappahannock River Stock has the fastest growth. Tissue dry weights of this slock increased significantly over lime at both sites. Contents of glyco- gen, protein, and lipid increased with growth. All stocks sampled from May to July had low P. mariiuts infection and prevalence. Mortality was low in all stocks and lower in the Gulf of Mexico than Chesapeake Bay populations. No significant differences were noted in levels of plasma protein and lysozyme among stocks. Currently we are analyzing oysters sampled in August and Sep- tember. Correlation between growth, physiological, biochemical, and defense condition and P. mariiuts infection among oyster stocks will be discussed. This research was funded by the NOAA- Vir'jinia Sea Grant-Oyster Disease Research Program. A UNIFIED INFORMATION SYSTEM FOR SHELLFISH RESTORATION. P. Comar, L. Kracker. P. Bauersfeld, and M. Meaburn. Center For Coastal Environmental Health and Biomo- lecular Research. National Ocean Service. NOAA. 219 Fort Johnson Road. Charleston. SC 29412. The Shellfish Information Management System (SIMS) is an intergovernmental data system designed to provide a current cen- tral source of information on shellfish safety, resource, and habitat useful to multiple users at local, state, regional, and national levels. SIMS is being developed as a GlS-enabled, web-accessible rela- tional database of shellfish harvest water survey, classification, and resource information. Most of the data in SIMS is provided by state agencies, and SIMS will allow more extensive access to and integrative analysis of that information. In 1999. the National Ocean Service. Center for Coastal Environmental Health and Bio- molecular Research in Charleston. South Carolina, began partner- ing with a growing number of coastal states in the design and applications for SIMS. This spatially enabled. Oracle database is designed with extensive query functionality, visualization, and analytical capabilities for a wide range of shellfish safety, water quality, resource, and restoration concerns. Shellfish restoration is a new component being developed for incorporation into SIMS so that trends in restoration can be quantified and visualized. Water quality, benthic and habitat suitability, shellfish resource, presence of disease agents, social and economic factors, and other influ- ences impact shellfish restoration decisions and actions. Discus- Shellfish. Restoration. Hilton Head Island, SC Abstracts. December 2002 893 sion will include the rationale and means for collecting and inte- grating such restoration data layers into SIMS. EFFECTS OF PEARL NET STOCKING DENSITY ON SUR- VIVAL, GROWTH. AND GONADAL MATURATION OF BAY SCALLOPS. M. Davidson,' L. Hoist.' H. Bokuniewicz." C. Smith,-' and K. Tetrault,-' 'Nysdec, 205 North Belle Mead Road. East Setauket. NY 1 1733. "Marine Science Research Cen- ter, Suny Stony Brook. NY 1 1790; and 'Dornell Cooperative Ex- tension Marine Program, 3059 Sound Avenue, Riverhead. NY 11901. The stocking densities under which bay scallops are reared can have long-term effects on survival, growth, and spawning success that may not be evident while the scallops are in culture. To investigate the influence of stocking density on scallop production, hatchery reared bay scallops were stocked in pearl nets at three different densities during the summer. In the fall, bay scallop sur- vival and shell heights were recorded. The animals were trans- ferred to lantern nets and stocked at two different densities, grouped by their initial densities in the pearl nets, and over win- tered. Bay scallops raised at high densities exhibited lower survival and slower growth than those raised at lower densities. Regardless of density in the lantern nets, growth and survival still showed the negative effects of initial crowding in the pearl nets. Two-way analyses of variance revealed significant differences among the peari net and lantern net treatments in scallop survival and growth. Gonadal indices show that all the bay scallops, regardless of treat- ment, spawned at the same time. At the time of spawning there were no significant effects of density on gonadal index. Bay scal- lop restoration efforts should ensure that scallops are reared under conditions that maximize survival and growth. PROBIOTIC APPROACH TO ENHANCE HEALTH OF HATCHERY PRODUCED SHELLFISH SEED. R. A. Elston,' R. M. Estes,^ A. Gee,' R. P. Herwig,^ K. Kinnan,^ and S. Rensel,' 'Aquatechnics/Pacific Shellfish Institute, P.O. Box 687, Carisborg, WA 98324; "School of Fisheries, University of Wash- ington, 3707 Brooklyn Ave. N.E., Seattle, WA 98105-6715; 'De- partment of Biology, Pacific Lutheran University, Tacoma. WA 98447-0003; "'School of Fisheries, University of Washington, 3707 Brooklyn Ave. N.E., Seattle, WA 98105-6715; ^Aquatechnics/ Pacific Shellfish Institute. P.O. Box 687, Carisborg, WA 98324; and ^'Department of Biology, Pacific Lutheran University, Tacoma, WA 98447-0003. Bacterial diseases of intensively cultured larval and juvenile shellfish cause significant losses in hatcheries and nurseries. In addition, chronic bacterial infections are a significant cause of bivalve seed losses postplanting. From commercial hatchery case histories, a number of virulent juvenile oyster bacterial pathogens have been isolated, characterized, and pathogenicity confirmed by challenge procedures. Prevention and control strategies for bacte- rial pathogens in hatcheries and nurseries must include routine sanitation of system surfaces, water filtration, brood stock sanita- tion, and maintenance of low dissolved organic levels. Antibiotics have been used in experimental settings but are not routinely used on production scale systems because of cost as well as risk of producing resistant strains. A program to select and test probiotic strains of bacteria, as an alternative to antibiotic use, is underway, and results to date will be presented. Bacterial pathogens were first screened by comparing whole cell fatty acid profiles. Based on this evaluation, most pathogens were consistent or close to the Vibrio genus, but probiotic candidates represented a variety of bacterial genera. Selected representative isolates were further characterized using biochemical criteria and 16s rDNA sequencing. Candidate probiotic bacteria are first tested in agar plate inhibition tests. Strains showing inhibition to isolated pathogens are tested for haemolytic activity and pathogenicity to shellfish seed. Candidates passing these tests are then tested for inhibition of mortality and morbidity response in laboratory pathogen challenges. This re- search was supported in part by Saltonstall-Kennedy program (Na- tional Marine Fisheries Service, U.S. Department of Commerce) grant to Pacific Shellfish Institute. Olympia, Washington. MANAGEMENT BY SIZE LIMIT OF THE WHELK BUC- CINUM UNDATUM FISHERY IN THE SOUTH WEST IRISH SEA. E. Fahy, Marine Fisheries Services Division, Marine Institute, Abbotstown, Castleknock, Dublin 15, Ireland. Whelk landings in the south west Irish Sea increased from 56 t in 1990 to 6,575 t in 1996 after which they stabilized between 3,600 and 4,600 t annually. At its peak, the fishery supported approximately 80 vessels but this number has halved since this time. In 1994, a size limit of 50 mm was introduced for conser- vation purposes. Age-based assessments of the landings were con- ducted in 1994, 1996, 1997, and 1999, for which purpose the fishery, ranging from 52°10' to 53°30', is divided into four sectors. Landings to the four sectors display biological characteristics that indicate the occurrence of a number of stocklets rather than a single stock unit. Compliance with the size limit has been poor. From 20 to 33% of total landings (by number) in any of the assessed years have been less than the legal limit. Trends in cpue have been monitored since 1990. Some fishermen in the center sectors improved their yield between 1994 and 1998. Whelk have responded to a reduction in fishing effort since 1996, immediately after which averaged mortality coefficients (Z) were highest (0.79); they declined to 0.61 in 1999. The survival of the whelk fishery in the south west Irish Sea is attributed to the instability of the market which is dominated by a single customer. South Korea. A more effective size limit for this fishery would be 68 mm (83 mm in the northern sector), and this is considered unrealistic, suggesting that alternative management measures will have to be introduced. 894 Abstracts, December 2002 Shellfish, Restoration, Hilton Head Island, SC MANAGING THE FUTURE OF SOUTH CAROLINA'S OYSTERS: AN EXPERIMENTAL APPROACH EVALUAT- ING CURRENT HARVESTING PRACTICES AND BOAT WAKE IMPACTS. L. D. Coen and A. Fischer, Marine Re- sources Research Institute. SCDNR. Charleston. SC 29412. Oyster reefs provide an important intertidal habitat to the Southeastern United States. However, harvesting and recreational boating invariably impact these critical habitats and their associ- ated functions. In 1998, we began to experimentally evaluate the direct impacts of four harvesting practices (complete harvest, cull- in-place. clamming, and rake down) on intertidal oyster resources. Initially. 26 sites were sampled by quadrat to establish baseline assessments. Initial mean oyster size (SH) across sites ranged from 23-.^,-' mm. with initial densities ranging from 1.700-7,500 oys- ters/nr. Then, the above harvesting practices were simulated at replicated sites, each paired with an adjacent control site. Water quality (temperature. DO. salinity, chl a) was measured during the study period. Trays of shell were deployed at each site to evaluate oyster recruitment and growth. After approximately I year, inore than LI .^.000 oysters recruited to the 1.^0 deployed trays. This recruitment, a surrogate for larval supply/habitat quality, and the baseline assessments are analyzed and discussed. In 1999. we con- ducted experiments to understand how boat wakes compromise shell (cultch) deployments for oyster restoration and marsh erosion control. For this, we deployed stabilized (mesh) and unstabilized shell treatments, monitoring cultch retention after controlled boat wakes. In the first pilot experiment, unstabilized treatments lost 3.3.6'^ (7.7 cm) rnore shell than stabilized treatments after expo- sure to 32-controlled boat passes. In a second experiment, 22.4% (5.17 cm) more was lost after only 24 passes. Both harvesting practices and recreational boating wakes can potentially impact the growth, recruitment, and recovery of intertidal oyster resources. Additionally, oyster reefs that fringe marshes can serve as mod- erators of both marsh and bank erosion. Further studies with re- mote sensing technologies should be employed to monitor the oyster-marsh interaction. GENOMIC APPROACHES TO MARKER DEVELOPMENT AND MAPPING N THE EASTERN OYSTER, CRASSOS- TREA VIRGINICA. P. M. Gaffney,' K. S. Reece,- and J. C. Pierce,"' 'College of Marine Studies, Lewes, DE 19958; ^Virginia Institute of Marine Science. Rt. 1 208, Gloucester Point. VA 23062; and 'University of the Sciences in Philadelphia. 600 S. 43d St.. Philadelphia, PA 19104. In response to the dramatic decline in the Atlantic oyster fish- ery, efforts are underway to expand hatchery production of the Eastern oyster, for both commercial farming and for replenishment of disease-challenged natural populations. In particular, there is a strong demand for genetically improved oyster strains resistant to two common protozoan parasites, Deniio and MSX. The genetic improvement process will be enhanced by the development of molecular markers and a genetic linkage map. To facilitate future marker development in C. viiginica. we obtained 0.7 MB of ran- dom genomic sequence data from a small-insert fl kb) pGEM library. A modest number of significant BLASTX hits may prove valuable for designing type I markers for comparative mapping with the Pacific oyster. In addition, we the searched the sequence database for repetitive sequences. Several satellite DNA sequences were identified and compared with putative satellite sequences obtained by traditional cloning methods. Our database yielded use- ful information on the distribution of microsatellite loci. Dinucle- otide microsatellites were dominated by the AG motif (669<"). Tri- nucleotide microsatellites included all possible motifs in appar- ently equal frequencies. Tetranucleotide microsatellites were more common than trinucleotides and, unlike the other microsatellite classes, were frequently associated with repetitive sequences, with a strong tendency for certain tetranucleotide motifs to be associ- ated with particular repetitive sequences. This information will be useful for tetranucleotide microsatellite marker design, as well as interpretation of linkage mapping data. The repetitive sequence database will be used as an adjunct for designing new primers, to reduce the frequency of non-target amplification. COMMUNITY-BASED OYSTER RESTORATION: CASE STUDIES FROM CHESAPEAKE BAY. W.J. Goldsborougii,' R. D. Brumbaugii,' D. W. Meritt,- and J. A. Wesson,' 'Chesa- peake Bay Foundation, 162 Prince George Street. Annapolis. MD 21401; "University of Maryland, Center For Environmental Sci- ence, P.O. Box 775, Cambridge, MD 21613; and 'Virginia Marine Resources Commission, P.O. Box 756, 2600 Washington Avenue, Newport News. VA 23607. Public support for oyster restoration in the Chesapeake Bay region has increased in recent years, largely because of expanded opportunities for direct citizen involvement in restoration work The commercial value of oyster restoration is the most easily ap- preciated aspect of restoration, whereas associated benefits such as impro\ed fish habitat and water quality are only recently being more widely recognized. As opportunities for public participation have expanded, the support for restoration has increasingly been based on these associated ecosystem benefits, particularly in de- veloped areas where water quality may preclude commercial or recreational harvest of bivalves. One of the principal ways that the public now participates in oyster restoration is by growing hatch- ery-produced oysters using small-scale aquaculture techniques (i.e., "oyster gardening") for eventual transplanting onto brood- stock sanctuary reefs. Analyses of four local examples of citizen involvement in oyster gardening/restoration in the Chesapeake re- veal a general pattern of roles and responsibilities for successtui community-based restoration. Local leadership, sources for shell and seed, education, technical guidance, amenable government rules and regulations, media exposure, and funding emerge as key Shellfish, Restoration, Hilton Head Island, SC Abstracts, December 2002 895 