STUDIES ON THE MARKING OF COMMERCIAL SHRIMP WITH BIOLOGICAL STAINS ...J Biological DECU -1957 WOODS HOLE, MASS. SPECIAL SCIENTIFIC REPORT- FISHERIES No. 231 UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE EXPLANATORY NOTE Tlie series embodies results of Investigations, usually of restricted scope, Intended to aid or direct management or utilization practices and as guides for administrative or legislative action. It is issued in limited quantities for official use of Federal, State or cooperating agencies and in processed form for economy and to avoid delay in publication . United States Department of the Interior, Fred A. Seaton, Secretary Fish and Wildlife Service STUDIES ON THE MARKING OF COMMERCIAL SHRIMP WITH BIOLOGICAL STAINS By C . E . Daw son Bears Bluff Laboratories, Wadmalaw Island, S.C. Formerly of the Institute of Marine Science The University of Texas This study was supported by funds made available by the Saltonstall- Kennedy Act, under a contract between the U. S. Fish and Wildlife Service and the University of Texas. Special Scientific Report --Fisheries No. 231 Washington, D. C. September 1957 ABSTRACT To determine the effectiveness of biological stains as marking agents for the commercial shrimp Penaeus setiferus, P^. aztecus, and^. duorarum, experiments were conducted on captive shrimp held in storage tanks and aquariums supplied with circulating sea water. Some 26 stains were tested by immersion, injection, and feeding. Staining by immersion proved consistent- ly unsuccessful. Distinctive and fast abnormal coloration has been induced by injection with Fast Green FCF (National Aniline), Niagara Sky Blue 6B, Trypan Blue, and Trypan Red. Feeding with mullet (Mugil cephalus) previously stained with Trypan Red also results in abnormal coloration suitable for identification. The feeding technique constitutes a satisfactory method for marking shrimp under 80 mm . in length. Whether these stains will prove equally fast under natural conditions must be determined by field experiments. CONTENTS Page Introduction 1 Methods and Materials 2 Storage facilities 2 Salinity and temperature 2 Shrimp 2 Stains 3 Experimental Methods 3 Immersion 3 Injection 3 Feeding 4 Controls 4 Experiments 5 Mortality 17 Etiscussion 17 Acknowledgments 23 Summary 23 References 23 STUDIES ON THE MARKING OF COMMERCIAL SHRIMP WITH BIOLOGICAL STAINS INTRODUCTION The role of tagging- and marking as aids in determining growth, migration, fishing pressure, and mortality in a commercial -fish- ery population has been described by many authors (Rounsefell and Kask, 1945; Rounsefell and Everhart, 1953). With the exception of various lobster and crab investigations, tagging and marking programs have been confined to finfish. Commercial shrimp have been tagged, using the Petersen disk, by the Fish and Wild- life Service, by the Bureau of Fisheries, and by at least one of the Gulf States . Lindner and Anderson (1956) tagged a total of 46,532 shrimp from 1935 through 1947 and reported a gross return of 15.6 percent. Data presented show that recoveries decrease with decrease in size at tagging. No recoveries were obtained from shrimp tagged at a length of 80 mm. or less, and recovery of shrimp in the 85 to 110 mm. range was less than 10 percent. The authors present no details on the maximum period of tag retention, but growth analysis based on re- coveries was limited to a 60-day period. It would appear, therefore, that few recoveries were made of shrimp with more than 60 days' "out-time". McRae (1952) reported a 2.01-per- cent return on 1,137 shrimp tagged along the Texas coast during April, May, and June, 1951. Complete size data are not shown, but the small- est shrimp recovered was approximately 104 mm. long at time of tagging. The maximum period between tagging and recovery was 80 days. These programs have met with some success, but a more suitable tagging or mark- ing method is needed for shrimp under a length \J The term "tagging" is used to denote the application of a mechanical tag or other identif- ication device. "Marking" refers to the use of mutilation, staining, or other means of identifica- tion not requiring the attachment or insertion of mechanical devices. of 100 mm., and for use in long-term experi- ments on both juvenile and adult shrimp. The tagging or marking of shrimp is complicated by their frailty and small size, and by a high shedding frequency that precludes use of the exoskeleton for either marking or tagging. The massive population and a high mortality re- quire that large numbers be released, preferably over a short period, before a tagging or marking experiment can attain statistical validity. Since tags may affect the normal swim- ming ability of shrimp and physical damage accompanying attachment may be considerable, this study was directed toward investigating the practicality of marking shrimp with biological stains. Although tags would be superior for ob- taining individual growth and migration data, it is considered that field studies are directed to- ward the shrimp population rather than the individual . A distinctive and easily applied ab- normal coloration would serve the identification requirements for population studies . The use of biological stains for identifica- tion purposes has been tried with varying degrees of success on a number of marine and fresh- water animals. Loosanoff (1937) was successful in marking starfish, Asterias, for periods up to 10 months with Nile Blue Sulfate, and Vernon (1937) marked Asterias with Neutral Red. Feder (1955) has used these stains to mark the Pacific- coast starfish, Pisaster ochraceus. Gustafson (1953) and Klawe (1954) were generally unsuccess- ful in their efforts to stain the marine worms Glycera and Nereis although some 14 stains were tested. Dunn and Cocker (1951) and Carranza (1953) injected fish with a number of stains with varying success. Two studies of particular in- terest are those of Menzel (1955) and Racek (1955). Menzel injected white shrimp, Penaeus setiferus, with Fast Green and reported that staining was plainly visible for over 60 days, Racek apparent- ly achieved some success in marKing Australian penaeid prawns by immersing them in solutions of Trypan Blue and Nile Blue Sulfate . He considered Nile Blue Sulfate to be most promis- ing owing to the low staining mortality and the "long period" of stain retention. Since certain methods have been devel - oped which show sufficient promise to warrant large-scale field tests, the results of research conducted at the University of Texas Institute of Marine Science on the use of stains for mark- ing shrimp are presented here. METHODS