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Only the vertical components of movement are represented. Each verti- cal bar shows total net displacement upward or downward for 13 individuals over a 3-hour period when conditions on the rock sur- face changed from total exposure to total submersion on a rising tide, or from total submersion to total exposure on a falling tide. Numbers above and below each vertical bar show the numbers of animals whose net displacement during the period was upward and downward, respectively; numbers on the zero line indicate the numbers of animals remaining stationary, or showing only horizontal movement. Only one individual in the population homed consist- ently for the entire 45 hour period (Figure 4c). Inter- estingly enough, the homing limpet not only returned to its home site as the tide went out, but also during periods of high tide accompanied by surge. This type of behavior was also observed in homing Acmaea limatula studied in other locations. They would leave their home sites as the tide was rising, return when the tide was high and then either remain at home or leave again as the tide was falling. This behavior helps to emphasize the character- istic drop in activity during periods of high tide as shown in Figure 1. Although only one of 13 animals in the population whose activity was closely followed, homed consistently for a period of 4 low tides, the percentage of homing THE VELIGER 1200-1500 | Tidal Conditions EL IE. SE Ie Page 7 animals may be higher for the total population. In an- other field observation made on Mussel Point, 9 of 15 Acmaea limatula homed consistently for a 48-hour period. These limpets were located on a horizontal rather than a vertical surface, and although the number of animals observed is small, the findings suggest that homing may be more prevalent among populations of A. limatula which must cope with desiccation accompanied by direct sunlight. 1400-1530 2000-2130 1200-1350 1300-1450 “N 50 “N 40 30 20 10 10 Down 20 darkroom daylight am 30 Figure 3 Vertical movement in the laboratory under simulated conditions of incoming tides. DISTRIBUTION 1n RELATION to ALGAE The algae present seem to play a significant role in the distribution of Acmaea limatula. A good example of this can be cited from the population whose movements are shown in Figure 1. The surface of the vertical face on which the 13 limpets were located was occupied mainly by diatoms and microscopic green and blue-green algae on the left-hand upper side of the rock, and by encrusting red algae, mainly Hildenbrandia and some Peyssonelia, on the lower right-hand side. These two regions occupied approximately equal areas, and both areas bore occasional small clumps of Endocladia or Page 8 -— =1 inch LC = period of Ni High Tide exposure Flow f \ Ebb (Night) (Night) Ebb (Day) Ebb (Night) High Tide is Flow (Day) MN Ebb Flow (Night) (Night) Figure 4 a Typical displacement tracks for 3 individuals of Acmaea limatula. Observations were made at intervals of 14 hours over a period of Gigartina. Though Hildenbrandia and Peyssonelia dom- inated only about half of the rock surface (Figure 5), the 13 limpets observed spent a combined total of 500 hours in this lower right-hand region. During the 45-hour observation period, only 2 limpets ventured into the re- gion bearing mainly green and blue-green algae and diatoms, where they spent a total of 85 hours. Although individual Acmaea limatula can be found in widely differing regions within the intertidal of Mussel Point, the larger populations (up to 37 per square yard) are invariably located in regions where Hildenbrandia or Peyssonelia or both are abundant. In order to demon- strate this relationship betwen limpets and algae, a 3.4 square foot rock surface was chosen containing 14 A. limatula, a fairly abundant crop of the red encrusting alga Hildenbrandia, and a wide variety of other algal growths. A clear plastic sheet was placed over the region and the areas occupied by the various algae were out- lined with wax marking pencils. The locations of the 14 THE VELIGER Vol. 11; Supplement —— =1 inch (0 = period of exposure Figure 4 b 45 hours, May 2 to 4, 1966. Limpets did not always move between successive observation periods, even when submerged or awash. A. limatula were noted. The drawing was traced onto a large piece of graph paper and the different regions were cut out and weighed. By comparing these weights to the weight of 1 square inch of graph paper, it was possible to get a fairly accurate determination of the areas occu- pied by the various algae present. Figure 6 shows what more qualitative observations confirm, that, at least during low tide, A. limatula is often found in direct association with the red encrusting algae. This, of course, ‘is only an instantaneous glance at the distribution of A. limatula, but it seems pertinent when viewed in conjunc- tion with Figure 5 and the gut content analyses below. Although on Mussel Point large populations of Ac- maea limatula were found only on the red encrusting algae Hildenbrandia and Peyssonelia, this may not necessarily apply to other regions along the Pacific Coast. It was noted, for example, that on Point Lobos, just south of Carmel, California, fairly large populations of A. limatu- la seemed to thrive on low horizontal expanses of sand- Vol. 11; Supplement THE VELIGER Page 9 600 @ 500 I BS s ot = 00 Boal @ Ey Qa, Q ; wn Ss 7) ‘g 300 g< Fog = & 200 Ss oF os & 100 to) 2 aS 3 8 3 ce 25 3 © s gv Q ) Q oe a Re gb 5 an an Moy oo CS c . 3 Oo SS 2 g 1 100 a2) [ > S = ~ é BERS ) 2 25 A = & 2 8 B & s SLAs 9 2 ~ a SS Oy s 501 38S ° x = Figure 6 Distribution of 14 Acmaea limatula at low tide in relation to algae available within a 3.4 square foot area, mid-May, 1966. dia, Peyssonelia, Lithophyllum, and Lithothamnion. Strangely enough, A. limatula seems to ignore the en- crusting red alga Petrocelis (Figure 7g) even though it grows in relative abundance within the intertidal region inhabited by this limpet. The non-encrusting algae are rarely ingested except for tiny individuals of larger species, or species which form a short fuzz growing close to the rock surface (Table 1, III; Figure 7b, 7e, 7f, 7h). In such cases the thalli found were very small and capable of being swallowed whole. Only once was a fragment of a large, non-encrus- ting alga (not an entire thallus) found within the stomach, although many such algae were available to the individ- ; Supplement Vol. 11 THE VELIGER Page 10 “uOLsaI yoes ul pauturexa syoduy G 9} Ul paroyUNOoUS UOT}eIIvA jo asuevl oy} MOYs sIeq [eOT}IOA iaddn ay} JO spus oy} 3 soul] OUT, 996] ‘Ke ulog Jessnyy uo seaie JUaIeYIp g ul uayed POOF 0} I[QeII~Ae Poo} jo diysuonepoy L amsiy DIUuawog]0/) © pyauosshag sruoffip aidossos91f : 2 DISaYyyDIT wnssojsopoyy DUuty]D10/) 5 COTES) ay G8 cn = 9 - Creo: pipuv.quap]iH pyauosshag atdoosor yy uorlUmDyz0YINT pipuv.quap)}iH pyauosshag Pa pioydopoiy wnpydydoynT mnipue) o $1]9901]9g + 9 a - ° ~ a Comer pisfjoy stdoososoTu Dipub.quapyiH unssojsopoyuy stdodsor1n1y $YaI0Laq pyauosshag a co) a _ ° — a co ~ otdoosoxo Tur Dyaaag wnipyay pavpuy unpydyqoyny $019901]UI) wnipyay + oo Ey - fe) co ct or) + stdoosoro1fy DIppjI0puq ty wnpdyg oynT unssojsopoyy otdoososotu pyauosstag Duos o $9990149g pipuv.quapiiy vipuv.quaplA wnydygoynT Dada aay oe) ct - ° = a co) ap = oe) a = ° ™ on co ~ fos) a = ° ms nN for) + uayeq oes[y jo g[qepreay ses[y usjyeq oesty jo gqureay esTV uajeq oesty jo giqepreay ees,y afqearasqag, jo qunowy 9ANeeyY afqearesqag jo qUNOWTY 9ANRIDXY DEE SLO) of junoUy sAnePY qunoUry 9ANLIY qunoury dANvaY qunowy 9ANe[2yY Vol. 11; Supplement Table 1 Frequency of Occurrence of Algae Found in Gut of Acmaea limatula Number of Animals Type of Algae with Algae in Gut I. Microscopic Algae (small greens, blue-greens, and diatoms) 40 II. Encrusting Algae 1. Hildenbrandia occidentalis 25 2. Peyssonelia pacifica 25 3. Lithophyllum sp. 15 4. Lithothamnion sp. 10 5. Ralfsia pacifica 1 III. Low, Turfforming Algae (up to 3mm high) 1. Gelidium coulteri 10 2. Cladophora trichotoma 5 3. Leathesia difformis 5 IV. Other Algae Centroceras clavulatum Colpomenia peregrina Rhodoglossum affine . Fragment of a large brown alga a> CS eee uals analyzed. The item found ingested was a section of a large brown alga which could not be found in the surrounding area. Figure 7h provides an excellent example of Acmaea limatula’s tendency to avoid the larger non-encrusting algae as food sources. Although this limpet eats the en- crusting coralline algae Lithophyllum and Lithotham- nion, the non-encrusting form Corallina was completely ignored here, even though it dominated the region. FOOD NICHE SPECIALIZATION in Acmaea limatula AND Acmaea pelta Acmaea limatula and the very eurytopic limpet A. pelta may often be found occupying the same general areas and habitats in ‘the intermediate and low intertidal zones on Mussel Point. In order to substantiate the presence of overlapping populations, Peter Craig and the author mapped species distribution in 3 transects, each 3 feet wide, extending from the low to the high intertidal zones. Populations of the two species overlap broadly, and several places were discovered where both A. limatula and A. pelta were abundant (see also Cratc, 1968). With such an overlap in distribution on the part of two species in the same genus, one might expect consid- THE VELIGER Page 11 erable competition, especially for food since both species are herbivores and scrapers. Interestingly enough, little competition for food actually exists. The two species live side by side, A. pelta eating the larger, non-encrusting algal forms (Craic, 1968) and A. limatula ingesting primarily the encrusting red and coralline algae. SUMMARY — . Individuals of Acmaea limatula move actively only during periods when they are either splashed by water or submerged. The time of day or night at which the period of wetting occurs does not appear to influence the amount of activity. . Animals do not begin moving immediately upon being splashed, and some remain inactive for a time after they are first submerged. Activity reaches a high level as the tide is rising, characteristically drops during periods of high tide, and shows some increase again as the tide recedes. 3. With rising tides at night, a definite upward movement occurs. During the daytime hours, the displacement is characteristically in a downward direction during both rising and falling tides. The tendency to move down- ward during the daytime is proportionally greater during the receding tides than on the incoming tides. 4. Only one of the 13 limpets in the population studied in detail homed consistently for the entire 45-hour ob- servation period. However, on a large horizontal rock, 9 of 15 homed consistently for a 48-hour observation period. 5. Where the red encrusting algae Hildenbrandia and Peyssonelia are present, Acmaea limatula spends most of its time on these. 6. The main foods of Acmaea limatula are microscopic algae and the encrusting red and coralline algae Hil- denbrandia, Peyssonelia, Lithophyllum,and Lithotham- nion. The encrusting red alga Petrocelis is ignored, even though it is relatively abundant in the region inhabited by the limpet. Non-encrusting forms are not eaten un- less they are very short, growing close to the rock surface. 7. Although Acmaea limatula and A. pelta may often be found occupying the same general areas and habitats in the intermediate, and low intertidal zones of Mussel Point, there is little interspecific competition for food. Acmaea pelta eats the larger, non-encrusting algae, while A. limatula ingests primarily the encrusting red and coralline algae. nN Page 12 THE VELIGER Vol. 11; Supplement ACKNOWLEDGMENTS I would like to express my sincere thanks to the faculty and staff of Hopkins Marine Station, especially to Drs. Donald P. and Isabella A. Abbott for their advice and encouragement. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. LITERATURE CITED Craic, PETER CHRISTIAN 1968. The activity pattern and food habits of the limpet Ac- maea pelta. The Veliger 11, Supplement: 13 - 19; plt. 1; 5 text figs.; 1 table (15 July 1968) Giynn, PETER W. 1965. Community composition, structure, and interrelation- ships in the marine intertidal Endocladia muricata — Balanus glandula association in Monterey Bay, California. Beau- fortia (Zool. Mus. Amsterdam) 12 (148): 1-198; 76 figs.; 32 tables; 5 appendices Wieser, W. Rocers, DoNALD ALLEN 1968. The effects of light and tide on movements of the limpet Acmaea scutum. The Veliger 11, Supplement: 20 - 24; 4 text figs.; 1 table (15 July 1968) Ross, THomas LEE 1968. Light responses in the limpet Acmaea limatula. The Veliger 11, Supplement: 25 - 29; 3 text figs.; 3 tables (15 July 1968) Seapy, Rocer R. 1966. Reproduction and growth in the file limpet, Acmaea limatula CarPENTER, 1864 (Mollusca: Gastropoda). The Veliger 8 (4): 300-310; 7 figs.; 2 tables (1 April 1966) SEGAL, EarL 1962. Initial response of the heart-rate of a gastropod, Acmaea limatula, to abrupt changes in temperature. Na- ture 195 (4842): 674-675; 1 fig.; 3 tables SEGAL, Eart & Pau, Augustus DEHNEL 1962. | Osmotic behavior in an intertidal limpet, Acmaea lima- tula. Biol. Bull. 122 (3) : 417 - 430; 5 figs.; 1 table Test, Avery RANsom (GRANT) 1946. Speciation in limpets of the genus Acmaea. Con- trib. Lab. Vertebrate Biol., Univ. Michigan, Ann Arbor. No. 31: 1-24 1952. Investigations of the microfauna inhabiting seaweeds on rocky coasts. Journ. Mar. Biol. Assoc. U. K. 31: 35 - 44 Vol. 11; Supplement THE VELIGER Page 13 The Activity Pattern and Food Habits of the Limpet Acmaea pelta BY PETER C. CRAIG Hopkins Marine Station of Stanford University" Pacific Grove, California 93950 (Plate 1; 5 Text figures; 1 Table) Acmaea pelta EscHSCHOLTZ, 1833, Is ABUNDANT in the rocky intertidal zone along the California coast. De- scribed as the most eurytopic member of the genus Acmaea by Test (1945), A. pelta ranges in its inter- tidal habitat from the higher Endocladia to the lower Egregia association at Mussel Point, Pacific Grove, Calli- fornia. Studies of its biology to date have been concerned with its general ecology (Test, 1945), the effects of grazing on diatom populations (CASTENHOLZ, 1961), the reproductive cycle (FriIrcHMAN, 1961, 1962), and its ecological role in the Endocladia zone (GLYNN, 1965). The present study was conducted to provide more infor- mation on the behavior and foods of A. pelta. FIELD STUDIES In order to determine activity patterns of Acmaea pelta, 16 limpets were individually marked and observed at hourly intervals during high tides over a continuous peri- od of 4 days. Later, another 10 limpets were observed at 30 minute or hourly intervals for a 24 hour period. A small mark was painted on the substrate at each end of each limpet to indicate the animal’s original position; at each successive observation, measurement of the distance and angle of the limpet in relation to this point gave its new position. General movement, feeding activity, and degree of tidal exposure were noted at each observation. The degree of tidal exposure was indicated using the de- scriptive terms of GLyNN (1965): exposed —_ periods when the animals were exposed to air without wetting by waves or splash; awash — when the animals were wetted by the sea, but not for more than 50% of the time; sub- merged when the animals were wetted more than _ 50% of the time by splash or were continually immersed. ™ Permanent address: 2759 Birchwood, Deerfield, Illinois. The behavioral criteria used to determine the occurrence of feeding in the field were based on observations of Acmaea pelta under laboratory conditions, and are de- scribed later. General movement of the population is shown in Figure 1. Periods of movement in Acmaea pelta show a_con- sistent relationship with the tidal cycle. The limpets remain stationary when out of water. Movement usually does not occur until the end of the period when they are awash on an incoming tide. While only 3 of the limpets depicted in Figure 1 moved during the initial awash period of the higher high water, all moved during the early part of the period of submersion. During the fol- lowing period of lower high water (Figure 1), those animals too high on the rock to be submerged did not move at all, although they were awash for an extensive period. In general, A. pelta do not move until they are submerged, though after being submerged, some were ob- served to move upward on the rock into the zone still awash, climbing at a rate equal to that of the incoming tide. Acmaea pelta usually do not remain active for the high tide period and in several instances have been ob- served not to move at all (e.g. limpet “i”, Figure 1). Such stationary individuals usually move during the following high tide. Exact paths of movement were not plotted, but net displacement on the rocks between successive observation periods was noted for each limpet. This provides a meas- ure of minimum distance moved. The amount of move- ment and the area covered varies considerably with individuals; one limpet moved no more than 2 inches during any high tide period over a span of 4 days, another covered a distance of 6 feet during a single high tide period. Figure 2 shows examples of the types of paths taken by different individuals during a 4 day observation period. Page 14 THE VELIGER Vol. 11; Supplement (] exposed Re]submerged Key q moving NN awash Be 4— > feeding YS stationary a i —— SSN EET er ee NN Individual Limpets 1 ee ce NN ee NS SSS OOK “ 0200 0600 1000 1400 higher high water Se ENN SEN WN | a ee I BEE SS Sec se 0200 0600 1800 2200 lower low water Time of Day Figure 1 Movement and feeding activity of 10 Acmaea pelta observed at 30 minute or hourly intervals over a period of 24 hours, May 4 to 5, 1966. Limpets are indicated in order of their vertical positions on the rock surface; individuals lower on the rock were awash and submerged for longer periods. The extended time during which the two highest limpets were awash on the receding higher high water was due to downward movement on the rock of these animals. Path A shows rather random movement with the limpet coming to rest at a different spot at each exposure period. At some time during the 4 day study, 11 of the 16 limpets exhibited movement patterns like those of path B; they returned at least once to exactly the same loca- tion previously occupied. Homing, 1. e., returning to ex- actly the same spot and here adopting the same orienta- tion (Figure 2, path C), was noted in 4 of the limpets. One individual homed after moving 3 feet away from its spot. One limpet was observed to move into a spot and assume the same orientation as another limpet which had previously occupied that spot. Path D represents a type often observed. The majority of movement repeatedly occurs in a particular area, and the limpet returns to the same small area (approximate diameter 1 inch) with each exposure period but does not necessarily settle in exactly the same spot with the same orientation. VERTICAL MOVEMENT To test whether there was a general trend of vertical movement on the rock surface in relation to tidal expo- sure, the vertical component of movement of 10 limpets was observed at hourly intervals. The results reveal a specific pattern (Figure 3). When the tide rises at night a net upward displacement occurs, followed by a net downward displacement as the tide recedes. Similar movement patterns have been noted in Acmaea limatula (Eaton, 1968) and A. scutum (RoceErs, 1968). Com- parable data are not available for movements during the day. FEEDING ACTIVITY Studies were made to determine the frequency and dura- tion of feeding periods, and the relation of feeding to Explanation of Plate 1 A: Radula pattern of Acmaca pelta produced by a limpet feeding on a glass plate covered with a film of microscopic green algae and diatoms. B: Portion of a blade of Jridaca taken in the field. An Acmaea pelta was found on top of the radular marks, and Iridaca was almost exclusively present in its stomach. Tue VELIGER, Vol. 11, Supplement [Craic] Plate 1 Figure 1 Figure 2 SS Vol. 11; Supplement THE VELIGER Page 15 Representative tracks of 4 individual Acmaca pelta on a vertical rock surface over a period of 3 days, May 26 to 28, 1966. For each track, each type of line represents movement during one eee? . oe? . ee . ooee ater ee Nl ul sof {I ¢. Cc ae : , D B Figure 2 complete tidal cycle, and the limpets were exposed only at lower low water. Points enclosed by small squares are the places where the limpets remained stationary during periods of low water. Consistent homing behavior is indicated by the track in Figure 2 C. 40 up 20 20 Net Displacement down awash (flow) awash (ebb) a ea aa SS SS SS submerged Figure 3 Net vertical displacement of the 10 limpets in Figure 1 at succes- sive intervals during one nighttime higher high water, May 4 to 5, 1966. Each bar represents approximatcly the movement during one hour. Differences due to different vertical positions of the limpets have been compensated for by independently calculating the move- ment each animal underwent during the phases of tide indicated. tidal exposure. Acmaea pelta placed in aquaria or on glass plates covered with an algal film, and arranged so that radular movements could be seen in ventral view, revealed a characteristic feeding behavior. The head sways from side to side, completing a cycle in 1 to 2 minutes. This can be detected in a dorsal view, even in the field, by watching the cephalic tentacles. The mouth, if visible, is flattened and spread over the substrate. Forward loco- motion is relatively slow, in one case about 1cm in 5 minutes. Feeding behavior in the field and laboratory, as recorded by patterns scraped by the radula on larger algae or on alga-covered glass plates, corresponds with the above description. In such patterns, produced by radular action as the limpet’s head moves from side to side, each individual rasp of the radula is visible (Plate 1, Figure B). At greater magnification the marks of individ- ual radular teeth can be seen (Plate 1, Figure A). This pattern of feeding provides a moderately efficient cov- erage of the surface. It was often difficult to tell whether animals were feed- ing or not under field conditions, but some information on the feeding activity of Acmaea pelta was obtained. Of the limpets shown in Figure 1, 3 were not observed to feed during the period of higher high water, and 6 Page 16 THE VELIGER Vol. 11; Supplement showed neither feeding nor other movement at lower high water. While feeding may have occurred between observations, some limpets taken from the field as the tide receded had no food in their stomachs. It appears that limpets do not necessarily feed during every tidal cycle; for much of the period of activity the animals move about without showing clear evidence of feeding. The state- ment by Test (1945, p. 397) that Acmaea pelta “feeds at any and all times, regardless of whether the tide is in or out,” is not supported by the present study. FOODS oF Acmaea pelta Since Acmaea pelta is a very eurytopic organism, the question arises whether it is able to feed on a wide variety of plant material or feeds on a few forms which are widely distributed in the intertidal region. Published accounts (Trst, 1945; FrircHMAN, 1961) indicate that A. pelta eats a variety of algae, both microscopic and macroscopic, but quantitative information is lacking. A study was therefore made to determine the foods avail- able to A. pelta and the foods actually eaten, and to assess evidences of food selection. Acmaea pelta are most abundant in mid to upper inter- tidal regions that can often be characterized by the pres- ence of Endocladia, Pelvetia, Egregia, or Postelsia (Fig- ure 4). Four areas were chosen, each a region where a Endocladia Postelsia P16 Pelvetia 3 .8] shore o as} “Sw 5 SI L oe ss Egregia io) = 4 Sits = os Ora my 8 aS) ft § 2 2 a z 3 2 9S 1S) ‘3 gq rR) oe Figure 4 Horizontal and vertical distribution on a diagrammatic transect of a rocky shore, showing the main zones where Acmaea pelta occurs. Each zone is characterized by a predominating species of alga. Vertical ranges of the algae are based on SmirH (1944). different one of the above algae predominated. Since any alga present might represent a possible food source for A. pelta, an attempt was made to estimate the relative quan- tities of the different macroscopic algae present (Figure 5). Estimates are crude, for it is difficult to compare the availability of an alga with a thallus many feet long with the availability of encrusting forms. The abundance of microscopic algae was not determined. These forms, con- sisting of diatoms and unicellular and filamentous green and blue-green algae, occurred on otherwise bare rock surfaces and as epiphytes on many of the larger algae in all 4 areas. Very small juvenile individuals of larger algae were included with the larger algae. Twenty five Acmaea pelta whose guts retained food materials in recognizable states were collected from each of the 4 areas. A portion of the material from the stomach of each animal was microscopically examined. Identifi- cations of algae in both field environment and stomachs were made with the kind help of Dr. Isabella A. Abbott of the Hopkins Marine Station. An attempt was made to assess the relative amount of each alga present to within 10%. Materials which could not be identified in micro- scopic examination are listed as “unidentified debris.” The values obtained from the analyses of the stomach contents of the 25 A. pelta from each area were then averaged (Figure 5). The Endocladia region, between 6.0 and 3.0 feet above mean lower low water (SmitH, 1944; a somewhat greater range is indicated by GLynn, 1965), is characterized by macroscopic red algae. In this region Acmaea pfelta ingested a variety of algae (Figure 5A). Macroscopic algae (55%) were present in twice the volume of micro- scopic algae (26%). The macroscopic algae present in the gut are not minute, immature plants but consist of small fragments of much larger specimens. Field obser- vation suggests that these are probably obtained from plants which are growing in small crevices and which have been grazed repeatedly, and from the holdfasts of larger plants growing on the open rock surface. The brown alga Pelvetia characterizes a zone between 4.5 and 2.0 feet above mean lower low water (SMITH, 1944). This plant is the primary food of Acmaea pelta here (Figure 5 B). Most limpets in this region are found in the moist area under the blades of the Pelvetia, and except for a few small individuals they are not often seen on the blades themselves. They apparently feed on the holdfast where limpets are often observed during a period of submersion. Pelvetia appeared unusually macerated in the stomach of these limpets and is thought to consti- tute a large portion of the 33% of unidentified debris. The Egregia region occupies the 2.0 to 0.0 foot level in the intertidal zone (Smirn, 1944). Here, too, macroscopic plants form the major part (62%) of the diet of Acmaea pelta (Figure 5, C). As in the Pelvetia region, one brown alga predominates in the environment and in the gut contents. Limpets are often found directly on the holdfast Page 17 THE VELIGER ; Supplement Vol. 11 sep Pay nusprug) otdoosgrorur DUIJDL0/) wniu0j9I014s05 $Y3I0L9g unssopsopoyy DUlpLDSID IFPI | uolUiumDyzOYNT Dipubiquappiyy ‘vyauosskag TEL ED | B. Pelvetia Region ‘OINeWU OU 20 10 te) fe) 20 50 40 30 30 40 sugqep peynuepruy) py g sowoLaquy uoYyjODULIG dtdoosororut pidydsog pioydopnyiy CET EVERD) DavpiLy unssopsopoyy DUljLDaID) DUuIpJDL0/) DIpvjIOpUu pipubiquappiyy “pyauosskag A. Endocladia Region $199019q “OLOBUU OU 20 10 ie) 10 [o) a 40 30 30 40 ua}ea arqeyreae aes[y JO us.I0g D. Postelsia Region ° ° = oo) C. Egregia Region 30 ° Nn 20 EGE EME) DEIN UoYyj1ODWULIG sidosso19 1 59990194 Dis a1aq DippjI0puA DUDPLOYIOLIII FY vappiy By DE DINAA BS 3 | DUYJvI0) Postelsia < UOUUDYIOYRNT I 30 40 50 Sigs p spay Nus piu DIO1SSDL pyd1owmosaqug DipojIopuy piuamod jo) Figure 5 wunssojsopoyy o1dosso191Ud xipogsoyhyq COMES) ‘ore OU $9990119q DipUviguearirel pail) DUIY}VL0/) uolUuuDy}0Y jv pavpuy Duiivdiy Dis a3 ° ° ° ° iy nN 40 ° (oe) I a[qeyreae aes[y Jo jusI1eg pic algac. These areas may contribute a portion to the microscopic algae eaten; other microscopic algae occur as epiphytes on larger The foods available and the foods eaten in 4 regions, each charac- terized by a predominating species of alga, where Acmaea pelta is plants. abundant. ‘“‘No Macro.” refers to areas devoid of visible macrosco- Page 18 THE VELIGER Vol. 11; Supplement Table 1 Algae Eaten by Acmaea pelta (and the per cent of the 25 limpets per region in which each alga was found) Regions: 3 N 8 = 3°30 oe RQ AY RQ a a 84% 100% 100% 56% 85% I. Macroscopic Plants A. Green Algae 1. Prasiola meridionalis SETCHELL & GARDNER, 1920 12 8 5 2. Enteromorpha intestinalis (LINNAEUS) Linx, 1820 8 8 4 3. Ulva sp. = - 4 - 1 4. Cladophora trichotoma 4 - - - 1 B. Red Algae 1. Rhodoglossum affine (Harvey) Kyun, 1928 44 28 48 - 30 2. Endocladia muricata (Post. & Rupr.) J.G. AGARDH, 1847 48 20 20 - 22 3. Iridaea sp. 36 12 16 24 22 4. Gelidium sp. 32 8 5. Lithothamnion sp. 8 - 12 4 6 6. Gigartina sp. 20 - - - 5 7. Dermatolithon dispar (Fosuie) Fosuie, 1909 8 - - 8 4 8. Porphyra perforata J.G. AcarpH, 1883 8 - - 4 3 9. Peyssonelia pacifica Kyun, 1925 = 4 - - 1 C. Brown Algae 1. Pelvetia fastigiata (J.G. AcaRpH) De Tonr, 1895 - 92 - - 23 2. Egregia menziesii (TURNER) ARESCHOUG, 1876 - - 52 - 13 3. Postelsia palmaeformis RuPRECHT, 1852 ~ - - 32 8 4. Colpomenia peregrina (SauvacEau) HameEL, 1931-39 16 4 5. Heterochordaria abietina (Rupr.) SetcH. & Garp. 1924 = - 4 - - 1 D. Flowering Plants 1. Phyllospadix scouleri HooKER, - - 8 - 2 II. Microscopic Algae 1. unicellular green algae, 80 24 64 84 63 diatoms, blue-green algae 2. Dermocarpa 8 4 - - 3 3. Goniotrichum sp. - 4 4 2 4. Entophysalis deusta (MENEGH.) Drovet & Datry, 1948 8 = = = 2 5. Ectocarpus sp. 8 - 8 - 4 6. Hapalospongidion gelatinosum SAUNDERS, 1899 - 4 2 1 7. Pylaiella gardneri Cotiins, 1898 = - - 12 3 and stipes of Egregia plants, and scars apparently caused by extensive feeding are frequently found beneath them. This tendency of Acmaea pelta to ingest more macro- scopic than microscopic algae is reversed in the exposed and surf-swept Postelsia region (4.0 to 1.0 foot intertidal level; Smiru, 1944). Over half the limpets collected in this region were taken from Postelsia stipes, but Postelsia is not the major food found in their stomachs. They apparently feed mainly on diatoms and other epiphytic microscopic algae growing on Postelsia (Figure 5D). Limpets not directly on Postelsia also ingested quantities of Iridaea. Microscopic algae constitute 45% of the volume of stomach contents and macroscopic algae only 32% in this zone. Comparisons of plant foods available and stomach contents from the 4 regions show that Acmaea pelta does not feed at random but ingests significantly large quanti- ties of macroscopic algae. However, all the major phyla of marine plants are represented in the diet. JoBe (1968), in a study of the digestive enzymes of A. pelta, found Vol. 11; Supplement THE VELIGER Page 19 amylase activity marked, and that of fucoidinase and alginase somewhat less. The variety of foods eaten, shown in Table 1, may be an important factor influencing the ability of A. pelta to live in a wide range of intertidal conditions. Where different species of the same genus occupy the same general area and habitat, and are more or less sym- patric, the extent to which they compete for various re- quirements is always of interest. Acmaea pelta is often found in company with other species of Acmaea, espe- cially A. limatula. In a study of the foods of A. limatula, Eaton (1968) found that A. limatula ingests primarily the red encrusting algae, Hildenbrandia, Peyssonelia, Lithothamnion, and Lithophyllum. In contrast, A. pelta eats very little of these species. The dietary studies sug- gest that in situations where both species occur, there is relatively little competition for food. SUMMARY 1. Most movement and feeding of Acmaea pelta occurs while the animals are submerged and while they are being splashed during tidal ebb. At night, the popu- lation shows a net upward displacement when the tide rises and a net downward displacement as the tide recedes. All but 1 of 16 limpets observed over a 4-day period returned at least once to exactly the same loca- tion previously occupied, but only 4 of them consist- ently homed. 