factors. In the Chesapeake, a partnership approach based on co- operation between various combinations of citizens, schools, local businesses, local service organizations, watershed associations, academic institutions, state and federal agencies, conservation or- ganizations, and private foundations has successfully addressed these needs. It is becoming increasingly clear that the groundswell of public support for restoration resulting from community-based approaches is a key factor in generating increased public funding for restoration. MOLECULAR IMMUNE RESPONSES OF THE EASTERN OYSTER TO THE PARASITE PERKINSUS MARINUS. M. Gomez-Chiarri and P. Mufioz, Department of Fisheries, Animal and Veterinary Science, University of Rhode Island, 127 Wood- ward Hall. Kingston, RI 02881. Microbial pathogens and parasites like Peikinsiis marinus and Haplospnridiwn iielsniu place a large economic burden on oyster fisheries and aquaculture. Although there has been a steady in- crea.se in our knowledge on the pathology and epizootiology of the diseases caused by these parasites, relatively little is known about the molecular mechanisms involved in the response of oysters to infection. The goal of this project funded by the ODRP is to monitor systematically the induced expression of genes involved in the response oi Crassostreu virginica to infection by the parasite Perkinsiis marinus. An mRNA differential display technique coupled with stringent verification assays (reverse Northern blot) will be used to isolate oyster and parasite sequences expressed in a differential manner after challenge of oysters with cultured P. marbuis. Genes will be cloned and sequenced using standard mo- lecular techniques. The temporal and tissue patterns of expression of the candidate genes in oysters will be studied using Northern blot. Preliminary results from the challenge experiments will be presented at this meeting. FACTORS AFFECTING THE STRESS RESPONSE IN OYS- TERS ON THE WEST COAST: IMPLICATIONS FOR SUM- MER MORTALITY. G. C. Cherr.' C. S. Friedman,' F. J. Griffin.' A. Hamdoun,' J. Mitchell,' L. Righetti,' D. P. Cheney,^ R, A, Elston,^ and B. McDonald,^ University of Cali- fornia. Davis, Bodega Marine Laboratory. P.O. Box 247. Bodega Bay. CA 94923 and "Pacific Shellfish Institute. 120 State Ave. N.E. #142. Olympia, WA 98501. Summer mortality of Crassostrea gigas on the West Coast of the United States is an unpredictable phenomenon of unknown etiology but one that is hypothesized to be caused by multiple stressors. Previous research has identified a dinofiagellate (Gyiu- nodinium sanguineum). temperature, and low dissolved oxygen as possible contributors. We have attempted to delineate the indepen- dent effects of two of these suspected factors, phytoplankton and temperature, while conducting parallel field studies in California and Washington to determine the effects of seed stock lineage and seed planting times. Laboratory challenges confirmed that G. san- guineum can produce stress/mortality in the absence of other in- sults. Phytoplankton bloom events have coincided with field mor- tality; however, the species present were a Pseudonitzchia-Vike species and Proroccntrum spp.. not G. sanguineum. Previous re- search on temperature effects showed thai the ability of C. gigas to tolerate otherwise lethal temperatures occurred after sublethal ther- mal shock and induction of the heat shock protein 70 (HSP70) family. This is termed the heat shock response (HSR). We have examined the abilities of C. gigas from three different habitats (Toten Inlet, WA; Mud Bay, WA; Tomales Bay. CA) to mount a HSR and compared this ability with environmental and summer mortality data. Our current findings suggest that chronic sublethal environmental stressors such as heat and immersion can induce HSP70 expression and acquisition of thermal tolerance in C. gigas. However, these chronically stressed animals exhibit a compro- mised HSR; they do not tolerate post-heat shock temperatures as high as nonchronically stressed counterparts. This research was funded by National Sea Grant College Program Office: Oyster Disease Research Program. COMMUNITY-BASED OYSTER HABITAT RESTORA- TION AND ENHANCEMENT IN SOUTH CAROLINA. N. H. Hadley and L. D. Coen. Marine Resources Research Institute. SCDNR. P.O. Box 12559, Charleston. SC 29422. Oyster reefs provide important habitat for finfish, crabs, and shrimp; improve water quality; and. when located adjacent to Spartina marsh, form a natural bulwark to reduce erosion. Oyster habitats nationwide are threatened by adverse effects of coastal development. The majority of oysters in South Carolina occur intertidally. where they may be exposed for as much as 6 h because of the -2 meter tidal range. This makes them especially vulnerable to physical disturbances such as boat wakes. Substrates in South Carolina are typically soft mud and oyster shell provides one of the few hard surfaces for larval oyster attachment. Oysters readily recruit to shell placed in areas which otherwise may have no re- cruitment because of lack of suitable substrate. At sites with ap- propriate characteristics functional oyster reefs may be established in 3 to 5 years, with some attributes beginning earlier. This pro- gram will use community volunteers to establish multiple small- scale oyster habitats by planting oyster shell and covering it with stabilizing mesh. We will also develop a volunteer-based moni- toring program to evaluate restoration success. Community part- ners with existing volunteer contacts have been enlisted to assist in this program. An oyster shell-recycling program is being estab- lished to generate shell for future restoration projects. Schoolchil- dren will be involved through collaboration with the Charleston Math and Science Hub to develop classroom and field activities directly related to oyster habitats. Materials (pamphlets, a website. CD) will be developed to educate the public about oyster habitats and shell recycling. 896 Abstracts, December 2002 Shellfish, Restoration, Hilton Head Island, SC ESSENTIAL OR JUST OPPORTUNISTIC FISH HABITAT? UTILIZATION OF RESTORED COMPLEX SHELLFISH HABITAT BY FISH SPECIES. J. M. Harding and R. Mann, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary. Gloucester Point. VA 23062. Shellfish restoration typically creates complex habitat in re- gions where such habitat is limited or absent. Observations to date suggest that increasing habitat complexity supports more diverse representation in other trophic levels. Such observations have been used to argue for shellfish restoration sites in the wider context of essential fish habitat. We present temporal and spatial data on fish utilization of a dine of habitats from a complex, "restored"" site, through a two-dimensional but spatially complex site, to a mo- notonous sand bottom, and pose the question as to whether fish utilization of this cline suggests "essential"" or simply opportunistic utilization of the varying resource. EFFECTS OF CLIMATE VARIABILITY ON THE PREVA- LENCE AND INTENSITY OF DERMO AND MSX DIS- EASES IN EASTERN OYSTER POPULATIONS. E. E. Hof- niann.' J. M. Klinck.' E. N. Powell,- S. E. Ford." S. Jordan,' and E. Burreson,"* 'CCPO, Old Dominion Llniversity, Norfolk, VA 23529; "Haskin Shellfish Research Laboratory, Rutgers Uni- versity, Port Norris. NJ 08349; 'Sarbanes Cooperative Oxford Laboratory. 904 South Morris Street. Oxford, MD 21654; and ■^Virginia Institute of Marine Science. P.O. Box 1346, College Of William and Mary. Gloucester Point, VA 23062. With previous funding from the National Sea Grant Oyster Disease Research Program, we developed numerical models that simulate the annual cycles of intensity and prevalence of the dis- ea.ses, Dermo, caused by Perki)isiis mariiiKs. and MSX, caused by Hapliispordiiim iielsoni. in Eastern oyster (Cmssosiiea viri;inica> populations in Delaware Bay and the upper Chesapeake Bay. The host-parasite models consist of models for the growth of the para- sites, as well as a model for the growth and development of the oyster. The external forcing for the models is from time series of temperature, salinity, food supply, and total suspended solids. Our recent project has focused on combining the two disease models with the oyster growth model. Simulations w ith the combined two di.sease-oyster model provide insight into the effect of variability in environmental conditions in initiating and controlling epizootics of Dermo and MSX in Chesapeake and Delaware Bays. The com- bined model also provides a mechanism for investigating possible interactions between Dermo and MSX diseases that modulate the level of intensity and overall prevalence of the two diseases in oyster populations. Initial results suggest that there is only limited interaction between the two diseases in the host and that environ- mental conditions are the primary determinant of which disease is dominant at a given location, as long as the oysters are susceptible to both disease agents and that the dose of infective particles does not vary. FIELD TRIAL OF A BAY SCALLOP (ARGOPECTEN IRRA- DIANS) SPAWNER SANCTUARY. C. Smith,' S. Dumais,' L. K. Hoist,- and M. Davidson," 'Cornell Cooperative Extension of Suffolk County, Marine Program, 3059 Sound Avenue. Riverhead, NY 11901 and "New York State Department of Environmental Conser\ation. Division of Fish. Wildlife, and Marine Resources, 205 North Belle Meade Road, East Setauket. NY 1 1733. The populations of Bay scallop in New York waters have ex- perienced critical decline over the past two decades, caused in large part by the occurrence of Brown Tide algal blooms and its concomitant effects on habitat and shellfish health. Management efforts in the past have focused heavily on hatchery-produced stock, which has been free planted into the estuary. Frequently, follow-up investigations show no juvenile scallops at the release sites, and the ultimate fate of the seed stock is unknown, except through extrapolation of harvest data. New York State Department of Environmental Conservation, working with Cornell Coopera- tive Extension"s Marine Program, set out to evaluate "spawner sanctuaries"" as a management method to ensure that reproducing scallops are present in the system in densities sufficient to increase the population. Mature scallops (n = 15.000) that had been over wintered in a creek adjacent to Comelfs hatchery were stocked into lantern nets at a density of 100 animals per tier and deployed into Cutchogue Harbor in Peconic Bay. New York. A larval drift model and diver transects were used to calculate relative contri- bution of the sanctuaries to the 1999 year class set in Cutchogue Harbor. Results were further normalized to reflect differences in gonad weight between wild stock and hatchery-reared scallops. RESTORING CRITICAL HABITATS IN THE NIGER DELTA FOR SHELLFISH PRODUCTION. A. C. Ibe' and P. O. Abohweyere.- 'The Regional Coordination Centre Gef s Large Marine Ecosystem Project for the Gulf t)f Guinea and "United Nations Industrial Development Organization, Nigerian Institute for Oceanography and Marine Research, P.M.B. 12729, Victoria Island, Lagos, Nigeria. The Niger Delta is a fan-shaped piece of land located between 504'. 00 and 7040'. 00 longitude and stretching from the Benin Ri\er in the west to the Bonny River in the east, covering an area t)f about 16,340 knr. This low lying region, riddled with intricate water channels through which the river Niger empties into the sea, consists of three broad ecological zones: freshwater, mangrove, and the coastal sandridges. The mangrove ecosystems are prime areas for production, constituting spawning and nursery grounds for near shore, demersal and pelagic fish species including shell- fishes. Shellfish of economic importance found in the Niger Delta include the oyster Cnissastrea gnsar that settles as spat on man- grove aerial roots at intertidal levels, the periwinkles Tympanoto- lunts fuscatus and Pachymelia quadriserata. and Penaeid shrimps that generate over US $195,977.26 from 10,664 MT annually in foreign exchange. Anthropogenic activities in the form of defor- Shellfish. Restoration. Hilton Head Island. SC Abstracts. December 2002 897 estation. sand mining and nourishment, channelization, dredging, oil and gas exploitation, and rapid urbanization are presently re- ducing the aerial extent of the mangroves as well as degrading the mangrove environment. This has an impairing effect on the shell- fish production potential of critical habitats of the Niger Delta. This work thus advocates rational use of the mangrove ecosystem and reforestation of the mangrove swamp where possible such that one of the goals of the shellfish restoration effort of "restoration or enhancement of populations of commercially exploited shellfish depressed by over-harvesting and or reduced environmental qual- ity" could be achieved in the Niger Delta. STAKEHOLDER AND CONSENSUS— HOW DO WE MAKE THESE ELEMENTS WORK FOR EFFECTIVE AC- TION? M. Janowiez, New Brunswick Department of Environ- ment and Local Government. 364 Argyle Street. Fredericton. New Brunswick. E3B 1T9 Canada. One model for effective stakeholder insohement requires implementation of a consensus decision-making process. This al- lows each stakeholder to understand the context within which each participant is working and requires the development of respect among the participants. This is an age-old concept but in reality, few in 21st century North America can easily accept and work within it. This article will examine why stakeholder in\olvement is a necessary means to formulate approaches to shellfish restoration or any other local economic development and planning program. It will explore the foundations of consensus decision-making includ- ing examination of concepts of democracy, discussion on Native North American decision-making processes and other decision- making models. And. finally, it will identify some methodology for achieving an effective stakeholder, consensus decision-making process with the emphasis on developing a common ground of understanding. OYSTER BIOMASS AND ABUNDANCE IN NORTHERN CHESAPEAKE BAY: TRENDS IN RELATIONSHIP TO HARVEST. RECRUITMENT, PARASITIC DISEASES. AND ENVIRONMENTAL VARIATION. S. J. Jordan, K. N. Green- hawk, C. B. McCollough, and M. L. Homer, Maryland Depart- ment of Natural Resources, Sarbanes Cooperative Oxford Labo- ratory. 