AND MATERIALS materially complicated by the burrowing habit of shrimp. Salinity and temperature TTiree hundred and five daily observa- tions of salinity and temperature were made during the period from May lu, 1955, through May 31, 1956. Mean monthly tank salinities ranged from 25.9 o/oo to 38.7 o/oo and monthly means of tank water temperature range from 14.3°C. to 29.3''C. Storage facilities All experiments were conducted on the dock laboratory of the University of Marine Science at Port Aransas, Texas . Seven storage tanks with capacities ranging from 94 to 102 gallons were constructed of 3/4-inch marine plywood. Each was covered with 1/4 -inch hardware cloth and divided into from two to four compartments by hardware - cloth partitions. Five aquariums and four small tanks with capacities of from 8 to 10 gallons completed the storage facilities . Tanks and aquariums were provided with continuously circulating sea water pumped from a depth of approximately 6 feet below mean low water in Aransas Pass. Water flow averaged about 1 .8 gallons per minute to each large tank and 1 gallon per minute to each aquar- ium and small tank . Piping for the system consisted of galvanized pipe and bronze valves . Rubber hose, glass tubing, and one length of lead pipe were used in distributing water from the main lines to the various tanks . Although the possibility of lethal contamination of the water by copper and zinc from the piping caused some initial concern, a flourishing growth of coelenterates, annelids, moUusks, other Crusta- cea, and an occasional young fish caused this factor to be discounted. The rate of water ex- change apparently precluded lethal concentration of toxic metals within the tanks. The bottoms of several tanks were covered with about 1-1/2 inches of mud and sand in an effort to retard cannibalism . While there is some evidence of reduced cannibalism, problems of observation and counting were Shrimp- hi view of the fact that any tagging or marking method should be applicable to all com- mercial shrimp, no effort was made to separate experimental animals by species. White shrimp (Penaeus setiferus), brown shrimp (P. aztecus), and pink shrimp (P. duorarum) were used either mixed or' separately, depending upon their avail- ability. In experiments involving more than one species, no indication of specific differences in stain acceptance was apparent . Shrimp ranged in length from 30 mm . to 175 mm. and were, for experimental purposes, divided into rough size groups of small (30 to 80 mm.), medium (80 to 120 mm.), and large (120 to 175 mm.). In general, no regular measurements were made of individual shrimp either before or during any particular experi- ment. Most of the shrimp fell within the medium size classification and, unless specifically stated to the contrary, medium shrimp were used in all experiments . Shrimp were fed every other day with mullet (Mugil cephalus). Trash fish were used as food during the first 2 weeks of operations, but this practice was discontinued following an almost total mortality in one tank after feeding with Synodus foetens . The mullet, either fresh or frozen, was filleted and cut into pieces roughly 4 mm. square. These were dropped into each tank or compartment on the basis of one piece per shrimp. The size of the individual pieces was modified when feeding predominately large or small shrimp. Food supplied in this fashion is readily eaten and allows for even distribution. Tanks were checked at least once a day for the presence of dead individuals or evidence of shedding, and periodic counts were made of the shrimp remaining in each experiment. Ab- dominal and cephalothoracic exoskeletons were removed whenever found. Shedding shrimp were observed to eat the branchial cast and associated structures, and these were conse- quently left in the tanks . An indication of the number of shedding individuals was obtained by counting the cepholothoracic exoskeletons found in each tank. Since the entire exoskeleton as well as the newly shed shrimp may be cannibal- ized, this provided only a minimum figure. When bodies were found, shrimp which had died from the effects of experimental procedures or other causes were differentiated from those eaten by their fellows during or immediately after ecdysis by the presence or absence of a firm exoskeleton. Stains Stains were selected which complied with one or more of the following criteria: solubility in water or other nonlethal solvent, previous use as a vital stain, and a color so opposed to the natural coloration of shrimp as to be readily detected by the casual observer. necessary to first dissolve the stain in a small quantity of distilled water when preparing a sea -water solution. When a stain proved difficult to dissolve, the mixture was warmed and filtered through Whatman No. 1 filter paper. The per- centage of stain solution indicated for each experiment is based on the weight of dry stain to 100 milliliters of solvent. As no advantage was found in aging solutions, stains were usual- ly prepared just before use. EXPERIMENTAL METHODS The marking qualities of each stain were tested by one or more of three methods: immer- sion, injection, and feeding. After preliminary tests the laboratory procedure for each method was standardized as follows: A. Immersion: The shrimp sample was placed in an aerated stain solution for a prede- termined number of minutes. The shrimp were then transferred through two changes of fresh sea water at 5-minute intervals so as to remove excess stain adhering to the exterior of the animals or trapped within the branchial chamber. A final transfer was then made to an appropriate storage tank for observation. With a single exception all stains were obtained from the Hartman-Leddon Company (Harleco) or the National Aniline Division of the Allied Chemical and Dye Corporation . Wide variability in the characteristics of stains mar- keted by different manufacturers is common, and it was considered most practical to limit tests to material readily available from a few American producers . Stains certified by the Biological Stain Commission at Geneva, New York, were used when available. Pertinent data on the source and quality of each stain are shown in table 1 . The total dye content refers to the percentage of dye in the dry stain. The lot number is the manufac- turer's identification number of the dry stain. Complete information on each stain may be found in Conn (1953). Distilled water, filtered sea water, glycerine, mineral oil, and alcohol were used as solvents. Most of the dry stains were rela- tively insoluble in sea water, and it was usually