2. Feeding is not continuous during periods of activity, and apparently animals do not feed during every tidal cycle. 3. Acmaea pelta ingests a wide variety of algae, both microscopic and macroscopic. The most common mac- roscopic plants eaten are the red algae Rhodoglossum affine, Endocladia muricata, and Iridaea sp., and the brown algae Pelvetia fastigiata and Egregia menziesu. 4. Acmaea pelta and A. limatula often occur in close proximity. Dietary studies suggest that in such situa- tions there is relatively little competition for food. ACKNOWLEDGMENTS I would like to extend my sincere appreciation to Drs. Donald P. Abbott and Isabella A. Abbott of the Hopkins Marine Station for their kind help and dedicated interest. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. The photographs in Plate 1 were taken by Mr. S. E. Johnson. LITERATURE CITED CasTENHOLz, RicHarpD WILLIAM 1961. The effect of grazing on marine littoral diatom popu- lations. Ecology 42 (4): 783-794; 6 figs. Eaton, Cuartes McKENnpDREE 1968. The activity and food of the file limpet, Acmaea lima- tula. The Veliger 11, Supplement: 5-12; 7 text figs.; 1 table (15 July1968) FrirtcHMAN, Harry Kier, II 1961. A study of the reproductive cycle in the California Acmaeidae (Gastropoda). Part 3. The Veliger 4 (1): 41 to 47; plts. 9 - 14 (1 July 1961) 1962. A study of the reproductive cycle in the California Acmaeidae (Gastropoda). Part 4. The Veliger 4 (3): 134 to 140; plts. 30 - 32 (1 January 1962) GLYNN, PETER W. 1965. | Community composition, structure, and _ interrelation- ships in the marine intertidal Endocladia muricata — Balanus glandula association in Monterey Bay, California. Beau- fortia (Zool. Mus. Amsterdam) 12 (148): 1-198; 76 figs.; 32 tables; 5 appendices Jose, ALAN Hat 1968. A study of morphological variation in the limpet Acmaea pelta. The Veliger 11, Supplement: 69 - 72; plt. 6; 1 text fig.; 2 tables (15 July 1968) Rocers, DonaLp ALLEN 1968. The effects of light and tide on movements of the limpet Acmaea scutum. 20 - 24; 4 text figs.; 1 table The Veliger 11, Supplement: (15 July 1968) SmitH, GitBerT Morcan 1944. Marine algae of the Monterey Peninsula. Stanford Univ. Press, ix+622 pp.; illust. Stanford, Calif: Test, Avery RaANsom (GRANT) 1945. Ecology of California Acmaea. 395 - 405 Ecology 26 (4): Page 20 THE VELIGER Vol. 11; Supplement The Effects of Light and Tide on Movements of the Limpet Acmaea scutum DON A. ROGERS Hopkins Marine Station of Stanford University’ Pacific Grove, California 93950 (4 Text figures; 1 Table) THE LIMPET, Acmaea scutum EscHSCHOLTZ, 1833, in- habits the midtide zone on rocky shores along the Califor- nia coast. Test (1945) has noted some aspects of the ecology of this species, but detailed information is lacking on its activity pattern. The present study was undertaken to determine the movements of the Acmaea scutum population and the effects of tide and light on these movements. FIELD STUDY METHODS anp MATERIALS Except where noted, all studies were carried out on the rocky shores of Mussel Point, Pacific Grove, California. Two vertical rock surfaces, located on the leeward side of Mussel Point,were chosen for the first set of field studies. Both surfaces faced west and both were exposed to con- siderable surf. To facilitate the measurement of limpet movement, narrow horizontal lines were painted on both rock surfaces approximately one foot above the highest Acmaea scutum. One inch divisions were marked along this line. The A. scutum were individually marked by dots of paint on their shells. Marking was done at low tide on a warm day, and the animals were not removed from the rock. The horizontal positions of the marked limpets were indicated with reference to the divisions on the horizontal line; vertical positions were measured with a yard stick. Further painted lines on the rock were avoided because it was felt they might affect limpet movements. On April 27 and 28, 1966, the positions of 19 Acmaea scutum were recorded every hour or every two hours for a 24-hour period. On May 3 and 4, 1966, the positions of 11 other A. scutum were recorded hourly for a 24-hour period. The light conditions and the level of the tide were * Permanent address: 16 Los Cerros Drive, San Rafael, California. recorded with each observation. At each observation it was noted whether the limpets were submerged by the sea, awash, or exposed to the air. The term “awash” covers all conditions between the first dampening of the animals by splash to complete submersion. The positions recorded from the field observations were plotted to scale on graph paper. The resulting points for each limpet were connected with straight lines to provide a track indicating minimum net displacement in succes- sive intervals of time. Preliminary observations showed that the marked Acmaea scutum were underwater about 16 hours a day. A wet suit, snorkel, mask, and underwater light were used to observe the limpets when submerged. During the period of higher high water on both days the heavy surf conditions made observations impossible. INDIVIDUAL TRACKS Of the 30 Acmaea scutum marked in the 2 areas, 6 were either washed away or had their paint marks removed by the water. All of the remaining 24 A. scutum moved during the 24-hour observation periods. Earlier observa- tions had indicated that A. scutum moves very little when out of water. For this reason the intervals between suc- cessive observations were greater when the animals were out of the water. Figure 1 shows the trails of 2 A. scutum as plotted from positions recorded every hour or every 2 hours. In Table 1 I have tried to show the percentage error in my tracking method. Without continuous obser- vations one cannot be certain of the exact path the limpet followed to reach the new point. Table 1 shows that there is an apparent decrease of 15% in the trail length if one records positions every 2 hours rather than once every hour. Even with hourly readings the distances represented by the recorded tracks are probably less than the actual distances moved, but the limpets move so Vol. 11; Supplement Table 1 Differences in Trail Length Recorded When Positions of Limpets Were Measured Every Hour vs. Every Two Hours Individual Acmaea scutum Distance Tracked (Inches) Observed Observed every hour every two hours 1 28 23 2 35 29 3 76 65 4 53 42 5 23 20 6 16 16 7 30 23 8 25 24 Total: 286 242 242 es 85% slowly that the error is probably small. The trails of the 2 limpets shown in Figure 1 are characteristic of the trails of all the marked A. scutum. The trails demonstrate that individuals cross one another’s tracks during periods of activity. Figure 2 shows that between successive periods of lower low water A. scutum moves an average of 40 inches with extremes of 16 and 76 inches. © Start of observation period 3€ End of observation period AME AO oS ___ +1.0 foot i Se tide mark My Figure 1 _ Trails of two Acmaea scutum on a vertical rock surface during periods of awash and submersion from 1500, April 27, 1966 to ogoo, April 28, 1966. Each point represents the position of the limpet at the time of observation. THE VELIGER Page 21 While plotting the trails of the 24 limpets it was noticed that at low tide they returned to the vicinity of the spot they had occupied the previous low tide. The average net displacement of the 24 limpets from one low tide to the next was 5.5 inches. The largest net displacement for any one of the 24 limpets was 14 inches. Only one of the 24 limpets returned to exactly the same spot it had occu- pied the.previous low tide, and here it assumed its original orientation. Subsequent observations at Cypress Point, Pebble Beach, California, confirmed the general observa- Number of Limpets Co) | 16-24 | 25-32| 33-40] 41-48] 49-56 | 57-64 65-72] 73-80 Total Inches Traveled in 24 Hours Figure 2 Total distance moved by 24 Acmaea scutum on a vertical rock surface during a 24 hour period. tion that individuals tend to return to a spot near that occupied at the previous low tide. Combining the net dis- placement figures with the data in Figure 2 shows that Acmaea scutum moves an average of 40 inches between low tides,, but the average net displacement is 5.5 inches. MOVEMENTS 1n RELATION to TIDE AND LIGHT In an attempt to find a relationship between tide and light and the movements of Acmaea scutum, the total hourly movement and the vertical vector of the hourly movement were compared at different phases of the tide and under different light conditions. The total hourly movement is the distance between the positions occupied by a limpet before and after a one hour interval. The vertical vector of the hourly movement is the vertical separation of the limpet’s position before and after a one hour interval. The relation of total and vertical movement to light is represented in Figure 3 for the 2 Page 22 THE VELIGER eS =] Sessa essa =] (4 Sd Ws Ooo! 0063 Wl 0002 00g! + S ies . a JJ See 19497 [EPL quowlaAoul ATINOFY [eI0], — no data — Si S&S gS. o wort ro” (seyouy) (3023) JUaUIBAOUL ATINOF] [CIO], [94977 [EPL Hour of the Day Vol. 11; Supplement Hour of the Day ° ° ° ise) 8 © © =~ is (soyouy) peAour soue\sIq [eOnJaA, (sayouy) peaout sourysiq [eTI9A, Vol. 11; Supplement THE VELIGER Page 23 Figure 3 (<— on facing page) Comparison of light and tide conditions with total movement and the vertical component of total movement. Figure 3A refers to data collected April 27 to 28, 1966, from the movements of 16 limpets. Figure 3 B refers to data collected May 3 to 4, 1966, from the movements of 8 limpets. Time is represented on the horizontal axes of both graphs. The vertical axes of the upper graphs, showing total hourly movement, represent the total distance moved during the hour for all of the limpets. The vertical axes of the lower graphs, showing the vertical component of movement, represent the upward or downward displacement each hour of all the limpets from the position they had occupied at the beginning of the hour period. observation areas. The A. scutwm represented in Figure 3 A were uncovered by the tide only once during the 24- hour watch. The A. scutum represented in Figure 3 B were uncovered twice during the 24-hour watch. Figure 3 shows that Acmaea scutum moves most when it is awash. Each peak on the graphs showing total move- ment corresponds to a period when the animals were awash. Acmaea scutum does not move when out of the water and in sunlight, but the animal may move a short distance during the day when out of the water in the shade. When completely submerged, A. scutum moves sporadically, and the effects of light and darkness on movement under these conditions appear negligible. The graphs showing the vertical component of move- ment (Figure 3) demonstrate that Acmaea scutum moves upward when washed by the incoming tide and down- ward when washed by the outgoing tide. The light con- ditions during the period of wash appear to affect the distance A. scutum moves. In Figure 3 B, when the in- coming wash was accompanied by sunlight, both the total and the vertical movements were much less than when the incoming tide was accompanied by shade. The effects of light and darkness on the vertical movements of sub- merged animals are difficult to evaluate from these field studies. LABORATORY STUDIES To test the correlation of Acmaea scutum movements with the conditions of tide and light as found in the field, several laboratory experiments were made. Preliminary studies showed that when submerged in laboratory aquaria A. scutum generally moves upward. It was decided to test the effects of light and turbulence on this ~ movement. Turbulence was chosen as a variable, in addi- tion to light, because the field studies had indicated that A. scutum moves most when awash. A 20 inch by 7 inch glass plate was placed vertically in an 18 inch deep cylinder (diameter 8 inches). Light was provided by a 100 watt bulb placed directly above or directly below the glass cylinder. In all of the experiments air was bubbled through the water, but where turbulence was desired, 2 bubblers were placed in the cylinder and the compressed air flow was increased to the point where the surface of the water was vigorously agitated. In each ex- Conditions of Experiment SSOUyIeCd Ja}e@\Q WING aaoqy Wo ISI] Jaye JU[NqINy, aaoqy Woy IST Jaye WING JaqVM ING MOpPg Wo WYSTT JoyeAq JU[NGINT, Jaye\q JugTNqNy, MOpPg Woy WS] joy je} > » Gs} we § he} fel = 6 § 3 5, 2 oo 8 w 7 % at bottom Figure 4 Results of laboratory experiments. Each experiment lasted 2 hours. The top graph shows the percentage of limpets that were at the surface after the 2 hour period. The middle graph shows the percentage of limpets that were between the bottom of the glass plate and the surface after the 2 hour period. The lower graph shows the percentage of limpets that were still at the bottom of the glass plate after the 2 hour period. Page 24 THE VELIGER Vol. 11; Supplement periment 12 to 16 A. scutum were placed on each side of the glass plate about 1 inch from the bottom. Each experi- ment was run twice; a total of approximately 50 limpets were subjected to each combination of variables for a period of 2 hours. The results of the experiments are shown in Figure 4. The first 4 bars of the graph represent the combinations of variables most closely approximating those in the field. In the trial with light above the cylinder and calm water the fewest limpets reached the surface. This result corresponds well with the field observation that Acmaea scutum move upward only a short distance when they are awash on a rising tide during the day. Of the first 4 experiments, the greatest number of limpets reached the surface under conditions of darkness and turbulence. This result correlates well with the field observation that A. scutum move upward most rapidly when they are awash on a rising tide at night. Preliminary studies showed that, when submerged, Acmaea scutum exhibits a negative geotaxis, and moves upward. Ross (1968) found that submerged A. scutum also exhibit a negative phototaxis. In the first 4 experi- ments it appears that these two responses are operating. When animals were submerged in the light, the negative responses to light and gravity opposed each other, and only 40% of the limpets reached the surface in the 2-hour period. To test this interpretation the light was placed beneath the cylinder in the last 2 experiments. Under these circumstances the negative taxes to gravity and light reinforced one another and more than 80% of the limpets moved to the surface in the 2 hour period. SUMMARY 1. The effects of light and tide on the movements of the limpet Acmaea scutum were studied in the field and in the laboratory. 2. Acmaea scutum moves most when subject to the tur- bulence of the tidal wash during tidal ebb and flow, but continues to move when submerged at high water. 3. Acmaea scutum moves upward with the incoming wash during tidal flow and downward with the out- going wash during tidal ebb. 4. The movements of Acmaea scutum during periods of wash appear to be dependent on the light conditions; vertical and total movements are greater at night than during the day. 5. In the field situation Acmaea scutum moved an aver- age of 40 inches between 2 successive periods of lower low water but returned to within an average distance of 5.5 inches from the starting point. 6. In the laboratory Acmaea scutum moves upward when submerged; the rate is slowest when animals are illu- minated from above, higher under condition of dark- ness, and still higher when the animals are illuminated from below. These results generally confirm results obtained in the field, and are interpreted in terms of a negative geotaxis (stronger) and a negative photo- taxis (weaker) in limpets awash or submerged. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. The author also wishes to thank Dr. Donald P. Abbott for his advice and assistance in the execution of the work. LITERATURE CITED Ross, THoMAs LEE 1968. Light responses in the limpet Acmaea limatula. The Veliger 11, Supplement: 25 - 29; 3 text figs.; 3 tables (15 July 1968) Test, Avery RANsom (GRANT) 1945. Ecology of California Acmaea. Ecology 26 (4): 395 - 405 Vol. 11; Supplement THE VELIGER Page 25 Light Responses in the Limpet Acmaea limatula TOM L. ROSS Hopkins Marine Station of Stanford University ' Pacific Grove, California 93950 (3 Text figures; 3 Tables) PRELIMINARY OBSERVATIONS of the phototactic responses of the limpet Acmaea limatula CaRPENTER, 1864, led to the studies of spectral sensitivity reported in this paper. Mean response times of dark-adapted animals to light of different colors and intensities were determined, and attempts made toanatomically localize the photoreceptors. The results suggest the possibility of at least two photo- receptors and several visual pigments in this organisms. METHODS anp MATERIALS The animals used were all collected at Mussel Point, Pacific Grove, California, in the intertidal zone between +2 and +6 ft. The observations of responses were made in a plastic-lined, water-tight wooden trough, which could be continuously supplied with fresh sea water. At each end of the trough, a 150 watt tungsten lamp, en- closed in a sheet metal box, supplied the illumination. The boxes, painted flat black to reduce reflection, had 3” holes drilled in the face at the height of the lamp fila- ments. Each light, connected through a rheostat, could be independently operated for illumination from one, or both ends of the trough. Various areas of the spectrum were isolated by Corning glass filters, nos. 554, 401, and 244, corresponding respec- tively to blue, green, and red. Maximum transmittance with the blue filter was at 425 mp, decreasing to 10% transmittance at 500 mp, and cutting off completely above 540 mu. The green filter transmitted maximally at 520 mp, with no transmittance below 460 my or above 600 mp. The red filter transmitted all wavelengths greater than 600 my. A standard infrared absorbing filter, com- posed of acidic 0.5% CuSO: in distilled water, was also used. Light intensity was varied by adjustment of a rheostat. In experiments with white light the animals were placed 32 cm from the light source and the intensity at ' Permanent address: 2036 Bell Avenue, Sacramento, California. each rheostat voltage setting determined with a thermo- pile. Where color and intensity were varied, this thermo- pile reading was used to approximate equal intensities of the light at each color at any given voltage. This was ac- complished by varying the distance of the animals from the light source, as indicated by the thermopile readings. The difference in distance between the placement of animals in white light and the placement in any other color never exceeded 10 cm (in blue), and in some cases was as little as 2cm (in red). Because of the insensitivity of the gal- vanometer, accurate measurements were not possible below 60 volts. The equalizations of intensity are, there- fore, only approximate. Data were obtained on response (yes or no), response time, and response time as a function of color and inten- sity. The response time given is from initial illumination until a definitive turning response (described below) was ascertained. All animals used in the experiments were allowed to dark adapt for a minimum of one hour. RESULTS anv DISCUSSION In the initial work it was necessary to determine a defin- itive response of the limpet to illumination, such as a turning response (FRAENKEL & Gunn, 1940), or to changing illumination, such as in the clam Mya (HeEcut, 1919). Also it was necessary to determine whether the genus Acmaea would exhibit sufficiently consistent re- sponses on which to base a study. Ten animals each of the species Acmaea pelta EscuscHoLtz, 1833, A. scabra (GouLp, 1846), A. scutum EscHscHoLtz, 1833, A. digi- talis EscHSCHOLTZ, 1833, A. limatula CARPENTER, 1864, and A. asmi MippENporRFF, 1849, were collected and tested for light responses. The general response to light, when exhibited in any species, was found to be a combination of backward movement, sidewards movement, and a 180° reversal of orientation. During illumination the animal shows a characteristic extension of pallial and cephalic tentacles, while the shell is kept fairly close to the substrate. Page 26 THE VELIGER Vol. 11; Supplement Quantitative results (Table 1) show that both the percentage of animals responding to light, and the speed of this response, is greater in Acmaea limatula than in Table 1 Responses of Six Species of Acmaea to White Light. Animals 32cm distant from light source (rheostat at 110 volts) Ove~ nies 23 23 % oe 8a ene mites ni @ 8 3 vo 33 gg a a2 Species gs E ee g FS E g Z< Ze S85 as Acmaea scabra 10 4 173 22 Acmaea pelta 10 4 140 35 Acmaea scutum 10 4 101 56 Acmaea digitalis 10 2 201 18 Acmaea asmi 10 ; 0 _ - Acmaea limatula 10 9 84 21 the other species. This species, therefore, was chosen for continued study. It was decided that the reversal re- sponse, if exhibited within 4 minutes after illumination, would be scored as the definitive response. Responses after 4 minutes were scored as negative. Fifty-one Acmaea limatula were collected and tested, of which 50 showed positive responses with a mean re- sponse time of 76 seconds and a standard deviation of 26 seconds. The responses of these animals to illumination from the opposite direction were also tested. In only 7 out of 50 cases did the animal stop in its initial turning re- sponse when the illumination was changed from one end of the trough to the other. After an extended time those animals which did not initially respond to the second light would migrate away from it. These observations suggest some process of bleaching or adaptation to illu- mination. All the following studies were done with the 50 animals which exhibited the initial positive responses, regardless of the response to the second illumination. A control experiment was then done to determine the animal’s natural response to being removed from an aquarium and being placed, in total darkness, into the testing apparatus. Of 25 animals tested in the dark, only 2 showed behavior which might have been construed as a positive response had the animal been illuminated. The above findings show that the results obtained in all ex- periments are not significantly affected by the animal’s natural response to being moved, but depend only on illumination. In the next experiment 10 animals were placed in a dry trough to ascertain the effect on response time and percentage of responses. Six of the 10 animals showed responses with a mean time of 112 seconds and standard deviation of 36. This experiment shows that although the animals do respond to light when out of water, there is a significant decrease in both rate and number of re- sponding animals. In the next experiment correlations between size and response time were examined. Thirty animals were cho- sen; 10 each in size ranges less than 9mm, 9 to 16mm, and greater than 16 mm. All 3 groups showed at least 9 out of 10 responses, with respective times and standard deviations of 71=+22, 68-14, and 77-21 seconds. These results suggest no correlation between response to light and size of the animal. Spectral sensitivity of the response was then investi- gated, using the various color filters. The results of this experiment, shown in Table 2, indicate equal sensitivity in the visible spectrum at the maximum intensity of the lamp. Table 2 Responses of Acmaca limatula, Normal and Without Eye Spots, to Light of Varying Spectra. Intensity is about equal in all colors. o we ga 5 & ‘6 fe 7 td 2 5 9 23 Es Filter Soy Je) fe 3 Si A=) & 8 ao 6 & aa Sf o ¢ so zg Zi ik = 3s ns Normal Animals Infrared absorbing 10 10 57 8 Blue 10 10 51 12 Green 10 10 52 10 Red 10 9 61 31 Animals Without Eyespots Infrared absorbing 5 4 51 9 Blue 5 4 79 17 Green 5 1 56 0 Red 5 1 59 0 In the next experiment, attempts were made to localize the photoreceptors of Acmaea. The presumptive receptors are the eyespots, which in Acmaca limatula are greenish in color and are located at the base of the cephalic tentacles on the back of the head. Several attempts were made to remove the eye spots by cauterization with a hot needle. The 5 out of 18 animals which recovered completely had lost their cephalic tentacles as a result of the operation, Vol. 11; Supplement THE VELIGER Page 27 Table 3 Responses of Normal Acmaea limatula to Light of Different Colors and Intensities, and Responses of Eyeless Animals to Varying Intensities of Blue Light in Seconds. The numbers indicate mean response time in seconds, standard deviation, and (in parentheses) the number of responses versus the number of animals tested. Relative Response Time (Seconds) to Different Colors Intensity Normal Eyeless (Volts *) white blue green red blue 100 47+ 9 (5,5) 82 £13 (5, 5) 83: 26 (5, 5) 75 £25 (5,5) 81 +12 (4,5) 90 88 =21171|(55'5)) SiH 1 (OND) 80+11 (5,5) 57 £23 (5, 5) 115 +27 (4,5) 80 124 +18 (5, 5) 76 £25 (5, 5) 107 + 26 (5, 5) 119*31 (5,5) 138 +20 (2, 5) 70 127 +22 (4, 5) 141 £33 (4, 5) 124 +20 (4, 5) 115 +24 (4,5) 124 + 4 (2,5) 60 156+31 (5, 5) 79 +16 (5, 5) 153 £12 (3,5) 51+ 11 (5, 5) 0+ 0(0,5) 50 114 +24 (4, 5) 68+ 18 (5, 5) 240+ 0 (1,5) D116) (655) - 40 41+ 9 (5,5) 67 +17 (5, 5) 0+ 0 95 +22 (5, 5) - 30 121 +15 (3, 5) 130 +31 (3,5) - 154 +12 (2,5) - ' (rheostat setting in volts) and hence no indications of eyespots were apparent. These 5 animals were then tested for sensitivity to light of various spectra and intensity. The second part of Table 2 shows the eyeless animals react significantly only to white and blue light at maximum intensity. The last experiment run in this study determined the effect of varying intensity of white, blue, green, and red light on the responses of normal and eyeless Acmaea limatula. The results of this experiment are shown in Table 3 and Figures 1, 2, and 3. In normal animals, the response time in white, blue, and red light is biphasic, whereas in eyeless animals responses are only seen at high intensities of blue and white light. The results of the last two experiments suggest the 180 160 140 100 Mean Response Time 20 110 100 go 80 70 60 5° 40 30 Voltage (Intensity) Figure 1 Responses of Acmaea limatula to varying intensities of white light. Page 28 THE VELIGER Vol. 11; Supplement 240 Zo) % blue @® green 200 oy red 180 160 © ® oe o 3 ° y o B ia g = x @ B 20 ) 110 100 go 80 70 60 50 40 3° Voltage (Intensity) Figure 2 Responses of Acmaea limatula to blue, green, and red light of varying intensities. presence of at least two pigments in Acmaea limatula, with the eyespots probably most important at the lower intensities. The greenish color of the cyespot might ac- count for the fact that the animal exhibits poor responses to green light at lower intensities. The site of the other photoreceptor is completely undetermined. Two possible candidates are either the pallial tentacles, which normally are extended from under the shell edge, or, in A. limatula, the heavily pigmented side of the foot. Either of these possibilities would entail an amazing nerve nctwork to determine the source of light since both the pallial ten- tacles and the foot would be symmetrical sites for the photoreceptors as opposed to the normal condition of directional asymmetry of photoreceptors. SUMMARY Light responses in 6 species of the genus Acmaea (A. scabra, A. digitalis, A. scutum, A. pelta, A. asmi, A. limatula) were investigated. Acmaea limatula showed the strongest responscs to light. In all cases, responses when exhibited, were of a negative phototactic nature. Experi- ments varying color and intensity indicate the eyespots Vol. 11; Supplement THE VELIGER Page 29 of A. limatula are important as photoreceptors in colors o . . ee . ie blue, green, and red at high and low intensities. There is ee at least one other photoreceptor and pigment, which is functional only in blue light at higher intensities. 200 : ACKNOWLEDGMENT 100 160 The author wishes to express his thanks to Drs. Lawrence Blinks and David Epel of Hopkins Marine Station, for 16 their ideas and comments throughout this project. This E e work was made possible by Grant GY806 from the Under- Bi r) graduate Research Participation Program of the National § Science Foundation. a. @ wz 100 E Bi LITERATURE CITED 60 FRAENKEL, GOTTFRIED & DonaLp L. GUNN 1940. The orientation of animals. Oxford, Clarendon Press; vi+352 pp. 40 Hecut, SELIG 1919. Sensory equilibrium and dark adaptation in Mya arena- Ze 71a. Journ, Gen. Physiol. 