9045 S. Mon-is St.. Oxford. MD 21654. The Chesapeake Bay Program has committed to a 10-fold in- crease in the Bay"s oyster population. Oysters are patchily distrib- uted over about 1,500 km" of the Bay floor. Therefore, it is im- practical to assess their absolute numbers by direct means. Tradi- tionally, landings data, with their inherent inaccuracies and biases, have been the only means of estimating trends in the population. Maryland's monitoring program records relative numbers and size distributions of oysters annually at 43 fixed sites. By applying a length: weight equation to size-frequency data from this fishery- independent survey, we computed an index of relative biomass that varied from year to year in response to the relative abundance and size distribution of the oyster populations. The index reflects in- terannual variations in recruitment and growth, as well as mortality caused by the oyster parasites Haplosporidiiiiu iielsoni and Perk- insiis marinus. An index of market oyster (>72 mm shell height) biomass had a strong predictive relationship with annual harvests, but an index of sub-inarket oysters (<72 mm) was not a good predictor of harvests in subsequent years, probably because of high and variable rates of natural mortality due to parasitic diseases. Relative biomass is a sound indicator for measuring progress to- wards the oyster restoration goal, and has promising applications in fisheries-related stock assessment. PUBLIC AND PRIVATE OYSTER RESTORATION IN MARYLAND'S CHESAPEAKE BAY. C. J. Judy and E. Campbell, Maryland Department of Natural Resources. Shell- fish Di\ision. 580 Taylor Avenue. Annapolis. MD 21401. Oyster restoration is a shared venture between government and the private sector. Maryland oyster projects through the 1990s have been a collaboration between state, federal, and private groups. Projects have focused on the creation of oyster sanctuaries to protect broodstock and enhance benthic community diversity, restoration of habitat; and planting of seed oysters, primarily from hatcheries. In round numbers, the acreages for a cooperative proj- ect range from a few to over 10 and the number of oysters planted in a year range from about 10 million to over 50 million total. A wide range of participants constitutes the private sector: environ- mental groups, non-profit oyster restoration groups, community groups, private citizens, watermen and school groups. The number of projects by such groups has risen dramatically since the eariy 1990s and encompasses types of projects not normally conducted by State agencies alone. Other projects are more uniquely govern- mental. The long standing Maryland Department of Natural Re- source's seed and shell programs plant about 400 acres of seed and 800 acres of shell per year. The number of oysters planted as seed range between 120 million to over 800 million per year. These projects mostly produce market oysters, but environmental and broodstock benefits accrue from such mass plantings. The Feder- ally funded Reef Program conducted by the State restores oyster populations in sanctuaries using shell and seed resources. To- gether, public and private entities are working toward improving oyster habitat and oyster populations to improve the industry and the ecological role of oysters. MUSSEL CULTURE AND COCKLE FISHERIES IN THE NETHERLANDS: FINDING A BALANCE BETWEEN ECONOMY AND ECOLOGY. P, Kamermans and A, C. Smaal, Netheriands Institute For Fisheries Research. Centre for Shellfish Research. P.O. Box 77. 4400 Ab Yerseke. The Nether- lands. In the Netherlands, mussel seed is fi.shed in a coastal sea in the North of the country (Wadden Sea) and cultured in an estuary the 898 Abstracls. December 2002 Shellfish, Restoration, Hilton Head Island, SC South (Oosterschelde). Dredging for cockles takes place in the Wadden Sea, and two estuaries in the South (Oosterschelde and Western Scheldt). The Wadden Sea and the Oosterschelde are nature reserves where human activities are possible only when they do not cause negative effects. In 1993. a policy was formu- lated to ensure preservation of bird populations and restoration of mussel banks and seagrass meadows. As a result of this policy, fishing for mussel seed and cockles is not allowed in areas with a high potential for the development of mussel banks and seagrass fields. The location of these areas is based on CIS models. All vessels are equipped with a black box to control the closed areas. There is some debate about the closure because fishermen have the impression that fishing improves the sediment for settling of mus- sel larvae. Consumption-sized cockles and mussels are also the prefeiTed prey of oystercalchers and eider ducks. Therefore, the policy makes use of a quotum system in the cockle fisheries. Each year, basin-wide surveys take place to determine the total amount of cockles present. In years when cockle stocks are low an amount is reserved for the birds. Both fishermen and environmentalist question the calculated amounts needed by the birds. An overview of the viewpoints of the interest groups and the role of policy makers and scientists is given. AN ECONOMIC ANALYSIS OF PUBLIC GROUND OYS- TER REEF RESTORATION IN CENTRAL LOUISIANA DAMAGED BY HURRICANE ANDREW. R. J. Dugas,' W. R. Keithly," M. Bourgeois/ P. Meier,' D. Lavergne."* and A. Di- agne,'* 'Louisiana Department of Wildlife and Fisheries, Marine Fisheries Division, 1600 Canal Street, New Orleans, LA 701 12; 'Louisiana State University. Coastal Fisheries Institute, Wetland Resources Building, Baton Rouge. LA 70803-7503: 'Louisiana Department of Wildlife and Fisheries, Marine Fisheries Division, 1600 Canal Street. New Orieans, LA 70112; and ^Louisiana De- paitment of Wildlife and Fisheries, Socioeconomic Division, P.O. Box 98000, Baton Rouge. LA 70898-9000. In August 1992, Hurricane Andrew heavily damaged Louisiana coastal environments, particularly oyster, Crassostrea virginica. reef communities. The transport and transfer of tremendous amounts of sediment and vegetative matter resulted in massive oyster mortalities and extensive reef damage. The Louisiana De- partment of Wildlife and Fisheries received $5.\ million of federal funds for restoration of oyster habitats on both Louisiana public and private oyster grounds. Of these funds, $3.2 million were used in Terrebonne Parish, the area most severely impacted. Restoration efforts were comprised of sweeping buried reefs and depositing cultch material for oyster reef construction. Some 1,780 acres of waterbottoms were swept by commercial oyster harvesters with bag-less oyster dredges. Mined oyster shells/clam, Rangia. shell mixture were deposited (at a rate of approximately 1 32 cubic yards per acre) on 306 acres of waterbottoms in 1994 (42,576 cubic yards) and 553 acres in 1995 (70,902 cubic yards). Economic benefits associated with a restoration effort of this nature accrue to both oyster consumers and oyster producers. To consumers, the benefits reflect a reduction in price paid for the harvested product, which in tuni translates to an Increase in willingness to pay relative to what was paid (i.e., consumer surplus). To producers, the ben- efits reflect an increase In returns to the scarce resource, oyster population, used in the production process (i.e.. producer surplus). This study provides an estimate of benefits derived from the res- toration efforts and compares these benefits to costs. Overall, the results indicate a favorable benefit to cost ratio. THE REROUTING OF STORMWATER DISCHARGES FOR WETLANDS ENHANCEMENT, LEVEE PROTEC- TION, AND OYSTER HABITAT PROTECTION AND RES- TORATION. K. E. Landrum,' K. M. St. Pe,' B. Ache,- and F. Kopfler,'' 'Barataria-Terrebonne National Estuary Program. P.O. Box 2663, Nicholls State University. Thibodaux, LA 70310; "Bat- telle, 191 East Broad Street, Suite 315, Athens, GA 30601; and 'Epa/Gulf Of Mexico Program, Stennis Space Center, Building 1103, Room 202, MS 39529-6000. The Barataria-Terrebonne estuary is losing over 22 square miles of emergent wetlands each year because of erosion, saltwater intrusion, and natural and anthropogenically-induced subsidence. An extensive levee system has successfully halted overbank flood- ing of the Mississippi River, eliminating sustaining inputs of sedi- ments and freshwater to the Barataria-Terrebonne estuary. This situation represents not only the imminent loss of a nationally significant wetland resource but also threatens a unique culture, local infrastructure, and the region's significant contribution to the national economy. Runoff from rural and agricultural areas is col- lected in a borrow canal inside the back levee and then pumped into adjacent wetland areas by a series of stormwater pump sta- tions. Over 250 pump stations currently discharge stormwater. draining approximately 500,000 acres, in the Barataria-Terrebonne estuary. These pump discharges are generally directed into large, human-made canals to ensure that stormwater is quickly evacuated from the leveed area and they often flow directly to high-salinity bays through some of Louisiana's prime oyster growing waters. Redirecting discharges so that they are retained in adjacent wet- lands may maintain lower local salinities, provide a sediment source to subsiding wetland areas, and support plant growth, di- rectly benefiting the degrading wetland systems, especially those directly seaward of levees that protect property from storm surges and flooding. Retention of storm water may also produce corollary water quality benefits, such as nutrient uptake and pathogen die-off prior to encountering oyster-growing areas. The Barataria- Terrebonne National Estuary Program is leading an effort to moni- tor changes at pump station sites in the estuary to demonstrate the benefits of this unique process. Shellfish. Restoration. Hihon Head Island, SC Abstracis, December 2002 899 THE SHELLFISH CHALLENGE INITIATIVE: A COOP- ERATIVE SUCCESS STORY IN THE BARATARIA- TERREBONNE NATIONAL ESTUARY. K. E. Landrum. Barataria-Terrebonne National Estuary Program. P.O. Box 2663. NichoUs State University, Thibodaux, LA 70310. The Shellfish Challenge Initiative is an interagency and inter- state effort undertaken to establish progress on the Environmental Protection Agency's Gulf of Mexico Program Shellfish Challenge. With an overall goal of increasing Gulf shellfish beds available for safe harvest by ten percent, more than 200 experts in shellfish management, habitat restoration, and pollution control helped de- velop 32 shellfish restoration strategies targeting 24 watersheds in the Gulf of Mexico. A watershed implementation initiative was developed within the Barataria-Terrebonne National Estuary re- sulting in the identification of 61 oyster restoration opportunities, including geographically targeted projects to reduce inputs of fecal coliform bacteria, enhance shellfish habitat, revise shellfish man- agement procedures, and collect and analyze additional needed information to better assess project feasibility. The 61 candidate restoration projects were ranked by members of the Barataria- Terrebonne National Estuary Program Management Conference, and detailed implementation plans were developed for the four selected priority projects. The priority projects included the fol- lowing: the installation and improved use of marina pumpouts and dump stations; connecting poorly operating individual wastewater treatment systems to community level treatment systems; rerouting stormwater runoff to suitable wetlands; and revising the shellfish relay system. Implementation of the four projects is underway with active educational and interacfive workshop components designed for state and local officials and the general public. Funding allo- cations by local and state government attest to their involvement and acceptance of the implementation process and their agreement to promote active stewardship of an economically important re- source and conservation principal. OYSTER POPULATION RESTORATION IN CARAQUET, N.B.; PHASE I, POPULATION ASSESSMENT. T. Landry. M. Ouellette, and P. Cormier, Department of Fisheries and Oceans, GFC. P.O. Box 5030. Moncton N.B., ElA 4Y1 and De- partment of Agriculture, Fisheries and Aquaculture. 22 Boul. Saint-Pierre. Caraquet, N.B. EIW 1B6. Canada. A decrease in the productivity of oysters in Caraquet Bay. N.B. is generating some interest in restoration projects. The first phase of this initiative is to conduct a quantitative assessment of the distribution, abundance and population structure of the natural beds in this bay. which represents the most northern location with a sustainable oyster (Crassostrea virginica) population. The results from 1999 assessment is the fifth of a series of similar exercises conducted in 1974, 1979,1987, and 1991 but the first to use a geostatistical approach to data analysis. A comparison between the two assessment methods reveals that the geostatistical approach is more accurate and of greater use for the next phase of this project, w hich will look at identifying and characterizing the suitable oys- ter habitat of this bay for restoration efforts. The comparison among the five assessments over the past three decades is showing that the status of this population is approaching a critical state in tenns of recruitment and habitat quality and quantity. The resto- ration of this oyster population is of great socio-economical and ecological importance to this area. COMMUNITY-BASED INITIATIVES FOR IMPROVING WATER QUALITY IN SOUTHWESTERN NEW BRUNS- WICK, CANADA— AN UPDATE ON SUCCESS. K. L. LeBlanc, Eastern Chariotte Waterway Inc.. 17 Main Street. St. George, New Brunswick, E5c 3h9. Canada. The Southwestern New Brunswick Clam Resource Committee (CRC) was formed in 1997 to better understand the importance of the soft-shell clam iMya areiiaiUO fishery and to prevent further loss of shellfish growing areas due to monitoring cutbacks in Southwestern New Brunswick (NB). Soft-shell clams are the main molluscan shellfish harvested in the region. The committee has over 20 stakeholders that include nonprofit groups, industry and government and is chaired by Eastern Chariotte Waterways Inc. (ECW. a nonprofit group). The mandate of the committee is to preserve the clam fishery in Southwestern NB because of its long- term socio-economic importance to the region. In 1998. the CRC developed the Cooperative Bacterial Monitoring Program (CBMP), which allows industry and community-based contribu- tions for the maintenance and improvement of the classification of growing areas. Under this community-based initiative coastal wa- ter samples are collected over a 1,500-km length of shoreline over a two-year period. Presently, 67% of the growing areas are avail- able for the harvest of clams in Southwestern NB during all or selected times of the year, an increase of 32% over 3 years. How- ever, it is important to note that clean-up efforts in growing areas coupled with the CBMP are responsible for the improvements of water quality that have permitted an increase in soft-shell clam harvest. The CRC coordinates clean-up efforts through the Fundy Flats Remediations plan, a program managed under ECW. APPLICATION OF COMMERCIAL-SCALE OYSTER AQUACULTURE TO REEF RESTORATION. A. T. Leggett. R. Brumbaugh, W. Goldsborough, and A. McDonald, Chesa- peake Bay Foundation. 