Sleder (2) Acanthina punctulata (Sowerby, 1825) is a common predatory prosobranch gastropod in the upper intertidal zone at Mussel Point, Pacific Grove, California. It is very similar to Acan¬ thina spirata (Blainville, 1832), and the question of whether the two forms represent distinct species is still open. Pre¬ vious work on both snails has been reviewed by Abbott and Haderlie (1979). The main previous study of food and foraging in Acan¬ thina punctulata, by Menge (1974), was carried out at Santa Cruz Island, off Santa Barbara. Systematic studies of its dis¬ tribution, diet, activity patterns, and predatory activity in the Monterey Bay area, the nothern limit of its known geograph¬ ical range, are needed. This paper is a preliminary investigation into these aspects. The function of the strange tooth on the lip of the aperture of Acanthina's shell has long puzzled in¬ vestigators. Hewatt (1934) gave a detailed account of its use to prevent closure of the operculum in barnacles, but other in¬ vestigators have not confirmed this. This paper presents my ob¬ servations on the use of the tooth in the predatory activity of Acanthina punctulata. Studies were carried out on Mussel Point, close to the Hopkins Marine Station of Stanford University. The study site was an area 20 by 15 meters located in a relatively protected place that was accessible at high tide. Bare rocks and rocks bearing barnacles were prevelant in the study site; tidepools and crevices, and beds of the sea anemone Anthopleura elegant¬ issima (Brandt,1835) existed amongst these rocks. Vertical position Sleder (3) of animals in the intertidal zone was established using a surveyed benchmark near the study site. FIELD STUDIES OF DISTRIBUTION, ACTIVITY PATTERNS, AND DIE Field studies were made in the period May 1 to June 1, 1979. Snails used for observations of distribution, activity patterns and feeding were selected at coordinate points in the study site generated by random numbers. When a snail was seen feeding, a 10 cm radius circle was placed around it and the relative abundance of different prey was subjectively determined on a scale of 0 to 4 (0-none, 4=very abundant). To study the distribution of Acanthina punctulata on var- ious substrata, 50 quadrats, each 1 m, were selected in the study area at coordinates picked by use of a random number gen¬ erator. Within each meter squared plot, the percent of the total area represented by each type of substrate was measured with an accuracy of about + 5%, and the 50 plots were then averaged to¬ gether to yield the percent of each type of substrate for the total area. For a study of activity, 60 Acanthina punctulata in the 20 by 15 meter study site were selected by a random number coor¬ dinate point method. They were marked with red fingernail polish and followed over a 25 hour tidal cycle. Observations were made every 2 hours, of movement, feeding, and condition of emersion of each snail. A snail was classified as moving if over a 30 second time period it could be seen crawling with its tentacles Sleder (4) extended. With regard to condition of emersion, animals were classified as dry (totally exposed above water), awash (water was washing back and forth over the snail), or submerged (tot¬ ally under water). At low tide, numerous Acanthina were found in Anthopleura elegantissima beds, or submerged in tidepools. A snail in a tidepool that was being washed by the incoming tide was still counted as being in a tidepool. In determining feeding activity, I did not distinguish between drilling and eating. An Acanthina on a prey species (e.g., another snail), with a drill hole just beneath it, was counted as feeding. When the prey consisted of barnacles, (e.g., Chthamalus spp., Balanus glandula), feeding activity was harder to determine. If a snail was poised over one barnacle and looked as though it could be feeding, I carefully lifted the anterior portion of the snail up to see if I could actually see the proboscis withdrawn from the barnacle. Often I could. If I couldn't actually see the proboscis withdrawn yet the barnacle underneath was gaping open, I counted the snail as feeding. When any uncertainty existed, the snails were counted as not feeding. If the snail was feeding, the prey was marked with a small dot of fingernail polish so a de¬ termination could be made at a later time whether the snail was feeding on old or newly caught prey. Supplementary observations on feeding and movement were obtained at eight high tides and eight low tides. Sleder (5) SULTS AND DISCUSSION The results of the distribution studies are