1: 545 - 558 10) 110 100 90 80 79 60 Voltage (Intensity) Figure 3 Responses of Acmaea limatula, without eyespots, to blue light of varying intensities. Page 30 THE VELIGER Vol. 11; Supplement Orientation and Movement of the Limpet Acmaea digitalis on Vertical Rock Surfaces ALAN C. MILLER Hopkins Marine Station of Stanford University * Pacific Grove, California 93950 (18 Text figures; 4 Tables) At Musset Point, Pacific Grove, California, Acmaea digitalis EscuscHouitz, 1833, is abundant on granite rocks above the Endocladia-Balanus zone (see GLYNN, 1965). Preliminary observations showed that these lim- pets tend to orient with their heads facing downward on vertical or nearly vertical rocks during periods of low water. No previous studies on orientation in A. digitalis have been found. However, in studies on ciliary currents of Lottia gigantea SowerBy, 1843 by Appotr (1956), and the ecology of Acmaea dorsuosa GouLp, 1859 by ABE (1931), both authors found that the majority of these limpets were oriented head downward at low tide. Studies of the movements of A. digitalis, which might help ex- plain this orienting behavior, are lacking. Previous obser- vations of movement in this species have been related mainly to such matters as homing (VILLEE & Groopy, 1940; GaLBrairH, 1965), or to shifts in the distribution of populations over extended periods of time (FRANK, 1965). In the present study an attempt was made to determine the orientation and movements of Acmaca digitalis on vertical rocks at various stages of the tidal cycle and to gain some insight into the factors influencing them. FIELD STUDIES on ORIENTATION An initial field study was carried out to determine the orientation tendency of Acmaea digitalis on vertical sur- faces at low tide. Acmaea digitalis were observed on one large vertical rock, and the orientation of 136 limpets was recorded in terms of the position on a clock toward which the head of each was pointing. Animals with their anterior ends straight up were recorded at 12 o'clock, those with heads straight down at 6 o'clock, etc. The ' Permanent address: Box 1295, Trona, California 93562 results, shown in Figure 1, reveal a clear tendency, at low tide, to orient with the head downward from 4 to 7 o'clock. Later observations on other populations, includ- ing those in movement studies, confirm this tendency (Table 1). Young Acmaea scabra (Goutp, 1848) on Figure 1 Orientation of 136 Acmaea digitalis at low tide on a vertical rock surface. The per cent of the population with heads pointed in each clock direction, from 1 to 12, are: 3, 6, 9, 15, 20, 13, 15, 2 2, 4, 2, and g. 2 Vol. 11; Supplement THE VELIGER Page 31 Table 1 Orientation shown by a limpet population during the period from lower low water to higher low water on three successive days, May 3 to 5, 1966. o 12) ca & Per cent of Limpets (N = 152) mA Oriented in Each Clock Direction L 2 8 @ § © 7 8 © i@ il Ww Aprons Dry Lo og og 1 2 8 iG 8 8 4 Bi Wet) e« Damp AB ad ® WB HS le OD Fue “4 1 ORO > Splash S 9 8 © Bd 2 iO Fe 7 § ios = Damp So ee Some aOR 14 4e 7 Gh 11330 Dry 3 8 SB 10 Ae 7 QD mW 2 7 7 al iGo Dry, 3.14 2B 10 we 8 iw 8 H § 8 i Use > Dry 3 ie 2B lO 7 3 12 8 8 8 8 i Oekfo = Splash 3° 9 § i iy 8 10 1 2 8 © 8 iMileH Damp 2 ey is) PAS) I HS) Dry QW 2 i 2B 2} 12 10 8 8 7 SB We Damp ae 9) -B il wy G6 8 1G 1B 4 Bil Opto) 14 Damp 4 10° SB ll Be CG 71 8 7 Si Oxs 2 Damp “ai 8. iil AO 8S io 8 7g B i Obkto = Splash ASO nGn 1324 5) Sila 6) 16) 41) 31230 Damp & iQ 8 We ee 4 2 We @ 7 a il ilzeto) Dry 410 G We § 2 WM Gog & i iWeoo vertical surfaces appear to show a similar tendency, though detailed observations are lacking. Upon finding an orientation tendency at low tide, the next step was an investigation of orientation during a tidal cycle. All the following field observations on orien- tation were made on a vertical granite rock face (see Table 4, rock 1). On this rock surface 152 Acmaea digi- talis were individually marked. The shell of each limpet was painted with a patch of yellow paint upon which an identifying number was marked in India ink. Each lim- pet’s orientation was recorded approximately every 3 hours during lower low water (LLW), lower high water (LHW), higher low water (HLW), and the intervening mid-tides, on 3 successive days. No readings were taken at higher high water (HHW) due to heavy surf. The results, shown in Table 1, clearly indicate that changes in orientation take place during the tidal cycle. The net change in orientation for each individual be- tween successive observations was then computed, assum- ing that rotation in each case was accomplished by turning the minimum number of clock units necessary _ to account for the change (Figure 2). During the first tidal cycle the population showed more rotation upward with a rising tide and more rotation downward with a receding tide, but this was not repeated in the next two tidal cycles. The general activity of the population, as measured by the sum of the up and down rotation, and by the per cent of the population rotating (Figure 2), shows a peak asso- ciated with each high tide. The decrease in the height of these peaks of activity from one recorded high tide to the next is associated with a slight decrease in the heights of successive LHWs, and a resultant decrease in both the number of animals being effectively wetted and the dura- tion of the period of splash. Also, during the first recorded LHW a fog bank blocked the sunlight, while on later LHWs direct sunlight dried the rock surface sooner. The results shown in Figure 2 suggest that activity of the population is proportional to the degree of wetting of the limpet population and the rock surface at high tide. Since at some LHWs the whole population was effect- ively wetted and at others only the lower limpets received significant splash, for subsequent high tide studies the population on the rock face was divided into groups; a high group ranging from about 14 to 18 feet above mean LLW, and a low group located 9 to 12 feet above mean LLW. Hourly observations were made during a HHW Figure 2 Change in orientation in the limpet population shown in Table 1, during the period from lower low water to higher low water on three successive days, May 3 to 5, 1966. and a LHW period. The results are shown in Table 2 and Figure 3. The amount of rotation and the per cent of the population rotating are clearly related to the amount of splash received. The graphs in Figure 3 per- haps explain the differences in rotational activity shown Splash Page 32 THE VELIGER Vol. 11; Supplement Percent of Population Rotating S 75 Percent of Population Rotating : ona 50 Se 5 hae 0 —_a Bo Be 25 2 75 ey ER) 8 pe () Minimum Clock Units Limpets Rotated 200 Minimum Clock Units Limpets Rotated faleny Kolo} > 100 5 © ) «8» “Ep : ‘ ae ais ) 80 Number of Limpets Rotating Up and Down a, 100 60 3 i - bbe 40 z a 8 5) 4 ) 20 2 i m i U = Number of Limpets Rotating Up and Down g 20 2 8 fc) A 40 Ss By) a0) 60 5 g i ee es aa Seat BE ° Z : 0415 1045 19300500 1130 0245 0545 1230 1720 os = 5:4 4.2 Low HHW LHW 1930 2130 2330 0130 0930 1030 1130 Figure 3 Vol. 11; Supplement GE MELIGER Page 33 Table 2 Per cent of limpets oriented in each clock direction, during higher high water and lower high water, May 16 to 17, 1966. Condition on the rock surface: S = splash; W = damp; D = dry. Per cents of Limpets Oriented in Each Clock Direction High Limpet Population N=55 § g 3 2 ee 2 2 282: 2 e< Sa 2 & ae 88S 3 S 8 § § § Ww W 1 JY © 2B Bs © O° © 2 ie) Tk 1. BO 18 18 3 AR On On ON) An a 4 2 OG Gee 250253 3m Sil 5 NG) alley ike 6 AO Om on Omron 95) 7 Se OL ET wa manele w/in aah 8 a GN HE aI BG 9 Ae ee Opera ts. Si 25 10 Mil n eS Oc > gS 22g os = 4 A=} br ) 2 379 8 2 g 5 , CBE 2¢ g 6 ae gS e 2 es a 225 za a5 April 1966 26: 1050 14.75 34 - 27: 0930 16 40 40% 28: 0930 18.75 41 37% 29: 1030 20.25 43 46% May 1966 2: 1345 16.75 43 61% 3: 1645 13.5 34 57% 5: 0700 19.5 44 42% Bg GRY) 18.7 42 57% 6: 1100 16.75 41 46% 6: 1500 16.25 39 64% 6: 1850 17.25 42 54% 12: 1200 14.75 34 46% 14: 1500 14 37 53% 16: 1530 15.25 36 41% 17: 1100 10.25 26 65% 17: 1700 11.5 27 759% 19: 0100 8.25 22 56% 19: 0300 13 27 43% 26: 1300 15.25 37 41% 27: 1300 11.25 29 40% Observations of Acmaea digitalis in several different locations around the Monterey Peninsula indicate that, in general, clusters are found in more protected areas. They tend to occur in spots which receive more shade than the surrounding rock face due to a slight depression in the rock barely noticeable when the full sun is on the whole rock face. On the rock island where most of this study was carried out, between 1:30 and 3:00 PM. the cluster areas would be outlined by a shade pattern which covered them but left the rest of the rock in full sun- light. Clustering also tends to occur on surfaces at right angles to the sea which receive some surge and splash from waves but do not bear the full brunt of the breakers. In areas directly exposed to wave action, Acmaea digi- THE VELIGER Page 49 talis have not been observed to cluster, although their populations may be very dense. Here they remain ran- domly dispersed, although small, temporary clusters of 3 to 6 animals may aggregate in a crack or depression in the rock. The factors stimulating clustering are not fully known. Animals cluster when the tide is out and disperse when the tide is in, at night as well as during the day; thus light does not seem to be a direct and immediate causal factor. Tide level and wave action appear to play an important role. The limpets recluster when they are still being splashed on an ebbing tide and the area is still totally wet, as though the frequency of splash and the amount of water passing over them were being measured. The fact that the animals recluster when the rock is wet and may do so at night seems to indicate that clustering is not similar to the phenomenon of aggregation in sow bugs which keep moving until they end up in the dampest and shadiest spot available (ALLEE, 1926). It is also difficult to look at clustering as a purely random occur- rence because it is seen that limpets return to a fairly stable cluster area. Some observations made suggest that the height of the sea at high water and the degree of wave action may influence the height on the rocks where clustering occurs. Toward the end of the month that the cluster shown in Figure 1 was observed, the weather grew progressively more stormy, and wave action increased. During this period the cluster rose in its position on the rock (Figure 1), gradually occupying a higher area. Also, the cluster underwent progressive fragmentation with time. This behavior recalls the finding of Ape (1932) that clustering is seasonal, and that the clusters of Acmaea dorsuosa break up shortly after the winter storms begin. The means by which animals are able to find their ways back to the cluster area are not clear. There is a possibility that mucous trails are involved. Another ani- mal which occurs slightly above Acmaea digitalis in the intertidal region, Littorina planaxis Puiuppi, 1847, has been proven to follow mucous trails left on rocks by members of its own or other species (Mryamoro, 1964; Peters, 1964). Whatever the mechanism, it may be similar to that involved in “homing” in limpets. During the month that the clusters were observed 4 of the animals were seen to return to exactly the same location and orientation at successive low tides for a period of 2 or 3 days. They then changed resting spots and repeated Figure 3 THE VELIGER Vol. 11; Supplement y l l l Y Z 1500 Incoming Limpets, Not Present on the Previous Day 77, Individuals Remaining Z from the Previous Day Present on the Previous Day Turnover rate of limpets in cluster membership between successive observations. Dates and times are the same as those in Figure 1. this behavior for several more days. Homing in some A. digitalis was also noticed by MILter (1968). SUMMARY Acmaea digitalis populations often cluster on intertidal rocks at low tide. Clustering occurs in areas which receive some shade and some protection from direct wave action. Animals disperse on a rising tide. Reclustering occurs during tidal ebb while rocks are still wet, and takes place both day and night. A cluster varying in size from 22 to 44 individuals occupied an average of 15 square inches (range 8 to 20 square inches) each day over a 32 day period. Daily shifts in cluster position occurred; total cumulative area occupied was 77 square inches. Only 17 members of the original cluster remained after 32 days while a total of 22 new members entered. ACKNOWLEDGMENTS I would especially like to thank Dr. Donald P. Abbott of Hopkins Marine Station for his assistance and guid- ance during this study. I also wish to thank Galen Hil- gard and Raymond Markel who helped me obtain equip- ment, and ‘Tudy Balesteri for keeping an eye on me when field work was necessary at high tide and access to my offshore rock was difficult. My sincere appreciation goes to Susan E. Fitzpatrick for the lettering on the original text figures. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. LITERATURE CITED ABE, Nosoru 1932. The colony of the limpet (Acmaea dorsuoso Goutp). Outgoing Limpets Which were Vol. 11; Supplement THE VELIGER Page 51 Number of Animals 20me279 201120) GON 12) 3) 4) 5 67, April May Days \/ ~~ A DS Soe oN ~ \ \ B \ en we 4 Tioecoones oO ‘ eQ 1S 9 oO 8 9 10 If 12 19 14 15 16 17 18 I9 Figure 4 Composition of the cluster at each observation period. Line A represents the total number of limpets present in the cluster during each observation. Line B represents the number of limpets present which were members of the cluster when the study began; some individuals left the cluster for a few days and later rejoined. Line C represents limpets present which were not charter members but joined the cluster after the study began. Tohoku Imp. Univ. Sci. Reprts., 4th ser. (Biol.) 7: 169 - 187 ALLEE, WARDER CLYDE 1926. Studies in animal aggregations: causes and effects of bunching in land isopods. Journ. Exp. Zool. 45: 255 - 277 Frank, PETER WOLFGANG 1965. The biodemography of an intertidal snail population. Ecology 46 (6): 831 - 844; 8 figs.; 6 tables MILLER, ALAN CHARLES 1968. Orientation and movement of the limpet Acmaea digi- The Veliger 11, Supple- (15 July 1968) talis on vertical rock surfaces. ment: 30 - 44; 18 text figs.; 4 tables Miyamoto, ALan C. 1964. Clustering in Littorina planaxis. Unpubl. student res. paper, Hopkins Marine Stat., Stanford Univ. Peters, RonALp S. 1964. | Function of the cephalic tentacles in Littorina planaxis PuitipP1 (Gastropoda: Prosobranchiata) . The Veliger 7 (2): 143 - 148; 10 text figs. (1 October 1964) Page 52 THE VELIGER Vol. 11; Supplement Studies of Homing Behavior in the Limpet Acmaea scabra BY WILLIAM F. JESSEE Hopkins Marine Station of Stanford University * Pacific Grove, California 93950 (4 Tables) THE TENDENCY OF MEMBERS of the species Acmaea scabra (GouLp, 1846) to return repeatedly to a specific location on their rock substratum, i. e. homing, has been reported from time to time. The first such study on the Pacific species of Acmaea, by Wetts (1917), gives the impression that homing is a highly individual trait, even among members of the single species A. scabra. However, the tendency to home was apparently not studied in more than a few represent- atives of each species, and the frequency of homing behavior was not evaluated. Two attempts at such an evaluation were subsequently reported (Hewatt, 1940; ViLLEE & Groopy, 1940). Hewatr’s study of 31 Acmaea scabra on granite rocks revealed homing behavior in all 31 individuals. On the other hand, VILLEE & Groopy concluded from their study of 86 members of this species on a sandstone substratum, that there was no evidence for homing behavior. This latter study, however, was restricted to observations made principally at low tide, a time when there is little or no activity in the limpet population. All animals that had not been observed away from their homesite during the period of study were ignored. In a more recent quantitative study by Brant (1950), 298 marked Acmaea scabra were observed over a period of 24 days. Homing behavior was reported in 98.7% of this test population. Preliminary observations by the author confirmed these findings. The principal object of the present study was to in- vestigate the mechanism involved in homing behavior. In addition, two other aspects of homing which have received little investigation are included in this study. These are the homing behavior of animals from high and low elevations in the intertidal, and the behavior of different size classes of limpets. ' Present address: University of California at San Diego Med- ical School, San Diego, California 92037 METHODS anp RESULTS The observations and experiments described below were made at Pescadero Point on the Monterey Peninsula, California during April and May, 1966. The first series of experiments performed was designed to detect any differences in the homing ability of high and low populations, and in that of large and small animals. One hundred individuals of intermediate size, between 10 and 15mm in length, were measured, and the animal and its homesite marked with red nail polish. Fifty of these animals were on a horizontal granite sur- face with a median elevation of +2.4ft; this population will be referred to as the “low” group. The other 50 animals, hereafter called the “high” group, were on a gently sloping rock with a median elevation of +/7.1ft. After waiting 3 days to insure that the position marked was indeed the animal’s home, a procedure that was followed in all subsequent experiments, 30 in each group were displaced randomly 3 to 4. cm from their homesites. The other 20 in each area served as controls. The animals were observed after 24 hours, and as shown in Table 1, Table 1 Homing Behavior of Acmaea scabra Populations at High and Low Intertidal Locations. Observations Made 24 Hours After Displacement of Experimental Animals. Total Home NotHome Missing Low Animals 30 25 2 3 Displaced Low Control 20 19 0 1 Animals High Animals 30 27 1 2 Displaced High Control 20 20 0 0 Animals Vol. 11; Supplement no significant difference was seen in the homing ability of the high and low groups. A similar experiment was then carried out in an area of intermediate elevation, +3.9ft, to compare the homing behavior of large animals, greater than 15 mm in length, with that of small ones between 6 and 10 mm long. Here, too, no significant differences were observed, as can be seen from Table 2. However, a less complete study sug- gested that animals smaller than 6mm usually do not home. Table 2 Homing Behavior of Large (greater than 15mm in length) and Small (6 to 1omm in length) Individuals of Acmaea scabra. Observations Made 24 Hours After Displacement of Experimental Animals. Total Home NotHome Missing Large Animals 20 19 0 1 Displaced Large Control 15 15 0 0 Animals Small Animals 40 36 2 2 Displaced Small Control 30 27 2 1 Animals Experiments in the laboratory, utilizing glass plates over which limpets had been allowed to move, revealed no residual mucus in the form of trails as determined by the India ink test of PeTEers (1964). Nonetheless, field experiments were performed to investigate the possibility that mucus or some other chemical substance on the sur- face of the rock was the agent active in governing homing behavior. In the first type of experiment, a stiff fiber brush was used to scrub a 14” strip around the homesites of 20 previously marked animals. The areas surrounding 15 other animals, which served as controls, were left un- scrubbed. All animals were then displaced 4 to 5 cm from their homes to a position outside this strip. Their loca- tions after 24 hours were noted, and are presented in Table 3. A variation of this experiment was also carried out in which the home itself was included in the area scrubbed. This was to eliminate any possible attractant on the home, as well as “trails” leading to the home. As shown in Table 3, neither of these treatments completely destroyed the ability to home. A second group of experiments was performed to remove chemical substances. All animals in a one foot square area were removed and the rock painted with a THE VELIGER Page 53 Table 3 Homing Behavior of Acmaea scabra Following Scrubbing of Home or Surroundings or Both. Observations Made 24 Hours After Displacement of Experimental and Control Groups. Total Home NotHome Missing Surroundings 20 10 6 4 Scrubbed No Scrubbing 10 10 0 0 Home and Sur- 30 23 3 4 roundings Scrubbed No Scrubbing 20 16 0 4 solution of 32% NaOH. The surface was then rinsed repeatedly with copious quantities of seawater until the pH of the wash water was near neutrality. The animals were then replaced within 4cm of their homes. Five animals outside the treated area were also displaced a similar distance and utilized as controls. At the end of the customary 24 hour waiting period, 8 of the 10 animals in the experimental group had returned to their homes. Having established that the removal of chemical sub- stances on the rock had no noticeable effect on the homing ability, experiments were begun in which the topography of the rock surface surrounding the home was altered. A geologist’s pick and a chisel were used to create a strip 1” wide around the homes of 25 animals. The topography of the rock in this strip was totally changed. The animals were then replaced 4cm from their homes, and outside the chiseled area. Fifteen other animals were similarly displaced, but the topography of their surroundings was left unaltered. This latter group served as a control. Within 24 hours only 4 of the 25 experimental animals had homed. Seventeen of the 25 were not in their homes, the remaining 4 having disap- peared. Some additional evidence suggesting the involvement of topography in the homing mechanism resulted from an experiment performed with a small group of animals at China Point. Seven animals were marked and displaced the customary 4 to 5 cm from their homesites. A hammer and chisel were then used to create a strip about 1” wide between the animal and its home. As the tide came in, the animals were observed to move to the edge of the chiseled area, then travel along the edge of this area until they reached its end. At this point they resumed their travel toward the homesite, 6 of the 7 returning by the next low tide. This reaction to territory made unfamiliar by alteration suggests that recognition of some element of topography may be involved in the homing mechanism. Page 54 THE VELIGER Vol. 11; Supplement DISCUSSION If populations at the high and low levels are studied, using animals of comparable size, there is no significant difference in the homing ability of the two groups. Nor is there any significant difference in the homing ability of animals of different sizes at the same intertidal level. These observations confirm earlier ones by Haven (1966). However, it was also observed that animals of less than 5 to 6mm in length usually do not home. Such animals are found mainly in the lower intertidal. The inclusion of these extremely small animals in earlier studies of homing behavior may account for some of the discrepancies in the findings of this study as compared to these earlier works. The results of the experiments designed to elucidate the mechanism of homing suggest that it is related to the perception of the topography of the rock surface in the vicinity of the limpet’s homesite. The fact that the ani- mals would not cross an area of unfamiliar topography, but went directly home upon reaching familiar territory points to a “memory” of the convolutions of the rock surface. It is, however, possible that some other factor in the environment which was not investigated is respon- sible, at least in part, for homing. Light or its polarization pattern, for instance, could be such a factor. The proba- bility that the mechanism is indeed involved with topo- graphy is in good agreement with the tentative results reported by GatpraitH (1965) on the mechanism of homing in Acmaea digitalis EscuscHoitz, 1833 and Lottia gigantea Sowersy, 1843. The high percentage of animals which homed after treatment of the rock with sodium hydroxide argues against the possible use of some chemical agent as a homing guide, as the concentrated alkali would be ex- pected to dissolve or denature any substances deposited by the limpets. While other external factors, such as polarized light or water currents, have not been ruled out, it is doubted that the animal possesses the sensory equipment to utilize such highly variable sources for orientation. Work should, however, be carried out to examine these possibilities. The implications of a homing mechanism based on a knowledge of topography, in an animal such as the limpet, demand additional investi- gation. ADDENDUM Two additional series of experiments were completed later in an effort to determine what portion of the sensory apparatus is involved in homing behavior. The first experiments, designed to determine the role of the cephalic tentacles in homing, were performed at Pebble Beach, San Mateo County, California in January and February, 1967. In these experiments 60 animals were marked in the customary manner and left for 3 days to insure that the position marked was the home spot. Then a small typewritten number was affixed to the shell of each animal, and the same number placed adjacent to its home using Duco cement. The animals were then brought into the laboratory, where all were anesthetized in a solution of magnesium chloride isotonic with seawater. Both cephalic tentacles were then excised from 40 animals, distal to the eye spot. The remaining animals were uninjured and were utilized as controls. All animals were then placed in a salt water aquarium for a period of 4 to 5 days to allow recuperation of the operated animals. The animals were then returned to the beach during a period of low tide, and placed within 3 to 4cm of their own homesites. The locations of the animals at the end of 24 hours, or two high tides, were observed and are summarized in Table 4. Of those animals which could be located at the end of the 24 hour period, 29% of the experimental group had homed, as compared to 75% of the controls. Table 4 Homing Behavior of Acmaea scabra Following Excision of Both Cephalic Tentacles or Bilateral Destruction of the Eyespots. Observations Made 24 Hours After Replacement of Experimental and Control Groups. Total Home NotHome Missing Cephalic Tentacles 40 9 22 9 Excised Controls 20 12 4 4 Eycspots Destroyed 40 28 4 8 Controls 20 11 4 5 The second series of experiments was performed at Moss Beach, San Mateo County, during April and May, 1967, and was designed to determine whether or not the eyespots are utilized in homing. Sixty animals were marked and individually numbered as before. After the usual waiting period the limpets were brought to the laboratory, where all were anesthetized with the mag- nesium chloride solution. A dissecting needle which had been heated to redness was then employed to cauterize both eyespots on 40 of the animals, the remainder being utilized as controls. After a 4 to 6 day period of recovery Vol. 11; Supplement in the aquarium all the animals were returned to the field and replaced within 3 to 4cm of their homes as before. The positions of these animals were observed at the end of 24 hours, and are also summarized in Table 4. The eyespot is apparently not utilized in homing. Ex- amination of the 4 experimental animals which failed to home indicated that in these, substantial damage had been done to the cephalic tentacles. In those animals in which no damage was observed, there was 100% homing. The loss of the cephalic tentacles appears to have a Statistically significant effect on homing. This observa- tion is, however, open to question. It is a distinct possi- bility that the decrease in the instance of homing was a result of trauma or other factors. The fact that some 29% of the experimental group returned to their homes despite the loss of the tentacles seems to substantiate this possibility; the presence of alternative systems which may be utilized in homing is another possible explanation. SUMMARY 1. Homing behavior of Acmaea scabra was studied in the field with respect to the intertidal height of the population, the size of the individuals, and the mech- anism involved in homing movements. 2. No significant difference was found in the homing ability of populations in the high intertidal region as compared to those at lower levels. 3. The size of the animals was found to be unimportant in determining their ability to home, except that ex- tremely small animals, less than 5 to 6 mm in length, were usually found to be non-homing. 4. The possibility that chemical substances on the rock surface are the agents of homing behavior appears highly unlikely. 5. Although a few other environmental factors were not experimentally eliminated, the evidence presented indicates that the topography of the rock surface is utilized in homing by Acmaea scabra. 6. (From Addendum) Excision of the cephalic tentacles significantly reduced the tendency to home, while THE VELIGER Page 55 destruction of both eyespots had practically no effect on the incidence of homing. ACKNOWLEDGMENTS The author wishes to express his thanks to Drs. J. H. Phillips and D. P Abbott, and to Mr. Stoner L. Haven for their advice and assistance; the advice and keen inter- est of the late Professor V.C. Twitty was also deeply appreciated. Thanks are extended to the Monterey Foundation and Del Monte Properties, Inc. for per- mission to work in the Pescadero Point area. This work was made possible by Grant GY806 from the Under- graduate Research Participation Program of the National Science Foundation, and by a grant from the Arizona Academy of Sciences. LITERATURE CITED Brant, DaniEL HosMER 1950. A quantitative study of the homing behavior of the limpet Acmaea scabra. Unpubl. Spec. Prob. Reprt. Dept. Zoology, Univ. Calif: Berkeley GALBRAITH, RoBERT T. 1965. | Homing behavior in the limpets Acmaea digitalis and Lottia gigantea. | Amer. Midland Natural. 74 (1): 245-246 Haven, STONER BLACKMAN 1966. Personal communication Hewatt, WILuis GILLILAND 1940. Observations on the homing limpet, Acmaea scabra Goutp. Amer. Midland Naturalist 24 (1): 205 - 208; 1 fig. PETERS, RONALD S. 1964. Function of the cephalic tentacles in Littorina planaxis Puiuippi1 (Gastropoda: Prosobranchiata) . The Veliger 7 (2): 143 - 148; 10 text figs. (1 October 1964) VILLEE, CLAUDE ALVIN & THomas Conrap GRroopy 1940. The behavior of limpets with reference to their homing instinct. Amer. Midland Naturalist 24 (1): 190-204; 25 figs.; 3 tables WELLS, Morris M. 1917. The behavior of limpets with particular reference to the homing instinct. Journ. Anim. Behav. 7 (6): 387 - 395 Page 56 THE VELIGER Vol. 11; Supplement Occurrence and Behavior of Hyale grandicornis, A Gammarid Amphipod Commensal in the Genus Acmaea BY SAMUEL E. JOHNSON II Hopkins Marine Station of Stanford University’ Pacific Grove, California 93950 (Plates 2 and 3; 4 Text figures) IN THE couRSE of preliminary studies of the genus Acmaea at Hopkins Marine Station, Pacific Grove, Cali- fornia, mottled grey-green amphipods were frequently encountered under the shell of A. digitalis EScHSCHOLTZ, 1833, A. limatula CARPENTER, 1864, A. pelta Escu- SCHOLTZ, 1833, A. scabra (GouLp, 1846), A. scutum EscHscHoLTz, 1833, and Lottia gigantea SoweErRsy, 1843. Dr. J. Laurens Barnard of the Smithsonian Insti- tution has identified the amphipods as immature speci- mens of Hyale grandicornis (KRoOyYER, 1845) (Plate 2, Figure 1). No mature amphipods have been found in association with any of the above limpets. Dr. Barnard (personal communication) states that he found mature specimens on cobbles and with Ulva in Carmel Bay, Cali- fornia. An examination of the algae Endocladia, Gigarti- na, Ulva,and Iridaea growing in areas adjacent to the Ac- maea populations yielded no amphipods resembling those found with Acmaea, although an unidentified species of Hyale, mentioned by GLtynn (1965), does occur here and has been found in the present study. This species ' Permanent address: 15300 Hume Drive, Saratoga, California. differs from immature H. grandicornis in the pattern of its dorsal markings and in having brown rather than black, silver-spotted eyes. The H. grandicornis found under Acmaea species, averaging 2 to 3mm in length (the range is 1 to 6mm) is much smaller than this unidentified Hyale, which has an average length of 6mm. Hyale grandicornis occurs under individuals of Acmaea species in many different localities along the coast of the Monterey Peninsula. Population studies on this amphipod were carried out at Pescadero Point, on the open coast just north of the northern edge of Carmel Bay, Califor- nia, from 25 April to 30 May, 1966. The intense wave action in this area, the presence of vertical granite sur- faces ranging to 30 feet above the level of mean lower low water, and the varying conditions of exposure and protection afforded by large boulders and sheltered pools provide a variety of different habitats. The sites selected for study (Plate 2, Figures 2 and 3; Text figure 1) were not exposed to direct wave action, being situated either obliquely to the line of waves or on the shore side of large boulders. The sites chosen were divided up into zones (Plate 2, Figures 2 to 4) based on both plant and animal Explanation of Plate 2 Figure 1: An immature Hyale grandicornis, showing dorsal and lateral markings. The scale represents 1 mm. Figure 2: Zones A, C, and D. Pescadero Point, Carmel Bay, California. Taken on 23 May 1966 at 10:00A.M. Arrow points to marker representing an elevation of +5.2 feet. The scale represents 2 feet. Figure 3: Zone B. Pescadero Point, Carmel Bay, California. Taken on 23 May 1966 at 10:00 A. M. The scale represents 2 feet. Figure 4: Zones A, D, and E. Pescadero Point, Carmel Bay, Cali- fornia. Taken on 23 May 1966 at 10:00 A. M. The scale represents 2 feet. Tue VELIGER, Vol. 11, Supplement [Jounson] Plate 2 Figure 1 Figure 4 Vol. 11; Supplement Zone A Microscopic algae and diatoms in the upper region, with the Balanus — Chthamalus association near the lower margin Zone B_ Microscopic algae and diatoms on the underside of large boulders, shaded and protected from desiccation Zone GC Leathery encrusting algae, primarily Peyssonelia, Hildenbrandia, and Petrocelis Zone D Macroscopic algae, mainly Endocladia, Gigartina, and Iridaca Zone E Encrusting algae, mainly Peyssonelia and Ralfsia, associated with the barnacle Tetraclita. Ulva appeared at the end of the field study Below this zone the dominant plants on the rock face are encrusting and branched coralline algae. THE VELIGER Page 57 Pacific Grove, California RICKETTS Dory . 