B. Injection: A 1-cc. tuberculin syringe equipped with a No. 25 by 1/2 -inch needle was most practical for laboratory use. Holding the syringe in the right hand, a shrimp was grasped with the left so that its head was pointed toward the left wrist and with its abdomen held in a flexed position by the left thumb and forefinger (fig. 1). The needle was then introduced through the articular membrane of the sixth abdominal joint slightly to the left of the middorsal line and at an angle approximating 45 degrees. The needle was inserted to a depth of from 2 to 4 millimeters until stain was visibly entering the blood-vascular system through the dorsal abdominal artery. After injection the shrimp were examined for signs of physical damage or excessive weakness. Shrimp killed or incapacitated by overinjection or obvious physical damage were replaced be- fore transfer to final observation tanks. Volume of individual injections generally ranged from 0.01 cc. to 0.05 cc. and averaged about 0.03 cc . Injections of greater volume frequently resulted in rapid death and did not produce more vivid or durable staining. The 1/2 -inch No. 25 needle Table 1. --Stains tested Company Identification Lot Stain Commission Percentage Stain number - ■ -Manufacturer No. certificate No . dye content Alizarin 1027 Harleco 30 Alizarin Red S 1034 Harleco 42 LAr-5 Alizarin Yellow GG 36 Harleco 14 Alizarin Yellow R 40 Harleco 11 Alphazurin 2G 712 Harleco 1 Bismarck Brown Y 331 Nat'l Aniline NN-11 60 Brilliant Cresyl Blue 877 Nat'l Aniline NV 41 60 Brilliant Vital Red 456 Harleco 12 Chlorazol Fast Pink 353 Harleco 2 Cresyl Violet (acetate) Nat'l Aniline NW 19 56 Diamond Black 299 Harleco 1 Dianil Blue 2R 465 Harleco 3 Erythrosin Bluish 773 Harleco 22 LEr-4 83 Fast Green Extra Bluish 691 Harleco 4 Fast Green FCF Harleco 7 NGf-7 97 Fast Green FCF Nat'l Aniline NGf-13 92 Fast Yellow 16 Harleco 5 Janus Black 134 Nat'l Aniline 14358 Janus Green B 133 Nat'l Aniline NJ-18 50 Malachite Green 657 Harleco 68 LMg-8 96 Methyl Green (hydrochloride) 657 Harleco 13 Methyl Green 684 Curtin Methylene Blue (chloride) 922 Harleco 80 LA-21 86 Neutral Red 825 Nat'l Aniline NX-17 69 Niagara Sky Blue 6B 518 Harleco 3 Nigrosin (water soluble) 865 Harleco 6 LNi-4 Sudan IV 258 Harleco x-10-1 85 Trypan Blue 477 Harleco 30 Trypan Blue 477 Nat'l Aniline 15814 Trypan Red 438 Harleco 12 & 16 Trypan Red 438 Nat'l Aniline 14184 Victoria Blue B 729 Harleco 15 Wool Green S 737 Harleco 3 permitted easy injection without excessive drip and allowed for ready control of penetration depth . supplied for the duration of the experiment. Controls G . Feeding: Mullet, cut as for normal feeding, was placed in a stain solution for 15 to 20 minutes and agitated to ensure even staining. The solution was then decanted, and the food was washed in sea water to remove excess stain . The stained food was then dropped into the tank following normal feeding practice. After a number of stained feedings, normal food was The prime requisite of a shrimp stain is that it must provide a fast and easily distinguished color. Most of the experiments were designed to test staining characteristics, and regular con- trols were not established. Simultaneous controls were run with the more successful stains. In control experiments an equal .lumber of untreated shrimp were held in a separate compartment in the Figure 1 . --Recommended method for handling the individual shrimp for injection. The needle is inserted through the articular mem- brane of the sixth abdominal joint. same observation tank as the stained shrimp. A further check on experimental mortality was provided by what might be termed the natural mortality within the storage system. This is the total mortality of nonexperimental shrimp and was estimated to average about 15 percent a week. EXPERIMENTS blue -green. Abdominal fading and cephalothor- acic concentration was evident within 24 hours and complete within 72 hours. Although there was some fading, the stain remained through at least one ecdysis and a pale -green color was retained by the one shrimp surviving through 160 days. Though readily apparent to a careful observer, the color was not sufficiently distinct for field use . Each stain, with the exception of four that proved insufficiently soluble in aqueous solution, was tested by at least one of the three staining techniques and frequently in a number of concentrations and solvents . The num ber of individuals used in each test ranged from 5 to 60. Of the 30 stains tested, 26 were found unsatisfactory as shrimp -marking agents. A summary of experiments with these unsuccess- ful stains is presented in table 2. Tests involving stains that are considered suitable shrimp- marking agents are treated in some detail below. The products of two manufacturers aie com- pared in three of these stains. Fast Green FCF (Harleco) Four injection experim ents were com- pleted using this stain in concentrations of 0.75, I.O, and 8.0 percent. Each solution was pre- pared with sea water. Ten shrimp irtjected with a 0.75 -percent solution showed an immed- iate pale -green general coloration. Fading from the abdominal region and simultaneous concentration in the cephalothorax, especially in the branchiae, was evident within 5 hours. Coloring was pale, however, and within 3 days it was not sufficiently obvious for identification purposes. There was an immediate general blue to blue -green coloration in 2 lots of 15 and 20 shrimp injected with the 1 -percent solution. Abdominal fading was rapid, and the stain was usually concentrated in the heart and branchiae within 72 hours. Some fading was evident, but a pale -green color was retained in the branchiae of 2 shrimp remaining after 84 days. This col- oration had remained fast through at least one ecdysis. Fifteen shrimp injected with an 8 per- cent solution were immediately colored a deep Ten shrimp were immersed for 15 min- utes in a 0.75 -percent solution made up with sea water. One shrimp died within 8 hours. There was some slight stain acceptance in the anterior digestive tract, branchiae and in irregu- lar areas of the abdominal exoskeleton . Loss of abdominal color was complete within 24 hours. Rapid fading progressed and after 6 days only a very light green tint was detectable in 2 of the remaining 9 shrimp. Fifteen shrimp were given three feedings stained with an 8 -percent solution prepared with sea water. Stomach and digestive tract were stained a moderate to deep blue which persisted for 8 days after the final stained feeding. There- after, fading was relatively rapid with only a trace of abnormal coloration remaining in 13 days. Fast Green FCF (National Aniline) Two injection experiments were completed. Fifteen shrimp injected with a 1 -percent solution made up with 10 ml. of distilled water and 90 ml. of sea water showed an immediate general blue- green coloration. One died within 18 hours. Except for a green spot at the injection site, color was rapidly concentrated in the cephalothor- ax. General cephalothoracic tinting and the spot at the injection site faded slowly, but coloring in the branchiae and heart remained fast for 120 days and through at least one ecdysis. The color was very distinctive and could be readily detected by even a casual observer. Twenty shrimp were injected with a 1- percent solution m ade up with an unknown solvent. this solution was supplied by Dr. Winston Menzel, and was the same as that employed in his experi- ments (Menzel, 1955). 