shown in figures 1 and 2. In vertical distribution at low tide, (Fig¬ ure 1), the snails occured 2 to 5 feet above mean lower low water (MLLW), with the majority at 2.5 to 4 feet in a zone including some red algae (Gigartina papillata (J. Aghardi 1848), and some Endocladia muricata (J. Aghardi 1847)). The snails found at the 2 foot level were down among tidepools and the ones found at 5 feet were high upon bare rocks covered with scattered barnacles. Figure 2 shows the types of substrata upon which Acan¬ thina punctulata were found. For example, of the total area of 50 square meters examined, 49% was covered either with bare rock or rock bearing Chthamalus or Balanus. A correspondingly large number (105) of Acanthina appeared on this substrate. The sandy bottoms of tidepools accounted for approximately 23% of the total area sampled and a large number (89) of Acanthina were found on this substrate. It is interesting to note that while Anthopleura elegantissima beds accounted for only 3% of the total area sampled, a comparatively large number of Acan¬ thina were present among the beds. Figure 3 shows the activity of the Acanthina population over a 25 hour tidal cycle. Under conditions of low tide, many snails are exposed and dry. As the tide rises, these snails be¬ come awash or submerged by the incoming water. Snails which are in the moist environments of A. elegantissima beds and tidepools Sleder (6) at low tide, crawl up onto the rocks and barnacles now washed or covered by the incoming tide. The lower portion of Figure 3 shows that as the tide rises, the number of snails high on rocks and barnacles 4.5 feet or more above mean lower low water also rises, and then lowers again as the tide goes down. Figure 3 also shows that at low tide, when a majority of the snails are in an exposed condition, there is little movement. Of the snails moving during periods of low tide, almost all are awash or sub¬ merged. As the tide rises and more snails become awash or sub¬ merged, the number moving increases. This relation can be seen across the 25 hour tidal cycle. Feeding occurs according to the same pattern. The number of snails seen feeding increases as the tide rises, and decreases as the tide falls. Interestingly, of those snails feeding at low tide, a large proportion of them are feeding on prey caught during the previous high tide, in¬ dicating that hunting occurs at high tide. This is shown more clearly in Figure 4, depicting the number of new prey caught as a function of tical height during the 25 hour study. A regression line for the data points has a slope of .9 and a p-value less than .Ol, showing that the number of new prey caught increases significantly as the water level rises. Supplementary data on feeding and movement, appearing in Figure 5, show more movement and feeding at high tide than at low tide; of the snails moving and feeding, most were either awash or submerged. My data show clearly there is a marked increase in activity and feeding as the Acanthina punctulata population becomes awashed or submerged by the incoming tide. This phenomenon was reporte Sleder (7) by Glynn (1965) who states that at high water, more Acanthina move up into the Endocladia-Balanus belt and commence to feed. J.L. Menge (1974) in her study on the prey selection and foraging activities of Acanthina punctulata at Santa Cruz Island, found that the population there forages at low tide but sometimes continues to feed over the high tide. It seems possible this discrepancy could be explained by differences in physical nature of the respective study sites. If Menge's site experienced more wave action than my protected locality, this might have re¬ stricted foraging at high tide. Figure 6 shows the diet of the Acanthina population in relation to the relative abundance of the prey. The abundant barnacles Chthamalus spp. and Balanus glandula, were consumed most frequently. Unlike the Santa Cruz Island population re¬ ported by Menge (1974), a surprisingly large number of Acanthina at Pacific Grove were found feeding on Tegula funebralis (A. Adams, 1885), which were also abundant in the field. Most of these captures occured near tidepools. Two smaller gastropods, Littorina scutulata (Gould,1849) and Littorina planaxis (Philippi, 1847), were also consumed readily in the field. Littorina planaxis normally exists in a zone above the Acanthina population; usually the individuals preyed upon are those knocked down by wave action into the zone of Acanthina. Its interesting to note that while the limpets Collisella digitalis (Rathke, 1833), and Collisella