1946 CALIVN 1939 -6 Littorina planaxis I Acmaea Prasiola digitalis fj Bal Crustose ‘alanus Red glandula Niro —-4 2 : BAe ye Endocladia : Littorina ° scutulata : Porphyra Be D Pelvetia Mytilus Tegula re: E -2 — funebralis Gigartina : Mytilus : Tridaea : Pollicipes eve 5 Tegula Egregia ; brunnea ae (0) Hydroids Corallina Sponges Galliarthia 4 Figure 1 Description and Vertical Position of Zones at Pescadero Point, and Correlation with the Zones of RickETTS & CaLvIN (1939) and Doty (1946). —- indices, and following natural groupings of organisms on the rocks. Correlation of these zones with those of RIcKETTS & Catvin (1939) and Dory (1946) is shown in Text figure 1. Intertidal elevations were determined by measurement from a United States Geological Survey bench mark on Pescadero Point and were checked against the theoretical tidal heights as determined from _ the United States Coast and Geodetic Survey Tide Tables for the Pacific Coast, using the time and height correc- tions for Monterey. 25 April to 30 May 1966. The population densities of the 5 species of Acmaea studied were determined by dividing the surface of the sampling sites into quadrats of 400 cm’, and recording numbers of each Acmaea species present. The total area of each zone studied in the sampling sites ranged from 24 to 3m’. After determining the distribution and numbers of Acmaea species, the distribution and frequency of occurrence of Hyale grandicornis was determined by sampling of Acmaea species in the individual zones. Attempts were made to collect representative numbers Page 58 THE VELIGER Vol. 11; Supplement & Oy Ne Y y Y » ay Se Fe es & Y oxo YY & ¢ OY o & ® yy? Ye re YX A 5.72 4.45 20 24 0.47 0.19 0.81 0.08 : ee See Ba 14% 2570 26% 17% at 28 23 8 2.76 ge 0.15 = 9176 =< 45 Jo 26% _ 47% 61% 39 38 33 29 58 0.03 0.05 0.96 0.13 0.36 D ci sos eal % ai 16% 3272 30% 2 4 73 22 a1 .0 i 0.74 0.55 B68 E 715% 37% 69% 80% 43 49 35 5 Figure 2 Distribution of Acmaea Species and Immature Hyale grandicornis at Pescadero Point, California, 25 April to 30 May 1966. Figures inside or to the right of the white squares indicate the average number of Acmaea per 400 cm?. The size of the square is pro- portional to this number. The black area within the squares is proportional to the percent of Acmaea hosting amphipods; all percentage figures shown refer to the percent of Acmaea bearing one or more amphipods. The numbers below the boxes show the total number of limpets of each species examined from each zone. of the 5 species studied from each zone but certain species in some areas were extremely scarce or non-existent. Extreme care was needed in collecting, for the amphi- pods often jump away when the limpet is lifted from the substrate. Each limpet collected was then identified, and its shell length measured. The amphipods present with each limpet were counted and sorted into 3 size groups (0.5 to 2mm; 2+ to4mm; 4+ to 6mm). Text figure 2 shows the population density of cach species of Acmaea for each zone, and the percentage of each Acmaea species which served as hosts to Hyale grandicornis. In Zone A, no amphipods occur even though suitable hosts are present. The Acmaea species of Zone B, slightly lower in the intertidal but overlapping Zone A, bear a small population of amphipods. The large boulders characterizing this zone shade and protect it from desiccation, keeping it moister than Zone A. From this point down to the lowest populations of Acmaea examined, the population of amphipods increases, with the exception of Zone D. Zone D is characterized by the macroscopic algae Endocladia, Gigartina, and Iridaea, while Zones C and E are distinguished primarily by the encrusting algae Hildenbrandia, Peyssonelia, Petrocelis, and Ralfsia. Ulva was just beginning to grow in Zone E when field studies were discontinued. The present studies do not indicate any clear preference on the part of the amphipods for any particular species of Acmaea. Moreover, for those limpets which did house Tue VELIGER, Vol. 11, Supplement [Jounson] Plate 3 Figure 5 a Amphipod orientation and feeding position in Acmaea scabra. The scale represents 5 mm. Vol. 11; Supplement amphipods, there appears to be no clear correlation be- tween the shell length of the limpet and the number of amphipods present (Text figure 3). However, for limpets Average Number of Amphipods per Limpet Limpet Shell Length in mm Figure 3 Correlation Between Limpet Shell Length and the Number of Amphipods Housed, for those Limpets which bore Amphipods. with a shell length of 8 mm or more there does appear to be a slight positive correlation between shell length and the percent of the population bearing amphipods (‘Text figure 4). Limpets less than 8 mm long did not accom- modate amphipods. 50 2 40 % of Limpets hosting one or 30 more Amphipods 20 8-15 16-25 26-35 36+ Shell Length Size Groups in mm Figure 4 _ Correlation Between Limpet Shell Length and the Incidence of Amphipods. THE VELIGER Page 59 NATURAL HISTORY Field examinations carried out at Pescadero Point and at Mussel Point during day and night and at high and low tide periods indicate that the amphipods do not leave the limpet at any time unless the latter is removed from the substratum. These observations are supported by the absence of free-living immature Hyale grandicornis in areas adjacent to the limpet populations. The position of the amphipods under the shell was determined from laboratory observations using aquaria in which field conditions were approximated. During the day the amphipods are found behind the head in the nuchal cavity or deep in the groove between the mantle fold and the foot. Sometimes an amphipod may be ob- served at the edge of the mantle fold, but this behavior is rare in the daytime. In conditions of near darkness and splash or submergence they are found lying on their sides, in contact with the pallial tentacles of the limpet at the mantle margin (Plate 3). In this position the amphipods groom themselves, and from it they also feed, reaching around the edge of the shell or moving com- pletely out onto its dorsal surface and scraping up the algae growing there. Gut contents were unidentifiable, but the material on the shell is composed of numerous varieties of diatoms and several types of blue-green algae, primarily Enteromorpha. Occasionally small growths of Ulva are found on the shells. The amphipods seem never to leave the limpet, but seek cover under the shell when disturbed. If an amphipod is trapped outside and is unable to crawl under the host’s shell again, it either presses itself closely against the edge of the shell and remains there, or moves away to a nearby limpet. Other types of shelter, either in field or laboratory, appear to be ignored. When forced to swim, Hyale grandicornis moves very rapidly at first, but quickly slows and appcars to seek shelter. If repeatedly disturbed, it soon ceases all movement. It is of particular interest that Dr. Barnard found mature specimens of Hyale grandicornis in Ulva, and that the present study has revealed only juvenile individ- uals in the mantle groove and nuchal cavity of Acmaea. Ulva occurs mainly in the warmer months in the region studied, and at the end of the present study it was just beginning to grow. Perhaps the immature forms of H. grandicornis migrate to the Ulva as they attain sexual maturity and as the alga appears each summer, and possibly the juvenile amphipods survive the winter and spring under Acmaea shells. The author plans to continue research on the problem. Page 60 SUMMARY 1. Immature specimens of Hyale grandicornis (KROYER, 1845) are found in the nuchal cavity and pallial groove in 5 species of Acmaea and in Lottia gigantea. 2. The percentage of limpets hosting immature Hyale grandicornis increases with decreasing height in the intertidal region. 3. The amphipod shows no clear preference for partic- ular limpet host species. 4. No amphipods were found in any limpets less than 8 mm in shell length. For limpets above this size there is a slight correlation between shell length and the percent of limpets bearing amphipods. However, for those limpets which housed amphipods, there was no correlation between shell length and number of am- phipods borne by each limpet. 5. Immature Hyale grandicornis remain in contact with their limpet hosts under all prevailing conditions of tide and light. They appear to feed on algae growing on the surface of the limpet shells. 6. No mature Hyale grandicornis have been found in association with any limpets. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. Dr. J. Laurens Barnard of THE VELIGER Vol. 11; Supplement the Smithsonian Institution identified the amphipods as Hyale grandicornis. The author is very grateful for his help and suggestions. Appreciation is also expressed for pertinent literature supplied by Dr. Peter W. Glynn of the Institute of Marine Biology, University of Puerto Rico. Discussions with David A. Egloff of Hopkins Ma- rine Station proved helpful in suggesting approaches to field and taxonomic problems. Special thanks must go to Dr. Donald P. Abbott of Hopkins Marine Station, Stan- ford University for his time and effort spent in discussing the project with the author, and particularly for his criticism and editorial comments on this paper. The Monterey Foundation authorized the use of Pescadero Point for field studies. LITERATURE CITED Doty, MAxwELL STANFORD 1946. Critical tide factors that are correlated with the verti- cal distribution of marine algae and other organisms along the Pacific coast. Ecology 27: 315 - 328 GLYNN, PETER W. 1965. | Community composition, structure, and interrelation- ships in the marine intertidal Endocladia muricata — Balanus glandula association in Monterey Bay, California Beau- fortia (Zool. Mus. Amsterdam) 12 (148): 1-198; 76 figs.; 32 tables; 5 appendices Ricketts, Epwarp F. & JAck CALvIN 1962. Between Pacific tides. 3¢ ed., rev. by J. W. HepcPETH xiii +502 pp.; illust. Stanford Univ. Press, Stanford, Calif. Vol. 11; Supplement THE VELIGER Page 61 Factors Affecting the Attraction of Acmaea asmi to Tegula funebralis LANI LEE ALLEMAN Hopkins Marine Station of Stanford University ' Pacific Grove, California 93950 (1 Text figure) INTRODUCTION Acmaea asmi (MippEnporFF, 1849) lives almost exclu- sively as a commensal on the shell of Tegula funebralis (A. Apams, 1854). Previous workers found this associa- tion to be quite specific, A. asmi preferring T. funebralis to T. brunnea (Puuipri, 1848). Test (1945) suggested that T. funebralis released a chemical attractant. The source of this attractant was considered to be the shell by Raprorp (1959), whereas EIKENBERRY & WICKIZER (1964) concluded that both animal and shell were necessary. The following study attempts to settle these differences and to provide further information on behavioral and chemical aspects of this association. MATERIALS anp METHODS Most organisms used in this study were collected in inter- tidal areas near Hopkins Marine Station, except for Tegula brunnea which was collected in the Pebble Beach area, Carmel Bay, California. All experiments were run over night in pyrex dishes kept at 12° to 13°C in a darkroom. Preliminary experiments showed that if no choice were offered, Acmaea asmi would climb onto any shells tested. Therefore, to measure relative preferences, the limpets were allowed to choose between two different substrates. In the test 5 limpets were placed in about 2cm of water in the center of a 24cm pyrex pie plate. Ten test shells were placed equidistantly around the periphery of ' Permanent address: 2401 North Rosewood Avenue, Santa Ana, California. the plate. There were 5 of each type to be compared, and they were placed alternately in the circle. To permit washing of the Tegula shells with various solvents, the operculum was sealed with canning wax (Parowax). The animals survived this sealing treatment, and most importantly, were apparently unaffected by such solvents as alcohol or distilled water. In all experiments the dishes were then placed over- night in a darkroom at 12° to 13° C, and the number of limpets on the test shells determined the following morn- ing. RESULTS ann DISCUSSION Raprorp (1959) found that the shells of Tegula fune- bralis were not preferred by Acmaea asmi if the shells were boiled in alcohol for 15 minutes. The results shown in Figure la indicate that simple room-temperature wash- ing in ethanol for 1 hour also removes any attractant on the shell. In this experiment the limpets were given a choice between parowax-sealed shells washed in alcohol and control parowax-sealed shells. In 5 trials (25 limpets) the control shells attracted 90% of the limpets, while the alcohol-washed shells attracted 10%. The attractant is also partially removed by distilled water. If sealed shells containing Tegula funebralis are washed for 2 hours in distilled water and compared with normal, sealed shells, only 25% are found on the washed shells versus 65% on the control shells (Figure 1b). (Where the total percentage does not equal 100% the difference represents those animals not found on any shell.) However, distilled water washing is not as effective as alcohol washing. As seen in Figure 1c, 60% of the Page 62 THE VELIGER Vol. 11; Supplement animals preferred the water-washed shells as compared to only 15% on the alcohol-washed shells. Although Acmaea asmi is rarely found on Tegula fune- bralis shells inhabited by Pagurus spp., the limpets do not discriminate between these two types of shells. Thus, using the usual test system, equal numbers of limpets = © BS ° lop) = fc) I r= ° ie) inhabited Tegula shell (80%) Control (65%) Water-treated (25%) Water-treated (60%) Pagurus (in Tegula shells) 45% Tegula (in Tegula shells) 50% uninhabited, old shells (47%) Pagurus - control (45%) Alcohol-treated Tegula (43%) Pagurus-alcohol (20%) Alcohol-treated (10%) Alcohol-treated (15%) uninhabited, old shells ( 0%) Figure 1 Preference of Acmaea asmi for various substrates. Twenty-five Acmaea asmi were tested in each experiment. Each test (A to G) reprsents the percentages of animals found on the indicated substrate (percentages of those not responding are not shown). were found on sealed T: funebralis shells containing Pa- gurus spp. as on parowax-sealed T: funebralis shells con- taining its normal host (Figure 1d). Similar to normal shells, the attraction of Pagurus-inhabited shells is lost when treated with alcohol (Figure le). A final preference test series was made with old unin- habited Tegula shells found on the beach. Figure 1f shows that Acmaea asmi prefers normal inhabited shells to these old shells. However, as shown in Figure 1g, treatment of normal shells with alcohol renders them as unattractive as the old shells. The above experiments suggest that the “attractant” is completely removed or destroyed by alcohol, and partially removed or destroyed by distilled water. Furthermore, it is found on Tegula shells inhabited by either T: funebralis or Pagurus spp., but is not present on uninhabited shells found on the beach. The “attractant,” then, could be an alga or bacterial film associated with the shell, which is removed or destroyed by alcohol or distilled water. How- ever, the major algal epiphyte found on T: funebralis is also found on the shells of T. brunnea and Acanthina Spirata (BLAINVILLE, 1832) (EIKENBERRY & WICKIZER, 1964), which are not the normal hosts for Acmaea asmi. BEHAVIORAL OBSERVATIONS The above results on preference are consistent with a dif- fusible “attractant” emanating from the Tegula shell. Behavioral observations, however, indicate that the pref- erence might be made at the tactile rather than the olfactory or chemosensory level. Continuous observations were made of the selection process in a test situation where normal, sealed Tegula funebralis shells were alternated with alcohol-treated ones. As soon as the Acmaea asmi were placed in the center of the Tegula funebralis circle, they extended their ten- tacles and began to feel the substrate. These tentacles are thin and almost as long as the shell when fully extended. They are moved in a tapping manner from side to side as the animal crawls in a seemingly random fashion across the bowl. When a specimen touched another A. asmi with its tentacles, it felt the shell and then climbed immediately onto it. If a limpet crawled between two Tegula funebralis shells, one alcohol-treated and the other not, it would tap each shell with its tentacles and then, in every case ob- served (11), climb onto the untreated shell. If, however, the Acmaea asmi encountered only one shell, whether alcohol-treated or not, it would generally climb on. Once on a shell, the A. asmi continued to move and sometimes changed shells. Out of 25 animals tested, 9 were at one time or another on alcohol-treated shells. After 8 hours, when the experiment was concluded, only 2 limpets were still on these shells. From these latter observations, it seems that Acmaea asmi is not reacting to some diffusible chemical attract- ant from the shell, but rather testing the substrates with its tentacles. It will crawl on the first curved surface encountered, but if other choices are available, will even- tually end up on the preferred substrate. It is interesting to note that Test (1945) stated that the diffusible attract- ant was sensed at 7 mm and Raprorp (1959) felt the range to be 10 mm. If an A. asmi is placed this close to a Tegula funebralis, it can generally touch it with its ten- tacles and would, therefore, react to its presence. Vol. 11; Supplement SUMMARY The behavioral basis of the association between Acmaea asmi and Tegula funebralis has been investigated. The observations indicate that A. asmz is not attracted by a diffusible substance, but senses its substrate through con- tact with its tentacles. The critical substance(s) on the shell is (are) easily destroyed or dissolved by ethanol and slightly removed by distilled water. These factors are also present on Pagurus-inhabited Tegula shells, but are not found on uninhabited shells found on the beach. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the THE VELIGER Page 63 National Science Foundation. I also wish to thank Dr. David Epel, Dr. Donald P. Abbott, and Mr. Roger Kingston for offering helpful advice. LITERATURE CITED EIKENBERRY, ARTHUR B., Jr. & Diane E. WickIZzER 1964. Studies on the commensal limpet Acmaea asmi in rela- tion to its host, Tegula funebralis. The Veliger 6, Supplement: 66 - 70; 11 text figs. (15 November 1964) RapForp, RUTH 1959. A study on Acmaea asmi. honors paper. Unpubl. Stanford Univ. Test, FREDERICK HaroLtp 1945. Substrate and movements of the marine gastropod Acmaea asmi. Amer. Midland Naturalist 33 (3): 791 - 793 Page 64 THE VELIGER Vol. 11; Supplement Shell Damage and Repair in Five Members of the Genus Acmaea P TODD BULKLEY Hopkins Marine Station of Stanford University’ Pacific Grove, California 93950 (Plate 4; 2 Tables) SHELLS OF THE COMMON LIMPETS of the genus Acmaea found in the rocky intertidal of the California coast often show evidence of having sustained and repaired extensive damage. Studies of shell damage and repair were made on Acmaea scabra (Goutp, 1846), A. digitalis Escu- SCHOLTZ, 1833, A. pelta EscHscHoLTz, 1833, A. limatula CarPENTER, 1864, and A. scutum EscHSCHOLTZ, 1833. PepparD (1964) studied growth and repair in the shells of Tegula funebralis (A. ApAms, 1854) and studies of normal growth over a 3 year period were made of Acmaea digitalis, A. pelta and A. paradigitalis Fritcu- MAN, 1960 by Frank (1965). SEApy (1966) reports on the growth of A. limatula over a period of one year. FRETTER & GRAHAM (1962) discuss the general growth in prosobranch mollusks and include a chapter on the shell. No previous work, however, has been done on repair in the 5 species of Acmaea under consideration here. Specimens of each of the 5 species showing evidence of shell damage were collected from Pescadero Point, Monterey County, and Mussel Point, Pacific Grove, Ca- lifornia on May 24 to 26, 1966. All levels of the inter- tidal area were covered, and both protected areas and regions exposed to very heavy surf were included in the survey. One hundred animals showing evidence of having repaired shell damage were collected; all but two of these occurred where the wave action was heavy and where debris such as rocks and large shells were tossed and shifted about by the waves at high tide. Even in the latter areas animals with damaged shells constituted a very small minority of the total population. Of several hundred Acmaea digitalis examined only 3 were found to be damaged significantly. Acmaea scabra * Permanent address: 40 Woodley Road, Winnetka, Illinois. and A. pelta populations showed the same small propor- tion of damaged shells, although in the areas studied A. pelta was not as abundant as the other species. Many more damaged shells of A. scutum and A. limatula were found, but they comprised less than 10% of the observed populations of these species. Often several A. scutum or A. limatula with damaged shells were found in the same small area. Such groups of injured animals were not found in the other species. The numbers of damaged animals collected and the types of damage are shown in Table 1. As can be seen, injury to the edge of the shell, with pieces of the margin chipped off, is the most common (Plate 4, F). Repair of the margin can be recognized readily not only by the newness of the repaired portion but by the ridge which is produced where the new shell material meets the old. Table 1 Frequency and Types of Natural Shell Damage in 100 Limpets ~ = s ra o es | 5 28 ,2 2 Es 2% = Species gs 5 o a3 s 38 3 5 g rat (acerca (S) Sg Se GA Acmaea scutum 60 53 1 4 0 0 2 Acmaea pelta 3 2 0 0 0 0 1 Acmaea digitalis 3 0 0 0 2 1 0 Acmaea scabra 2 0 0 0 1 0 1 Acmaea limatula 32 26 3 2 1 0 0 Vol. 11; Supplement In many cases pieces up to 20% of the width of the shell have been broken off and replaced. Often the entire mar- gin of the shell has been broken off and a ring of newer shell material can be seen clearly all around the original shell. In Acmaea limatula this new shell material has the same characteristic file-like appearance as the original shell. In 4 of the animals collected a portion of the top of the shell near the apex had been crushed (Plate 4, A). Pieces of the shell had been pushed inward, pressing down the viscera. Repair had been achieved by the laying down of new nacreous layers below these pieces of shell, cementing together the crushed fragments of the top of the old shell and partially embedding them to form a solid unit. Any holes left by missing pieces of shell had been covered over on the inside by new shell material (Plate 4, B). Where the shell had been cracked severely from margin to apex, new nacreous material had been laid down over much of the interior of the shell, binding the pieces into a solid unit. In some cases quite extensive damage of this sort has been repaired (Plate 4, C). Four of the shells each contained a small hole from 1} to 34mm in diameter. Although the cause of these holes is unknown, they appear too irregular in outline to be the work of predatory boring snails. The holes had been covered over on the interior with new nacreous material. Two of the shells of Acmaea digitalis, one of A. lima- tula and one of A. scabra had been very severely eroded by a fungus growing in the shell (Plate 4, D). The erosion was heaviest at the apex and extended down the sides to varying degrees. The surface of these shells was much softer than that of normal shells and had a spongy appearance when viewed under the dissecting micro- scope, Bonar (1936) reported finding an ascomycete, Didymella conchae, in the shells of Acmaea, and noted that among A. digitalis uninfected shells are actually rare. Examination of populations of A. digitalis bore this out, but only in unusual cases was the damage particular- ly noticeable. One A. digitalis shell was found with one large and several small barnacles, Balanus glandula Dar- win, 1854, living on it. There were deep pits in the shell, not caused by Didymella conchae, which were clearly formerly inhabited by barnacles and may actually have been eroded by them (Plate 4, E). In conjunction with observations of natural damage, laboratory studies of repair of artificially induced damage were undertaken. Three animals of each species were chosen with normal, undamaged shells. Slots were drilled in the shell with a high speed dental drill. All the slots _ were of uniform width, approximately 14mm, and ex- tended varying distances from the margin towards the apex. In no case did the slot extend up to the attachment THE VELIGER Page 65 of the shell muscle. Care was taken not to damage the mantle. The animals were placed on rocks in aquaria with constantly circulating seawater and aeration and kept submerged throughout the experimental period (May 16 to May 23). They were removed each day, placed on a glass slide, and the extent to which they had repaired the slots observed under a compound micro- scope and measured with an ocular micrometer. The results are presented in Table 2. Table 2 Repair of Artificially Damaged Limpet Shells -< = eis Pe an 8 4 &p Species oe a E 5 ‘g ae oO > nv As <2 Acmaea scutum 25 2 0.075 25 4 0.047 28 8 0.000 Acmaea pelta 22 2 0.05 24 5 0.07 28 10 0.000 Acmaea digitalis 14 1 0.001 14 3 0.000 14 5 0.000 Acmaea scabra 20 2 0.001 19 4 0.000 20 6 0.000 Acmaea limatula 21 2 0.023 21 4 0.025 23 8 0.000 In each case where repair took place, it began with the laying down of a thin transparent layer at the interior end of the slot. This layer was gradually extended to- wards the margin of the shell and thickened from be- neath. Often this new shell broke off. After about 4 days the new shell became opaque as it continued to thicken. The mantle, whose margin usually conforms exactly to the margin of the intact shell, did not contract locally to a shape conforming to the margin of the slot until after about 2 days. In no case did repair begin until this had taken place. In those animals with deeper slots (see Table 2) the mantle margin was unable to contract enough to conform to the margin of the slot and no repair took place. The high incidence of evidences of natural shell dam- age and repair in Acmaea limatula and A. scutum is prob- ably the result of 3 factors. First, both species inhabit the lower areas of the intertidal (RickETTS & CaLvin, 1952) Page 66 where wave action is heaviest, and where loose rocks and debris which might be pounded against their shells are more prevalent. Secondly their shells are both flatter and thinner than those of the limpets of the higher inter- tidal areas, thus presenting a larger and weaker surface. Finally, both species show a much more rapid rate of repair than either A. scabra or A. digitalis. Thus an A. scutum or A. limatula with a damaged shell would be more likely to be able to repair any damage to its shell before a predator could take advantage of the weakened shell. Limited observations on shell growth in undamaged animals maintained in laboratory aquaria from April 27 to May 21, 1966, suggest that addition of new shell at the margin occurs more rapidly in A. scabra than in the other 4 species, and that new shell may be added at very uneven rates at different regions of the shell margin in this species. Acmaea scabra is known to inhabit a par- ticular “‘scar” on the substrate to which its shell conforms exactly and to which it consistently returns (HEwatt, 1940; Test, 1945). The rapid and uneven marginal growth it exhibits might enable animals to achieve con- formity of the shell to a new spot on the rock more rapidly. However, shells of A. scabra with slots drilled in the shell margin showed a slow rate of repair. SUMMARY Shells of Acmaea scabra, A. digitalis, A. pelta, A. scutum and A. limatula found in the field showed a variety of types of natural damage. Damage through chipping at the edge of the shell predominated, but shells were found in which regions near the apex had been crushed; in which the tops and sides were croded duc to an ascomycete, Didymella conchae; in which cracks ex- tending from the margin to near the apex were present; in which the tops were eroded apparently due to bar- nacles, Balanus glandula, living on the shell, and in which small holes of unknown origin occurred near the apex. Repair of such damage in all cases had been accomp- lished by the laying down of new nacrcous material on the interior of the shell below the damaged portion. Rates of repair of artificially damaged areas on shell THE VELIGER Vol. 11; Supplement margins were much higher than rates of growth on ad- jacent undamaged shell margins. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. Help in the project was given by Dr. Donald P. Abbott, Hopkins Marine Station. The photographs in Plate 4 were taken by Mr. Samuel E. Johnson. Permission to collect in the Pescadero Point area was kindly granted by the Monterey Foundation. LITERATURE CITED Bonar, LEE 1936. | An unusual ascomycete in the shells of marine animals. Univ. Calif: Publ. Bot. 19: 187 - 194 Frank, PETER WOLFGANG 1965. Growth of three species of Acmaea. 7 (3): 201 - 202; 1 fig.; 1 table FRETTER, VERA & ALASTAIR GRAHAM The Veliger (1 January 1965) 1962. British prosobranch molluscs, their functional anatomy and ecology. London, Ray Soc. xvi+755 pp.; 316 figs. Hewatt, Wiis GILLiLaND 1940. Observations on the homing limpet, Acmaea scabra Goutp. Amer. Midland Naturalist 24 (1): 205 - 208; 1 fig. PEPPARD, MARGARET CAROLINE 1964. Shell growth and repair in the gastropod Tegula funeb- ralis (Mollusca: Gastropoda) The Veliger 6, Supplement: 59 - 63; 3 text figs. (15 November 1964) Ricketts, Epwarp F. & Jack CALvIN 1962. Between Pacific tides. 3¢ ed., rev. by J. W. HepcPeTH xiii+502 pp.; illust. Stanford Univ. Press, Stanford, Calif. Srapy, Rocer R. 1966. Reproduction and growth in the file limpet, Acmaea limatula CarPENTER, 1864 (Mollusca: Gastropoda). The Veliger 8 (4) : 300 - 310; 7 figs.; 2 tables (1 April 1966) Test, AVERY RaNsoM (GranT) 1945. Ecology of California Acmaca. 