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D 0) " •o to 0) '3 i- s ■= XI o 2 -3 C "3 m bo cn X 5 o 01 ■s a — o 2 O OJ " § i .s Q o " > DO 12 R T3 . u Q E E 3 CO M « col c < O E (2^ 13 « B Vh CO O ^ a o c ■-' to •s- a ■ to* v 2 1^ ^ bo m rt 2 i! -J a e V a v-i 2 c en V-I 01 'J bo CO (3 ^ tJ O' CO O O ? 6 0) o o "3 s: o ■s QJ > a CO bO B ■B V CO 60 B XI o OJ B CO S 0) B ■a 6 M (U .s >, 'S ID 2 fi a u 0 II 2 Q P < — I csl col 13 within 3 days . A green spot at the injection site was retained for varying periods up to 53 days . The green staining of the branchiae and cephalothorax showed some gradual fading, but it was retained through at least one ecdysis and remained very evident in the four shrimp surviving after 84 days. Niagara Sky Blue 6B Fourteen shrimp were injected with a 1 -percent solution made up with 10 ml. of dis- tilled water and 90 ml. of sea water. Immed- iate coloration ranged from an overall medium to a blue spot at the injection site; all showed some branchial concentration. Three shrimp died within 4 days . Gradual abdominal fading was evident, and staining was confined to a blue coloring of the branchiae and heart in 37 days . After 103 days, three surviving shrimp showed well-defined dark-blue coloration of these areas. In a similar test on 30 shrimp, the results were generally the same. Three shrimp died in 3 days; cannibalism was high and only four shrimp survived after 52 days. Twenty -five shrimp were given 6 feed- ings stained with a 1 -percent solution as above. By the third feeding there was a dark -blue col- oration of the stomach and digestive tract, and several showed a poorly defined pale blue- green in the branchial region. One shrimp showed a bright blue -green staining of the bran- chiae which was retained until termination of the test 26 days after the initial stained feeding. Examination of casts showed that much of the branchial coloration was lost at ecdysis. Trypan Blue (Harleco) Ten shrimp were injected with a 1 -per- cent solution prepared with 5 ml. of distilled water and 95 ml. of sea water. This resulted in pale -blue coloring of the branchiae and at the injection site; several showed an overall bluish tint. This initial staining was not spec- tacular and at first appeared to be insufficiently vivid for identification purposes. However, within 24 hours a dark -blue concentration was evident in the branchiae. Although there was some tendency to fade to a dark blue -grey in about 30 days, this color remained fast and was readily observed in two individuals surviving through 141 days. Staining remained through at least two shedding cycles. Among 20 shrimp given injections aver- aging 0.05 cc. of a similar solution, several showed signs of weakness and one died within 8 hours. The remainder recovered and showed a moderate to dark blue in the injection area; branchial and abdominal tinting occurred in several. There was a dark -blue to blue -green concentration in the branchiae within 55 hours with simultaneous fading from other areas . Of the three shrimp remaining through 179 days, all retained dark -blue branchiae and two showed a bluish tint at the injection site. Of 15 shrimp injected with a 1 -percent solution made up with 10 ml. of distilled water and 90 ml. of sea water, 3 died within 5 minutes and 5 within 24 hours. This is presumed to have resulted from too great an injection. Owing to the initial absence of bright coloration, there is a tendency to overinject when using this stain. A general dark -blue branchial concentration resulted within 24 hours, and this remained dis- tinct in the two shrimp surviving through 220 days. Food acceptance was poor among 10 shrimp given three feedings stained with a 1 - percent solution prepared with distilled water. The digestive tract retained a pale bluish tint unsatisfactory for identification purposes. Five shrimp were immersed for 15 min- utes in a 0.4 -percent solution made up with 10 ml. of distilled water and 990 ml. of sea water. This resulted in an indistinct tinting of the cephalo- thorax, but no evidence of abnormal coloration could be seen after 24 hours. Trypan Blue (National Aniline) Twenty- six shrimp were given injections averaging 0.06 cc. of a 1 -percent solution made up with 10 ml. of distilled water and 90 ml. of sea water. This stain was less soluble in sea water than the Harleco product, and the solution was filtered before use. Immediate coloration was detectable only at the injection site, in the dorsal abdominal artery, and in a pale tinting 3f the branchiae. Within 24 hours there was a general branchial concentration, and several 14 shrimp showed blue coloration of the abdomen and injection site. The abdominal color faded slowly but remained distinct in some for over 60 days. The blue to blue -gray branchial staining was retained for the duration of the 120 -day experiment. Thirty additional shrimp were injected with a 1 -percent solution as above. Initial staining was similar to that detailed in the pre- ceding test. Cannibalism accompanying ecdysis was high, and only 7 individuals remained after 52 days. Fifteen ahrimp given 5 feedings stained with a similar solution showed a temporary blue coloration of the digestive tract. All evi- dence of staining disappeared within 3 days after the final stained feeding. Ten shrimp, divided into 3 lots, were immersed for periods of 7, 5, and 3 minutes in a 1 -percent solution prepared with 25 ml. of distilled water and 275 ml. of sea water. Two of 3 shrimp immersed for 7 minutes were dead at completion of the staining period. In the re- mainder, abnormal coloration was confined to a slight bluish tinting of the branchiae and an- terior margins of the antennal scales . Normal coloration was attained within 48 hours. Ten shrimp injected with a 1 -percent solution made up with 5 ml. of distilled water and 95 ml . of sea water showed an immediate general pink coloring and definite branchial concentration. The general coloration persisted in several individuals for at least 70 days, and a bright-red branchial staining was very evident in 4 shrimp which survived 234 days. Eleven shrimp injected with a 1 -percent solution prepared with 10 ml. of distilled water and 90 ml. of sea water showed similar colora- tion on injection . Abdominal staining faded within 16 days, but the red branchial concentra- tion was fast in the one shrimp remaining after 220 days. Eleven shrimp were given injections, averaging 0 . 