142 W. York Skeet. Suite 318, Norfolk, VA 23510. Oyster reef restoration projects in the Chesapeake Bay increas- ingly involve the addition of broodstock to enhance localized oys- ter spawning activity. Since 1996. more than 4 million adult oys- ters have been transplanted onto sanctuary reefs in Maryland and Virginia waters. Volunteers and school students have grown and transplanted a significant number of hatchery-produced oysters, in collaboration with state management agencies. In an effort to in- crease the numbers of hatchery-produced oysters being trans- planted onto sanctuary reefs, the Chesapeake Bay Foundation has 900 Abstracts. December 2002 Shellfish, Restoration, Hilton Head Island. SC initiated a commercial-scale grow out operation in the lower York River with a annual production goal of I million adult oysters. Oysters produced by this program will approximately double the number of oysters available for transplanting onto sanctuary reefs in the lower Chesapeake Bay each year. A new cage system was developed in accordance with existing regulations in Virginia gov- erning shellfish aquaculture. and was used to maximize the number of oysters produced over a relatively small acreage of leased oyster grounds. Data on growth and mortality as well as water quality parameters are collected as a routine part of the operation. Early monitoring results showed a 78% increase in size (measured volu- metrically) of -1-8 mm seed, and a 63% increase in 8-12 mm seed one week after deployment in mid-July. The oysters produced by the operation will not only be used to enhance broodstock popu- lations on sanctuary reefs, but will also be used as "natural capital" to enticing further public and private investment in oyster resto- ration. analysis of Gulf coast oysters, clams and shrimp has shown stimu- lation of a unique anti-molluscan protein that varies in MW 4 to 22 kd and has anti-V'//)/7V) viiliuficus activity. When the protein and phage are used together, eradication of Vibrio vidiiificus occurs to more than 8 logs. Analysis by Edman degradation of the 22-kd oyster protein revealed a unique N-terminal 16 amino acid frag- ment, as did analysis of two cyanogen bromide gel purified frag- ments. The proteins were not detected in Japanese or Olympia (Washington) oysters (kindly provided by Dr. C. Kaysner. FDA I. Finally, a new rapid assay to study the effect of temperature and brief bacterial exposure has been developed, suggesting the pos- sibility that Vibrio viiliiificiis could undergo a non-culturable state under certain conditions (K. Johnston, pers. comm.). Our intention is to isolate the genes expressing the 4- and 22-kd proteins; then express them in large amounts to use with the specific phage in a biodepuration procedure. This work was supported by SK Grant #NA97FD0062 to RBL from NOAA. DOLLARS AND SENSE OF OYSTER RESTORATION: AN EXAMINATION OF NITROGEN REMOVAL BY A RE- STORED OYSTER REEF. M. Luckenbach.' F. O'Beirn,' P. Ross,' J. Nestlerode.- and L. Sorabella." 'Virginia Institute of Marine Science, College of William and Mary. Wachapreague. VA 23480 and "Virginia Institute of Marine Science. College of William And Mary. Gloucester Point. VA 23062. Arguments for the conservation and restoration of oyster reefs, often at the expense of fisheries exploitation, include water quality benefits derived from feeding activities of the oysters and reef- associated fauna. Yet, there has been limited basis for directly evaluating the water quality improvements associated with con- servation or restoration of oyster reefs and for comparing those benefits to the economic value derived from oyster fishery pro- duction. Using data from oyster populations developing on experi- mental reefs near the mouth of Chesapeake Bay. we model the nitrogen uptake and release attributable to the oysters and develop nitrogen budgets for the reefs on an area-normali/ed basis. We then explore the potential effects of fisheries exploitation of these reefs by modeling the harvest of market-sized oysters and exam- ining the effects on nitrogen removal. Finally, we consider the economic returns from oyster harvesting in relation to the costs associated with alternative nitrogen removal. The results give con- text to water quality benefits to be derived from oyster reef sanc- tuaries and should help to guide fisheries management decisions related to balancing conservation and exploitation. REDUCTION IN THE VIBRIO VULNIFICUS LOAD OF OYSTERS BY A NOVEL SHORT-TERM COMBINATION BIODEPURATION TREATMENT. R. B. Luftig and W. Pelon, Department of Microbiology, LSU Health Sciences Center. New Orleans. LA 701 12-1393. Based upon an improved method. Vibrio vulnificus phage can be maintained and stored at high titer. Further, mass spectroscopic COMING SOON TO A RESTORATION SITE NEAR YOU: THE INVADING. PREDATORS ORIENTAL GASTROPOD RAPANA VENOSA. R. Mann and J. M. Harding. School of Marine Science. Virginia Institute of Marine Science. College of William and Mary. Gloucester Point. VA 23062. Rupana venosa Valenciennes 1846 ( Neogastropoda. formerly Muricidae. currently Thaididae) is a predatory gastropod native to the Sea of Japan. Yellow Sea. East China Sea. Bohai Sea. and Taiwan. The species has been introduced to the Black Sea. Adri- atic Sea. and Aegean Sea. where it is generally considered to be responsible for decimation of local commercially valuable mollusc species. It was first reported in the Chesapeake Bay in 1998. Bal- last water transport of larval stages from the eastern Meditcrtanean or Black Sea is the suspected vector of introduction. To date over 1200 specimens of adult Rapaini have been collected from Hamp- ton Roads and a limited region of the Southern Chesapeake Bay. Population demographics, records of Rapana egg cases in the field and our ability to culture early life history stages at prevailing temperature and salinity strongly suggest active breeding in this receptor location. Temperature and salinity tolerance data for Ra- panii suggest that it can both invade the higher salinity regions of most East Coast estuaries and survive on exposed shorelines from Cape Cod, MA to Charleston. SC. Dispersal is facilitated by pe- lagic development, and may be exacerbated by ballast water trans- port of larval stages originating in Hampton Roads. Hard substrate habitat, typical of many curtent shellfish restoration efforts, ap- pears optimal for post settlement stages, but larger adults may invade soft sediments. Predation has been demonstrated on a range of commercially valuable shellfish species including Mercenaria incrceiiaria. Crassostrca viraiuica. Myii urcnaria and Mytiliis edu- lis. Shellfish. Restoration. HiUon Head Island. SC Abslracts. December 2002 901 UP CLOSE AND PERSONAL: A SUGGESTED QUANTITA- TIVE APPROACH TO BROODSTOCK ENHANCEMENT ON SHELLFISH RESTORATION SITES. R. Mann, School of Manne Science. Virginia Institute of Marine Science. College of William and Mary. Gloucester Point. VA 2.^062. Shellfish broodstock are typically added to restored habitat to facilitate rapid recruitment by aggregating spawning adults and thus increasing fertilization efficiency. Although this is conceptu- ally attractive there exist few data on which to build quantitative guidelines to optimize the practice. For example, published size versus fecundity relationships for oysters are based on data that has both methodological and size limitations. Similarly, fertilization models are based on sea urchin studies from flow regimes that are arguably quite different from shellfish restoration sites. A quanti- tative approach is proposed wherein a variety of size-fecundity and fertilization models are proposed for examination in building guidelines to optimize both size and density of placement of shell- fish used in broodstock enhancement. The biological and eco- nomic aspects of these alternatives are compared. LINKING PUBLIC AND PRIVATE PARTNERS FOR RES- TORATION AQUACULTURE IN MARYLAND'S SEASIDE BAYS. D. W. Webster' and D. W. Meritt,^ 'University of Mary- land. Wye Research & Education Center. PC Box 169. Queen- stown MD 21658 and "Shellfish Aquaculture Specialist, Univer- sity of Maryland Center for Environmental Science. Horn Point Lab. Po Box 775, Cambridge, MD 21613. Traditional harvest of the bard clam (Menenaria ineicenaria) in Maryland's seaside bays utilizes the hydraulic escalator dredge. This method has raised concern among environmental groups due to its perceived impact upon eel grass [Zostera spp.) in shallow estuarine waters. Meanwhile, an increase in demand for hard clams and strong wholesale prices have caused many clammers who normally harvest softshell clams (Mya arenaria) in the Chesa- peake Bay to shift harvest to the seaside bays, placing increasing pressure on that area. Hard clam aquaculture is well known and may provide an alternative to harvesters, who will likely come under increasingly restrictive regulations. Differences in culture methods for the clam have evolved in many states to take advan- tage of local conditions. In Maryland, the clam aquaculture indus- try has only recently begun, with few persons currently investing in the technology to produce these animals through husbandry. During 1998, investigations were begun with cooperation from university and private companies to develop seed production and evaluate grow out techniques. The Maryland Industrial Partnership (MIPS) program has funded development of a hard clam nursery/ grow out operation. University of Maryland Sea Grant Program (UMSG) funded a survey of the hard clam disease QPX to assess background levels of this potential problem. University of Mary- land Cooperative Extension (UMCE) funded an extension project to assess grow out techniques, as well as conduct outreach educa- tional programs designed to bring the technology to those who can use it. These cooperative studies are described as well as future directions for the project. PRODUCTION OF DISEASE-FREE OYSTER SEED USING SHALLOW WATER NURSERIES IN THE MID- CHESAPEAKE BAY. D. W. Meritt and S. Tobash, University of Maryland, Center for Environmental Science. Horn Point Labo- ratory, Po Box 775, Cambridge. MD 2161.^. Recently in Maryland, there has been an emphasis on the pro- duction of disease-free oyster spat for use in oyster restoration. Spat produced using traditional methods utilize sites where oyster parasites are common and are typically infected at the time of relay to the grow-out site. As part of the Action Plan for Oyster Recov- ery in Maryland, the Maryland Oyster Roundtable established oys- ter recovery zones in several major tributaries of Chesapeake Bay into which only disease-free oysters can be introduced. Given the problem with producing disease-free oyster .seed using natural methods, hatcheries have been employed for disease-free seed pro- duction. Since 1994, the University of Maryland's Horn Point hatchery has produced over 90 broods of spat using in-water nurs- ery systems. Dermo, the disease caused by the parasite Perkinsus marinus, is of greatest concern in these systems due to low salini- ties. Only one brood of spat has tested positive for Dermo since 1994. Based on trials conducted over the past six years, we have demonstrated that it is not only possible but also likely that unin- fected oyster seed can be produced using hatcheries and shallow- water nursery systems. Disease-free seed are being used to test the idea that by prohibiting the movement of parasites into upstream portions of the oyster producing rivers, dermo will be naturally purged from oyster populations in those regions. Early data sug- gest that there is some validity to this concept. A MULTIFACTORIAL APPROACH FOR DESCRIBING THE RELATIONSHIP BETWEEN THE CLASSIFICATION OF SHELLFISH HARVESTING WATERS AND ADJA- CENT LAND USE IN MURRELLS AND NORTH INLET, SOUTH CAROLINA. K. A. Nelson and G. I. Scott. National Ocean Service. 218 Fort Johnson Road. Charieston. SC 29412. Urbanization poses a particular threat to the coastal areas of the southeastern United State, where the lands surrounding the wet- lands are still relatively undeveloped compared with other regions. Fecal coliforms. including Escherichia coli. are important indica- tors of public health since human and/or animal feces may come in contact with and contaminate drinking water supplies or filter- feeding shellfish. The measurement of the concentration of fecal coliforms is the current criterion for deciding when and if shellfish harvesting should be approved. Predictive models that would cor- relate information on land use change and development would be useful so that downgrades in water quality can be predicted before 902 Ahslrculs. December 2002 Shellfish. Restoration. Hilton Head Island. SC they occur. The approach used for this study involved an historical comparison of land use change and fecal coliforni bacterial den- sities on Murrells Inlet (Ml) (urbanized site) and North Inlet (Nl) (pristine site). Both MI and NI are bar-built estuaries are located on the northern coast of South Carolina near Myrtle Beach. The mi- crobiological and water quality data used in this research covers the period of 1967-1995 and the following parameters were uti- lized: date of sampling, most probable number of fecal coliforni bacteria, salinity, rainfall, and water temperature. The regressions models utilized the above parameters and a change in trend term that accounted for both instantaneous and gradual changes in water quality that may arise from a particular intervention. For MI, the 1980 intervention consisted of both the construction of a jetty and the conversion from septic tanks to a main sewer line. For NI, the 1973 intervention was the construction of Baruch Laboratory. For MI, the intervention, controlling for other parameters, was found to be significant at the alpha = 0.05 level. This means that there was a significant decrease in the increasing trend of bacteria for MI and that the conversion to the sewage line had a beneficial effect on water quality and probably dominated the jetty effect. For Nl, the laboratory construction had no impact on water quality so back- ground natural sources of bacteria probably dominated the small increase from human sources. These findings indicate that the use of Intervention Analysis may provide coastal managers with an effective process to evaluate landscape changes on bacterial water quality in shellfish harvesting