395 - 405 Ecology 26 (4): Explanation of Plate 4 A. Shells of Acmaca scutum showing crushed tops. B. Interior of shell shown in A. Note how new nacreous material has been laid down. C. Shell of Acmaca scutum showing severe cracking. D. Shell of Acmaca digitalis showing erosion due to a fungus in the shell. E. Shell of Acmaea digitalis with barnacles growing on it. Note deep pits. F. Shell of Acmaca limatula showing damage to edge and sub- sequent repair. [BuLKLEy] Plate 4 Tue VE.icErR, Vol. 11, Supplement Vol. 11; Supplement THE VELIGER Page 67 Some Observations of Predation on Acmaea Species by the Crab Pachygrapsus crassipes DEXTER CHAPIN Hopkins Marine Station of Stanford University" Pacific Grove, California 93950 (Plate 5) SPECIES OF THE GENUS Acmaea are distributed through- out the rocky intertidal zone of the California coast in association with a variety of animals potentially preda- ceous on these limpets. Predation on Acmaea by the fish Gibonsia elegans Husgs, 1929 was observed by MitcHett (1953). A continuation of this work by JoHNsToN (1953) indi- cated that Oligocottus snyderi GREELEY, Gibonsia metz Husss, 1929, and Gobiesox maeandricus (G1rARD, 1858) were also occasionally found with limpets in their gut. Frank (1965) has presented evidence that Leptoplana may be predaceous on some species of Acmaea on the Oregon coast. The other major predator reported in the literature is the starfish (BuLLocK, 1953; FepeEr, 1959). Recorded here are observations of predation by the crab Pachygrapsus crassipes RANDALL, 1839, a previously un- recognized predator of these mollusks. MATERIALS The following limpet species were included in the study: Acmaea scutum EscuscHottz, 1833, A. digitalis Escu- SCHOLTZ, 1833, A. limatula CARPENTER, 1864, A. pelta EscHscHoLtz, 1833, and A. scabra (Goutp, 1846). These animals and Pachygrapsus crassipes were collected between Mussel Point and Cypress Point on the coast of Monterey County, California, and studied both in the field and in the laboratory. RESULTS anp DISCUSSION Pachygrapsus crassipes was never observed attacking Ac- maea in the field. In the laboratory, when left undis- ' Permanent address: 2027 Hillyer Place, Washington, D. C. 20009 turbed, the crab made sometimes as many as 5 attacks in 3 hours. While all limpets were preyed upon, crabs prefer- entially attacked A. limatula. Pachygrapsus crassipes had 2 methods of attacking its prey. The first was simply to pry the limpet off the rock with its cheliped. This method was successfully used if the limpet did not have its shell clamped to the rock surface and the crab could get underneath the edge. Animals attacked in this manner showed a characteristic chipping of the edge of the shell. This method was used most often on Acmaea scutum and A. digitalis. The second method of attack was never directly ob- served. However, the result of this method could be as- sessed by the examination of shells. Shells of limpets sub- jected to this form of attack had lost the peak of the shell above the muscle scar (see Plate 5). A total of 17 shells with the tops removed were taken from the aquaria con- taining the crabs. Such shells were never found in any other tanks. Several shells were found that had deep scratch marks on them that were possibly made by the crab’s chelipeds exerting pressure on the shell. The scars were randomly oriented on the shell and were found distributed over the entire surface of the shell. Attempts were made to mechanically duplicate the posible squeezing action of the chelipeds by means of a pair of needle-nose pliers suspended from a stand above the limpet, so that only a lateral pressure was exerted between the points. These experiments indicate that there is a fracture zone or weak area that encircles the shell just above the point of attachment of the shell muscles. This zone seems to be present in all Acmaca species studied, but it is most pronounced in the shells of A. lima- tula where, if a pressure of just 2 pounds was applied with the pliers, the shell might break. The maximum Page 68 pressure needed to break an A. limatula shell along the shear zone was 17 pounds at the tips of the pliers. Studies on factors influencing the shell breakage showed it is important to exert the pressure exactly on the very narrow shear zone. The shells were most easily broken if the tips of the pliers were placed on the longitudinal axis rather than the lateral axis. When the pressure was exerted below the shear zone, one Acmaea limatula shell withstood a pressure of 45 pounds. The same shell broke when a pressure of 12 pounds was exerted at the shear zone. If the shells were artificially broken in this manner, they did not always produce a clean circular break. The remaining shell fragments, however, could very easily be broken back to the shear zone but no farther so that a very even break could be achieved. A second set of experiments was run in which a cheli- ped that had recently been removed from a crab was used to exert pressure on the shells in the same manner that the pliers were used. The results of these experiments indicated that the cheliped could puncture the shell at the fracture zone as easily as, or more easily than, the pliers although no exact pressures were recorded. The cheliped did not suffer any damage when the pressure was applied. By having the crab squeeze a piece of balsa wood, and then duplicating the damage with pliers with about the same squeeze area, it was possible to make a very crude determination of the pressure that could be exerted by the crab, With this method, it was found that the pres- sures exerted by the crab exceeded 23 pounds, which is more than any pressure needed to artificially break a limpet shell at the shear zone. Surveys of the intertidal area on Mussel Point indicate that shells from which the peaks have been removed make up about 14% of the total Acmaea shells cast up on the beach. Such shells were often not highly eroded and the loss of the peak did not appear to be due to erosion after the death of the limpet. Explanation A: Acmaea shells broken by various means. From left to right: (1) Acmaea limatula shell found in an aquarium containing only Pachygrapsus crassipes as a predator. (2) Acmaea digitalis shell artificially broken with needle-nose pliers. (3) Acmaea limatula shell artificially broken with needle-nose pliers. (4) Acmaea limatula shell found in an aquarium with Pachygrapsus crassipes. THE VELIGER Vol. 11; Supplement SUMMARY The crab, Pachygrapsus crassipes, has not previously been recognized as an important predator of limpets. Laboratory observations and experiments suggest that the crab can remove the tops of the shells of some limpets by squeezing with the cheliped, thus making the viscera available for food. The results of a survey of the shells cast up on the beach, and the number of limpets appar- ently attacked by this method in the laboratory, suggest that these animals may be responsible for a significant mortality in limpet populations. ACKNOWLEDGMENTS The author gratefully thanks Dr. John Phillips and Ray Markel for their patience and assistance in carrying out this study and preparing the manuscript. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. LITERATURE CITED BuLLock, THEODORE HoLMEs 1953. Predator recognition and escape responses of some intertidal gastropods in presence of starfish. Behaviour 5 (2): 130-140 Feper, Howarp MITCHELL 1959. The food of the starfish Pisaster ochraceus along the California coast. Ecology 40: 721 - 724 Frank, PETER WOLFGANG 1965. The biodemography of an intertidal snail population. Ecology 46 (6): 831-844; 8 figs.; 6 tables JouHNsTon, RicHarp FouRNESS 1954. The summer food of some intertidal fishes of Monte- rey County, California. Calif: Fish & Game Bull. 40 (1): 65 - 68 MitcHELL, D.F 1953. An analysis of stomach contents of California tide pool fishes. Amer. Midland Natural. 49 (3): 862 - 871 of Plate 5 B: An artificially broken Acmaea limatula shell. The very smooth surface of a break in the cleavage zone is seen. The notch on the left was made by the tips of the pliers when the pressure was applied slightly below the cleavage zone. C: A shell of Acmaea sp. found cast up on the beach. This illustrates the effects of the slight erosion often found in such shells. Tue VELIGER, Vol. 11, Supplement [CuaPIn] Plate 5 Figure 1 Figure 3 Vol. 11; Supplement THE VELIGER Page 69 A Study of Morphological Variation in the Limpet Acmaea pelta ALAN JOBE Hopkins Marine Station of Stanford University ' Pacific Grove, California 93950 (Plate 6; 1 Text figure; 2 Tables) Acmaea pelta EscHscHoLTz, 1833 is a low intertidal limpet that is widely distributed on the North American west coast. A great variation in shell appearance has been noted, especially when comparing large and small specimens. Preliminary observations indicated a possible correlation between habitat and morphology, which led to a study of the distribution of A. pelta with respect to substrate and algal association. These field studies, re- ported below, indicated definite relationships between morphologic types and habitat, leading to a study of digestive enzyme potential. FIELD STUDIES Field observations were done at Mussel Point, Pescadero Point, and Cypress Point on the Monterey Peninsula, California. Three morphologic forms of Acmaea pelta were observed, differing in color, shape, and length to height ratios of the shell. These different forms of A. pelta were positively identified as being variations of the 1 Permanent address: 505 Pacific Avenue, Solana Beach, California species using standard radula length and teeth deter- minations as well as the classification by jaw shape pro- posed by WaLKER (1968). These various morphologic forms are described below in terms of color, shell shape, and length to height ratios as measured with a vernier caliper. MORPHOLOGIC FORMS Brown Form: The typical large brown Acmaea pelta is characterized by a convex slope from the high peaked shell apex to the periphery and is often infected with the fungus common to shells of littoral animals of the Pacific coast (Bonar, 1936). These brown-colored speci- mens are generally over 30mm in length, and have a length to height ratio of 2.3 as seen in Table 1. Illustra- tions of the brown shells are shown in Plate 6, Group A. Black Form (Pelvetia): Two separate populations of the small black Acmaea pelta were observed, on the mussel beds and on and under Pelvetia fastigiata (J.G.AGARDH) De Toni 1895. Those found associated with Pelvetia (Plate 6, Group C) had a straight to concave slope from Table 1 Length to height ratios of the different forms of Acmaea pelta as related to position in the intertidal zone. ° E sth Se Bs & £3 Z2 & 3 Form of Animal 9 Location gs ge qe vn Zo 5 nd Brown Acmaea pelta 30 High Intertidal 24 2.32 + 0,246 Black Acmaea pelta 15 Under Pelvetia 22 2.86 + 0.270 Black Acmaea pelta 15 Under Pelvetia 20 2.93 + 0.268 Black Acmaea pelta 15 Mussel beds 12 2.53 + 0.295 Black Acmaea pelta 10 to 15 Mussel beds 27 2.89 £01272 Black Acmaea pelta 10 Mussel beds 23 3.21 + 0.209 Ne nna Page 70 the apex to the edge and a characteristically cleaner shell than any other A. pelta form. The shell shape was flatter than the brown A. pelta, and had a uniform length to height ratio for the entire size range encountered. This flat shell often had a rather hooked apex and a more wavy peripheral edge than the black form seen in mussel beds (see below). Black Form (Mussel beds): Acmaea pelta was found on the mussel bed areas generally on mussels, Mytilus cali- fornianus Conrap, 1837, and on the goosenecked bar- nacles, Pollicipes polymerus SowErBy, 1833. These were generally black with length to height ratios which varied with size, as seen in Table 1. No specimens over 20 mm were found in the beds. The profile of the members of this form (Plate 6, Group D) approaches that of the large brown A. pelta. The shells of this form are not as clean and as shiny as those found under Pelvetia, and the shell periphery is smooth. Green Form: The third group of Acmaea pelta, inter- mediate in size between the black A. pelta on Pelvetia and the large brown A. pelta form, is shown in Plate 6, Group B. This group shows a great variation in shell shape and often displays the growth ring type of change in the exterior shell. Most specimens are greenish-black in color. Some, however, are almost white and others are ribbed or checkered. HABITAT A survey of the intertidal zone of the Monterey Peninsula revealed differences in the density of the Acmaea pelta populations. Mussel Point and Pescadero Point had rich populations while Point Pifos was sparsely populated. The reason for this uneven distribution was not investigated. Brown Form: This form of Acmaea pelta occupies a high inshore intertidal position from +4 to above + 6ft. The brown organisms were found predominantly on more barren rocks and were exposed to more sun and arid conditions than the other types. They were generally found on vertical rather than horizontal faces. The brown limpets were found in an apparently random association with encrusting algae and algal filmsas well as Endocladia muricata (PosTELS & RuprecHT) J.G.Acarpu, 1847, high Pelvetia fastigiata, Rhodoglossum affine (Harvey) THE VELIGER Vol. 11; Supplement Kyin, 1928, and other algae. Occasionally one was found on bare rock close to sand with no macroscopic algae evident. Black Form (Pelvetia): At low tide 25% of this form was on the holdfast or stipe of Pelvetia. An obvious scar was found under these limpets, indicating that this alga may be a primary food source. The remaining 75% were attached to the rock substratum under the Pelvetia. The Pelvetia beds provided moisture and shelter from the sun. In more exposed areas, such as Pescadero Point, where Pelvetia is not found in large beds, the black Acmaea pelta were less numerous and randomly distributed. Here again they were to be found in locations offering shelter and moisture. Black Form (Mussel beds): Acmaea pelta in the mussel beds were not associated with conspicuous algae. This form could be found exposed to the sun at low tide, as opposed to the Pelvetia form which was never found exposed to direct sunlight. The mussel beds provided a damp, sheltered environment that is subjected to splash except at very low tide. Green Form: The middle-sized green Acmaea pelta had a habitat virtually identical with that of the black Pel- vetia form. Text figure 1 shows the percent distribution of these different types of A. pelta as related to position and habitat in the intertidal. LABORATORY STUDIES The intertidal distribution, along with Craic’s (1968) observations on feeding habits suggested that, since the different forms were eating different algae, possibly there was a difference in digestive enzymes in the Acmaea pelta forms. The digestive enzyme potential of the different forms of A. pelta was assayed by measurement of reducing sugar released from purified polysaccharides (Netson, 1944). The polysaccharides studied were fuco- idin, laminarin, and alginic acid from brown algal sources, agar and K-carrageenin from red algae, and starch. Since some of these carbohydrates contain sul- fated groups, particularly fucoidin, sodium hydroxide was substituted for barium hydroxide (NELSON, op. cit.) to avoid possible precipitation of the sulfated sugar frag- ments produced by enzymatic hydrolysis, A 10% extract of the entire digestive system including esophagus, buc- Explanation of Plate 6 Different varieties of Acmaea pelta encountered on the coast of the Monterey Peninsula. Group A: Brown form; Group B: Green form; Group C: Black form (Pelvetia); Group D: Black form (mussel beds). Tue VELIcER, Vol. 11, Supplement [Jose] Plate 6 Figure 2 Figure 3 Figure 4 Vol. 11; Supplement Little 80% + Algae Present Endocladia Pelvetia 60% 40% 20% ee aa aia —g = ee = = about +6 ft. 6 to 5 ft 5 to 4 ft THE VELIGER Page 71 Gigartina Fucus Iridaea Mussel Beds Egregia 4 to 3 ft. 3 to 2 ft. below +2 ft. Figure 1 Percent distribution of the three morphologic types of Acmaea pelta (each type = 100%) as related to position and habitat in the intertidal zone. Black Acmaea pelta (231 animals) shown in solid bars; brown (123 animals) in open bars; and green (168 animals) in cross hatched bars. cal salivary glands, and hind gut was prepared from animals of the black and brown morphologic forms. The tissues were chilled on ice and homogenized in ice cold homogenizing medium. Each 1 ml of tissue was ho- mogenized in a medium containing 1 ml of an aqueous streptomycin solution (5mg per ml) to inhibit bacterial growth, 1 ml of a saturated solution of ovomucoid iso- lated by the method of Frepricg « Deutscu (1949), and 7ml of buffer (0.2M Tris buffer at pH 7.2 and 0.2M acetate buffer at pH 5.5). Ovomucoid was used as a trypsin inhibitor because of problems encountered with proteinases in the enzyme extract. The ovomucoid in- creased activity approximately two-fold. One half ml of enzyme extract, one half ml of a 0.5% solution of puri- fied polysaccharide and 14 ml of buffer were incubated at 20° C for one hour. The amount of polysaccharide in a one ml aliquot was equivalent to approximately 200 micrograms of reducing sugar on complete hydrolysis. The colorimetric determinations were made with a Klett- Summerson photo-electric colorimeter using a green fil- ter, and values were corrected for reducing sugar present in enzyme and substrate controls. The corrected values are expressed in terms of micrograms of glucose per hour per ml aliquot of reaction medium, and are equivalent to 0.02 ml of gut tissue. RESULTS Marked amylase activity, particularly at pH 5.5, was _ demonstrated in extracts from large brown Acmaea pelta and the black form found on Pelvetia. Table 2 gives the quantitative results. Higher activity was observed in ex- tracts from the brown animals. Low levels of fucoidinase and alginase were consistently demonstrable at pH 7.2. No enzymatic hydrolysis of K-carrageenin, laminarin, or agar was observed at either pH. Table 2 Enzyme activity expressed in micrograms of reduced sugar per 0.02 ml of gut tissue. Any value less than 2 micrograms is considered inconclusive. Black Acmaea pelta Brown Acmaca pelta (Peluctia) Polysaccharide pHs mpHes.2 pH5.5 pH7.2 Starch 52pg 24ug 112yg 50ug Agar 0 lug 0 lug K-carrageenin 0 0 0 0 Laiminarin 0 0 0 0 Fucoidin Sug 4ug 0 Sug Alginic Acid 0 8ug 0 Sug DISCUSSION The field work demonstrated a difference in shell shape of the Acmaea pelta found in different habitats in the intertidal. It appears that shell growth and the resulting shell shape is probably a response to the substrate upon which the animal lives. The high peaked convex shell of the black variety found in the mussel beds may be an adaptation to attachment to a substrate such as is pre- sented in the mussel beds where large flat surfaces are at a minimum. The flatter A. pelta under Pelvetia have an excess of flat but rough rock surface to grow and move Page 72 on. The wavy shell periphery is an indication of an adaptation to this environment. This seems to be a re- action to the substrate similar to but not as extreme as that of the limpet A. scabra whose shell grows to conform to irregularities of the home site (Hewat, 1940). Ac- maea pelta is characteristically a high, almost center peaked limpet. This is true when exposed and of reason- able size, as in the brown form and the larger specimens from the mussel beds. Since no large specimens were found in the mussel beds and small ones were abundant, mortality may increase with age, the larger limpets being unable to survive on this substrate. Substrate and expo- sure therefore appear to be large factors in determining shell morphology. The reason for the different colors seen in Acmaea pelta is not clear, but a possible correlation with different algal food is plausible. The reported experiments do not seem to indicate that digestive potential is the critical factor. They also show that alginic acid and fucoidin could be utilized only slowly in comparison with utili- zation of starch. Although extracts prepared from brown forms displayed more activity than those obtained from black ones, this difference may be simply related to the ease of dissecting and the resulting greater purity of the extracts prepared from the larger brown limpets. Although not related to digestive potential, the differ- ences in color of peak and peripheral portions of shells suggest possible changes in diet during the life of the animal. Long term experiments will be required to explore this relationship. SUMMARY Populations of Acmaea pelta EscHSCHOLTZ, 1833 exhibit variation in shell shape and color. Field studies showed correlations of these variables with algal association, sub- strate, and exposure. Shell shape seems to be dependent upon substrate and exposure. Shell color may be related to diet, but preliminary investigations of gut carbohy- THE VELIGER Vol. 11; Supplement drases failed to reveal any distinctive quantitative dif- ferences in the amylase, fucoidinase, and alginase activ- ities found in the A. pelta forms assayed. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. Thanks to Dr. J. Phillips, my advisor, who isolated and purified the polysaccharides, agar, K-carrageenin, laminarin, fucoidin, and alginic acid for the digestive enzyme assays. Thanks to Miss Cathy Walker who helped in giving positive identification to the Acmaea pelta forms. LITERATURE CITED Bonar, LEE 1936. An unusual ascomycete in the shells of marine animals. Univ. Calif: Publ. Bot. 19: 187 - 194 Craic, PETER CHRISTIAN 1968. The activity pattern and food habits of the limpet Ac- maea pelta. The Veliger 11, Supplement: 13 - 19; plt. 1; 5 text figs.; 1 table (15 July 1968) Frepricg, EucENE & Harotp Francis DeuTscH 1949. Studies on ovomucoid. Journ. Biol. Chem. 181: 499 - 510 Hewatt, WILits GILLILAND 1940. Observations on the homing limpet, Acmaca scabra Goutp. Amer. Midland Naturalist 24 (1): 205 - 208; 1 fig. Netson, Norton 1944. A photometric adaptation of the Somogyi method for the determination of glucose. Journ. Biol. Chem. 153: 375 - 380 WALKER, CATHERINE GENE ~ 1968. Studies on the jaw, digestive system, and coelomic de- rivatives in representatives of the genus Acmaea. The Veliger 11, Supplement: 89-97; 13 text figs.; 1 table (15 July 1968) Vol. 11; Supplement THE VELIGER Page 73 Anatomical and Oxygen Electrode Studies of Respiratory Surfaces and Respiration in Acmaea ROGER S. KINGSTON Hopkins Marine Station of Stanford University" Pacific Grove, California 93950 (Plate 7; 6 Text figures; 1 Table) THE LIMPETS OF THE GENUS Acmaea are found in abun- dance in the rocky intertidal zone on the shores of the Monterey Peninsula, California. Individuals of the spe- cies Acmaea scabra (Goutp, 1846) and A. digitalis EscHscHOoLtz, 1833, are found high in zones one and two described by Ricketts & Cavin (1962). Other species common to this area, A. pelta ESCHSCHOLTZ, 1833; A. limatula CarRPENTER, 1864; A. asmi (MiIDDEN- porFF, 1849); and A. scutum EscuscHoLtz, 1833, are found under water much of the time lower down in zones three and four. These species differences in exposure suggested that Acmaea must be capable of differing degrees of respi- ration in air and water. The aim of this study was to determine the site(s) of the specialized respiratory sur- faces of Acmaea, and to list their respiratory effectiveness when exposed to air and when submerged. I. ANATOMY Initially the anatomy of the circulatory system was in- vestigated. It was assumed that gas diffusion could occur across any body surface and that a specialized respiratory area might be one where gas exchange was facilitated by blood flow close to the external body surface. Individuals of the species Acmaea scutum, A. pelta, and A. digitalis from low, middle, and high intertidal zones, respectively, were taken as samples. The principal method of study was injection of colored substances into the circulatory system. Injections were carried out with 26-gauge hypodermic needles or fine glass needles pulled from soft glass tubing. A number of different injection fluids was tried, including latex, vital stains dissolved in alcohol or water, and 1 Permanent address: 611 West Acacia Street, Salinas, California. commercial inks. Best results were obtained with an aqueous colloidal suspension of carbon. The tissues re- mained relaxed during the carbon injections and the particles did not diffuse out of the vessels. Successful injections were made on both live, fresh animals and on animals relaxed in a solution of MgCl. isotonic with sea water. Large areas of the circulatory system were colored by injections into the heart or visceral cavity. For study auricle -——— > _ ventricle post. eff. ves. ant. eff. vess. ; 3 ea Saas ; visc. cavit. = 7 buccal sinus ctenidium mantle circumpallial vess. ant. aff. ves. Figure 1 Circulatory system of Acmaea. Solid lines represent known relationships; broken lines represent supposed relationships of localized areas, injections were made into local vessels. Blood flow direction was determined by observation of the vessels during injections of a dilute carbon suspension. Gross blood flow, determined by the above methods, is shown in Figure 1. The results indicate two separate and distinct areas — the ctenidium and the mantle - where there is a large amount of blood flow close to the animals’ external surface. The first of these areas, the ctenidium, is generally considered the respiratory organ of Acmaea. Colloidal carbon injections of this organ colored the major efferent Page 74 THE VELIGER Vol. 11; Supplement and afferent vessels and the capillaries of the right and left thirds of the ctenidial filaments, but not the central portions of the filaments. The filament vessels, however, were stained in their totality with vital dyes. These obser- vations indicate that capillaries sufficiently fine to pre- vent the passage of colloidal carbon connect the efferent and afferent vessels. The other area where a large amount of blood flow was found close to the surface was the ventral side of the mantle fold facing the mantle groove. Blood enters the mantle fold from the visceral cavity through vessels which pass through the shell muscle fibers. In the area between the base of the mantle groove and the circum- pallial vessel these vessels anastomose. The density of interconnecting vessels in this network is extraordinary (see Plate 7, Figures 1 to 3). Near the circumpallial Ventral view of blood flow and respiratory surfaces in Acmaea aav = anterior afferent vessel cav = ctenidal afferent vessel cev = ctenidial efferent vessel cg = ctenidial gills cpv = circumpallial vessel fr = foot removed me = mantle edge msv = mantle supply vessel myn = mantle vessel network nc = nuchal cavity pev = posterior efferent vessel pv = pallial vessels vc = visceral cavity vcr = visceral cavity removed Explanation Figure 1: Colloidal Carbon Injection of Mantle (beginning injection) 1. glass needle 2. mantle supply vessel 3. side of foot 4. bottom of foot 5. edge of mantle Figure 2: Colloidal Carbon Injection of Mantle (finished injection) Respiratory vessel network is filled. Note the “standard” venation in the foot (1). vessel (Figure 2) the vessels of this network come to- gether to form a highly branched pattern. Blood in these vessels goes to the edge of the mantle, servicing the glands and pallial tentacles, returns to the anterior af- ferent vessel, and thence to the heart. As in the ctenidium, there were fine capillaries in the mantle fold near the circumpallial vessel which could not be filled with sus- pended colloidal carbon, but which could be colored by dissolved stains. The above observations show that the ctenidium and the mantle fold are two areas through which blood passes just before returning to the heart (Figure 1). Since, in most organisms, blood is oxygenated at the respiratory surfaces just prior to its return to the heart, these observa- tions suggest that both the mantle fold and the ctenidium are respiratory surfaces. The possible role of the mantle fold as a respiratory surface suggested that the ciliary mantle currents, earlier described in Acmaea by YoncE (1962) and believed to be cleansing currents, might also serve a respiratory role. To observe these currents, carmine particles suspended in sea water were placed in the mantle groove of over- turned animals. A circular current in the mantle groove was found, moving in a plane perpendicular to the side Figure 3 Respiratory ciliary current in the mantle groove. Solid lines represent blood flow direction; broken lines represent ciliary current direction. cpv = circumpallial vessel f = foot vc = visceral cavity m = mantle of Plate 7 Figure 3: Roof of nuchal cavity partially injected. Injection was made in mantle groove at left. Note network pattern of vessels (1), anterior afferent vessel (2), and head pinned back against foot (3). Figure 4: Functional End of the Oxygen Cathode 1. recess filled with distilled water 2. glass cover 3. platinum wire THE VELIGER, Vol. 11, Supplement [KincsTon] Plate 7 Figure 1 es : Figure 2 TVITh. TF TV Figure 4 Figure 3 Nestea bevay Vol. 11; Supplement THE VELIGER Page 75 of the foot and to the animal’s substratum. This ciliary current moved opposite to the direction of blood flow in the mantle and was a counter-current of the type often associated with respiratory surfaces (see Figure 3). A sim- ilar ciliary current was found in the nuchal cavity moving opposite to the direction of blood flow in the ctenidium. This mantle counter-current found in Acmaea is similar to that found in the mantle groove of Lottia (Axxorr, 1956). Because of the large amount of blood flow close to the surface, the type of venation, the position relative to the heart in terms of blood flow, and the ciliary counter- currents, both the ctenidium and the mantle are here suggested as respiratory surfaces in Acmaea. II. FIELD OBSERVATIONS Acmaea scabra and A. digitalis from zones one and two, A. pelta and A. limatula from zone three, and A. scutum from zone four were observed in the field to record the behavior of the mantle and ctenidium under dry and wet conditions. The study was qualitative; observations were made of animals on dry rocks, on splashed rocks, and in quiet pools with the aid of a ten-power magni- fying lens. When an individual had been out of the water for a long enough period to have adjusted to the lack of water, the following characteristics were usually evident. The shell was pulled down onto the surface of the rock sub- strate( though not tightly clamped), the mantle fold was wet (as found in Lottia [Appotr, 1956]), and when the animal was disturbed it clamped down tightly with some water often exuding from the mantle groove. If the animal was taken from the rock and turned over, the wet area of the mantle fold between the foot and circum- pallial vessel was noticeably swollen, and the vessels appeared dilated and gorged with blood. The ctenidium was found to be withdrawn in the nuchal cavity, and in Acmaea scabra which had been dry for many hours and whose nuchal cavity was no longer filled with water, the ctenidium was hardly visible. The above characteristics were observed in members of all species but were partic- ularly evident in A. scabra and A. digitalis. When an Acmaea individual was under water, its shell was elevated one to three millimeters off the substrate, its mantle protruded somewhat beyond the edge of the shell, and a ciliary current in the nuchal cavity, demon- strated with carmine particles in a quiet pool, flowed counter to ctenidial blood flow. When an animal was overturned and compared with a member of the same species from a dry area, the wet animal’s mantle appeared flatter and the mantle vessels seemed smaller in diameter. In the laboratory, where the ventral side of submerged Acmaea could be observed through aquaria walls, the ctenidia of A. scutum, A. pelta, and A. limatula were con- sistently found extended and lying partially in the right mantle groove. In A. scabra and A. digitalis taken from high, dry areas and kept under water in aquaria for 48 or more hours, the ctenidia were never seen to extend more than one-half the distance from the back to the front of the nuchal cavity. They are, therefore, appar- ently not sufficiently long to extend to the mantle groove, and are much reduced in size compared to the ctenidia of A. pelta, A. limatula, and A. scutum. These field studies indicate that the mantle is the site of increased blood flow when the animal is out of the water, whereas the ctenidium is usually the site of similar increased flow when the animal is under water. Under water, the elongated, filamentous ctenidium is a principal wetted surface. Out of water, however, the ctenidium contracts and the surface of the mantle fold is kept wet at the expense of water in the nuchal cavity, suggesting that the mantle fold is the chief respiratory site in dry environments. The laboratory and field observations show that ctenidial elongation and mantle swelling may be controlled by the water or air environment, and also suggest an evolutionary