scabra (Gould,1846), were both very abundant in the study area, none were observed being preyed upon. Acanthina in laboratory Sleder (8) aquaria showed the same general preferences as in the field, but in the labratory they selected a wider varity of prey items including the gooseneck barnacle Pollicipes polymerus (Sowerby, 1883), the limpets Collisella digitalis and Collisella scabra, and the bivalve Mytilus californianus (Conrad,1837). PREDATORY BEHAVIOR When observed in lab, Acanthina punctulata approaches potential prey with cephalic tentacles extended, swinging its shell to the left and right on its longitudal axis as it moves along. The orientation of the shell often places the marginal tooth directly in front of the snail, and cephalic tentacles can often be seen extending out from either side of the tooth. When an Acanthina contacts another snail as prey, it usually contacts the prey first with its tentacles. The predator then places the anterior portion of its foot upon the prey and starts to mount it, as if trying to pull the prey off the sub¬ stratum. This behavior was also noted by Bigler (1964) and Menge (1974). Once the prey is positioned underneath the foot of the Acanthina, drilling commences. Drilling involves a secretion from the Acessory Boring Organ (ABO) located in the propodium, which somehow dissolves or softens the shell of the prey; drilling then involves mechanical rasping by the radula of the softened portion of the prey's shell (Hemingway, 1973). Most snail prey are drilled at the same place, the thickest part of the shell outside the area where the columellar muscle attaches to Sleder (9) the columella (Menge, 1974). My studies indicate that consum¬ ption of an individual snail takes anywhere from 2 hours to a day depending on the size of the prey. Observations of Acanthina feeding on meat removed from Littorina planaxis and placed in a deep glass vial provided a good view of the extended pro¬ boscis. The proboscis is semi-transparent and the odontaphore, radular sac, and esophageal tube could be distinguished. Meat is detached from the prey in relatively large chunks and passed rapidly along the esophagus. A special attempt was made to observe the use of the mar¬ ginal spine of Acanthina punctulata. Paine (1966) studied sev- eral species of Acanthina and reported no use for the tooth ex¬ cept perhaps as a brace or wedge when drilling through prey. I have often seen the tooth wedged up against the inside lip of the prey's operculum when Acanthina is drilling the shell of another snail. Hewatt (1934) describes another use of the spine in preying on barnacles. He says, "When attacking a barnacle, the snail assumes a position above the opening of the barnacle shell so that this spine is directly above the line of contact of the closed scutes of the barnacle. The Acanthina usually takes this position when the tide is out and the barnacle thus is closed. When the water returns over the area, the natural re¬ action of the barnacle is to open up and begin the feeding act¬ ivities. As soon as this occurs, the snail quickly inserts its spine into the opening between the scutes, the proboscis everted, Sleder (10) and the soft parts of the barnacle are consumed." MacGinitie and MacGinitie (1968) make the same general observation. My own observations, while incomplete, also indicate that Acan¬ thina punctulata uses its spine to help it feed on barnacles. Four times, twice in the laboratory and twice in the field, I have observed the use of the spine over the top of a barnacle. Both times in the field, the action occured when the snail was awash on an incoming tide. The Acanthina crawled upon a Chtha¬ malus, sat there for a minute or so with no apparent motion. Then the Acanthina raised the anterior portion of its shell (its foot still firmly on the side of the barnacle), and in a ham¬ mering motion, brought it down on top of the opercular scutes of the barnacle. It then lifted its shell and repeated the same motion. I was not able to see whether the spine was actually separating the scutes and entering the mantle cavity of the bar¬ nacle. Nevertheless, once the spine was over the operculum opening, it stayed in this position for approximately 2 minutes. After this time period, the snail took its spine off the bar¬ nacle and settled over the prey in a feeding position. I let the snail stay on the barnacle for 15 minutes, after which time I lifted it up to reveal the proboscis everted between the opercular openings. When the Acanthina was removed from the prey, the bar¬ nacle was left gaping open as if it