03 cc, of a 1 -percent solution pre- pared as above but warmed to facilitate dissolv- ing of the dry stain. Abnormal coloration was essentially similar to that in the previous ex- periment. On the thirty -ninth day two shrimp showed loss of equilibrium and rested on their side rather than in the normal position. When stimulated, both showed normal swimming ability but lost balance shortly after coming to rest. Both were cannibalized within 7 days. Five shrimp surviving 46 days retained abnormal branchial color as previously described. Trypan Red (Harleco) Ten shrimp were injected with a 1 -per- cent solution made up with 5 ml. of glycerine and 95 ml. of sea water. A slow diffusion re- sulted in a general pink color with bright -red concentrations in the branchiae and injection area . Rapid abdominal fading followed, but distinctive branchial coloration remained fast in 4 shrimp surviving 245 days . Two lots of 10 and 15 individuals were injected with a 1 -percent solution prepared with 10 ml. of distilled water and 90 ml. of sea water. A mortality of 18 shrimp within 72 hours was attributed to excessive injection vol- umes. Staining was essentially the same as in the preceding test, but the general coloration was persistent, together with the branchial con- centration, in the 3 shrimp remaining through 124 days. Thirty-three shrimp given injections, averaging 0.05 cc, of a I -percent solution pre- pared with sea water showed pink abdominal and red branchial colorations. A high mortality re- sulted in the loss of 25 individuals within 10 days. Abdominal fading was slow but the branchial color was fast in the remainder and was evident in the one shrimp surviving 175 days. Ten shrimp were given four feedings, stained with a 1 -percent solution made up with 10 ml. of distilled water and 90 ml. of sea water, at 4-, 2-, and 4-day intervals. Red branchial concentration was apparent after the third feed- ing, and several showed diffuse red staining of the entire cephalothorax. Branchial color was retained through at least three shedding periods and was very evident in the one individual sur- viving 215 days after the final stained feeding. Fifteen shrimp given three feedings stained with a 2 -percent solution prepared with 15 distilled water all showed red branchial color- ing before the final stained feeding and several had accepted various degrees of abdominal staining. Three died within 7 days. There was irregular retention of abdominal coloring but the bright-red branchial concentration showed no indication of fading through 79 days following the final stained feeding. An identical test on II large shrimp produced similar results. Three individuals surviving 65 days after the final stained feeding all showed bright-red branchiae . Twenty-five shrimp were given three feedings stained with a 2 -percent solution, as above, which had been warmed and filtered. With the exception of two individuals which had apparently not accepted stained food, all showed red branchial coloring before the final stained feeding. Of 7 shrimp surviving 183 days, 6 showed distinctly red or pink branchiae and one showed no evidence of abnormal coloring. Fifty shrimp were given feedings stained with a 1 -percent solution prepared with 45 ml. of distilled water and 5 ml. of sea water . The majority showed red to pink branchial coloring after three feedings and several showed general pink coloring. After the fifth feeding, the 2 0 most deeply stained individuals were isolated and subsequently given normal food; those re- maining were given four additional stained feedings . Since no difference could be detected in intensity of coloring, the two groups were again combined 24 days after the final stained feeding. Seven shrimp remaining after 127 days all showed bright -red branchiae. Since can- nibalism was high, it was impossible to deter- mine differences in mortality between the five -feeding and nine -feeding groups. Fifteen small shrimp were given eight feedings stained as for the preceding experiment. The majority showed red branchial concentration following the third feeding and this held fast in the six individuals surviving 127 days after the final stained feeding. Immersion tests on two lots of 10 shrimp in 0.2 -percent and 1 -percent solutions were un- successful. In general all abnormal color faded within 3 days. One shrimp accepted some red coloration in the branchiae, and this remained fast for the 17-day duration of the experiment. Several fruitless attempts were made to duplicate branchial staining following immersion. Trypan Red (National Aniline) Twenty -five shrimp injected with a filtered 1 -percent solution made up with 10 ml. of distilled water and 90 ml. of sea water showed an immediate pale -red branchial concentration and several showed general pink coloration. Initially this did not appear as bright as that pro- duced by the Harleco product but it intensified to a bright red within 8 days . Eventually, all but one showed a diffuse pink coloring of the en- tire cephalothorax, and abdominal staining was retained when present. There was a high rate of cannibalism following ecdysis, but a bright- red color remained in the branchiae of four shrimp surviving 79 days. The majority of 50 shrimp showed a bright -red branchial color after two feedings stained with a 2 -percent solution made up with distilled water . This abnormal coloring was evident in all after five feedings. Cannibalism was great, but the branchial color remained fast in 7 individuals througji 7 days following the final stained feeding. Sixty shrimp were given five feedings stained with a 2 -percent solution as above except that it was warmed to facilitate dissolving of the dry stain. Various degrees of abnormal coloring from a pale -pink tinting of the branchiae to a deep- red general coloration were observed following the third stained feeding. Six shrimp, all with the general coloration, died within 5 days, and on the fifth day two individuals with similar color exhibited a loss of equilibrium as noted previously. One of these died within 3 days, while the other survived 27 days and shed once without recover- ing normal balance. Again cannibalism was high, and after 58 days the 10 individuals remaining all retained bright -red branchiae. Of 25 small shrimp given two feedings stained as above, all but 4, which had apparently not eaten, showed bright -red branchiae. Two died within 6 days. There was no general stain- ing, and no loss of equilibrium was observed. 