areas. INFLUENCE OF OYSTER REEF STRUCTURE ON FISH ASSEMBLAGES: DOES THE PLACEMENT OF ARTIFI- CIAL SUBSTRATE ENHANCE FISH POPULATIONS? J. A. Nestlerode.' M. W. Luckenbach,- P. G. Ross,^ and F. X. O'Beirn," 'Virginia Institute of Marine Science, College of Wil- liam & Mary, Gloucester Point, VA 2.3062, "Virginia Institute of Marine Science, College of William & Mary, Wachapreague, VA 23480. The objectives of oyster reef habitat restoration within Chesa- peake Bay are not only the enhancement of oyster stocks but also to restore the physical structure and ecological function of these systems. We revisit the artificial reef fisheries attraction vs. pro- duction issue by comparing transient nekton community structure among reef structures constructed of different substrates. The sub- strate materials (oyster shell, surf clam shell, and pelletized coal ash) used in this study offer the opportunity to examine how habi- tats with various degrees of structural complexity contribute to differences in habitat u.se in terms of microhabitat availability, predation risk, and diversity and abundance of prey. Results indi- cate that oyster shell reefs, which have best supported the devel- opment of an oyster population, offer the highest degree of struc- tural complexity and support a more diverse community of both resident and transient nekton. Furthermore, increased availability of nesting sites (empty articulated oyster shells) and a high abun- dance of benthic prey items support the thesis of increased fish production rather than simply attracting t"ish to the reef structure from nearby habitats. The patterns observed here provide evidence that proper reef architecture and subsequent reef community de- velopment lead to increa.sed finfish production and should give context to the importance of substrate selection in similar restora- tion activities. SHELLFISH RESTORATION IN IRELAND: THE NEED FOR NOVEL PARTNERSHIPS. M. Norman, Taighde Mara Teoranta (Marine Research Limited), Cama Co.. Galway Ireland. Historically shellfish restoration projects in Ireland have been undertaken for a singular reason and driven by a single user group. Frequently, the goal has been increased commercial production of shellfish, and the restoration has been pursued by a grouping of fishermen or aquaculturists. The.se projects have a narrowly de- fined objective, profitability, and frequently "fail" when they do not achieve this. However, some recent restoration attempts have been performed by partnerships. These projects are driven by mul- tiple user groups and thus have a range of goals. It is postulated that this partnership approach has more chance of success as the partners support each other in "staying the course" and as the goals are broader, makinc success more achievable. REPRODUCTIVE BIOLOGY OF THE NORTHERN QUA- HAUG, MERCENARIA MERCENARIA, IN PRINCE ED- WARD ISLAND, CANADA. M. Ouellette, M. Hardy, T. Lan- dry, N. G. MacNair, and A. Boghen, Department of Fisheries and Oceans, Science Branch, Gulf Fisheries Centre, P.O. Box 5030, Moncton. New Brunswick, EIC 9B6; Department De Biologie, Universite De Moncton, Moncton. Nouveau-Brunswick, ElA 3E9; and Department of Fisheries and Tourism, PC Box 2000, Char- lottetown. Prince Edward Island. CIA 7N8. Canada. The northern quahaug, Mercenuria mercenaria. is an important species for both the commercial and recreational fisheries as well as for aquaculture purposes in Prince Edward Island. The manage- ment strategy of the quahaug resource is largely based on the minimum legal size of 50 mm. At the same time, there is a growing concern regarding the sustainability of the clam industry and hence, an evolving interest in stock enhancement. Effective brood- stock management, however, requires basic information about the animal's reproductive biology. Sexual maturity, ovocyte size, go- nado-somatic ratios, and time of spawning were established for quahaugs sampled from two sites in West River, PEL Histological methods and physiological condition indices are used to determine of the spawning activities. Findings revealed that the minimum size at sexual maturity was 25 mm and 30 mm (shell length) for males and females respectively. Furthermore, there was a positive correlation between ovocyte size and shell length. Seasonal varia- tion coincided with spawning predictions based on conventional Shellfish. Restoration. Hilton Head Island. SC Ahsuactx. December 2002 903 physiological condition indices. As well, the gonado-somatic con- trihiition increased as a function of length. Both histological and condition index data support the likelihood that a major spawn occurs in mid-June. The study provides useful information on the reproductive biology of M. meixenaria and could contribute to- wards a reassessment of existing management and grow-out strat- egies. Establishment of reproductive sanctuaries is also being in- vestigated as a method to increase the annual recruitment success in this study bay. CONSIDERATIONS FOR OYSTER RESTORATION IN MARYLAND: DISEASE, GENETICS, DENSITY. REPRO- DUCTION, AND HABITAT CREATION. K. R. Paynter. .Jr.. Department of Biology. University of .Maryland. College Park. MD 20742. Over the last 2 years, we have conducted numerous experi- ments and monitored several State and Federally funded restora- tion projects in the Maryland portion of Chesapeake Bay. A sum- mary of the results of these activities will be presented. Field experiments have revealed that oyster seed cohorts from different broodstocks appear to have differing resistances to disease. Video- graphic observations from the field have shown that high-density oyster plantings result in significant community enhancement leading to diverse benthic ecosystems. Laboratory studies have shown that benthic fishes such as gobies and blennies prefer natu- ral clumps of oyster shell compared to equal volumes of loose oyster shell. In addition, other laboratory studies have shown that eggs introduced into the water column more than a few centimeters from introduced sperm will have little chance of becoming fertil- ized. However, other studies have shown that high densities of oysters (>400/m") may result in deleterious effects on oysters themselves. Many aspects of oysters and their ecosystem must be considered when planning restoration projects. Those projects seeking to restore ecological function should bear in mind the complex relationships between oysters, the habitat they create as biogenic reef builders, and the water column in which they reside. CULTURE TECHNIQUES APPLIED TO WILD BIVALVE BEDS IN GALICIA, NW SPAIN. P. J. Pazo, Delegacion Ter ritorial Conselleria De Pesca CI Palma 4. 36202 Vigo Spain. The region of Galicia is located in NW Spain. It has a coastline of 1.195 km. Galicia is the first producer of molluscs in Spain, taking advantage of natural oceanographic conditions: a seasonal upwelling and existence of positive estuary bays (Rias). Molluscs have been exploited in Galicia since prehistoric times. The present shellfishing situation is developed in two ways: the gathering of molluscs on foot, raking the substratum for macroinfaunal bivalves in the intertidal belt. The other type of shellfishing exploits the subtidal molluscs beds and involves the use of small boats. In order to maintain and enhance bivalve production in intertidal wild beds. a series of culture techniques are applied, acting on the bivalve population (lowering high densities, enlarging area beds, sowing and repopulating new areas), fighting against predators and com- petitors (starfishes, drilling gastropods), removing green algae of the bed surface to avoid deleterious effects in young bivalves, changing substrate granulometry by adding coarse sand to areas with mud and silt condition, and by other means. To act on the recruitment problems of two very valued species: butterfish clam {Riulitapes decussata) and European flat oyster (Ostrea edulis), a plan was established by the Fisheries Department of Galician Re- gional Government: "Plan Galicia". This Plan began in 1997 and was aimed to gradually transform traditional intertidal shellfish gathering into a professional activity by enhancing both the inter- tidal wild molluscan beds and the social organization of the mainly female population of shellfish gatherers. This Plan is presently developing and relevant achievements are being achieved, mainly in the social area. THE INFLUENCE OF ENVIRONMENTAL FACTORS IN .JAPANESE OYSTERS HEALTH CONDITION CULTI- VATED IN THE SOUTH OF PORTUGAL. A. L. Pereira,' F. A. Ruano.' L. Chicharo." and D. Matias.' 'IPIMAR, Reseaich Institute for Fisheries and Sea, Av. De Brasilia 1449-006 Lisbon, Portugal; "UCTRA. Algarve University, Campus De Gambelas, 8000-062 Faro, Portugal: and 'CRIPsul, South Research Center of Ipimar, Av. 5 De Outubro, 8500 Olhao, Portugal. The effect of environmental parameters as well as the organic contamination, heavy metals and tribulytlin (TBT) upon the de- velopment of diseases in cultivated Japanese oysters Crassostrea gigas (Thunberg, 1793) was studied over 6 months. The results were also related with the condition index, growth and mortality rates. The study was performed in two different sites on a coastal lagoon in the South of Portugal. One site ("Elisatnar") is located in a clean area whereas the other (Olhao). due to its proximity to urban areas and to an important fishing harbour, is exposed to higher contamination levels. In Olhao. nosological examinations showed the higher levels of lesions and the greatest incidence of parasites. Two ciliates. Ancistniin sp. and Tiichodina sp.. were the most abundant at the two areas. The intensity of the infections and the lesional picture observed at the two sites, didn't seem to affect significantly the condition, growth and mortality of the studied animals. However, the differences in the infection intensity regis- tered in both sites could be an indicator of a lack of defensive response from the individuals submitted to more intense stressful conditions. THE "EEEOHM" (EASTFIELDS' ENVIRONMENT EN- HANCING OYSTER HOLDING MODULE). P. Perina and D. Perina, Eastfields Farms, Box 275, Mathews, VA 23109. The "EEEOHM" (Eastfields' Environment Enhancing Oyster Holding Module) was originally developed to be used commer- 904 Abstracts. December 2002 Shellfish. Restoration. Hihon Head Island. SC cially, but the system is extremely versatile and is adaptable to a small garden size operation. The emphasis in developing the EEEOHM was on "Keep It Simple. Keep It Cheap." The EEEOHM module consists of three ADPI square-sided oyster cages (also called bags). Each cage has attached four 2-liter soda bottles for flotation. The cages are strung together with a 13-foot piece of crab pot rope or clothes line running laterally through their centers. The modules can also be separated into single floats as the needs of the grower dictate. The reason for using just three cages per module is simply "ease of handling". Whether employing the system from a dock or a skiff, it's a lot easier to detach and lift just three cages at a time than to struggle with maybe 40 or more cages all attached to a single rope. No heavy lifting. The EEEOHM can be floated under or along side of a dock, tied between posts, or deployed in rows secured to two parallel ropes anchored to the bottom at each end. The latter usually requires the use of a skiff. During the last 14 years, we at Eastfields have tried many ways of growing oysters. We've found the EEEOHM to be one of the most efficient and cost effective systems of off bottom culture. The ADPI cages last for many years, and the soda bottles are free. We at Eastfields Farms are proud to have developed the EEEOHM and would appreciate the opportunity to answer any questions concern- ing this environment enhancing system. A BIOCHEMICALLY-BASED MODEL OF THE GROWTH AND DEVELOPMENT OF CRASSOSTREA GIGAS LAR- VAE. E. A. Bochenek,' E. N. Powell,' J. M. Klinck." and E. E. Hofmann,' 'Haskin Shellfish Research Laboratory. Rutgers Uni- versity. Port Norris, NJ 08349 and "CCPO. Old Dominion Uni- versity. Norfolk, VA 23529. A biochemically-based model was developed to simulate the growth, development, and metamorphosis of larvae of the Pacific oyster. Crassostrea iiii;(is. This model, which is the first of its type, defines larvae in terms of their gross biochemical compositiim; protein, neutral lipid, polar lipid, carbohydrate, and ash content. The model includes parameterizations for larval filtration, inges- tion, and respiration, which determine growth rate, and processes controlling larval mortality and metamorphosis. Changes in the initial ratios of protein, carbohydrate, neutral lipid, and polar lipid occur as the larva grows and in response to the biochemical com- position of available food. The model results show increased larval survival when low protein food sources are available. High-protein food sources result in insufficient carbohydrate and neutral lipid to cover metabolic costs and to permit successful metamorphosis. The result is lar\ ae that are unable to successfully complete meta- morphosis. Thus, food quality as well as food quantity appear to be primary controls on the ability of Crassostrea gigas larvae to reach the body condition needed for metamorphosis. Other simulations show that initial egg size (lipid content) controls the ability of the larva to sustain itself until it reaches a size where it can effectively filter and assimilate food. Large eggs produce larvae that are more able to withstand food-poor environments, suggesting that egg size variability may account for the range of larval sizes at which metamorphosis is attempted. A FISHERIES MODEL FOR MANAGING THE OYSTER FISHERY DURING TIMES OF DISEASE. J. M. Klinck.' E. N. Powell.