reduction in the ctenidium and a corresponding increase in mantle capacity from low to high intertidal species of Acmaea. III. POLAROGRAPHIC STUDIES Polarographic methods were used to substantiate the respiratory functions of the ctenidium and mantle in- dicated in the previous studies. Electrodes of the recessed type first described by Brink «& Davies (1942) were used to measure the difference in the oxygen tensions in different parts of the body of Acmaea. } to ammeter and recorder Figure 4 Circuit for oxygen electrode Page 76 THE VELIGER Vol. 11; Supplement Experimental apparatus consisted of several platinum cathodes, one silver - silver chloride anode, a 0.8 volt D. C. power source, a Keithley ammeter of 10° ampere sen- sitivity, and a chart recorder. Cathodes were made by sealing 26 gauge (B&S) platinum wire in hand-pulled soft glass capillaries. The wire was first heated to white incandescence in a flame to drive off the surface-ad- hering gases, and the capillary then fused around it. Next the capillary was cut to extend one millimeter beyond the polished end of the platinum wire. The recess thus formed was filled with distilled water and covered with a collodion membrane to prevent the entry of proteinace- ous material into the recess. After construction each cathode was calibrated. First the interval required for equilibration of oxygen within and without the electrode’s recess was determined by comparing successive readings from a constant environ- ment. Next the optimum duration for voltage application was sought, this being the shortest interval yielding a linear amps. vs./O2/relationship. By comparing the elec- trode output at different time intervals in sea water amps x 10° Co) 3 6 9 12 15 18 21 seconds Figure 5 Recorder tracings at three different oxygen tensions. ‘Ni Peo OL/ie B=) 1cci@3/ 1; C =0.5cc O,/1. The dotted line at 18 seconds indicates the amperage value used for construction of the O, calibration curve. samples of different oxygen concentrations, the reading at 18 seconds was found to be the shortest time yielding such a linear relationship (Figures 5 and 6). In the final calibration step, an amps. vs. /O2/curve (Figure 6, line C) was determined for each cathode, using 3 to 5 sea water samples of known O- content (by Winkler method). These cathodes were very stable, with little or no changes evident in the calibration curve from day to day. p fe) amps x 10°° RN te) 0.5 1.0 1.5 2.0 oxygen concentration Figure 6 Output of the oxygen electrode at different O, tensions and different times after closing of circuit. A = 3 seconds; B = 9 seconds C = 18 seconds All experiments and calibrations were performed at 21° C. To compensate for minor temperature fluctua- tions during an experiment (a one degree change resulted in as much as a 10% change in current flow), the O: tension at 2 to 3 different sites on the animal were simultaneously measured with 2 to 3 different cathodes. In each experiment the animal, previously kept in the desired environment for a minimum of 4 hours, was placed upside-down and the cathodes were inserted into the desired tissue or circulatory vessel, through holes pre- viously made with a dissecting needle. The cathodes were left in place and supported in small ring stands, whereas Vol. 11; Supplement THE VELIGER Page 77 the anode was placed on the tissue only at the time of measurement. In the first experiments, comparisons were made of blood oxygen in the visceral cavity, the anterior afferent vessel, and the pericardial sinus. The results (Table 1) show that the O. tension is higher in the pericardial sinus in both aerobic and aquatic conditions. Comparing the visceral cavity with the anterior afferent vessel showed that the O:. tension was higher in the latter area. Because blood flows from the visceral cavity to the anterior af- ferent vessel through the vessels of the mantle fold, gaseous exchange must occur at the mantle-fold surface. In the next group of experiments the ctenidial respi- ration was eliminated, and the respiratory efficiency of the mantle fold determined. Small lead clamps, cut from thin sheets of lead and bent in “V” shapes, were pinched around the ctenidial afferent and efferent vessels of ani- mals relaxed in MgCl:. The treated animals, kept over- night in either wet or dry environments, were tested during the following days and then sacrificed and ex- amined to ascertain whether the clamps were still in place and functioning. Of 20 animals kept under water, 19 were still alive one day after the clamping operation. Testing of 10 of these animals showed (Table 1) the O. tension of the pericardial sinus to be somewhat higher than that of the visccral cavity, although the differences were less than in normal, unclamped animals. The second day after the operation the remaining 9 limpets kept under water were dead. This experiment was performed twice, and each time all the animals were dead by the second day. All 20 animals with clamped ctenidia kept under dry conditions were still alive one day after the clamping operation. Testing of 10 of these animals showed the O: tension of the pericardial sinus to be higher than that of the visceral cavity, this difference being greater than in submerged animals with clamped ctenidia, and about the same as in normal animals under dry conditions. The second day after the clamping operation 2 of the 10 dry animals had died. Testing of the remaining 8 showed the O2 tension of the pericardial sinus to still be greater than that in the visceral cavity, although the absolute tension in both was slightly lower than the previous day. DISCUSSION Four lines of evidence indicate that the mantle fold serves a respiratory role in the limpet Acmaea. These are (1) the presence of a capillary system close to the surface of the mantle fold, (2) a counter-current cili- ary system passing over the mantle fold, (3) the dilation of the mantle fold with blood, and the concomitant de- creased size of the ctenidia, when the animal is dry, and (4) the polarographic evidence that the O: tension of the blood is higher after passage through the mantle fold, and before passage through the ctenidium. These results demonstrate that better gaseous exchange occurs at the mantle surface in air than in water, and Table 1 Mean Oxygen Tensions (cc/1) : : a 3 Eg ic Oe i) SF os am” 1S) NS sal ctenidium A. scutum 14 and mantle dry A. pelta 4 A. limatula 1 ctenidium wet A, pelta 6 and mantle A. limatula 2 mantle only dry A. pelta 7 1s* day A. limatula 3 mantle only dry A. pelta 3 OND A. scabra 3 day A. digitalis 2 mantle only wet A. pelta 8 1st day A. limatula 2 Z : =| el BS) hfs sss eSSe 5 eases i a>o8 108210139) 2 G7 10143) 9 2) 140) = 10:28 0.55 + 0.20 2) 0185) =="0!07 2) 1:60) = 0:21 1.00 i es) 0.83 + 0.44 COG iSO SI 4 57220154 139) ==s 052i 2 1.80 + 0.14 1:70) = 0:36 7 2.24 + 0.48 1.50 + 0.28 32:20) =E10'28 1.37 + 0.03 3 ies OO 8 isto) se Og 1.23 + 0.13 3 1.60 + 0.10 1.60 + 0.14 2 OOH sO 1 Be 202 3m==10193 8) 2290)==1 0195, 1.60 + 0.70 2) ) -1°80)==70:80 Page 78 THE VELIGER Vol. 11; Supplement that Acmaea has physiological and behavioral adapta- tions which allow it to better expose the mantle in air and the ctenidium in water. That Acmaea uses both ctenidium and mantle fold as respiratory organs is evolutionarily interesting, pointing to similarities with the related limpets Lottia gigantea Sowersy, 1843, which respires with ctenidium and palli- al gills (Aspott, 1956), and Patella, which has no cten- idium and respires solely with ciliated flaps fringing the margin of the circumpallial vessel (YoNcE, 1962). SUMMARY The circulatory system of Acmaea was injected with colloidal carbon. Two areas — the ctenidium and the mantle — were found where a large amount of blood flows close to the animals’ external surfaces. Blood flows through one or the other of these surfaces immediately before it returns to the heart. A ciliary counter-current was found associated with each of these surfaces. When observed in the field, the mantle fold was found to expand and the ctenidium to contract when the animal was out of water. Conversely, the ctenidium elongates and the mantle fold flattens under water. Low intertidal species of Acmaea have larger ctenidia and smaller mantle respiratory capacities than higher intertidal species. Oxygen polarography was used to measure the oxygen tensions of the blood in different parts of Acmaea. These measurements indicate that both the mantle and cten- idium are respiratory surfaces, and that the mantle is more effective in aerial conditions and the ctenidium more effective in submerged conditions. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. The author also wishes to thank Drs. David Epel and Donald Abbott. for their encouragement and help during the study and Dr. Lawrence Blinks for his suggestions and materials used in the polarographic study. LITERATURE CITED AxsBoTtT, DonaLpD PUTNAM 1956. Water circulation in the mantle cavity of the owl limpet Lottia gigantea Gray. The Nautilus 69 (3) : 79 - 87, 1 fig. Davies, PHitip WYNNE & FRANK BRINK, Jr. 1942. Microelectrodes for measuring local oxygen tension in animal tissues. Rev. Sci. Instrum. 13 (12): 524 - 533 Ricketts, Epwarp F. & Jack CaLviN 1962. Between Pacific tides. 34 ed., rev. by J. W. Hepcreru xiil+502 pp.; illust. Stanford Univ. Press, Stanford, Calif. YONGE, CHARLES MAURICE 1962. Ciliary currents in the mantle cavity of species of Acmaea. The Veliger 4 (3): 119-123; 2 figs. Vol. 11; Supplement THE VELIGER Page 79 Manometric Measurements of Respiratory Activity in Acmaea digitalis and Acmaea scabra SIMEON BALDWIN Hopkins Marine Station of Stanford University' Pacific Grove, California 93950 (3 Text figures; 2 Tables) Botu Acmaea digitalis EscHscHOLTZ, 1833 and Acmaea scabra (Goutp, 1848) spend a considerable proportion of the day exposed to the desiccating elements of the environment. Several field studies have shown that, al- though the two species are always found in close proxi- mity above about the +5 foot level of the intertidal, A. scabra is usually the only species found on exposed hori- zontal rock surfaces (Haven, 1964). This difference in ex- posure suggests possible physiological differences between the two that might be revealed by respiratory studies. To determine such differences, laboratory measurements of oxygen consumption were made under varying condi- tions of temperature and dehydration. The results showed a significant difference in metabolic rate between the two species, as well as differences in submerged and aerial respiration rates. METHODS Respiration rate was measured with Warburg-Barcroft manometers in a refrigerated water bath. Carbon dioxide was absorbed in the vessel side arm using 0.3 ml of a 30% KOH solution, with a wick of starch-free What- man no. 40 filter paper. The vessel constants for each run were determined for the conditions of the experi- ment, the variables being the experimental temperature, volume of flask content, and the volume of the flask plus manometer arm. The latter was determined by Umbreit’s method of calibration with water (UmpreiT, 1945), and the appropriate vessel constants then found from Dixon’s nomogram (Drxon, 1951). During each run the vessels were agitated at the rate of approximately 60 to 70 oscil- lations per minute. The experiments were done during April and May, 1966. ' Permanent address: 1359 Cliff Drive, Laguna Beach, California The organisms used were medium sized Acmaea scabra and A. digitalis collected about 1 hour before low tide from the +4 to +6 foot region of the intertidal zone near Hopkins Marine Station. They were then trans- ferred to aquaria supplied with running seawater at 15°C where they were allowed to equilibrate for at least 24 hours before being tested. Submerged and damp runs were made with animals temperature-equilibrated for 3 hours before the experi- ment in a finger bowl in the water bath. Animals used for determining submerged respiration rates were then placed in the Warburg vessels and covered with 6 ml of millipore filtered seawater. Animals used for determining damp respiration rates were shaken to remove excess filtered sea water and then placed in dry flasks. Animals used in aerial runs (no water included in Warburg vessel) were placed foot down on a stack of 6 paper towels for 34 hours at room temperature (approximately 22° C) before being placed in the dry vessel. With this procedure they were still able to attach to the sides of the vessels. For each experimental variable, animals were collected under the same conditions and subjected to the same treatment in order to minimize variables. Five separate vessels were used containing 3 limpets per vessel. Each run was repeated a minimum of 3 times using different animals so that each rate is an average of approximately 45 similar-sized animals taken from the same intertidal area. All respiratory measurements reported were made in the dark, since preliminary runs measuring aerial respira- tion of Acmaea scabra showed that photosynthesis of algal growths on the animals’ shells was producing sig- nificant amounts of oxygen. Measurements of respiration of empty limpet shells in the dark showed that the volume of oxygen consumed was about 0.05% of the total amount used by limpets during a 2 hour run. This Page 80 negligible amount was therefore not taken into consider- ation in the final rate calculations. In all experiments shell sizes of animals used were 14 to 17mm in length. Dry weights were determined by removing the shell and drying the animal to constant weight. Rates are expressed in pl O2 per mg dry weight. RESULTS Respiration rates of the two species as related to the state of their environment are shown in Figure 1 and Table 1. This depicts average oxygen consumption at 30 minute intervals during the 2 hour measuring period. It is seen that the damp and aerial respiration rate of Acmaea 1.6 pl Acmaea scabra eeee=m Acmaea digitalis 1.4 pl 1.2 pl = “Bp i S e “ & ‘ & £ “ ro) y “ 1.0 pl a © 7 a > 4 5 0,8 of a. OY e a ES $ kd fo So! ¢ 2g S 64 on = ra of 08 pl 2 e & e S i Er a ae os “7 5 Be 3 e , ~ o? Ce n e ? ern? 8 Pig Pad Dae 0.6 pl o oe” o” a C0 @# e e) ope of? 4, 4 Rie ” ey a? oe ao eo g o of 0.4. pl g ao ASE ka of e ke A i 5 Mee se e “ee 190 0.2 pl j (90 bo ZF } yy, a0 66 go 120 Time (Minutes) Figure 1 Oxygen consumption of Acmaea digitalis and Acmaea scabra at different degrees of hydration THE VELIGER Vol. 11; Supplement scabra is considerably less than the submerged rate, the submerged rate being 2.25 times greater than the damp rate, and 4.36 times greater than the aerial rate. By con- trast, it is apparent that the respiration rate of A. digi- talis is not so markedly affected by these conditions, the submerged rate being only 1.12 times greater than the damp rate and 1.42 times greater than the aerial rate. It is also seen that although the rate of submerged A. scabra is slightly greater than the submerged A. digitalis rate, the damp and aerial rates of A. digitalis are greater than the damp and aerial rates of A. scabra. Figures 2 and 3 and Table 2 depict respiration rates of submerged Acmaea scabra and A. digitalis as a function of temperature. In these experiments, the same group of animals was used for the different temperatures, and 1.0 pl = bo.8 pl § Ey me} ep 5 5 y o 9 0.6 pl & oo eo) = fe} = 9 fe} § 49 Qa, 0.4 ul SI ° B so g S Oo 0.2 pl 30 60 go 120 Time (Minutes) Figure 2 Oxygen consumption of Acmaea scabra at different temperatures they were equilibrated for 3 hours at the experimental temperature before being placed in the Warburg vessels. In A. scabra (Figure 2), it is seen that although respira- tion rate generally increases with temperature, it is not very pronounced, the initial rate increasing only about 60% in going from 10°C to 25°C. It is also seen that in A. digitalis (Figure 3), the respiratory rate is again rela- tively insensitive to temperature, and between 20°C and 25°C. it actually drops to a rate less than was found at Vol. 11; Supplement Time (Minutes) : Acmaea digitalis THE VELIGER Table 1 Oxygen consumption at different degrees of hydration 30 Acmaea digitalis Aerial 0.241+0.019 Acmaea digitalis Damp 0.297 + 0.056 Submerged 0.280 + 0.033 Acmaea scabra Aerial 0.127 + 0.092 Acmaea scabra Damp 0.216+0.034 Acmaea scabra Submerged 0.380 +0.081 60 90 120 0.404+0.024 0.587+0.042 0.728+0.071 0.538+0.097 0.701+0.155 0.955+0.205 0.544+0.049 0.818+0.145 1.148+0.155 0.142+0.091 0.258+0.088 0.335+0.039 0.334+0.048 0.466+0.089 0.6444 0.121 0.712+0.136 1.132+0.226 1.476+0.314 Page 81 Values are pl O, consumed per mg dry weight and each is an average of approximately 45 animals 10°C. This same drop of rate between 20°C and 25°C was observed whether the first measurements were made at 10°, then at 15°C, etc., or whether the first measure- ments were made at 25°, then at 20°C, etc. DISCUSSION Respiration Under Varying Exposure to Air The data indicate that in both species the maximum rate of respiration occurs under submerged conditions and the minimum rate under aerial conditions. In both species the rate during damp conditions falls between that of submerged and aerial. Acmaea scabra showed a much lower rate of respira- tion than A. digitalis under damp and aerial conditions, a finding which suggests a possible mechanism for con- servation of food reserves while out of water. This also fits in with Wuite’s (1968) observations on glycogen content of high and low forms of A. scabra. All animals of both species were chosen with a similar shell size, but the mean weight (without shell) of Acmaea scabra was later found to be 23 mg as compared to 59 mg for A. digitalis. This could account for the finding that A. scabra had a higher submerged rate than A. digitalis in that it may be a reflection of a higher surface to volume ratio for the former, rather than a higher meta- bolic rate. Respiration Under Varying Conditions of Temperature The increased respiratory rate at higher temperatures was not as great as would be expected on thermorlynamic grounds. The reason for this is not known. The marked drop in submerged rate of Acmaea digitalis between 20° and 25°C is interesting, since A. digitalis has a lethal temperature during prolonged exposure of around 32°C (Harpin, 1968). It is possible, therefore, that the de- creased respiratory rate is related to physiological de- rangements leading to death. As no such decrease was observed in A. scabra, it would appear that it is better suited to withstand elevated temperatures than is A. digitalis. This finding also agrees with the field observa- tions on distribution, and the lower tolerance to high temperatures of A. scabra as compared to A. digitalis (Harpin, 1968). SourHwarp (1958), in a study on intertidal animals, found that during exposure to increas- ing temperature, the animal’s activity was the first and Oxygen consumption at different temperatures Table 2 Time (Minutes) : 30 Acmaca digitalis 10°C 0.215 = 0.060 Acmaea digitalis 15°C 0.217+0.061 Acmaea digitalis 20°C 0.259 +0.088 Acmaea digitalis 25°C 0.161+ 0.050 Acmaea scabra 10°C 0.187 =0.003 Acmaea scabra 15°E 0.243 +0.020 Acmaea scabra 20°C 0.258 +0.025 Acmaea scabra 25°C 0.265 +0.063 Values are »] O, consumed per mg dry weight and each is an average of approximately 45 animals 60 90 120 0.407+0.079 0.583+0.106 0.765+0.136 0.450+0.048 0.675+0.062 0.919+0.100 0.511+0.133 0.71140.145 0.942+0.171 0.352+0.081 0.535+0.121 0.767+0.235 0.371+0.001 0.510+0.009 0.672 +0.012 0.480+0.048 0.632+0.061 0.830+0.059 0.475 +0.046 0.639+0.052 0.829 +0.063 0.593 +0.131 0.743+0.081 1.094+0.097 Page 82 1.0 pl 2 Ay o 2 08 wl eB m9} on & o [ov 0.6 pl O & c Ss a ee 0.4 Z 6) O, 0.2 pl 30 60 Time (Minutes) go 120 Figure 3 Oxygen consumption of Acmaea digitalis at different temperatures most sensitive body function to be affected. This would be reflected in a decreased respiration rate as the critical mortality temperature was approached. Further measure- ments at closer increments from 15° through 30° should be made in order to determine the exact point of rate decrease and the maximum tolerable temperatures for both of these species. In these limited laboratory observations it was impos- sible to take into consideration daily and tidal rhythms in respiratory rates that almost certainly were present (SANDEEN, STEPHENS & Brown, 1958). Other uncon- trolled variables that might have influenced the observed results are body weight and nutritional state of the animals, since the intervals between last feeding and respiratory measurements were unknown. SUMMARY Respiration in two species of limpets, Acmaea digitalis EscHscHOoLtz, 1833 and A. scabra (GouLp, 1848), was studied under varying conditions by means of Warburg THE VELIGER Vol. 11; Supplement manometers. In both species it was found that the maxi- mum respiration rate occurs when the animal is sub- merged, and the least occurs when it is exposed to air. Under damp and aerial conditions A. scabra showed a much lower rate of respiration than did A. digitalis. Under conditions of increasing temperature from 10°C to 25°C, A. scabra increased its respiratory rate approx- imately 60%. Acmaea digitalis’ rate increased 23% from 10°C to 20°C but at 25° its rate decreased to less than that at 10°C. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. The author also wishes to thank the faculty, staff and graduate students of the Hopkins Marine Station for their unending encourage- ment and assistance. LITERATURE CITED Dixon, Matco_m 1951. | Manometric methods as applied to the measurement of cell respiration and other processes. Cambridge Univ. Press; vii - xii+161 pp. Harbin, DANE 1968. A comparative study of lethal temperatures in the lim- pets Acmaea scabra and Acmaca digitalis. The Veliger 11, Supplement: 83 - 88; 4 text figs.; 1 table (15 July 1968) Haven, STONER BLACKMAN 1966. Personal communication. SanpvEEN, M. I., G. C. STEPHENS « F A. Brown, Jr. 1954. Persistent daily and tidal rhythms of oxygen consump- tion in two species of marine snails. Physiol. Zool. 27: 350 - 356 SouTHWaRD, A. J. 1958. | Note on the temperature tolerances of some intertidal animals in relation to environmental temperatures and geo- graphical distribution. Journ. Mar. Biol. Assoc. U. K. 37: 49 - 66 Umprer, W. W, R. H. Burris « J. F StaurFER 1945. | Manometric techniques and related methods for the study of tissue metabolism. Burges Publ., Minneapolis; pp. 1 - 198 WuiteE, TIMOTHY JEFFERY 1968. Metabolic activity and glycogen stores of Acmaea scabra. The Veliger 11, Supplement: 102 - 104; 2 tables (15 July ’68) ZEUTHEN, ERIC 1947. Body size and metabolic rate in the animal kingdom with special reference to the marine microfauna. Compt. rend. Lab. Carlsberg. Sér. chim. 26 (3): 16 - 161 Vol. 11; Supplement THE VELIGER Page 83 A Comparative Study of Lethal Temperatures in the Limpets Acmaea scabra and Acmaea digitalis DANE D. HARDIN Hopkins Marine Station of Stanford University ' Pacific Grove, California 93950 (4 Text figures; 1 Table) INTRODUCTION THE IMPORTANCE OF TEMPERATURE as a limiting factor for the geographical distribution of intertidal animals is generally recognized (Moore, 1958). Many studies have been conducted in an effort to correlate the distribution of some intertidal organisms with either air or sea tem- perature, among them those of Hurcuins (1947), SouTtHwarp (1950), and SourHwarp & Crisp (1954). Secat (1956, 1961, 1962) has indicated that there is a difference between the highest and lowest intertidal mem- bers of Acmaea limatula CarPENTER, 1864 with respect to such body functions as heart rate and oxygen consump- tion. Intraspecific differences in these processes have also been detected in animals from different latitudes. SEGAL. has further demonstrated that these differences are a possible effect of temperature and that acclimation of these processes to different tidal levels takes place. Studies have also been conducted to determine the lethal temperatures of many intertidal animals (Mayer, 1918; GowantacH & Hayes, 1926; BROEKHUYSEN, 1940). However, these investigators have not dealt satis- factorily with intraspecific variation or niche difference. For instance, there may be a difference between lethal temperatures of members of a single species from the extreme boundaries of its vertical intertidal distribution at a single latitude. There may also be differences between different species which are found at the same intertidal levels. This study was undertaken in an attempt to answer three main questions: 1) Is there a difference in lethal temperatures of animals of the same species and body size taken from different tidal levels? 2) Is there a dif- 1 Present address: Cowell College and Division of Natural Sciences, University of California, Santa Cruz, California. ference between lethal temperatures of two species occu- pying the same intertidal levels? 3) How does an organism’s lethal temperature relate to the temperature of its microhabitat? The organisms used in this study, the limpets Acmaea digitalis Escuscuottz 1833, and A. scabra (GouLD, 1846), are ideal for answering these questions, since they occupy the same vertical range in the intertidal zone (+-2 to +10 feet) along the central California coast (Test, IGE) MATERIALS ann METHODS The members of each species were collected from areas visibly dominated by one or the other (at Point Pinos and Mussel Point on the Monterey Peninsula) in order that the results gained by laboratory experiments would be as representative as possible of the normal population. For the purposes of testing intraspecific differences in lethal temperatures with respect to differences in inter- tidal location, animals were taken from the extreme upper (above +7 feet) and lower (below +4.5 feet) limits of the species in that area, and from a region mid- way between. Collected animals were placed in running sea water at approximately 15°C in the laboratory and used within 24 hours. Two types of laboratory experiments were conducted to determine lethal temperatures. The first type was run with the animals submerged. High, mid, and low mem- bers of each species were placed in continuously acrated beakers of sea water in a water bath and allowed to equilibrate to constant temperature. The temperature was then raised at a rate of 1°C per 5 minutes, to allow for complete equilibration of the internal and external temperatures of the animals (SouTHWarD, 1958). Fif- teen high, mid, and low members of each species were Page 84 THE VELIGER Vol. 11; Supplement removed at 1° intervals at the indicated temperatures and replaced in running sea water at 15°C. The animals were allowed to recover for 6 to 12 hours and checked for survival by pricking the mantle fold with a needle. If no response was elicited, the animal was considered dead. The second type of experiment tested the animals’ abilities to survive prolonged exposure to higher than normal temperatures in air. Members of each species from each of the three intertidal levels were placed in desiccators in the water bath. The bottom of each desic- cator was filled with a nearly saturated solution of am- monium chloride and potassium nitrate, resulting in a relative humidity, as determined with a Honeywell rela- tive humidity readout instrument, of 85 to 90% for each trial. Again the temperature in the containers was raised at the previously mentioned rate until the desired temperature was reached. The animals were held at this temperature (+0.3°), with 15 animals from each species and each intertidal level being removed at 5, 10, and 15 hours. Trials were run at 29°, 31.5°, and 34°C. After each time period the removed animals were replaced in running sea water at 15°C, and tested as above after 6 to 12 hours. Field temperatures encountered by the two species were taken on several days between noon and 4:00 pm at Mussel Point. Temperatures were taken with a portable thermistor (model 43TD Yellow Springs Tele-Thermo- meter). Five readings were recorded: 1) air tempera- ture 2 to 3 cm above the animal; 2) rock temperature 100 Acmaea scabra % Survivors next to the limpet; 3) the temperature on the surface of the animal’s shell; 4) the temperature beneath the animal’s foot; and 5) the temperature within the lim- pet’s mantle cavity. The air, rock, and shell temperatures were taken with a banjo-type probe, model 409, which was kept shaded to prevent heating by direct sunlight. The foot and mantle cavity temperatures were taken with a */:s inch flexible probe, model 402. The foot and mantle cavity temperatures were taken in situ. Each animal was lifted from the rock, the probe was placed under the foot or in the mantle cavity, and the animal returned to the spot from which it was taken. The limpet was then held in place with the blade of a knife and the temperature read-out recorded. RESULTS Figure 1 shows the results of the submerged temperature trials. Acmaea scabra consistently survived better than did A. digitalis, and the higher members of each species consistently survived better than did the lower intertidal animals. The results of the prolonged temperature trials are shown in Figures 2 and 3. There is no figure for the 34° trial as no individuals of either species survived this temperature. Although Acmaea scabra continued to sur- vive better than A. digitalis at 29° and 314°, the intra- specific differences seen in the submerged trials are less evident. Acmaea digitalis a9) ° 40 ° Atos 42° 43 °° Temperature in Degrees Centigrade Figure 1 Submerged Exposure to High Temperatures =high intertidal animals; = mid-intertidal animals; = low intertidal animals Vol. 11; Supplement THE VELIGER Page 85 % Survivors Acmaea scabra 5 10 15 Acmaea digitalis Hours of Exposure at 29.0° C Figure 2 Prolonged Exposure to 29° C Air Temperature O =high intertidal animals; @= mid-intertidal animals; X = low intertidal animals To check any possible correlations between size and survival, shell dimensions of all animals used in the sub- merged temperature experiments were determined with vernier calipers readable to 0.1 mm. Twenty individuals from each group were randomly chosen (by random selection of pieces of paper containing data on each in- dividual), and the respective size distributions are shown in Table 1. The data show a marked tendency for the low intertidal group to be smaller than the high intertidal group. 