16 All nine shrimp surviving 43 days exhibited fast branchial coloring. Two lots of approximately 35 small shrimp were given three feedings stained with a 1 -percent solution prepared with distilled water. All chowed bright-red branchiae after the final stained feeding, and this color re- mained fast in all individuals surviving the 11- day test. MORTALITY The available mortality data for each test with these more successful stains are presented in table 3. Mortality data on simul- taneous staining and control experiments are summarized in table 4. Mortality A refers to deaths known to have been accompanied by ecdysis and cannibalism. Mortality B indicates the percent mortality Known to have occurred without concurrent ecdysis and may be ascribed to experimental procedure, cannibalism, or other unlcnown causes. Shrimp listed as "un- determined" were completely cannibalized, and cause of death could not be ascertained. It was observed that isolated stained and un- stained shrimp, when held for extended periods, could succumb 2 or 3 days after ecdysis . In such cases the exoskeleton did not attain normal firmness, indicating a deficiency of some un- known corfiponent in the environment . In group experiments such mortality could not be recog- nized and, owing to the absence of casts, these deaths would be included under Mortality B. DISCUSSION In 11 experiments with 8 stains, the marking of shrimp by immersion was consist- ently unsuccessful. Though temporary colora- tion was attained in several cases, fading was always rapid and was usually complete within 5 days. One instance of more permanent mark- ing, using Trypan Red, could not be duplicated in subsequent trials . Immersion marking with Nile Blue Sulfate or Trypan Blue, suggested by Racek (1955), was unsatisfactory. Racek gives no data on the source of his stains, and differ- ences in stain characteristics may account for contrasting results. The possibility of differen- tial stain acceptance between the Australian and American shrimp cannot be wholly discounted, however . Injection with Fast Green FCF, Niagara Sky Blue 6B, Trypan Blue, or Trypan Red has resulted in fast abnormal colorations, and these show potential value as shrimp -marking agents. Injection is a rapid and efficient method of marking medium and large shrimp, and can be used under most field conditions. Although tuberculin syringes were used in laboratory studies, automatic constant -volume syringes are indicated for most efficient field operation. Fast Green FCF was originally suggested as a shrimp -marking agent by Menzel (1955), but he published no data on the source or con- centration of the stain used. Menzel later reported (personal correspondence) that the stain was a National Aniline product in a 1 -per- cent solution, but unfortunately no information was available on the solvent. A sample of this solution was kindly supplied by Dr. Menzel for inclusion in the present experiments. Injection with Fast Green FCF results in an immediate bright-green to blue-green general coloration . Abdominal fading is rapid, and this, together with simultaneous green branchial concentration, is usually complete within 72 hours. Menzel reported branchial con- centration following ecdysis, but in the present experiments this occurred either with or without shedding. One -percent solutions proved most satis- factory, as less concentrated solutions did not provide a fast stain, and no advantage was found in using more concentrated solutions. Experi- mental injection volumes ranged from 0.015 cc . to 0.2 cc. per shrimp, but 0.03 cc. was found adequate for medium shrimp. Mortality within the 24 -hour period fol- lowing injection average 4 percent with this stain, and no difference was observed in initial mortal- ity between injections of the National Aniline and Harleco products. It was difficult to arrive at the experim ental mortality in view of the preva- lence of cannibalism and deaths from unknown causes. Reference to table 4 will show the max- imum difference in total mortality of control and stained animals to be 15 percent over 84 days. Comparison 6f staining characteristics shows the National Aniline product to be superior to that of Harleco for present purposes . Although 17 Table 3 . - -Summary of available mortality data on experiments with Niagara Sky Blue 6B, Fast Green FCF, Trypan Blue, and Trypan Red No. of Stain shrimp Imm . Method- Inj. Percent Percent Total Total mortality mortality mortality days A- B- in percent Remarks 4/ Fast Green FCF (H) 15 20 XX 44 87 13 - XX 84 25 35 100 1 -percent solution in SW. 10 XX -- -- 7 -- 10 10 0.75-percent solution in SW; 15 min. immersion. 10 XX 4 0 0.75-percent solu- tion in SW. 15 XX 160 21 43 93 1 -percent solution in SW; 1 lost, 4 undetermined. 15 ~ ~ 3 ~ ~ 22 47 47 93 8 -percent solution inSW. 90 1 -percent solution in SW; 6 undeter- mined. Fast Green FCF (N. A.) 15 20 XX 120 20 XX 84 15 10 1 -percent solution 90 in 10%DW, 90%SW; 13 undetermined. 80 Menzel's 1 -percent; 1 1 undetermined. Niagara Sky Blue 6B 14 25 30 XX 103 36 29 37 32 28 XX 52 47 23 1 -percent solution 79 in 10%DW, 90%SW; 2 undetermined. 76 1 -percent as above; 4 undetermined. 87 1 -percent as above; 5 undetermined. Trypan Blue (H) 10 10 XX 141 60 10 23 20 1 -percent solution 70 in 5%DW, 95%SW; 1 lost. 5 XX - 2 0 0.4-percent solu- tion in 1%DW, 99%S 15 min. period. 20 - XX 179 15 25 85 1 -percent solution in 10%DW, 90%SW; 9 undetermined. 15 - XX 220 20 60 87 1 -percent solution as above; 1 unde- termined. 20 1 -percent solution in DW. 18 Table 3. --Summary of available mortality data on experiments with Niagara Sky Blue 6B, Fast Green FCF, Trypan Blue, and Trypan Red- -Continued. Stain No. of shrimp Method i^ Imm. F. Ini. Total days Percent mortality Percent mortality Total mortality in percent 4/ Remarks- Trypan Blue (N.A.) 26 XX 120 4 12 73 1 -percent solution in 10%DW, 90%SW; 15 undetermined. 15 5 14 6.5 1 -percent solution as above; 1 un- determined. 30 XX 52 47 77 1 -percent solution as above; 9 un- determined. 10 XX -- 3 — 20 20 1 -percent solution in8.5%DW, 91.5%SW Trypan Red (H) 10 XX 245 -- 10 60 1 -percent solution in 5%Gly.,95%SW; 5 undetermined. 