- J. N. Kraeuter,- and S. E. Ford," 'CCPO. Old Do- minion University, Norfolk, Virginia 23529 and "Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, NJ 08349. In Delaware Bay. market-size oysters have been produced by moving oysters from the seedbeds to planted grounds in higher salinity, where oysters increased in size and meat condition. In 1989. the oyster disease Dermo became active on the planted grounds and lower .seedbeds. The increase in disease reduced the desirability (loss of shell resource from seed areas) and profitabil- ity (high mortality) for moving oysters into higher salinity areas. As such, oyster production has focused on two production schemes: ( 1 ) direct harvest of oysters produced on the lower seed- beds, essentially a wild fishery and (2) the transplantation of oys- ters from the mid-estuary seed beds to the lower estuary seedbeds. Transplanted oysters are then harvested after the meat condition improves. One of the consequences of these approaches is the need to estimate the allowable production from the seedbeds each year, which is equivalent to setting a yearly quota. It seems clear that present oyster populations are below pre-disease levels, and that continued high disease levels will prevent recovery to pre-disease levels. The presentation describes a model developed for the man- agement of fished oyster populations that lie over a salinity gra- dient and for which disease mortality is a controlling influence. We will present a review of the Delaware Bay stock assessment for 1998 and 1999. We will then describe a model developed to ad- dress management issues when B,„„ and K are not appropriate options and apply it to Delaware Bay oyster populations. SHELLFISH DATA MANAGEMENT AND REPORTING SYSTEM (SDMRS). J. Power' and D. B. Walker,^ 'Environ- ment Canada, 224 West Esplanade. North Vancouver. British Co- lumbia. V7M 3H7, Canada, and "E-Carta Services, 419 N 18th Ave. E, Duluth. MN 55812-1352. The objective of this work was to design a user- friendly inter- face to digital watershed maps and hydrographic charts, thematic layers such as clam harvesting areas, farms, shellfish leases, and closures, and databases relating to pollution sources, water quality sampling, shellfish growing areas, locations of marine mammals and seabirds, fish farms and weather, for Canada's West Coast. Such an interface will allow scientists in the shellfish program of Environment Canada, Pacific and Yukon Region to readily gen- erate data reports including maps detailing shellfish closures and shoreline assessments. The Shellfish Data Management and Re- porting System (SDMRS) is an ArcView CIS application linked to Shellfish. Restoration. Hilton Head Island. SC Abstracts, December 2002 905 an Access database, which requires no special knowledge of SQL queries of databases or GIS. From ArcView the user chooses the sector to be mapped and the additional area around the sector to display map information from drop down menus. The system then generates the appropriate base map by clipping out the underlying hvdrographic charts and/or watershed maps. Then the user selects the themes and data sets to add to the map. again by choosing from drop down menus. The system adds the themes, uses ODBC to query the database and retrieve the appropriate data sets, converts them to the proper projection, and adds them to the map. When completed the map is then exported to the report document. RESTORING OYSTER REEFS FOR FISH: ESTIMATING ENHANCED SECONDARY PRODUCTION OF RESTORED OYSTER REEFS. S. P. Powers. C. H. Peterson, and J. H. Grabowski, Institute of Marine Sciences. University of North Carolina at Chapel Hill. 34.^1 Arendell Street. Morehead City, NC 28557. The recognition that oyster reefs provide an important resource, but also provide habitat for a variety of other species, has encour- aged restoration of oyster reefs as a method to enhance production of finfish and shellfish within estuaries. Here, we describe an approach for determining the augmented secondary production of bottom areas that were sand/mud flats and restored to oyster reefs. First, through a review of published and unpublished studies, we compared densities of animals on oyster reefs to nearby areas without biogenic structure. We divided those species that showed enhanced densities on reefs into three groups: ( I ) species that recruited exclusively to reefs, (2) species that had higher recruit- ment to reefs, but still recruited and used non-structured habitats, and (3) species that showed higher aggregations around reefs, but were not limited by reef habitat based on diet and life-history analyses. For this first group, all production is attributed to the reef. Because some proportion of the enhanced density of species belonging to this second group would probably have recruited to other habitat, the production attributed to the reef is adjusted by a coefficient of reef-habitat exclusivity (CRE) that we developed using diet analysis and life history information. For the final group of animals, the reef only receives credit for that fraction of growth that is enhanced by the presence of the reef, this determination is made through the application of a CRE. Applying this approach to proposed oyster reef restoration in Tampa Bay, FL, we estimated the augmented secondary production of 10 m" of oyster reef to be 2.57 kg y-'. RESTORING THE LITTLENECK CLAM RESOURCE FOR NATIVE AMERICAN SUBSISTENCE USE IN THE PRINCE WILLIAM SOUND, ALASKA. R. RaLonde, Univer- sity of Alaska, School of Fisheries and Ocean Sciences, Anchor- age, AK 99508-4140. Natural and man caused disasters decimated the littleneck clam (Protnthaca stammea) populations on the intertidal beaches of Pruice William Sound. Alaska. Subsequently. Native American Villages have been unable to harvest clams to meet their subsis- tence needs. Since 1995. the Quteckak Native Corporation has been actively pursuing restoration of the clam populations by de- veloping seed production technology, conducting site selection studies, and managing growout trials. The initial success of their restoration efforts now enables communities to harvest clams from the restored beaches and broaden the program. This presentation will describe the research and restoration results of the project and the human impact of the restored resource on the Native Villages of Prince William Sound. Alaska. DEVELOPMENT OF CRASSOSTREA VIRGINICA MICRO- SATELLITE MARKERS FOR A GENETIC LINKAGE MAP AND GENETIC MONITORING OF RESTORATION PRO- JECTS. K. M. Reece, VV. L. Ribeiro. K. L. Hudson, and S. K. Allen Jr., Virginia Institute of Marine Science, The College of William and Mary. Gloucester Point, VA 23062. Dermo and MSX have had significant impacts on natural popu- lations of the eastern oyster Crassostrea vir}>inica and have been a detriment to oyster aquaculture development. A potential solution to this problem is genetically improved disease-resistant strains ot C. virginica that can grow to market size despite disease challenge. Traditional selective breeding programs have resulted in strains of oysters that are being assessed for disease resistance. One means of accelerating selective breeding programs is to identify genetic markers associated with traits such as disease resistance or growth rate. A goal of this project is to develop genetic markers for con- structing a linkage map and to identify markers associated with disease resistance to use in marker-assisted selection programs. In addition, markers developed in the genomics project are being surveyed for use in genetic monitoring of reef restoration projects. To assess relative genetic contributions of wild and planted stocks to restored reefs; markers are being identified that are able to genetically distinguish selected strains and nattiral populations in Chesapeake Bay. C. virginica microsatellite markers are being developed in an ODRP funded genome mapping project. Several di-, tri-, and tetranucleotide repeat sequences have been identified. Primers for use in the polymerase chain reaction have been de- signed to anneal to regions flanking 39 microsatellites and ampli- fication reactions for 21 loci have been optimized. Fl individuals from four reference families have been screened at twelve micro- satellite loci for generating a genetic linkage map. Microsatellite allelic profiles of selected strains and natural populations are being examined. OYSTER REEF RESTORATION RESEARCH IN MOBILE BAY, ALABAMA. D. B. Rouse,' R. K. Wallace,- and F. S. Rikard,^ 'Department of Fisheries and Allied Aquacultures, Au- burn University. Auburn. AL 36849 and "Mobile, AL 36615. Oyster reef restoration in Mobile Bay has consisted primarily of shell planting on active reefs in the lower sections of the bay. 906 Ahstmcts. December 2002 Shellfish, Restoration, Hilton Head Island, SC Efforts are now underway to restore reefs in the mid-bay area. Studies are being conducted to determine why these reefs are no longer productive and what should be done to restore them to a productive state. Bottom surveys were performed to quantify cultch availability. Sediment traps were deployed to determine rates of sediment accretion and spat collectors were used to evalu- ate natural oyster set. Spat were deployed on the bottom and on platforms 20 cm and 40 cm above bottom. Data loggers were deployed on bottom and 40 cm above bottom to measure tempera- ture, salinity and oxygen concentration. Surveys revealed hard bottoms but little exposed cultch on non-productive reefs. Sedi- mentation was high and consisted mainly of silt with more than 10% organic matter. Single peak oyster sets occurred in the fall. Oysters at the three experimental levels grew to approximately 60 mm in the first year. During the second year, total mortality was observed at all three levels when oxygen levels dropped to 0 mg/L for 3 consecutive days. Similar periodic low oxygen events may be occurring at the study site and on other relic reefs that will hinder their successful restoration. Water quality studies suggest that cultch mounding will be necessary to elevate oysters above anoxic bottom conditions. TRANSPLANTING BROODSTOCK OYSTERS, CRASSOS- TREA VIRGINICA, ONTO RECONSTRUCTED OYSTER REEFS TO INCREASE SPAT RECRUITMENT IN THE PIANKATANK RIVER. D. C. Sherwood, Sandston. VA 2,^150. Virginia oyster reef restoration in the form of three- dimensional structures began in the Piankatank River. Virginia in 1993. From 1993 to 1998. fifteen artificial reefs were built in tributaries of the Chesapeake Bay. In December 1996. reproduc- tively active broodstock oyster from Tangier and Pocomoke Sounds were transplanted to the Shell Bar reef in the Great Wicomico River. Virginia. Surveys in the fall of 1997 indicated a high spat recruitment both on the reefs and nearby oyster bars. With this success, broodstock oysters from Tangier and Pocomoke Sounds were transplanted in December 1997 to two of the four reconstructed reefs in the Piankatank River. Since total quantity as well as density of broodstock was believed to be limiting factors for recruitment in this river, adding stock was expected to raise spat recruitment. Spat recruitment data was collected via dive sur- veys on the reefs, and via dredge and patent long surveys on the natural oyster bars. The recruitment of spat to both reefs and bars was significantly higher in 1998 (P < 0.001 ) than in the previous four years and a positive interaction {P < O.OO.'i) was seen between the reefs and the year 1998. Based on these data, stock enhance- ments in the Piankatank River successfully improved recruitment and suggest oyster restoration may be facilitated in other areas of the Chesapeake Bay by strategic enhancement ol spawning stocks. HARMFUL ALGAL BLOOMS AND SHELLFISH RESTO- RATION: CAN THEY CO-EXIST? S. E. Shumwav. Depart ment of Marine Sciences. University of Connecticut, 1080 Shen- necossett Road, Groton, CT 06340 Habitat quality is an obvious and important consideration for all shellfish restoration efforts including aquaculture and reseed- ing. Harmful algal blooms (HABs). worldwide threats to habitat quality, are naturally occurring phenomena and their number and frequency are increasing. These blooms impact ecosystem integ- rity, species interactions, aquatic animal health, population growth, hinnan health, economy, industry, and ecology and often pose a threat to wild and cultured shellfish populations. While algal spe- cies that impact human health receive the most attention, there are numerous HABs that cause the destruction and demise of shellfish beds and aquaculture operations. These HABs can also dictate the successful citing of restoration efforts. This presentation will re- view our knowledge of harmful algal-shellfish interactions world- wide including threats associated with the presence of recently identified problem species, e.g., Pfwsleria spp. and Helerocapsa. and discuss ways in which shellfish restoration efforts may be undertaken successfully in the face of these imposing threats. Miti- gation of these HABs has become an important focus for coastal research. Data will be presented on recent studies on the use of clay as a means of alleviating the impacts of HABs. Careful man- agement-science interaction, not eradication, remains the only safe and functional means of mitigation currently available. A COMPARISON OF TWO OYSTER {CRASSOSTREA VIR- GINICA ) STRAINS FOR PRODUCTIVITY AND SUITABIL- ITY FOR USE IN OYSTER REEF Rt;STORATION EF- FORTS. L. A. Sorabella,' M. W. Luckenbach," and F. X. O'Beirn," Virginia Institute of Marine Science. College of Will- iam and Mary. P.O. Box 1346. Gloucester Point. VA 23062. Vir- ginia Institute of Marine Science, College of William and Mary. P.O. Box 350, Wachapreague, VA 23480. Over the past ,5 years, eastern oyster (Cnisso.strt'u virgiiuca) restoration efforts in Virginia have focused on constructing reef structures to act as sanctuaries. Increasingly, shell plants are stocked with hatchery-produced brood stock oysters that spawn and increase recruitment to the reefs. This involves rearing hatch- ery-produced seed oysters in floating containers and out-planting them onto reefs when they reach an appropriate size (20-30 mm). To assist with the labor involved in this process, we recruited citizen and student oyster-gardeners who raised the bulk of the oysters for transplant onto the sanctuaries. Two strains were hatch- ery-reared for transplantation onto these reefs: CROSBreed oysters and wild-caught oysters from the lower Chesapeake Bay. The CROSBreed strain has been selectively bred since 1962 for resis- tance to the parasite Haplosporiduium nelsoni that causes MSX. and has more recently been bred for resistance to Perkinsus mari- nus. that causes Dermo. The second strain were large wild-caught Shellfish. Restoration. Hilton Head Island. SC Abstracts. December 2002 907 oysters collected from the Lynnhaven River (Chesapeake Bay, VA) where the oysters were surviving under presumed high pres- sure from both parasites. The objective of this research is to evalu- ate the performance of the CROSBreed stock and wild stock oys- ters deployed on sanctuary reefs in the Lafayette River (Chesa- peake Bay, VA). Evaluation compares the two strains based on female fecundity, growth, survival, and incidence of Perkinsus marinus and Haplosporiduium nelsoni infection measurements. As large-scale restoration projects proceed, it will be fundamentally important to assess which stock is most appropriate for use in the oyster restoration effort. OY.STER RESTORATION AND THE UNIVERSITY OF MARYLAND: INTERACTIONS BETWEEN RESEARCH, INDUSTRY, AND THE PUBLIC. S. M. Tobash and D. W. Meritt, University of Maryland. Center for Environmental Sci- ence, Horn Point Laboratory. PC Bo.