100 Acmaea scabra 5 10 15 The mean temperatures for 10 randomly selected lim- pets are represented in each graph of Figure 4. Acmaea scabra consistently exhibited higher microhabitat tem- peratures than did A. digitalis at similar air temperatures. It can also be seen that in all cases the actual temperature of the limpet (mantle cavity temperature) is higher than the microhabitat temperatures. No significant tem- perature differences of any of the measured variables were found between the upper and lower areas (the tem- peratures were taken from +3 to +7 feet). Hours of Exposure at 31.5° C Figure 3 Prolonged Exposure to 31.5° C Air Temperature © =high intertidal animals; @ = mid-intertidal animals; X = low intertidal animals Page 86 Table 1 Mean Shell Dimensions for 20 Randomly Selected High, Mid, and Low Intertidal Members of Each Species. All measurements are in centimeters, and the standard deviation is indicated in parentheses. Microhabitat and Size of Limpets Length Width Height Acmaea scabra High 1.35 (+0.26) 1.05(+0.21) 0.47 (+0.12) Mid 1.34(+0.17) 1.03(+0.15) 0.45 (+0.10) Low 1.27(+0.19) 0.98(+0.16) 0.36 (+0.05) Acmaea digitalis High 1.46(£0.19) 1.11 (40.15) 0.51 (+0.10) Mid 1.37 (+£0.19) 1.03(+0.18) 0.45 (+0.10) Low 1.20(£0.17) 0.95(+0.15) 0.40 (+0.05) DISCUSSION In all experiments Acmaea scabra survived high temper- atures, in both air and water, better than did A. digitalis. These differences between the two species correlate with the higher mean microhabitat temperatures which were found. Haven (1964) has observed that A. scabra is seen in greatest abundance on surfaces which are more hori- zontal than the areas of highest A. digitalis concentra- tion. This also correlates well with the observed lethal 27 26 25 24 23 22 Temperature in Degrees Centigrade On Clear Day THE VELIGER Vol. 11; Supplement temperatures, since A. scabra would therefore receive more and stronger sunlight than would A. digitalis. The observed intraspecific differences in ability to sur- vive high temperatures possibly result from temperature acclimation. Thus, even though the high and low mem- bers within each species experience much the same tem- peratures, the higher members would experience any high temperatures for longer periods of time than their lower counterparts. An alternative explanation, not in- volving temperature acclimation, is that the greater re- sistance of the higher forms is a consequence of their greater size or age or both. This is suggested by the results in Table 1, showing a continuum in size, with higher animals having larger shells than lower animals, and the work of Frank (1965), which showed that Acmaea digitalis move higher in the intertidal zone with age. However, these results do not establish a causal relationship between size and ability to withstand high temperatures, and further research is planned. The intraspecific differences in ability to survive high temperatures were much less clear in the prolonged tem- perature trials. This was especially evident with Acmaea digitalis, and can possibly be traced to the fact that, even though the A. digitalis population presents a size conti- nuum (with the smallest animals lowest in the intertidal zone and the largest animals highest), this species is probably more mobile than A. scabra. This results in Acmaea digitalis On Overcast Day Figure 4 Microhabitat and Body Temperatures The midline of each bar indicates the mean of 10 measurements, and the portion of each bar above and below the midline indicates the standard deviation. a = air; r = rock; s = shell; f = foot; mc = mantle cavity Vol. 11; Supplement THE VELIGER Page 87 part from the greater percentage of homing behavior found in A. scabra than in A. digitalis (Haven, 1964; JeESSEE, 1968; MILLER, 1968). It can be expected, there- fore, that the effects of acclimation would be more clearly defined in a population of animals which remain in rather fixed positions. This agrees with the greater intraspecific differences in survival at high temperatures seen in A. scabra. SUMMARY Lethal temperatures of the limpets Acmaea scabra and A. digitalis were studied. Acmaea scabra was found to survive high temperatures better than A. digitalis. These results correlate with field studies which showed that A. scabra experiences higher microhabitat and body tem- peratures than does A. digitalis at similar temperatures. It was also found that the internal temperatures of lim- pets are consistently above the external surrounding temperatures. Members of the species coming from the highest inter- tidal ranges of the species were found to survive high temperatures better than members from the lowest inter- tidal ranges of the species. These intraspecific differences may be a result of acclimation to the length of exposure to high environmental temperatures. There are indica- tions that the size or age, or both of these variables, may have an effect on the ability to survive high temperatures. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. My sincerest thanks are given to the faculty and staff of Hopkins Marine Station of Stanford University for allowing me the opportunity to do this study, and especially to Dr. David Epel who advised and directed me in my research. I am also gratefully indebted to Dr. A. Todd Newberry of Cowell College at the University of California, Santa Cruz, without whose encouragement and advice this work would not have been possible. LITERATURE CITED BroEKHuysEN, C. J. 1940. A preliminary investigation of the importance of desic- cation, temperature and salinity as factors controlling the ver- tical distribution of certain marine gastropods in False Bay, South Africa. Trans. Roy. Soc. South Africa 28: 255 - 292 FRANK, PETER WOLFGANG 1965. The biodemography of an intertidal snail population. Ecology 46 (6): 831 - 844; 8 figs.; 6 tables Gowan acu, J.N. & ER. Hayes 1926. Contributions to the study of marine gastropods. I. The physical factors, behavior and intertidal life of Littorina. Contr. Canad. Biol., N.S. 3: 133 - 166 Haven, STONER BLACKMAN 1964. Habitat differences and competition in two intertidal gastropods in central California (abstract). Bull. Ecol. Soc. Amer. 45: 52 Hutcuins, Louis WHITING 1947. The bases for temperature zonation in geographical dis- tribution. Ecol. Monogr. 17: 325 - 335 JessEE, WILLIAM FLoyp 1968. Studies of homing behavior in the limpet Acmaea scabra (Goutp, 1846). The Veliger 11, Supplement: 52-55; 4 tables. (15 July 1968) Mayer, A. G. 1918. | Ecology of the Murray Island coral reef. Tortugas Lab. 9: 1 - 48 Papers MILLer, ALAN CHARLES 1968. Orientation and movement of the limpet Acmaea digi- talis on vertical rock surfaces. The Veliger 11, Supple- ment: 30-44; 18 text figs.; 4 tables (15 July 1968) Moore, Hitary BRooKeE 1958. Marine ecology. John Wiley & Sons; vii+ 493 pp. SEGAL, EARL 1956. Microgeographic variation as thermal acclimation in an intertidal mollusc. figs.; 5 tables 1961. Acclimation in molluscs. 235 - 244; 5 figs.; 4 tables 1962. Initial response of the heart-rate of a gastropod, Acmaea limatula, to abrupt changes in temperature. Na- ture 195 (4842): 674-675; 1 fig.; 3 tables SouTHWarD, A. J. 1950. Occurrence of Chthamalus stellatus in the Isle of Man. Nature 165 (4193): 408 - 409 1958. Note on the temperature tolerances of some intertidal animals in relation to environmental temperatures and geo- graphical distribution. Journ. Mar. Biol. Assoc. U. K. 37: 49 - 66 SouTHwarp, A. J. « D. J. Crisp 1954. The distribution and abundance of certain intertidal animals around the Irish coast. Proc. Roy. Irish Acad. 57 (B): (1) 29 pp. Test, Avery Ransom (GRANT) 1945. Ecology of California Acmaea. 395 - 405 Biol. Bull. 111 (1): 129-152; 8 Amer. Zoologist 1 (2): Ecology 26 (4): Page 88 THE VELIGER Vol. 11; Supplement Studies on the Jaw, Digestive System, and Coelomic Derivatives in Representatives of the Genus Acmaea CATHERINE GENE WALKER Hopkins Marine Station of Stanford University‘ Pacific Grove, California 93950 (13 Text figures; 1 Table) PREVIOUS AND CONTEMPORARY WORKERS have noted some dietary differences between California species of Acmaea (e.g., Test, 1945, 1946; Craic, 1968; Eaton, 1968), and have noted some species differences in the radula and radula strap (e. g., Test, 1945, 1946; FrircuMman, 1960, 1961), which also may be related to diet. No similar attempts have been made to compare the digestive tracts or jaws of California limpets, though these too might be expected to show some variation possibly related to food habits. In the present work, the gross morphology of the gut has been compared in the following species: A. asmi (Mippenporrr, 1849) ; A. digitalis EscHScHOLTZz, 1833; A. limatula CarPENTER, 1864; A. pelta EScHSCHOLTZ, 1833; A.scabra (GouLp, 1846) A.scutum EscHSCHOLTz, 1833. The jaws have been studied in these six species and also in A. insessa (Hinps, 1842) ; A. paradigitalis Frircu- MAN, 1960; and Lottia gigantea SowersBy, 1843. Addi- tional observations have been made on the development of the digestive tract in very young Acmaea, and on the interconnection of the coelomic cavities in the 6 species. METHODS All specimens examined. were collected on Mussel Point, near the Hopkins Marine Station, Pacific Grove, Califor- nia, and at Point Pinos and Point Joe nearby on the Monterey Peninsula, during April and May, 1966. Dis- sections were made on animals killed in 95% alcohol. Since this type of preservation, making tissues more pliable, introduced distortion of certain structures, comparisons were made with fresh material also. Injection of Ward’s latex suspension was used to determine placement of gland ducts and intestinal loops. Starvation of the animals for 3 to 5 days before injection proved helpful in clearing ' Permanent address: 305 Parkridge Lane, Bellevue, Washington. the digestive tract, though injection of the entire tract was never successful. GENERAL DESCRIPTION oF THE DIGESTIVE SYSTEM The gross anatomy of the digestive tract of the Acmaea species studied (Figures 1 and 2) is similar to that of A. cpt ct st Figure 1 Generalized dorsal view of an Acmaea with shell and mantle removed: an— anus cpt — cephalic tentacles ct — ctenidium dg - digestive gland e — eyespot ft — foot gn — gonad ret — rectum rs — radula sac sm — shell muscle st — stomach Vol. 11; Supplement virginea (MULLER) as described by FRETTER « GRAHAM (1962; pp. 149 - 239, 477-509), and Lottia gigantea (see FisHerR, 1904). The mouth opens ventrally on the head. The lips completely encircle the elliptical opening with a wide wrinkled border. The mouth leads into the buccal or oral cavity which contains a series of pouches, grooves, the radula, and the jaw. The radula sac begins just behind the buccal mass and cavity. Passing straight Figure 2 Generalized dorsal view of the digestive tract of an Acmaca with the dorsal body wall, kidneys, gonad, digestive gland, and pericardium removed: dgd - digestive gland duct pro — proventriculus rs — radula sac an — anus es — esophagus j - jaw ret — rectum back over the head muscles, it enters the anterior side of the visceral mass. It passes between the intestinal loops and stomach, curves to the right and forms a loop sur- rounded by the digestive gland, and extends anteriorly again, rising up over the top of the gland where often it can be seen when the shell and dorsal body wall are removed. Reentering the head cavity at the same point at which it left, the sac ends in a fleshy knob or caecum which secretes the radula (FRETTER & GRAHAM, Of. cit., p. 173; RunHaM, 1963). The jaw, a pliable chitinous structure, is positioned anterodorsal to the oral cavity (Figures 2 and 6). It pro- tects the upper lip from the scraping movement of the THE VELIGER Page 89 radula teeth, and prevents food from escaping the buccal cavity (FRETTER & GRAHAM, Op. cit., p. 168). In Acmaea and Lottia gigantea there is a single symmetrical jaw, with 4 wing-like extensions that are opaque, white, and fairly flexible. The smaller anterior wings overlap the posterior wings on the dorsal side. Muscles attached to the bases of the wings run to the body wall and buccal mass. Across the anterior mid-portion of the jaw runs a harder band, which becomes reddish-brown in older animals. Behind the oral cavity lie the pharynx dilation and the esophagus. The latter extends backward to enter the vis- ceral mass slightly to the left of the midline. Curving to the right it passes upward through the digestive gland and ends just under the dorsal surface of the gland in a mid-dorsal position. The greater and lesser folds of the esophagus follow the course described by FisHER (1906, pp. 10-11) in Lottia. The lesser fold begins in the mid- ventral region of the pharynx, twists counter-clockwise as viewed from the rear, and finally reaches a dorsal position just before it ends. The greater folds, which begin dorsally, are twisted so they come to lic ventrally at the posterior end of the esophagus. The twisting of the eso- phagus is the result of torsion in the veliger stage. Lateral pouches or sacculations are found between the greater and lesser folds. They become smaller and finally disap- pear as the esophagus curves to the right in the mid- visceral region (Figure 7). The paired buccal glands lie along or under the mid- esophagus. Their ducts, whicl overlie the head muscles and empty into the oral cavity on dorso-lateral folds, are white and prominent. The numerous posterior salivary or esophageal glands lic on either side of the pharynx dilation and esophagus. They are conspicuous, small, finger-like processes that extend out laterally and, in some species, ventrally around the esophagus. These glands open into tiny pockets, which in turn are divisions of the lateral sacculations of the esophageal wall. Like the esophageal folds, the glands and pharynx dilation appear twisted as a result of torsion. The esophagus passes into the proventriculus or fore- chamber of the stomach, which opens into the stomach by way of a contractable aperture encircled by numerous small folds. The stomach forms a wide loop, encircling the digestive gland. In sexually mature individuals the gonad is sometimes visible laterally beyond the stomach margin. The digestive gland occupies the central region of the visceral hump, slightly overlapping the inner margin of the stomach and almost completely covering the right lateral portions. A single digestive gland duct enters the Page 90 THE VELIGER Vol. 11; Supplement stomach near its junction with the esophagus. The volume and color of the gland vary somewhat with species and with the size of the animal. The hind gut makes a series of 4 loops before it empties into the rectum (Figure 2). Loops 1 and 4 circle clockwise, while loops 2 and 3 circle counter-clockwise. The rectum begins where loop 4 crosses the anterior portion of the stomach. Constriction of the feces into linked pellets occurs in the latter portion of loop 4 and the rectum. The pellets are readily broken up, making them easily washed out of the nuchal cavity when the animal is splashed. Often if the animal has not been ex- posed to water for some time, fecal pellets may fill both sides of the mantle cavity. In the comparative study of the gut in species of Acmaea, differences between species were observed in the length and placement of the radula sac in the body, the jaw, the salivary glands, and the gut loops. LENGTH anp PLACEMENT or tut RADULA SAC The radula sac of all species except Acmaea scutum extends only to the mid-visceral region (Figures 3 and 4). The radula sac of A. scutum passes farther to the left and extends to the posterior visceral region before passing Figure 3 Dorsal view showing the placement of the radula sac in the visceral cavity of Acmaea limatula (shell length 25mm; ratio of radula length to shell length 1.11). Placement of the sac here is similar to that in A. pelta, A. scabra, A. digitalis, and A. asmi. es — esophagus rc — radula caecum rs — radula sac st — stomach Figure 4 Dorsal view showing the placement of the radula sac in the visceral cavity of Acmaea scutum (shell length 25mm; ratio of radula length to shell length 1.9). rc — radula caecum st — stomach es — esophagus rs — radula sac anteriorly again (Figure 4). The radula sac of A. scutum is almost twice the length of the shell, while in the other species it rarely exceeds 1.5 times the shell length (Table 1). THE JAW For species comparison, the jaw was removed, the muscle scraped away from the lower side, and the jaw then ex- amined microscopically. When jaws were mounted in wa- ter on slides, the cover slips were slightly propped up to avoid breaking the jaws or bending them severely. Ap- proximately 10 jaws, taken from animals ranging from small to large, were examined for each species (Figure 5). The jaw remains the same size, relative to the size of the animal, as growth occurs. With age, however, the Figure 5 (on facing page —>) Dorsal view of the jaws of large adult limpets. The jaws are partially flattened out on slides and drawn with a camera lucida. Dark areas represent the most opaque areas. Vol. 11; Supplement THE VELIGER Page 91 Acmaca Acmaea Lottia scutum paradigitalis gigantea Imm Acmaea Acmaea Acmaea limatula digitalis pelta I mim Timm Acmaea Acmaea scabra insessa Acmaea asmi Page 92 Table 1 Sz z Ze, 8 a.§ gS Rati radula length a a atio of ———_—>— ES 5 oo & shell length Species ZReS HG mean Range Acmaea limatula 10 2.0 - 3.5 1.06 0.90 - 1.29 Acmaea insessa 10 1.5 - 2.0 1.07 0.97 - 1.33 Acmaea digitalis 10 1.5-2.5 1.13 0.90 - 1.25 Acmaea paradigitalis 10 1.0- 2.0 1.19 1.00 - 1.40 Acmaea scabra 10 1.0 - 2.5 1.20 1.00 - 1.40 Acmaea asmi 10 0.5-2.5 1.23 1.06 - 1.37 Acmaea pelta 10 1°3'-=\3°5 124 1.06 - 1.50 Lottia gigantea 10 3.0 - 5.6 1.29 1.07 - 1.58 Acmaea scutum 14 1.5 - 4.0 1.90 1.71 - 2.00 anterior band gets harder and darker. The median ridges of the band become more prominent, possibly as a result of wear caused by scraping of the radula. In Figure 5 the jaws of large adult specimens of the species studied are arranged according to an increase in the irregularity of the anterior band. Acmaea asmi, A. limatula, and A. scutum show little or no marking on the band, and the anterior margin is smooth and unridged. Acmaea scabra, A. digitalis, and A. paradigitalis have small ridges in the medial region of the band, causing the margin to be slightly uneven. In A. insessa, A. pelta, and Lottia gigantea the anterior band bears a conspicuous median tooth with smaller ridges running along it. This characteristic is most apparent in A. pelta, which also has a darker anterior band. The undulating anterior bor- der of the anterior band in the jaw of Lottia is rather distinctive. Species differences in shape of the lateral wings of the jaw are less apparent, though variations occur in wing shape and in the relative size of the anterior and posterior wings. ANTERIOR anp POSTERIOR SALIVARY GLANDS The anterior and posterior salivary glands show some variation in placement and size in the species examined. In Acmaea digitalis, A. limatula, and A. scutum the two buccal or anterior salivary glands extend along the eso- phagus, their posterior ends lying on the esophagus to the rear of the posterior salivary glands (Figure 6). The ducts of the buccal glands of A. pelta, A. scabra, and A. asmi are shorter and wind back and forth several times across the head muscles (Figure 7). In these species the distal extremities of the buccal glands terminate near the posterior margin of the buccal mass on either side. In Acmaea digitalis, A. scabra, and A. limatula the very numerous posterior salivary glands extend straight THE VELIGER Vol. 11; Supplement out from the pharynx dilation and anterior esophagus (Figure 6). In A. pelta and A. scutum the pharynx dila- tion is wider than in the above species and the twist of the esophagus more apparent (Figure 7). The glands here are slightly smaller, and curve ventrally around the foregut. In A. asmi the posterior salivary glands are very small, and light green in color, while in the other species they are white. A count of glands was attempted, but an accurate number was difficult to obtain. The left side of the esophagus has about 65 glands, while the right side has a slightly smaller number, the number varying some- what with size of the animal and the species. Figure 6 Dorsal view of the anterior end of Acmaea digitalis, showing the location of the anterior (buccal) and posterior salivary glands. Semi-diagrammatic; based on 15 adult individuals. bg — buccal gland bgd — buccal gland duct es — esophagus gn — gonad j — Jaw pd — pharynx dilation psg — posterior salivary glands rs — radula sac _— st — stomach LOOPING or tHE GUT The length and placement of the gut loops in 6 species of limpets is shown in Figure 8. Some variation in the exact placement of the loops occurs within the individual spe- cies depending on the size of the animal and the degree of maturity of the gonad. However, there is a character- istic placement of the loops in each species. In Acmaea pelta, A. scutum, and A. scabra loop 1 circles farther posteriorly. Loop 2 is considerably smaller in A. digitalis, A. scabra, and A. asmi. In A. asmi the anterior portions of loops 1 and 2 extend farther anteriorly relative to the stomach. In A. digitalis, and A. scabra loop 3 does not Vol. 11; Supplement Figure 7 Dorsal view of the anterior end of Acmaea pelta, showing the location of the anterior (buccal) and posterior salivary glands. Semi-diagrammatic; based on 15 adult individuals. bg — buccal gland bgd - buccal gland duct gf — greater folds j - jaw If — lesser folds pd - pharynx dilation psg — posterior salivary glands rs — radula sac sac — sacculations lie as far to the left as in the other species. Dorsoventral thickness of the visceral mass is difficult to show in the drawings; in A. limatula and A. scutum, both rather flat species, the loops lie beside or just beneath one another. In the other species with taller shells the loops pass back and forth from ventral to dorsal regions as they twist through the digestive gland. The digestive tracts in A. pelta, A. limatula, and A. scutum are somewhat greater in diameter than those of the other species; this is a con- sistent difference, which appears related to diet but shows up whether the gut is full or empty. The stomach of A. Figure 8 (see page 94) Diagrams of the stomach, intestine, and rectum of individuals of six species of Acmaea, based on dissections of 25 to 30 animals of each species. Exact placement of the loops is subject to slight variation within each species; the diagrams show the typical condition in large adults. ret — rectum st — stomach THE VELIGER Page 93 pelta often contains sizable fragments of larger algac, and this species has been shown by Craic (1968) to feed mainly on larger red, brown, and green algae. The gut in A. limatula, which feeds mainly on flat encrusting algae (Eaton, 1968) contains smaller fragments. The gut con- tents of A. scutum observed in the present study re- sembled those found in A. limatula. In contrast A. digitalis, A. scabra, and A. asmi are known to eat microscopic green and blue-green algae and diatoms (CasTENHOLZ, 1961; FritcHMAN, 1961; Haven, 1965). The gut contents of these three species consists of very finely divided material. Looping of the gut is less complex in the smallest limpets. In order to determine at what stages the small limpets develop the adult pattern of intestinal coils, small limpets, 0.5 to 5.0mm in shell length, were examined. Animals 3 to 5 mm long could be identified to species. They were preserved and dissected in the usual way. Results showed that the digestive tract was present in essentially the adult condition. Still smaller individuals (1 to 2mm in shell length) probably representing Ac- maea scabra and A. digitalis were collected in a small splash pool in the high intertidal zone, which contained only large A. scabra and A. digitalis. Minute specimens (0.5 to 2.0 mm in shell length) of these species and prob- ably A. pelta as well, were also collected in the crevices among Mytilus californianus Conran, 1837, Tetraclita squamosa Darwin, 1854, and Pollicipes polymerus Sow- ERBY, 1833. These tiny limpets were killed and preserved in 70% alcohol, dehydrated in an alcohol series (the shells being removed in 95% alcohol), and cleared in cedar wood oil. The foot and mantle were removed by dissection, and the animals mounted on slides in cedar wood oil. The limpets had been feeding, and the digestive tract was dark and clearly distinguishable through the remaining more transparent tissues. The results of these studies are shown in Figures 9 to 11. Limpets 0.5 to 1.5mm in shell length have a rela- tively short stomach and intestine, and intestinal loops 1, 2, and 3 are not evident, though there are twists in the gut. Loop 4 and the rectum are in the normal position. Limpets 1.5 to 2.5 mm in length have loops 1, 2, and 3 but these are not fully developed (Figure 12). When the shell reaches 2.5 to 3.5 mm in length the digestive tract is almost the same as that in the larger animals (Figure 13). Placement of the inner loops 1, 2, and 3 may be slightly out of regular orientation, but this displacement is random. The radula sac is easily seen in its normal position. THE VELIGER Vol. 11; Supplement rt loop 2 Acmaea pelta ret loop 4 loop 4 Acmaea digitalis Acmaea limatula Acmaea scabra Acmaea scutum Acmaea asmi Figure 8 Vol. 11; Supplement rect st loop 4 Figure 9 Generalized diagram of Acmaea digitalis and A. scabra, showing the development of the digestive tract in dorsal view. ret — rectum st — stomach rct loop 4 Figure 10 Generalized diagram of Acmaea digitalis and A. scabra, showing the development of the digestive tract in dorsal view. ret — rectum st — stomach THE VELIGER Page 95 loop 2 loop 4 Figure 11 Generalized diagram of Acmaea digitalis and A. scabra, showing the development of the digestive tract in dorsal view. ret — rectum st — stomach COELOMIC SYSTEM The coelomic system consists of 4 interconnected cavities in the visceral mass: the right and left kidneys, the peri- cardium, and the gonad. The points of interconnection lie anteriorly on the right side of the visceral mass, just behind the muscular wall of the nuchal cavity (Figure 12 The right kidney, easily identified by its dark green to brown color, encircles the whole visceral mass, with its distal end just posterior to the pericardium. Internally its surface is quite irregular. The large right renal-genital pore has thick muscular lips and is just to the right of the anal opening. The pore on Acmaea scabra is much larger than that in the other species, and even exceeds the size of the anus. The left kidney is much smaller and difficult to distinguish from the muscular wall separating the pallial and visceral cavities. It is situated just back of the nuchal cavity, between the midline and the anus, and overlies part of the rectum (Figure 12). Acmaea digitalis and A. scabra both have relatively large left kid- neys. The left renal pore is a narrow slit opening on the anterior surface of the middle of the kidney (Figure 13). Only in A. digitalis are there very small bulbous lips on each side of the slit. Page 96 Figure 12 Generalized dorsal view of the coelomic system in Acmaea, with the dorsal body wall, digestive system, and posterior wall of the nuchal cavity removed. gn — gonad Ik — left kidney rk — right kidney rrp — right renal pore an — anus p — pericardium The pericardium occupies the space above the gonad in the left dorsal anterior region of the visceral mass. From it the reno-pericardial canal (Figure 13) crosses the body from left to right and, as in Lottia gigantea (see FisHer, 1904), divides just under the left kidney, one canal going to the left kidney and one under the rectum to the right kidney. The gonad is situated in the hollow above the foot beneath the digestive system. During the breeding season when it is greatly enlarged it extends up around the gut. The duct is merely an extension of the thin epithelium that surrounds the gonad (Figure 13). It arises on the antero-dorsal wall of the gonad in the midline and ex- tends to the right to open into the right kidney. Gametes pass from the gonad to the right kidney, and leave the kidney through the right renal-genital pore. SUMMARY 1. Placement and extent of the radular sac, the form of the jaw, the arrangement of the salivary glands, the looping of the gut, and the interconnections of coe- lomic derivatives are described and illustrated for Ac- maea: A. asmi; A. digitalis, A. limatula, A. pelta, A. scabra, A. scutum, A. insessa, A. paradigitalis. Some notes on Lottia gigantea are included. THE VELIGER Vol. 11; Supplement 2. The radula of Acmaea scutum is almost twice the length of its shell, while in the other species the radula ranges from 1 to 1.5 times the length of the shell. 3. Limpet jaws vary primarily in the shape and character of the hard anterior band. The jaws of Acmaea pelta, A. insessa, and Lottia gigantea bear an anterior median tooth. rk Figure 13 Enlarged view of the right upper corner of Figure 12, showing the interconnection of the coelomic cavities. Kidneys and anus open into the nuchal cavity. ct — ctenidium gn — gonad gnd — gonad duct Irp — left renal pore pre — pericardial renal canal rk — right kidney rrp — right renal pore an — anus 4. The limpets studied can be separated into two groups on the basis of the salivary glands. Acmaea digitalis, A. limatula and A. scutum have buccal glands that extend down the esophagus posterior to the esophageal glands, while in the other species they terminate near the pos- terior margin of the buccal mass. In A. pelta and A. scutum the posterior salivary glands are slightly smaller, and curve ventrally around the foregut, while in the other species the glands extend straight out from the pharynx and esophagus. 5. The pattern of gut loops shows minor but consistent differences between Acmaea species. Development of the adult pattern of loops is essentially completed be- fore animals attain a shell length of 4mm. The gut tends to be thicker and heavier in species feeding on Vol. 11; Supplement larger erect or encrusting algae (Acmaea pelta, A. limatula, A. scutum) than in species feeding on films of microscopic algae (A. digitalis, A. scabra, A. asmt). 6. The coelomic cavities and ducts are described and dia- grammed. Both right and left kidneys of Acmaea dig- italis and A. scabra are relatively larger than those of other Acmaea species. An excellent paper by Ricut (1966), published after the present study was completed, describes the internal anatomy of the Brazilian species of Acmaea and pro- vides illustrations permitting comparisons to be made between Brazilian and Californian species. Ricut also finds that the shape of the jaw and the looping of the gut provide means of distinguishing Acmaea species. ACKNOWLEDGMENTS My sincere thanks go to Dr. Donald Abbott for his encouragement and guidance throughout this project. Thanks also are extended to the staff of Hopkins Marine Station. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. LITERATURE CITED CasTENHOLZz, RicHaRD WILLIAM 1961. The effect of grazing on marine littoral diatom popu- lations. Ecology 42 (4): 783-794; 6 figs. Craic, PETER CHRISTIAN 1968. The activity pattern and food habits of the limpet Ac- maea pelta. The Veliger 11, Supplement: 13 - 19; plt. 1; 5 text figs.; 1 table (15 July 1968) Eaton, CHarLES McKENDREE 1968. The activity and food of the file limpet, Acmaea lima- tula. The Veliger 11, Supplement: 5-12; 7 text figs.; 1 table (15 July1968) THE VELIGER Page 97 FisHeR, WALTER KENRICK 1904. The anatomy of Lottia gigantea Gray. (Anat. Ontog.) 