25 XX 124 12 76 88 1 -percent solution in 10%DW, 90%SW. 10 XX 234 30 10 60 1 -percent solution in 5%DW, 95%SW; 2 undetermined. 11 XX 220 91 1 -percent solution in 10%DW, 90%3W; 10 undetermined. 10 4 223 20 60 90 1 -percent solution as above; 1 un- determined. 11 XX 46 18 9 36 1 -percent solution as above; 1 un- determined. 15 3 83 40 13 66 2 -percent solution in DW; 2 undeter- mined. 11 3 69 30 40 70 Stain as above. 25 3 183 16 40 72 Stain as above, filtered; 4 undeter- mined. 33 XX 175 3 83 91 1 -percent solution in SW; 2 undeter- mined. 50 9 127 34 16 86 1 -percent solution in 10%SW, 90%DW; 18 undetermined. 15 8 127 6.5 26 60 1 -percent solution as above; 4 un- determined. 10 XX - - 4 0 S min. period in 0.2^5 solution; 5%DW, 95%SW. 19 Table 3 . --Summary of available mortality data on experiments with Niagara Sky Blue 6B, Fast Green FCF, Trypan Blue, and Trypan Red- -Continued. Stain No. of shrimp Method 1/ Percent Percent Total Imm. F. Inj. Total mortality mortality mortality days A— B— in percent 4/ Remarks- Trypan Red (H) 10 XX 17 40 40 4 min. period in 1 -percent solution; 10%DW, 90%SW. Trypan Red (N.A.) 35 11 2 -percent solution in DW; 1 lost; 27 50 5 -- 60 28 13 87 undetermined. 25 XX 79 28 8 84 1 -percent solution in 10%DW, 90%SW; 12 undetermined. 60 5 58 28 28 83 2 -percent solution in DW; warmed; 16 undetermined. 25 2 43 4 8 64 1 -percent solution in DW; 13 undeter- mined. 35 3 11 14 48 1 -percent solution as above; 10 un- determined. 37 Stain as above; 9 undetermined. \/ Imm . -immersion; F -feeding (figure in feeding column indicates number of stained feedings); Inj . -injection; (H)-Harleco; (N.A.) -National Aniline; Gly. -glycerine; DW -distilled water; SW-sea water. 2/ Mortality known to have accompanied ecdysis and cannibalism . 3/ Mortality known to have occurred without ecdysis and cannibalism. 4/ Complete cannibalism. 20 Table 4. --Summary of mortality data on simultaneous staining and control experiments Percent Percent Total No. of Method Total mortality mortality mortality Stain shrimp Imm . F. Inj . days A B in percent Remarks- Control Fast Green FCF 20 20 20 XX 84 15 10 80 11 undetermined; Menzel's solution. XX 84 25 35 90 6 undetermined; 1 percent Harleco, SW. 84 10 75 12 undetermined. Trypan Blue Niagara SKy Blue 6B Control 30 30 30 XX 52 47 XX 52 47 23 52 53 9 undetermined; 77 1 percent Nat'l. Aniline; 10%DW, 90%SW. 83 5 undetermined; 1- percent solution; 10%DW, 90%SW. 8 undetermined. Trypan Red 60 Control 60 XX 58 28 16 undetermined; 28 83 2 percent Nat'l. Aniline in DW. 58 23 10 77 26 undetermined. 1/ DW = Distilled water; SW= Sea water. both have proved relatively fast and can be dis- tinguished for more than 120 days, the National Aniline stain results in a much more vivid coloration. Niagara Sky Blue 63 produced distinctive abnormal coloration when injected in a 1 -per- cent solution prepared with sea water and a small quantity of distilled water. The resultant blue branchial concentration is fast for more than 100 days. With this stain, the injection quantity is critical, and if injection volumes ex- ceed 0.03 cc . with medium shrimp initial mortality may be high . Initial mortality excepted, available data indicate no significant mortality increase over that of uninjected shrimp. Injection of Trypan Blue, either National Aniline or Harleco, results in distinctive blue branchial coloring which is easily identifiable over a period of at least 220 days. Immediate coloration ranges from a general pale -blue tint to a slight bluish staining of the branchiae. Gradual branchial concentra- tion results in a dark -blue staining of these structures within 48 hours . Subsequently the branchiae may attain a bluish -gray coloration, but this change is slight and does not serve to complicate the differentiation of stained and normal shrimp. Owing to the inconspicuous nature of the initial coloring there is a definite tendency to overinject with this stain. Excessive injection volumes result in high initial mortality, and experience has shown that injections should not exceed 0.04 cc. of a 1 -percent solution with medium shrimp. Racek (1955) abandoned Trypan Blue in- jections owing to excessive mortality. With the exception of high mortality in one test following overinjection, no excessive mortality was ascribed to Trypan Blue in the present study. In a 52 -day 21 controlled experiment (table 4), total mortality of injected shrimp was 6 percent less than that of the controls. If a blue marking agent is desired, the choice is about equally divided between Trypan Blue and Niagara Sky Blue 6B. Trypan Blue ap- pears to cause slightly less mortality, but abdominal coloring associated with Niagara Sky Blue 6B is retained for several weeks and m.ight prove an asset in short-term marking programs. Injection with Trypan Red, National Aniline or Harleco, results in distinctive red branchial coloring which has remained fast for 245 days and through at least two ecdyses. In- jection usually results in an immediate general pink coloration which is followed by branchial concentration within 24 hours and subsequent variable abdominal fading. A 1 -percent solution prepared with one part of distilled water and nine parts of sea water has proved most satisfactory. Glycerine was used in one experiment in place of the dis- tilled water, but this offered no advantage. Overinjection causes a high mortality which may continue for several days, and losses of equil- ibrium occurring more than a month after staining can probably be attributed to excessive injections. Individual injections should cease as soon as a general pink coloration is observed, and should not exceed a volume of 0.03 cc. with medium shrimp. With the exception of deaths attributed to overinjection, total mortalities have been low considering the long holding periods . There is Uttle doubt that some deaths have resulted from the suspected environmental deficiency as previously noted. A number of stains tested by the feeding technique produced various degrees of tempor- ary abnormal coloration, but only Trypan Red caused lasting and distinct coloring suitable for field identification. Feedings stained with Try- pan Red resulted in a bright-red branchial color which remained fast for more than 233 days and throu^ a minimum of three ecdyses. Marking was successful in all sizes of shrimp from 30 mm. through 175 mm. Excessive feeding, indicated by various degrees of pink to red general staining, results in high mortality with deaths frequently preceded by loss of equilibrium . With controlled staining, however, mortality compares favorably with that of normal