\ 775. Cambridge. MD 21613. Maryland oyster populations have been declining for many years. The LIniversity of Maryland is committed in its involvement to assist in restoration programs, placing an emphasis on the eco- logical contribution that oysters provide to the overall health of the Chesapeake Bay. Research efforts are focused on diseases, genet- ics, culturing techniques and basic oyster biology and ecology. The efforts of the Maryland Oyster Recovery Partnership and other concerned groups, along with increasing public involvement has enabled the oyster hatchery at the University of Maryland Center for Environinental Science Horn Point Laboratory to increase pro- duction of disease-free hatchery seed. These seed oysters are then used in a variety of restoration, education, and outreach activities statewide. Educational programs are targeted to a wide audience, ranging from nonprofit organizations of school-aged children to commercial watermen. Outreach projects promote the involvement of concerned citizens like the Chesapeake Bay Foundation's Oys- ter Gardening Program. Cooperation between commercial fisher- men, management agencies, and concerned groups and combina- tion of resources are key to a successful restoration program. The University of Maryland represents an important link in this pro- cess. The ongoing commitment of UMCES Horn Point Laboratory together with cooperative programs will continue to strengthen the future of the Chesapeake Bay oyster restoration effort. EFEECTS OF WATERSHED ALTERATIONS ON OYSTER POPULATIONS IN SOUTHWEST FLORIDA ESTUARIES: AN ECOLOGICAL APPROACH. A. K. Volety, M. Savarese, and S. G. Tolley, College of Arts and Science. Florida Gulf Coast University. 10501 Fgcu Boulevard South, Fort Myers. FL 33965. Southwest Florida is one of the country's fastest growing re- gions. Consequently, watersheds are heavily managed to accom- modate development. Studies on the effects of altered watershed in this region involving valued ecosystem components, like oysters. are lacking, but clearly necessary. Using the oyster, Crassostrea virfiiuica. as an indicator species, we are investigating ecosystem- wide health effects of watershed management practices in altered (Faka-Union. Henderson Creek, and Caloosahatchee River) and pristine (Blackwater River) estuaries. Measurements of oyster spa- tial distribution, condition index, spat recruitment, energy reserves, and disease prevalence of Perkinsus imiriiuis are underway using a "spatial homologue approach" (among-estuary comparisons at hydrologically and geoniorphically similar locations along the sa- linity gradient). Preliminary results indicate that in summer months, depending on the location, mean prevalence of P. iiuinmis infection in oysters varied between 33-73%, whereas the mean condition index varied between 2.4-4.7. The distribution of reefs, regions of maximum living density, and maximum oyster produc- tivity are shifted seaward in altered relative to pristine systems when water management practices are supplying excessive fresh- water to estuaries. These populations, however, exhibit lower P. iiHiriiiKs prevalence because of the greater freshwater influence. This project represents the first study of watershed alteration on oysters in Southwest Florida and will help provide target environ- mental conditions for restoration efforts. THE VIRGINIA OYSTER HERITAGE PROGRAM. J. A. Wesson' and L. B. McKay," 'Virginia Marine Resources Com- mission. 2600 Washington Ave.. 3rd Floor. Newport News. VA 23607-0756 and "Virginia Department of Environmental Quality, 629 E. Main St., Richmond, VA 23219. The Virginia Oyster Heritage Program (VOHP) is a bold ini- tiative to capitalize on recent advances and consensus on strategies for oyster restoration. The VOHP seeks to re.store oyster popula- tions and oyster reef habitat, and to. thereby, improve ecological function, water quality and the oyster industry in Virginia's portion of Chesapeake Bay and its seaside bays. Initiated in 1999 by the Department of Environmental Quality and Marine Resources Commission, the VOHP is a partnership of Federal. State, and private entities. Phase One focuses on the Lower Rappahannock River, with a goal of rebuilding 8 to 10, three-diinensional, oyster sanctuary reefs and restoration of more than 200 acres of oyster beds for direct harvest. The combination of funding from all sources totaled more than ,$1,500,000 in the first year, resulting in six reefs constructed, and 85 acres of harvest area restored. The Lower Rappahannock River, closed to harvest for six years, is an area of high salinity with consistent exposure to oyster diseases. Annual monitoring during this closure demonstrated that a signifi- cant proportion of the native oysters survived and grew, and that spatset became dependable. We believe that the lack of cultch has limited the increase in oyster populations and that enough natural broodstock has accunuilated to colonize these restored areas. With proper management of sanctuary and harvest areas, we also believe that a sustainable fishery can be established while simultaneously 908 Abstracts. December 2002 Shellfish. Restoralion. Hilton Heud Island. SC increasing the standing stock of oysters. We anticipate comparable ties for shellfishemien as well as other marine-based job opportu- funding in 2001. and the expansion of this model into other areas. nities. ENHANCING AND SUSTAINING NORTH SHORE SHELL- FISHING THROUGH AQUACULTURE. J. J. Whitten, Mer rimack Valley Planning Commission. 160 Main .Street, Haverhill. MA 01830. Soft-shell clamming has long been a vital commercial fishery on the North Shore of Massachusetts. Earlier in the century, the Merrimack River Estuary alone produced up to 100.000 bushels of soft-shell clams per year ( among the highest of shellfish harvesting communities on the East Coast). During the past decade, the fish- ery has seen significant declines and wide fluctuations in produc- tivity. Landings have plummeted as much as 809r along the North Shore. The dramatic downturn in this once prominent industry is due to a combination of factors such us: natural "boom-and-bust" reproduction cycles, predation from non-native species such as the green crab, over harvesting, as well as regulatory closures of pre- viously undocumented contaminated areas. The wide, unpredict- able fluctuations in bed producti\ ity make it impossible to ensure a sustainable and predictable harvest. The seeding of Massachu- setts' North Shore shellfish beds with hatchery-reared soft-shell clams offers the potential to moderate this cyclical and declining nature of productivity. Efforts to improve shellfishing through res- toration/enhancement efforts in other states ha\e demonstrated several methods by which a more consistent and sustainable har- vest can be achieved. This presentation examines regional efforts conducted by the Merrimack Valley Planning Commission (MVPC) in collaboration with the coastal communities of Glouc- ester. Ipswich and Rowley Massachusetts to enhance and sustain soft-shell clam (M\a arenana) populations. Ultimately a more sustainable harvest provides more secure employment opportuni- COMMUNITY-BASED OYSTER RESTORATION IN AN URBAN ESTUARY: DEVELOPING AN OYSTER CUL- TURE AND REEF RESTORATION PROGRAM IN THE HUDSON-RARITAN ESTUARY. A. Willner. NY/NJ Bay keeper. Building 18, Sandy Hook, Highlands. NJ 07732. Goals, progress, and challenges to development of a restoration program led by a nonprofit organization in an urban estuary will be discussed. The Hudson-Raritan Estuary supports a sparse oyster population that has collapsed since the beginning of the 1900s. Major natural oyster beds were exhausted by commercial shell- fishing in the eariy I800"s. since the closure of the industry in the 1920s the population has continued to decline to the current state of small groupings of individuals located in disparate reaches of the system. Prompted by the success of oyster restoration in other estuaries, NY/NJ Baykeeper, with guidance from a scientific ad- visory board, has begun to investigate the feasibility of restoring reefs to provide habitat for larval settlement and population recov- ery. Spat surveys conducted in 1998 demonstrating minimal re- cruitment, lead to the 1999 experiment of placing a large amount of shell on the historic footprint of an oyster population in New York Harbor to provide a greater area to increase probability of recruitment. Preliminary results demonstrate the potential for reef construction to increase oyster recruitment. To stock the reef in an effort to further improve recruitment success, a community-based oyster-culturing program has recently been established involving schools, marinas, civic groups and families. Oyster restoration in estuaries with pollution concerns such as this provide the oppor- tunity to investigate factors affecting population recruitment and maintenance as well as challenges to public education and out- reach in urban areas. THE NATIONAL SHELLFISHERIES ASSOCIATION The National Shellfisheries Association (NSA) is an international organization of scientists, manage- ment officials and members of industry that is deeply concerned and dedicated to the formulation of ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man- asement of shellfish resources. The Association has a membership of more than 1000 from ail parts of the USA. Canada and 18 other nations; the Association strongly encourages graduate students' mem- bership and participation. WHAT DOES IT DO? — Sponsors an annual scientific conference. — Publishes the peer-reviewed Joimuil of Shellfish Research. — Produces a Quarterly Newsletter. — Interacts with other associations and industry. WHAT CAN IT DO FOR YOU? — You will meet kindred scientists, managers and industr>' officials at annual meetings. — You will get peer review through presentation of papers at the annual meeting. — If you are young, you will benefit from the experience of your elders. — If you are an elder, you will be rejuvenated by the fresh ideas of youth. — If you are a student, you will make useful contacts for your job search. — If you are a potential employer, you will meet promising young people. — You will receive a scientific journal containing important research articles. You will receive a Quarterly Newsletter providing information on the Association and its activities, a book review section, information on other societies and their meetings, a job placement section, etc. HOW TO JOIN —Fill out and mail a copy of the application blank below. The dues are 65 US $ per year ($35 for students) and that includes the Journal and the Newsletter! NATIONAL SHELLFISHERIES ASSOCIATION-APPLICATION FOR MEMBERSHIP (NEW MEMBERS ONLY) Name: __^_ For the calendar year: Date: Mailing address: -^ Institutional affiliation, if any: . Shellfishery interests: Regular or student membership: Student members only — advisor's signature REQUIRED: Make checks {MUST be drawn on a US bank), international postal money orders or VISA for $65 ($35 for smdents with advisor's signature) payable to the National Shellfisheries Association and send to Nancy Lewis, Bookkeeper, PO Box 350. V.I. M.S. Eastern Shore Lab. Wachapreague. VA 23480. USA. INFORMATION FOR CONTRIBUTORS TO THE JOURNAL OF SHELLFISH RESEARCH Original articles dealing with all aspects of shellfish re- search will be considered for publication. Manuscripts will be judged by the editors or other competent reviewers, or both, on the basis of originality, content, merit, clarity of presentation. and interpretations. Each article should be carefully prepared in the style followed in prior issues of the Journal of Shellfish Research before submission to the Editor. Papers published or to be published in other journals are not acceptable. Title, Short Title. Key Words, Abstract: The title of the paper should be kept as short as possible. Please include a "short running title" of not more than 48 characters including spaces, and key words. Each manuscript must be accompanied by a concise, informative abstract, giving the main results of the research reported. The abstract will be published at the beginning of the article. No separate summary should be in- cluded. Text: Manuscripts must be typed double-spaced throughout on one side of the paper, leaving ample margins, with the pages numbered consecutively. Scientific names of species should be underlined or in italics and, when first mentioned in the text, should be followed by the authority. Common and scientific names of organisms should be in accordance with American Fisheries Society Special Publications 16 and 17; Common and Scientific Names of Aquatic Invertebrates from the United States and Canada: Mollusks and CSNAIUSC: Decapod Crus- taceans, or relevant publications for other geographic regions. Abbreviations, Style, Numbers: Authors should follow the style recommended by the sixth edition ( 1994) of the Council of Biology Editors [CBEJ Style Manual, distributed by the American Institute of Biological Sciences. All linear measure- ments, weights, and \olumes should be given in metric units. Tables: Tables, numbered in Arabic, should be on separate pages with a concise title at the top. Illustrations: Line drawings should be in black ink or laser print and planned so that important details will be clear after reduction to page size or less. No drawing should be so large that it must be reduced to less than one third of its original size. Photographs and line drawings should be prepared so they can be reduced to a size no greater than 17..^ cm x 22.7 cm, and should be planned either to occupy the full width of 17.3 cm or the width of one coliniin, 8.4 cm. Photographs should be glossy with good contrast and should be prepared so they can be reproduced without reduction. Originals of graphic materials (i.e., line drawings) are prefened and will be returned to the author. Each illustration should have the author's name, short paper title, and figure number on the back. Figure legends should be typed on separate sheets and numbered in Arabic. Digital Figures: Authors may provide digital figures (they are not required); they must be accompanied by hardcopy fig- ures of equal quality, which the printer will use for comparison and backup. If digital figures are supplied, please note the following instructions; • Each piece of art should be saved as its own file. • Files must be one of the following fonnats; TIF, EPS, or JPG. • Each file should be named according to its figure number and format (e.g., ■■fig2b.tif"). • Figures must not be embedded in a word-processor or spreadsheet document; the printer cannot use images stored in Word. WordPerfect. Excel. Poweipoint, etc. • Resolution; line shots: 1000 dpi; halftones/grayscales: 300 dpi if no lettering, 500 dpi if figure contains lettering. • Color figures; save the files as CMYK-encoded TIF images (preferred) or CMYK-encoded EPS or JPG images. Color figures have the same resolution requirements a BAV. above. Color illustrations will not be accepted unless the author agrees to cover the cost of associated reproduction and printing. Literature Cited: References should be listed alphabeti- cally at the end of the article. Abbreviations in this section should be those recommended in the American Standard for Periodical Title Abbreviations, available through the American National Standard Institute, 1430 Broadway, New York. NY 1 00 1 8. For appropriate citation format, see examples below; Journal: Watts, R. J., M. S. Johnson & R. Black. 