20 (1): 1-66; 14 figs.; 4 plts. FRETTER, VERA & ALASTAIR GRAHAM 1962. _ British prosobranch molluscs, their functional anatomy and ecology. London, Ray Soc. xvi+755 pp.; 316 figs. Zool. Jahrb. FrITCHMAN, Harry Kier, II 1960. Acmaea paradigitalis sp. nov. (Acmaeidae, Gastro- poda). The Veliger 2 (3): 53-57; plts. 9-12. (1 January 1960) 1961a. A study of the reproductive cycle in the California Acmaeidae (Gastropoda). Part I. to 63; 1 table; pit. 10 (1 January 1961) 1961b. A study of the reproductive cycle in the California Acmaeidae (Gastropoda). Part 2. The Veliger 3 (4): 95 to 101; 1 table; plts. 15 - 17 (1 April 1961) 1961c. A study of the reproductive cycle in the California Acmaeidae (Gastropoda). Part 3. The Veliger 4 (1): 41 to 47; plts. 9-14 (1 July 1961) Haven, STONER BLACKMAN 1965. Field experiments on competition between two ecolog- ically similar intertidal gastropods (abstract). Bull. Ecol. Soc. Amer. 46: 160 RicuHt, G. 1966. On the Brazilian species in the Acmaea subrugosa complex (Gastropoda: Prosobranchia: Patellacea) . Mala- cologia 4 (2): 269-295; 27 figs. RunuaM, N. W. 1963. = 4513 X = 522.15 X = 214.9 S.D. = 177.25 S.D. = 111.9 S.E. = 8.8625 S.E. =5.233 __ S.E. diff = 10.29 X diff/S. E. diff = 29.8 P = < 0.001 in the limpets from higher portions of the intertidal zone also suggests an increased storage potential in these animals. The glycogen and respiratory data also permit calcu- lation of the minimum time these higher populations could survive between feedings, if glycogen were the sole respiratory substrate. Assuming 6 moles of oxygen con- sumed per mole of glucose utilized, this could permit submerged maintenance for 60 hours. The lower popula- tion of Acmaea scabra, with its higher respiratory activity and lower glycogen store, could maintain itself for 17 hours. Since BALDwin (1966) has found that the aerial respiration rate in A. scabra is several times less than the submerged rate, the above values are certainly minimal for aerially breathing limpets. THE VELIGER Page 103 Table 2 Metabolic Activity of High and Low Populations Oxygen Consumption as pl O,/1 gm body weight/3 hrs. Acmaea scabra +6ft Acmaea scabra +2ft 440 115 132 230 222 194 190 292 165 242 77 230 129 200 160 156 120 148 194 238 167 157 176 378 220 315 144 124 143 202 150 200 176 330 315 240 318 296 164 250 180 450 315 278 234 206 336 175 172 222 332 550 254 317 130 322 150 440 139 570 108 435 92 480 416 = = 6244 = = 9398 X = 195.12 X = 284.78 S.D. = 81.04 S.D. = 120.11 S.E. = 2.531 S.E. = 3.67 S. E. diff = 4.45 X diff/S. E. diff = 20.01 P= < OM Lower metabolic activity and higher glycogen content are not the only characteristics of high populations that suggest adaptations for survival in the higher portions of the intertidal. Hewatr (1940) has observed that the shells of Acmaea scabra from higher intertidal areas are higher and thicker, while the shells of this species living Page 104 THE VELIGER Vol. 11; Supplement in low, moist areas are thinner and flattened. In addition, the study of JessEE (1968) indicates that A. scabra of the higher intertidal shows a greater tendency to home than A. scabra found in the low intertidal. Animals with a strong homing tendency present a greater irregularity of shell shape that is complementary to the home site. The resulting better fit of animal to substratum may decrease susceptibility to desiccation. A third adaptation of the higher forms, shown by the study of Harpin (1968), demonstrates that higher populations of A. scab- ra can withstand greater temperatures than populations lower in the intertidal. SUMMARY 1. Metabolic activity and glycogen content of popula- tions of Acmaea scabra from the higher and lower por- tions of the intertidal area were determined. 2. Animals from +6 feet showed lower metabolic acti- vity and larger glycogen stores than animals from +2 feet. 3. The possible survival value of this difference is dis- cussed. ACKNOWLEDGMENTS This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. I am also happy to ack- nowledge the guidance of Dr. John Phillips. LITERATURE CITED BALDWIN, SIMEON 1968. | Manometric measurements of respiratory activity in Acmaea digitalis and Acmaea scabra. The Veliger 11, Supplement: 79 - 82; 3 text figs.; 2 tables (15 July 1968) Dusors, M. 1956. Colorimetric method for determination of sugar and related substances. Anal. Chem. 28: 350 Harpin, DANE 1968. A comparative study of lethal temperatures in the lim- pets Acmaea scabra and Acmaea digitalis. The Veliger 11, Supplement: 83 - 88; 4 text figs.; 1 table (15 July 1968) Hewatt, Wiis GILtitanp 1935. Ecological succession in the Mytilus californianus hab- itat as observed in Monterey Bay, California. Ecology 16: 244 - 251 1940. | Observations on the homing limpet Acmaea scabra GouLp. Amer. Midland Naturalist 24: 205 - 208 Jessee, WILLIAM FLoyp 1968. Studies of homing behavior in the limpet Acmaea scabra (Gouxp, 1846). The Veliger 11, Supplement: 52-55; 4 tables. (15 July 1968) SmirH, RatpH INGRAM & JOHN HENRY WELSH 1949. Laboratory exercises in invertebrate physiology. Burgess Co., Minneapolis; 179 pp.; 41 figs. Vol. 11; Supplement THE VELIGER Page 105 Laminarinase and Hexokinase Activity during Embryonic Development of Acmaea scutum BY ANDREW V. MUCHMORE Hopkins Marine Station of Stanford University ' Pacific Grove, California 93950 (2 Text figures; 1 Table) THIS REPORT DESCRIBES some enzyme activities related to carbohydrate metabolism during embryonic develop- ment of the gastropod Acmaea scutum ESCHSCHOLTZ, 1833. Data are presented on activities of enzymes re- leasing free glucose from various polysaccharides, on hexokinase? (an enzyme common to both the pentose shunt and glycolysis), glucose-6-phosphate dehydrogenase (an enzyme of the pentose shunt), and isocitrate dehydro- genase (an enzyme of the citric acid cycle). MATERIAL anno METHODS Obtaining and Culturing Gametes: Adult Acmaea scutum were collected from an area on Cypress Point in Pebble Beach, California. The animals were removed from their shell, and the dorsal body wall ruptured near the posterior end of the animal, with care taken not to puncture the nearby digestive gland. Parts of ovaries from 5 females were removed with forceps, agitated in alkaline sea water to remove the mature eggs, and then treated as described below to remove the chorion. Sperm were obtained by agitating the testes in 25 ml of normal filtered sea water, and filtering the suspension through a double layer of cheesecloth. Removal of the chorion by the alkaline sea water treatment of WoLFsoHN (1907) was necessary to obtain fertilization and normal development. For Acmaea scu- tum, best results were obtained by 90 minutes incubation in alkaline sea water (2.5 ml of 0.1N NaOH in 100 ml of filtered sea water, final pH 9.2). The alkaline sea ' Permanent address: 720 Napoli Drive, Pacific Palisades, Cali- fornia. 2 The following abbreviations will be used: HK — hexokinase; G6PDh - Glucose-6-phosphate dehydrogenase; TEA — tri-ethan- olamine buffer; TPN -triphosphopyridine nucleotide (NADP), and G-6-P — glucose-6-phosphate. water was then removed, the eggs re-suspended in fil- tered sea water, and 1 ml of sperm suspension added. After 2 hours the eggs were agitated, allowed to settle, and the supernatant water containing most of the sperm removed and filtered sea water added. After about 36 hours, the embryos were gently centrifuged, and re-sus- pended in freshly filtered sea water. Above operations were all performed at 15° to 17° C. Enzyme Preparation: Assays of one large group of eggs were made before fertilization, 24 hours after fertilization at the trochophore stage, and 48 hours after fertilization at the veliger stage. In initial experiments, enzyme insta- bility was noted, which suggested the possibility of de- gradative enzymes (such as proteases). Therefore, in some experiments 6 to 9 mg ovomucoid prepared by the method of Frepricg & DeutscH (1949) were added to one half of the samples as a specific trypsin inhibitor. The eggs, trochophores, or veligers were divided into two parts and centrifuged. One sample was suspended in 2.5 ml buffer (0.05M TEA, pH 7.6), the other in 2 ml buffer-0.5 ml of ovomucoid solution (also suspended in buffer), and both samples then disrupted in a Dounce homogenizer at 0°C. The volumes of embryos used ranged from 0.05 to 0.1 ml packed cells per 2.5 ml. All prepara- tions were assayed for enzyme activity at 26° C. Fluorometric Assays: Because of the small amounts of embryos obtainable, sensitive fluorometric techniques, based upon the absorption of TPNH at 366 my and its fluorescence at wavelengths greater than 420 mp were utilized to measure enzyme activity. Using incubation mixtures described below, glucose-6-phosphate dehydro- genase (G6PDh) and isocitrate dehydrogenase activity were determined by direct measurement of TPNH for- mation; hexokinase (HK) activity was measured by coupling its product, glucose-6-phosphate, with excess G6PDh, to yicld TPNH; and carbohydrases releasing free Page 106 THE VELIGER Vol. 11; Supplement Set ein da a ee oo en Desde ee eee er glucose measured by coupling glucose production to HK and G6PDh in the presence of excess ATP. (1) Glucose-6-phosphate dehydrogenase: The 2 ml reaction mix contained, per ml, 43 pumoles TEA buffer (pH 7.6), 0.25 moles TPN, 5 umoles MgCl, and 4 pmoles glucose-6-phosphate (G-6-P). (2) Hexokinase: Same as (1), except 10 pmoles/ml glucose were substituted for G-6-P, plus 2 moles/ml ATP, and 0.5 ugm/ml G6PDh. (3) Carbohydrase: Same as (2), plus 2.5 pgm/ml HK, and in place of glucose were substituted either maltose (1 umole/ml), lactose (1 umole/ml), laminarin or starch (0.05%, final concentration). Negligible glu- cose was present in the starch, laminarin, or cell extract. Appreciable amounts, however, were found in the maltose and lactose. To eliminate this contribution, the reaction mix (minus cell extract) was incubated with sufficient substrate to remove all free glucose, and the reaction then initiated with cell extract. (4) Isocitrate dehydrogenase: Same as (1), except 2 pmoles/ml isocitric acid was substituted for the G-6-P. Other Assays: Unfertilized and veliger stages were also assayed for carbohydrases by measuring reducing sugars released, using the method of Somocyr as modified by Netson (1944). As a further check, amylase activity was also measured qualitatively by adding iodine to samples of extract and starch at 5-minute intervals, and using decolorization of the blue iodine-starch complex as an indicator of amylase activity. The fluorometric technique was capable of detecting the production of as little as 6x 10'' moles glucose per minute. The reducing sugar test of Somocy1 as modified by Netson could detect about 3 x 10° moles of reducing sugar. Protein was measured spectrophotometrically by both the method of WarsBurc & CurisTIAN (1941) and Lowry et al. (1951). Protein content was assumed to reflect enzyme content, since the observed rates were closely related to enzyme concentration (a twofold in- crease in extract approximating a two-fold increase in rate). The activities are therefore expressed as moles TPNH formed per minute per milligram protein. Commercial sources of enzymes and substrates: Hexo- kinase and glucose-6-phosphate dehydrogenase (Boehrin- ger und Sohne), isocitrate, glucose, TPN, and ATP (Sigma Chemical Company), lactose (Nutritional Bio- chemical Corporation), and maltose (Difco Labora- tories). The laminarin and starch were prepared by Dr. John Phillips of Hopkins Marine Station. RESULTS anp DISCUSSION Enzyme activities determined fluorometrically are listed in Table 1. The lack of amylase activity in unfertilized eggs and in veligers was also supported qualitatively by the lack of decolorization of the iodine-starch complex, and quantitatively by the reducing sugar test. This latter test indicated that, if amylase were present, it produces less than 30 x 10° moles reducing sugar per hour per mg protein. Enzyme extracts assayed for carbohydrase activity were prepared from embryos obtained from different females. For comparison of enzyme activity during development, eggs of 5 females were pooled, and enzyme activity was determined in the unfertilized egg, trochophore, and veliger stages. The rates of enzyme activity at these various stages are shown in Table 1 and Figure 1. Table 1 shows that by the veliger stage, regardless of previous activity, the embryos homogenized in ovomucoid show a greater activity than the extracts prepared without this Activity Egg Trochophore Veliger Stage Figure 1 Activity of Hexokinase During Development. Activity expressed as 10-'' moles TPNH/minute/milligram protein. trypsin-inhibiting compound. This could indicate the development of a trypsin-like protease. This conclusion is also supported by Figure 2, which shows that storage of HK at 0° C, without ovomucoid, results in considerable loss of activity, and that this loss does not occur with Vol. 11; Supplement THE VELIGER Page 107 Table 1 Enzyme Activities at Various Embryonic Stages Stage (3) § o 3, Enzyme 88.5 Hexokinase with mucoid She without mucoid 3.5 Laminarinase with mucoid 10+ without mucoid Maltase with mucoid 0 without mucoid 0 Galactosidase with mucoid 0 without mucoid 0 Amylase with mucoid 0 without mucoid 0 Isocitrate dehydrogenase with mucoid 63 without mucoid 33 G-6-P dehyrogenase with mucoid 42 without mucoid 52 3 f 3f Ba Ba Be Say iw oo » N ° 1 N Oo © w Hse Ow & Ee ea: Be 3 33 58: a $8 3.5 3.8 3.8 11 2.2 0.8 3.7 5 to 30 0 0 0 0 0 0 0 0 0 0 0 0 43 35 56 31 16 43 43 48 36 28 Enzyme rates expressed as 10-'' moles TPNH/minute/mg protein The table also shows enzyme activity after “aging” of extract at 0°C for 24 hours in the presence and absence of ovomucoid ovomucoid present. Similar stabilization of activity was also observed with laminarinase activity. Isocitrate de- hydrogenase activity in the unfertilized eggs (Table 1) shows anomalous behavior in ovomucoid which is unlike the activity of HK and ZF The reasons for this are unclear. Perhaps the most interesting discovery is that the eggs and early embryonic stages of Acmaea scutum possess an enzyme(s) capable of releasing glucose from laminarin. The rate of activity of this enzyme, however, was non- linear under the present assay conditions, and therefore no comparison of activity was attempted. The role of the embryonic laminarinase in the egg and early trochophore stages is difficult to visualize, since neither of these is a feeding stage. The veliger, however, does feed, and could certainly utilize a laminarinase-type enzyme for digestion of the large amount of algal lami- narin found near the surf zone in small fragments of brown algae. If the veligers are degrading laminarin to free glucose in their natural environment the sharp in- crease in HK activity (Figure 1), also found at this stage, could result from free glucose made available by laminar- inase. ZF activity, however, appears to remain relatively constant during development. Another possibility, which is presently being investi- gated, is that the observed laminarinase activity actually reflects B-1,3 glucanase activity involved in the break- down of cellular glyco-proteins. Page 108 THE VELIGER Vol. 11; Supplement Activity oO I 2 3 Time (Hours) Figure 2 Inactivation of Trochophore-Stage Hexokinase During Storage at 0° C. Activity expressed as 10-'' moles TPNH/minute/milligram protein. The top line is with ovomucoid, the bottom without ovomucoid. SUMMARY Activity of embryonic carbohydrases, hexokinase, glu- cose-6-phosphate dehydrogenase, and isocitrate dehydro- genase were analyzed fluorometrically in the unfertilized eggs, trochophores, and veligers of the limpet Acmaea scutum. Glucose-6-phosphate dehydrogenase and isocit- rate dehydrogenase activity was constant at all stages. Hexokinase activity increased almost threefold between the trochophore and veliger stage. No maltase, galacto- sidase, or amylase activity was detectable at any stage. A laminarinase type enzyme, capable of degrading algal laminarin to free glucose was found in all embryonic stages. The role of laminarinase in unfertilized eggs is puzzling since the substrate for this enzyme is not available until feeding begins at the veliger stage. ACKNOWLEDGMENTS The author is deeply indebted for help received from Dr. David Epel and Miss Sharon Proctor, both of Hopkins Marine Station. This work was made possible by Grant GY806 from the Undergraduate Research Participation Program of the National Science Foundation. LITERATURE CITED Frepricg, EUGENE « Haroitp Francis DrutTscH 1949. Studies on ovomucoid Journ. Biol. Chem. 181: 499 - 510 Lowry, Otiver Howe, N. J. Rosesroucu, A. L. Farr « R. J. RANDALL 1951. Protein measurement with the folin phenol reagent. Journ. Biol. Chem. 193: 265 - 275 Netson, Norton 1944. A photometric adaptation of the Somogyi method for the determination of glucose. Journ. Biol. Chem. 153: 375 - 380 Warsurc, O. & W. CHRISTIAN 1941. Protein estimation by ultraviolet absorption. Bio- chem. Zeitschr. 310: 384 WOoLFSOHN, JULIAN Mast 1907. The causation of maturation in the eggs of limpets by Biol. Bull. 3: 345 - 350 chemical means. Vol. 11; Supplement THE VELIGER Page 109 Nitrogen Excretory Products in the Limpet Acmaea WILLIAM H. BARIBAULT Hopkins Marine Station of Stanford University * Pacific Grove, California 93950 (1 Text figure; 2 Tables) INTRODUCTION EXAMINATION OF EXCRETORY PRODUCTS in the mollusks indicates that more terrestrial forms tend to be uricotelic and aquatic forms tend to be ammonotelic. This is espe- cially evident in NEEDHAM’s (1935) classic study of the littorinid snails where an ascending order of uric acid production was found to be directly correlated with in- creasing height in the intertidal zone. In this regard, members of the genus Acmaea also present an interesting continuum. On the Monterey Peninsula, populations of the five common species of this genus are found varying from +0.0 feet to +5.0 feet. To see if any correlations could be found between intertidal zonation and nitrogen excretory patterns, a comparative analysis of excretory products in this genus was made. METHODS anp MATERIALS The five species of Acmaea used in this investigation were A. digitalis EscHSCHOLTZ, 1833; A. limatula Car- PENTER, 1864; A. scutum EscHscHoLtz, 1833; A. pelta EscHscHoLtz, 1833; and A. scabra (Goutp, 1846). All species were collected from the intertidal area at periods of low tide on Pescadero Point in Monterey County, California. The same collection procedure was followed for all animals studied. Each animal was removed from the rock substratum, with care to avoid loss of fluid from the mantle cavity, and placed into a capped glass vial con- taining 5 ml of artificial sea water (Harvey, 1954). The animals were kept in the vials at sea water temperature for 2 to 3 hours. The artificial sea water (ASW hereafter) was decanted into a graduated centrifuge tube, the lim- pet removed, and each vial rinsed with 2 ml of ASW. Each animal was then gently squeezed, anterior end ' Permanent address: 232 Jesse Avenue, Glendale, California. down over the centrifuge tube, to empty the mantle ca- vity of any remaining fluid. The pooled fluid was brought to 10 ml with ASW, centrifuged to remove any particu- late matter, and the supernatant decanted and frozen until used for analyses. Total nitrogen, ammonia nitrogen, and urea nitrogen were assayed by the microdiffusion method of TERNBERG (1964). The total nitrogen was determined on a | ml sample after wet ashing with 1 ml of sulfuric acid saturated with copper sulfate. Ashed samples were di- luted with 5 ml of distilled water, and 4 ml of 10 M sodium hydroxide were substituted for the sodium car- bonate solution used in TERNBERG’s original method. Urea was determined as ammonia after a 30 minute incubation with 0.1 ml of a 50 mg per ml solution of urease (obtained from Matheson, Colman, and Bell) in distilled water. Uric acid was determined colorimetrically by the method of SosprinHo-Simoes (1965). Prior re- moval of proteins was found to be unnecessary. Gaseous excretion of ammonia was determined by placing the intact animal into a dry diffusion apparatus for 24 hours. Gaseous ammonia was collected by absorp- tion on alkaline filter paper, and analyzed for ammonia by TERNBERG’s procedure. The microdiffusion assay procedure was calibrated with ammonium sulfate solutions of known concentrations and found to be extremely reproducible (Figure 1) with trip- licate and duplicate determinations on the same sample revealing small standard deviations (e. g., Acmaea scutum NH: -N, 7.0 + 0.25 ug). RESULTS Ammonia, urea, and uric acid were found to be excre- tion products in all species of Acmaea investigated. Some species showed wider individual variations in amounts of certain excretory products than other species. For example, the values for ammonia nitrogen in A. digitalis Optical Density I 2 3 4 5 6 yg NH.N Figure 1 Calibration of diffusion apparatus with ammonium sulfate solutions using optical density as a measure of pg concentration ranged from 0.4 to 25 yg N, whereas for A. scutum the range was much less, varying from 6 to 10 wg N (Table 1). In terms of individual variation the greatest varia- bility in ammonia nitrogen was found in A. digitalis, fol- lowed by A. scabra, A. pelta, A. limatula, and A. scutum. THE VELIGER Vol. 11; Supplement Variation in uric acid was greatest in A. pelta, followed by A. scabra, A. limatula, A. scutum, and A. digitalis. The percentage distribution of the various nitrogenous compounds, based upon mean values, is shown in Table 2. The comparison between the five species indicates that relatively more ammonia is excreted by Acmaea digitalis and A. scabra, that less uric acid is excreted by A. digita- lis, and that A. scabra and A. scutum excrete less urea. It can also be seen that greater than 80% of the total non- protein nitrogen can be accounted for by these methods. Gaseous ammonia was found to be excreted in the five species of Acmaea examined with the dry diffusion tech- nique described above. Those forms found higher in the intertidal zone, A. scabra and A. digitalis, produced more of the gaseous product than the lower intertidal forms, A. scutum, A. limatula, and A. pelta. The mean value after 24 hours was 2.9 ug NHs-N for the higher species and 2.0 ug NH:-N for the lower ones. DISCUSSION The microdiffusion and colorimetric techniques, coupled with the collection procedure, have provided a means of analysis of individual specimens of the genus Acmaea. The extreme standard deviations obtained (e.g., in A. digitalis, NH:-N 8.9 + 8.3ug) indicate very large indi- vidual differences. The deviations did not result from the assay procedure, which, as previously indicated, was highly reproducible. These maximal and minimal compo- nents of variability, only seen in comparing individuals, would be masked by methods that measure pooled ma- terial. In particular, these results demonstrate the value of individual analysis. Although average values can be Table 1 Relative ug Nitrogen Excretory Products with Standard Deviations for Eight Animals for Each Species. Rest nitrogen is the difference between the total nitrogen and the sum of the three products. Nitrogenous Excretory Products in Acmaea Acmaea Acmaea Acmaea Acmaea Acmaea scabra digitalis limatula scutum pelta NH, N 10.6 +6.3 8.9+8.3 8.5+3.9 75+1.8 9.25+4.6 Urea N BND tote 5.9+4.2 Usa a} 7 46+19 11.1 44.2 Uric Acid N 14.6 +4.9 Ueyaeilo7y/ Wee/sechs} ieyjaeee) 2 il7/f3} Se(h7/ Rest N 5092535 5.0+2.9 2.8+2.7 5.7+4.2 5.75+4.7 Total N 33.0 +7.9 278+7.1 33.348.2 32.0+7.3 43.9 +9.1 Vol. 11; Supplement THE VELIGER Page 111 Table 2 Percentage Excretory Products Calculated From Mean Values of Table 1 Percentage Distribution of Nitrogen Products Acmaea Acmaea scabra digitalis NH, N 32 32 Urea N 11.4 21 Uric Acid N 44.5 28.5 Rest N V2 18.5 Total N 100 100 obtained, as in Table 2, the interspecific differences ob- served might therefore be misleading. Several hypotheses can be advanced to explain the large individual variation in the relative amounts of ex- cretory nitrogen. One is that enzyme content varies within the population, resulting in different distributions of excretory nitrogen products when comparing individuals. This is especially suggested by WiLi1AM’s (1966) work on biochemical variation within individuals of the same species. Another explanation is that individual variations in the diet would result in the observed distribution of nitrogen products. A third possibility is that the limpet changes its major products of nitrogen excretion as a result of exposure to air during the tidal cycle. This is suggested by NEEDHAM’s (1935) proposal that more ter- restrial animals tend to excrete uric acid, whereas more aquatic forms tend to excrete ammonia. This hypothesis is also suggested by the larger variations in the higher forms, which would have been exposed to longer, and more variable, periods of dryness. If correct, this could suggest a unique adaptation of the higher forms to secrete more uric acid when dry than when wet. Such a tran- sition would be of adaptive value, since ammonia and urea at high concentrations are toxic. Although urea was found to be excreted by all species examined, the metabolic mechanism of its formation is not clear. CAMPBELL (1966) found no arginase in the digestive gland of Acmaea spp., suggesting the absence of the ornithine cycle in this species. However, CAMPBELL only examined the digestive gland, leaving open the pos- sibility that the enzyme might be present in other tissues. While his results possibly eliminate the ornithine cycle, other pathways based upon purine degradation could be alternative mechanisms for urea production. For example, in fishes, products of purine breakdown from uric acid give rise to urea via the enzyme allantoicase (LAsKowskKI, 1951). This pathway is also suggested by preliminary Acmaea Acmaea Acmaea limatula scutum pelta 25.5 23.5 21 22 14.5 25.5 44.5 42.5 40.5 8 19.5 13 100 100 100 experiments indicating formation of urea by minced di- gestive gland in an ASW-uric acid solution. The finding that all three products are present, and the reported absence of arginase, suggests the possibility that the source of ammonia nitrogen is by protein catabolism, whereas the urea and uric acid products might result from purine catabolism. The nature of the “rest” nitrogen is not clear. Quali- tative tests with 0.25% ninhydrin solution show the pre- sence of peptides and amino acids in the samples from the limpets. This may account for some of the 13 - 18% unidentified nitrogen. SUMMARY Nitrogen excretory products were examined in the genus Acmaea by amicrodiffusion technique for urea and ammo- nia and a colorimetric technique for uric acid. Five species were examined, varying in vertical distribution. Similar concentrations of ammonia, urea, and uric acid were found in all five species. A large amount of intraspecific variation in the distribution of these compounds was noted, especially in the high intertidal forms A. scabra and A. digitalis. These variations might reflect individual differences in enzyme activity, individual differences in diet, or adaptive responses to the environment. ACKNOWLEDGMENTS I am indebted to Dr. John Phillips for his advice and encouragement during this investigation. Appreciation is extended to Dr. David Epel for his helpful recommen- dations in the preparation of the manuscript. The work was made possible by Grant GY806 from the Under- graduate Research Participation Program of the National Science Foundation. Page 112 THE VELIGER LITERATURE CITED CaMPBELL, JAMES WaYNE 1966. Distribution of arginase activity in molluscs. Comp. Biochem. and Physiol. 17: 259 - 270 Harvey, H. W. 1954. Chemistry and fertility of sea waters. Cambridge Univ. Press LaskowskI, M. 1951. The enzymes. Chapt. 27, ed. SuMNER & MyrsBAck, Acad. Press, New York, N. Y. NEEDHAM, JAMES GEORGE 1935. | Problems of nitrogen catabolism in invertebrates. Cor- relations between uricotelic metabolism and habitat in the phylum Mollusca. Biochem. Journ. 29: 238 - 251 SoBRINHO-SIMOEs, M. 1965. A sensitive method for the measurement of serum uric acid using hydroxylamine. Journ. Labor. Clin. Med. 65: 665 - 668 TERNBERG, JESSIE L. 1964. Colorimetric determination of blood ammonia. Jour. Labor. Clin. Med. 56: 766 - 776 WiiuaMs, RocEr JOHN 1966. Individuality in nutrition: Effects of vitamin A-defi- cient and other deficient diets on experimental animals. Proc. Nat. Acad. Sci. 55: 126 - 134 Vol. 11; Supplement THE VELIGER is open to original papers pertaining to any problem concerned with mollusks. This is meant to make facilities available for publication of original articles from a wide field of endeavor. Papers dealing with anatomical, cytological, distri- butional, ecological, histological, morphological, physiological, taxonomic, etc., aspects of marine, freshwater or terrestrial mollusks from any region, will be considered. Even topics only indirectly concerned with mollusks may be acceptable. It is the editorial policy to preserve the individualistic writing style of the author; therefore any editorial changes in a manuscript will be submitted to the author for his approval, before going to press. Short articles containing descriptions of new species or other taxa will be given preferential treatment in the speed of publication provided that arrangements have been made by the author for depositing the holotype with a recognized public Museum. Museum numbers of the type specimens must be included in the manuscript. Type localities must be defined as accurately as possible, with geo- graphical longitudes and latitudes added. Short original papers, not exceeding 500 words, may be published in the column “NOTES and NEWS’; in this column will also appear notices of meetings of regional, national and international malacological organizations, such as A. M. U., U.M.E., W.S.M., etc., as well as news items which are deemed of interest to our Members and subscribers in general. Articles on “METHODS and TECH- NIQUES” will be considered for publication in another column, provided that the information is complete and techniques and methods are capable of duplication by anyone carefully following the description given. Such articles should be mainly original and deal with collecting, preparing, maintaining, studying, photographing, etc., of mollusks or other invertebrates. A third column, entitled “INFORMA- TION DESK,” will contain articles dealing with any problem pertaining to collecting, identifying, etc., in short, problems encountered by our readers. In contrast to other contributions, articles in this column do not necessarily contain new and original materials. Questions to the editor, which can be answered in this column, are invited. The column “BOOKS, PERIODICALS, and PAMPHLETS” will attempt to bring reviews of new publications to the attention of our readers. Also, new timely articles may be listed by title only, if this is deemed expedient. Manuscripts should be typed in final form on a high grade white paper, not exceeding 814” by 11”, at least double spaced and accompanied by a clear carbon or photo copy. A pamphlet with detailed suggestions for preparing manuscripts intended for publication in THE VELIGER is available to authors upon request. A self-addressed envelope, sufficiently large to accommodate the pamphlet (which measures 51/2” by 814”’), with double first class postage, should be sent with the request to the Editor. EDITORIAL BOARD Dr. Donatp P. Asgort, Professor of Biology Hopkins Marine Station of Stanford University Dr. Jerry DononueE, Professor of Chemistry University of Pennsylvania, Philadelphia, and Research Associate in the Allan Hancock Foundation University of Southern California, Los Angeles Dr. J. Wyatr DuruaM, Professor of Paleontology University of California, Berkeley Dr. E. W. Facer, Professor of Biology Scripps Institution of Oceanography, La Jolla University of California at San Diego Dr. Cavet Hann, Professor of Zoology and Director, Bodega Marine Laboratory University of California, Berkeley Dr. G Darras Hanna, Curator Department of Geology California Academy of Sciences, San Francisco Dr. Jorn W. Hencretu, Resident Director Marine Science Laboratory, Oregon State University Newport, Oregon Dr. Leo G. HERTLEIN, Curator of Invertebrate Paleontology California Academy of Sciences, San Francisco EDITOR-IN-CHIEF Dr. RupotF Stoner, Research Zoologist University of California, Berkeley Dr. A. Myra KEEN, Professor of Paleontology and Curator of Malacology Stanford University, Stanford, California Dr. Vicror LoosanorrF, Professor of Marine Biology Pacific Marine Station of the University of the Pacific Dr. Joun McGowan, Associate Professor of Oceanography Scripps Institution of Oceanography, La Jolla University of California at San Diego Dr. Frank A. Pire.xa, Professor of Zoology University of California, Berkeley Mr. ALLyn G. Smiru, Associate Curator Department of Invertebrate Zoology California Academy of Sciences, San Francisco Dr. Ravpu I. Sorru, Professor of Zoology University of California, Berkeley Dr. Cuarzes R. STASEK, Associate Professor of Zoology Florida State University, Tallahassee, Florida Dr. Donato M. Witson, Professor of Biology Department of Biological Sciences Stanford University, Stanford, California ASSOCIATE EDITOR Mrs. JEAN M. 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