shrimp. Staining of individual shrimp usually accomplished in one or two feed- ings over a like number of days. Since aggres- siveness and appetite vary with the individual, it is impossible to stain a group of shrimp simply on the basis of a predetermined number of stained feedings. Before each stained feeding, shrimp should be examined for the presence of the char- acteristic bright -red branchiae, and individuals so colored should receive no further stained food. With a minimum of experience it is soon possible to separate properly marked shrimp with facility. Staining is equally pronounced with solu- tion concentrations of 1 percent and 2 percent, but the 1 -percent solution is favored as a further check against over staining. Since no advantage was found in using sea -water solutions, distilled water is the preferred solvent. Although differ- ences were inconsistent, the Harleco stain ap- peared to produce a slightly brighter color than the National Aniline product. All of the so-called fast stains described above exhibit a slight tendency to fade, but in every case the remaining color is quite distinct over the period indicated. Whether stained shrimp will retain abnormal coloration over similar periods under natural conditions cannot be ascertained without field testing. Under the conditions of the experiments, captive shrimp did not exhibit normal growth . Stained and normal shrimp held for extended periods showed little observable size increment even after several shedding cycles . Under con- ditions of normal growth, it is doubtful whether a 30- or 40-mm. shrimp could retain sufficient stain to enable identification as an adult. Despite these questionable factors, the use of the recommended stains and procedures offers a new and useful tool for shrimp fishery investigations. Marking by injection is a rapid method applicable to either sea or shore -based operations. The feeding technique, though probably restricted to use at shore stations, 22 would be most applicable to the marking of small shrimp. The number of individuals marked by this method at any one time would be limited only by the quantity of available shrimp and capacity of storage facilities. At present, only three colors, red, green, and blue, are available for use as shrimp -marking agents. Simultaneous use of identical colors on groups of shrimp of like size in neighboring areas ;ould lead to complete confusion of results. It is strongly urged that all shrimp -marking experiments be subject to judicious and cooperative planning by the vari- ous fishery-research agencies prior to commencement of field operations. ACKNOWLEDGMENTS The author is indebted to Dr. Gordon Gunter, Director, Gulf Coast Research Labor- atory, and the late Mr . William Herzog for valuable suggestions and assistance during the early phases of this study. The writer is also grateful for the courtesies extended by members of the Gulf Fishery Investigations, U. S. Fish and Wildlife Service, Galveston, Texas. SUMMARY 1 . Results of tests on the marking of commercial shrimp, Penaeus setiferus, P. aztecus, and P. duorarum, with biological stains are presented. 2 . Experiments were conducted on cap- tive shrimp held in storage tanks and aquariums supplied with circulating sea water . 3. Ninety-nine tests involving more than 26 stains were conducted during the period from June 1955 through June 1956. 4. Shrimp ranging in size from 30 mm. to 175 mm. were subject to one or more of three staining methods; immersion, injection, and feeding . 5. The immersion technique has been consistently unsuccessful with the stains used. 6. Fast Green FCF (National Anilin4 Niagara Sky Blue 6B, Trypan Red, and Trypan Blue have proved effective with the injection technique. Each of these stains has remained fast for more than 100 days and through at least one shedding period. Trypan Red and Trypan Blue have been retained for over 200 days . 7. Trypan Red is the only fast marking agent found among stains administered by the feeding method. It is effective throu^out the experimental size ranges, and is considered satisfactory for marking shrimp under a length of 80 mm. Distinctive coloration was retained for more than 233 days and through at least three ecdyses. 8. A high incidence of cannibalism a- mong captive shrimp makes determination of experimental mortality difficult. Evidence from control experiments indicates that when recom- mended procedures are followed there is little difference between mortalities of marked and normal shrimp. 9. Whether these stains will remain fast for extended periods under natural conditions can only be determined by field tests. 10. Since only three colors, red, blue, and green, have been found satisfactory as mark- ing agents, the value of these marking techniques in a field program will depend largely upon judicious and cooperative planning by the vari- ous shrimp investigations. REFERENCES Carranza, J. 1953. Metodos para marcar peces de pequeno tamano de utilidad en estudio de problaciones. Ann. Escuela National Cienclas Biol. 7{1-4):111-128. Conn, H . 1953. EXinn, A. 1951. Biological stains. 6th edn., 367 pp. Biotech Publications, Geneva, N.Y. and CM. Cocker Notes on marking live fish with bio- logical stains. Copeia 1951 (l):28-3l. 23 Feder, H. M. 1955. The use of vital stains in marking Pacific coast starfish. California Fish & Game 41(3):245-246. Gustafson, 1953. Klawe. W. 1954. A. H. Preliminary investigations in mark- ing marine worms. Res. Bull. Maine Dept. Sea and Shore Fish. 9:1-8. A method for marking marine worms. Science 120(3109): 187-188. Lindner, M. J., andW. W. Anderson 1956. Growth, migrations, spawning and size distribution of shrimp Penaeus setiferus. Fish. Bull. U.S. Fish and Wildlife Service, 106:555-645. Loosanoff, V. L. 1937. Use of Nile Blue Sulfate in marking starfish. Science 85(2208):412. McRae, E.D., Jr. 1952. Progress report on the shrimp in- vestigation. Ann. Kept. Mar. Lab. Texas Game Fish. Comm,., Sept.l, 1950, to Aug. 31, 1951, processed. Menzel, R. W. 1955. Marking of shrimp. Science 12 1(3 143): 446. Racek, A. A. 1955. Penaeid prawn fisheries of Australia with special reference to New South Wales. Indo -Pacific Fisheries Council (FAO) 6th Session, Tokyo, Sept. 30 - Oct. 14, 1955. Sym. 3, 18 pp., processed. Rounsefell, G. A., andW. H. Everhart. 1953. Fishery science - its methods and applications. 444pp., John Wiley & Sons, New York . Rounsefell, G. A., and J. L. Kask. 1945. How to mark fish. Trans. Amer. Fish. Soc. 73:320-363. Vernon, A. A. 1937. Starfish stains. Science 86(2220):64. 24 IHT.-DUP. SEC.. tASH., D.C.Ji.i.60 ."SL WHOI Libra, ^Sff..- ?<'rtals 5 WHSE 01166