1990. Effects of re- cruitment on genetic patchiness in the urchin Echinonietra mathaei in Western Australia. Mar. Biol. 105:145-151. Book: Claudi, R. & G. L. Mackie. 1994. Practical manual for Zebra Mussel monitoring and control. Boca Raton. FL; CRC Press. 227 pp. Chapter in Edited Book: Davio, S. R., J. F. Hewetson & J. E. Beheler. 1985. Progress toward the development of monoclonal antibodies to saxitoxin; antigen preparation and antibody detection. In: D. M. Ander- son, A. W. White & D. G. Baden, editors. Toxic dinoflagel- lates. Amsterdam; Elsevier, pp. 343-348. Page Charges: Authors or their institutions will be charged $100.00 per printed page. All page charges are subject to change without notice. A handling fee of $50 will be charged for all manuscripts accepted for publication. Proofs: Page proofs are sent to the corresponding author and must be corrected and returned within seven days. Alter- ations other than corrections of printer's errors may be charged to the author(s). Reprints: Reprints of published papers are available at cost to the authors. Information regarding ordering reprints will be available from The Sheridan Press at the time of printing. Cover Photographs: Appropriate photographs may be sub- mitted for consideration for use on the cover of the Journal of Shellfish Research. Black and while photographs and color illustrations will be considered. Corresponding: An original and two copies of each manu- script submitted for publication consideration should be sent to the Editor, Dr. Sandra E. Shumway, Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Rd., Groton, CT 06340. E-mail; sandra.shiunw ay@uconn.edu or sandrashumway@hotmail.com Membership information may be obtained from the Editor or the Treasurer using the form in the Jounud. Institutional subscribers should send requests to: Jounud of Shellfish Re- .search. P.O. Box 465. Hanover, PA 17331. Sergio Ragonese, Patrizia Jereb. and Earl Dawe A comparison of growth performance across the squid genus Ille\ (Cephalopoda, ommastrephidae) based on modelHng weight-at-length and age data 85 1 Chandrika Liyana-Pathirana, Fereidoon Shahidi, and Alan Wliittick Comparison of nutrient composition of gonads and coelomic fluid of green sea urchin Strongylocentroliis drnebachiensis °6 1 B. R. Moore, S. N. Kleeman, and R. J. G. Lester The development of a positive non-infectious control for the detection of Perkiiisii\ using the Ray test 87 1 S. J. Nichols and D. Garling Evaluation of substitute diets for live algae in the captive maintenance of adult and subadult freshwater unionidae 875 Abstracts of technical papers presented at the 4th International Conference on Shellfish Restoration. Hilton Head Island. South Carolina. November 1 5- 1 8. 2000 883 COVER PHOTO: Red crab {Gennii qmnquc dens) are found in deep water off of the coast of North America. These crabs were caught 140 miles south of New England aboard the Duimoiut Girl. Photo by Richard W. Dionne. Jr. with special thanks to the Sakonnet Times. The Journal of Shellfish Research is indexed in the following: Science Citation Index*. Sci Search®. Research Alert^. Current Contents«/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review. Environmental Periodicals Bibliography. Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts. Patrick Rice, Sammy M. Ray. Sherry D. Painter, and Gregg T. \'agle An intrinsic membrane protein in oyster sperm stimulates spawning behaviors in Crassostrea virtiiiiica: implications tor aquacultiire 715 Qiaoxiang Dong, Benoit Eiideline, Standish K. Allen, Jr., and Terrence R. Tiersch Factors affecting sperm motility of tetraploid Pacific oysters 719 Amy D. Nickens, Jerome F. La Peyre, Eric S. Wagner, and Terrence R. Tiersch An impro\ ed procedure to count Perkinsiis imiriiuis in Eastern oyster hemolymph 725 Stephen J. Jordan. Kelly N. Greenhawk. Carol B. McCollough. Jessica Vanisko, and Mark L. Homer Oyster biomass. abundance, and harvest in northern Chesapeake Bay: trends and forecasts 733 Andrew G. Jeffs. B. J. Dunphy, and R. M. G. Wells Experimental effects of water temperature on the gametogenic development of broodstock in the oyster, Oslreci cliilensis 743 Raymond E. Grizzle. Jamie R. Adams, and Linda J. Walters Historical changes in intertidal oyster iCnissosrrea vir^inica) reefs in a Florida lagoon potentially related to boating activities 749 Sonia Rodriguez-Astiidillo. Marcial Villalejo-Fuerte. Federico Garcia-Dominguez. and Rafael Guerrero-Caballero Biochemical composition of Spi'iulyliis Iciicacaiithus Broderip, 1833 (Bivalbvia: Spondylidae) and its relationship with the reproductiv e cycle at Isla Dan/ante. Gulf of Calitbrnia. Mexico 757 Ana Bratos. Jaksa Bolotin. Melita Peharda. and Jakica Sjire Seasonal distribution of the oyster Osrrea cJitlis (Linnaeus. 1758) larvae in the Bay of Mali Ston. .Adriatic Sea 763 Alan J. Power. Ellie Covington. Todd Recicar. Randal L. Walker, and Nelson Filer Observations on the egg capsules and hatchlmgs of the knobbed whelk. Biisyccm carica (Gmelin. 1791 ) in coastal Georgia 769 Dario Savini. Juliana M. Harding, and Roger Mann Rapa whelk Rcijuiini veiuisa (Valenciennes. 1846) predation rates on hard clams Menenaria meneiuiria (Linnaeus. 1 758) 777 N. Chaitanawisuti. S. Kritsanapuntu. and Y. Natsukari Economic analysis of a pilot commercial hatchery-based operation for spotted babylon. Biihylmiia areolata Link 1 807. juveniles in Thailand 781 P. T. Gibson. D. G. Worthington. C. Blount, and N. L. Andrew Incidental damage of blacklip abalone (Halintis nibnt) by commercial divers in New South Wales, Australia 787 Meegan E. Vandepeer, Patrick W. Hone, Jon N. Havenhand, and Robert J. Van Barneveld The effect of nonnutritive fillers on the digestibility of a manufactured abalone diet 793 Meegan E. Vandepeer, Patrick W. Hone, Jon N. Havenhand, and Robert J. Van Barneveld The digestibility of whole and dehulled lupms [Liiphnis niii^KstifhUus) fed to juvenile greenlip abalone, Haliotis liievifiata 799 C. D. Hawkins and J. B. Jones Lar\ al escape through abalone culture eftluent systems: an analysis of the risk 805 Qi Li, Choulji Park, and Akihiro Kijima Isolation and characteriz-ation of microsatelhte loci in the Pacific abalone. Huluilis discus luiiiiuii 811 Carolyn S. Friedman, Wendy Biggs, Jeffrey D. Shields, and Ronald P. Hedrick Transmission of withering syndrome in black abalone. Haliolis ciculicnHlii leach 817 Ma. Del Carmen Alvarez Tinajero, Jorge Cdceres-Mariinez, and Jose Guadalupe Gonzales Aviles Histopathological evaluation of the yellow abalone Haliaris comtgahi and the blue abalone Haliotis fidgens from Baja California. Mexico 825 Maria Georgina Gluyas Milldn, Casimiro Quinonez Velazquez, and Jesiis Talavera Maya Effect of El Nino 1 997-98 on the snail Astniea iiiiddsa (Wood) population along the Baja California western coast — 83 1 Martha E. Sandoval Quintero and Adolfo Gracia Reproduction of the spotted pink shrimp. Faifuntepenaeus brasiliensis ( Decapoda: Penaeidae) 835 R. Rosa and M. L. Nunes Influence of the reproductive cycle on the biochemical composition of deep-sea decapod Parapenaeus longirostris (Lucas, 1 846) in the Portuguese .south coast 843 CONTENTS CONTINUED ON PREVIOUS PAGE Sizhong Wang, Peter F. Duncan, Wayne Knibb, and Bernard M. Degnan Byssal attachment of Amusimn hallolt (Bernardi. 1861 ) (Bivahia: Pectinidae) spat 563 Maite Narvarte and Marina Kroeck Intraspecific variation in the reproductive cycle of the tehuelche scallop Aeqiiipectcii tchuclchiis (Pelecypoda. Pectinidae). in San Mati'as Gulf. Patagonia. Argentina 571 G. Roman, M. J. Campos, J. Cano, C. P. Acosta, Pilar Iglesias, and Oscar Garcia Reproductive and reserve storage cycles in Aequipeclen opercularis (L.. 1758) in Galicia. NW Spain 577 Gonzalo Gajardo, Margarita Parraguez. and Nelson Colihueque Karyotype analysis and chromosome banding of the Chilean-Peruvian scallop Argopecten pitrpunitiis (Lamarck. 1819) 585 Mohsin U. Patwary, Akelia Waiichope, Timothy W. Short, and Edward J. Catapane Molecular cloning and characterization of a fructose- 1.6-biphosphale aldolase cDNA from the deep-sea scallop Placopecteii iiicigellaiiiciis 591 Rosali'o Maldonado-Amparo and Ana M. Ibarra Comparative analysis of oocyte type frequencies in diploid and triploid catarina scallop (Argopecten ventricosiis) as indicators of meiotic failure 597 Martin LaFrance, Helga Guderley, and Georges Cliche Low temperature, but not air exposure slows the recuperation of juvenile scallops. Placopecten mageUanicus. from exhausting escape responses 605 Jose L. Riieda and Aad C. Smaal Selecti\e ingestion of pelagic versus henlhic algae by the cockle Ceraslodenna ediilc (Linne. 1758) 619 Donald Brown, Bernardita Campos, and H.-Jiirg Urban Reproductive cycle of the bivalve clams Semele solida (Gray. 1828) (Semelidae) and Gari soliJa (Gray. 1828) (Psammobiidae) from Chile 627 A. L. Heck, Jr., L. D. Coen, and D. M. Wilson Growth of northern \Meixenaria lucirciiaria (L.)] and southern \M. amipechiensis (Gmelin)] quahogs; influence of seagrasses and latitude 635 C Tirado, C. Salas, and J. I. Lopez Reproduction of Calllsia cluone L., 1758 (Bivalvia: Veneridae) in the littoral of Malaga (southern Spain) 643 S. Novoa, D. Martinez, J- Ojea, P. SoudanI, J.-F. Samain, J. Moat, and J.-L. Rodriguez Ingestion, digestion, and assimilation of gelatin-acacia microcapsules incorporating deuterium-labeled arachidonic acid by larvae of the clam Venerupis piilltislrn 649 William S. Arnold, Dan C. Marelli, Melanie Parker, Philip Hoffman, Marc Frischer, and John Scarpa Enhancing hard clam {Meixeiuuia spp.) population density in the Indian River Lagoon. Florida: a comparison of strategies to maintain the commercial fishery 659 Stefan Aki Ragnarsson and Gudriin G. Thorarinsdottir Abundance of ocean quahog. Arclica isUindua. assessed by underwater photography and a hydraulic dredge 673 Stefano Peruzzi and Ximing Guo Tetraploid induction by meiosis inhibition with cytochalasin B in the dwarf surfclam. Mtdiiiia lateralis Say: effects of temperature 677 Alexandra Leitdo, Raqiiel Chaves, Sara Santos, Pierre Boudry, Henrique Guedes-Pinto, and Catherine Thiriot-Quievreiix Cytogenetic study of Oslrea coiiclniphllii (Mollusca: Bivalvia) and comparative karyological analysis within ostreinae 685 Eric N. Powell, Kathryn A. Ashton-Alcox, Joseph A. Dobarro, Meagan Cummings, and Sarah E. Banta The inherent efficiency of oyster dredges in survey mode 691 E. Kenchington, C. J. Bird, J. Osborne, and M. Reith Novel repeat elements in the nuclear ribosomal RNA operon of the flat oysters Oslrea edulis C. Linnaeus. 1758 and O. aiigasi Sowerby. 1871 697 Maria Eugenia Valdez-Ramirez, Anne Donval, and Marcel Le Pennec Ultrastructural and histochemical criteria for determining normality in mature oocytes of the Pacific oyster Crassostrea gigas 707 2(28 057 CONTENTS CONTINUED ON PREVIOUS PAGE JOURNAL OF SHELLFISH RESEARCH Vol. 21, No. 2 December 2002 CONTENTS IN MEMORIAM Harold Haley Haskin (141 5-2002 ) 441 Eugene Burreson Honored Life Member: Jay Donald Andrews 445 Kenneth Chew Honored Lite Member: Neil F. Bourne 447 Loren D. Coen and M. Yvonne Bobo Honored Life Member: Vietor G. Bunell 449 Richard A. Lutz Honored Life Member: Herbert Hidu 45 1 George R. Abbe Honored Life Member: Sandra E. Shumway 453 F. H. Mackintosh and E. A. Smith Evakialion of Mist Alert '^' rapid test kits for the detection of paralytic and amnesic shellfish poisoning toxins in shellfish 455 Ana Maria Gayoso, Stacie Dover, Steve Morion, Mark Busman, Peter Moeller, Vanesa K. Fulco, and Lucie Maranda Diarrhetic shellfish poisoning associated with Pronicenlruiii limn (Dinophyceae) in Patagonian Gulfs (Argentina) 461 Graham C. Fletcher, Brenda E. Hay, and Margaret F. Scott Reducing neurotoxic shellfish poison (NSP) in Pacific oysters iCrassostrea ,?i,?rt.s) to levels below 20 mouse units • 1 00 g-' 465 F. M. Harper, E. A. Hatfield, and R. J. Thompson Recirculation of dinoflagellate cysts by the mussel, Mxlihis cdiilis L.. at an aquaculture site contaminated by Alexandrium fundyense { Lebour) Balech 47 1 Rodrigo Caspar Soria, Marcela Susana Pascual, and Victor Hugo Fernandez Cartes Reproductive cycle of the cholga paleta. Atriini seminuda Lamarck. 1819 (Bivalvia: Pinnidae) from northern Patagonia, Argentina 479 Qin-Feng Gao, Ka-Kei Mak, and Siu-Gin Cheung The physiological ecology of black-ribbed mussels, Septifer virgatus (Wiegmann) (Bivalvia: Mytilidae) on a sub-tropical wave-exposed shore in Hong Kong 489 Nestor Fernando Ciocco and Ana Maria Gayoso Microalgal food of the ribbed mussel Aiiliuomra atra (Molina, 1782) in Golfo Nuevo (Patagonia, Argentina) 497 John Brake, Jeffrey Davidson, and Jonathan Davis Triploid induction of Mylihis editlis using 6-dimethylanimopurine 503 Pauline Kamermans and Aad C. Smaal Mussel culture and cockle fisheries in The Netherlands: finding a balance between economy and ecology 509 Dirk A. Campbell and Maeve S. Kelly Settlement of Poimiloceros triqiieter (L.) in two Scottish lochs, and factors determining its abundance on mussels grown in suspended culture 519 Per Dolmer Mussel dredging: impact on epifauna in Limfjorden, Denmark 529 Katherine A. Ross, John P. Thorpe, Trevor A. Norton, and Andrew R. Brand Fouling in scallop cultivation: help or hindrance? 539 Laura Schejter, Claudia S. Bremec, Rut Akselman, Daniel Hernandez, and Eduardo D. Spivak Annual feeding cycle of the Patagonian scallop Zygoclilamys patagoiuca (King and Brodenp, 1832) in Reclutas bed (39S-55 "W), Argentine Sea 549 Karin B. iMhrmann, Andrew R. Brand, and Stephen W. Feist Comparison of the parasites and pathogens present in a cultivated and in a wild population of scallops (Argopecten piirptiiaHis Lamarck. 1819) in Tongoy Bay, Chile 557 CONTENTS CONTINUED ON INSIDE BACK COVER MBL WHill 1 IKRARY IdH lAAlJ A