were somehow paralyzed. The same general behavior was observed in the labratory. However, when the Acanthina lifted its shell up and gave the hammering blow with its spine, it thrust the spine between the Sleder (11) wall plates and the opercular plates of the barnacle. It re¬ peated this motion twice (the first time, the spine didn't reach the opercular opening and slid down the side of the bar¬ nacle). The snail stayed in this position for 2.5 minutes, and then took its spine out and settled over the barnacle as if to feed. At this point, I lifted the snail off; the barnacle was not gaping, and it gave a fast closing response when touched with a probe. After 45 minutes the barnacle still showed no signs of gaping. I replaced the snail near the barnacle. Almost im¬ mediately it moved over the barnacle in a feeding position, the anterior portion of the snail directly over the opercular plates of the barnacle; I could see its proboscis slowly retract from the gaping opercular plates. When the gaping barnacle was touched with a probe, the operculum slowly closed but not totally. I could see no damage to the plates and there was no bore hole present. My observations suggest the hypothesis that when preying upon barnacles, Acanthina punctulata uses some sort of fast acting toxin, and that the spine in some way helps the predator to inject or apply this toxin into the mantle cavity of the bar¬ nacle, causing paralysis. Toxic choline esters have previously been reported from the hypobranchial gland of Acanthina spirata (Bender, DeRiemer et.al., 1974; the species actually studied was probably Acanthina pun¬ ctulata) could be using choline esters from its hypobranchial gland as a poison to paralyze barnacles. Sleder (12) LABORATORY STUDIES Fresh Acanthina punctulata and barnacles (Chthamalus spp.) were gathered from the field before each experiment. To test the response of Chthamalus to two known choline esters, (benzoyl chloride and conbachol), a solution of 1 mg of choline ester per l ml of sea water was used. Hypobranchial gland ex¬ tracts used consisted of 4 hypobranchial glands macerated in 50 ml of sea water. A whitish-green mucus was often secreted from the hypobranchial gland when disturbed during dissections; this mucus was added to the hypobranchial gland extracts. The experimental control consisted of other tissue from the roof of the mantle cavity, in approximately the same mass as 4 hypo¬ branchial glands, ground up in 50 ml of sea water. Injections into barnacles consisted of 0.2 ml of extract; they were made with a fine needle and a 1 ml syringe. When I injected the solution in the tops of the barnacles, I waited until they opened to feed and then gently touched the needle inside the mantle cavity. For injections at the side of the operculum, I in¬ jected through the soft tissue lying between the opercular plates and the walls of the barnacles. LABORATORY RESULTS AND DISCUSSION Laboratory experiments showed that when barnacles were in¬ jected through opercular plates with solutions of known choline esters, paralysis occured within minutes after which the barnacle responded slowly or not at all to probing. The toxin was long acting, lasting anywhere from 3 to 24 hours. A group of control barnacles, injected with seawater through the opercular plates, Sleder (13) showed no paralysis. When a seawater solution of hypobranchial gland extract was injected through the opercular plates of barnacles, a paralyzing response was observed much like that noted for the injections of choline esters. The results are, summerized in Figure 7. The response of barnacles are observed at specified intervals over a period of 24 hours. The barnacles' responses to probing were graded on a scale of 0 to 5 (0-full, healthy response, l-tight closure of opercular scutes but no clamping down, 2-slow closure - couldn't close tightly, 3-very slow closure - couldn't close at all, 4-no response at all to probing, 5=death - determined after 24 hours of no response). Individual barnacles tested are listed along the Y-axis. Note that 4 in¬ dividuals (numbers 11 - 14) receiving the hypobranchial inject¬ ion through the top of the opercular plates had identical re¬ sponses. Six of the eight control barnacles (numbers 1 - 6) had the same healthy response (graded 0). Qualitatively, the amount of black shown in Figure 7 corresponds to the degree of paralysis observed in a barnacle. Note the apparent recovery of paralyzed barnacles around 4 to 6 hours after injection of hypobranchial gland extract. The results shown in Figure 7 suggest that Acanthina pun¬ ctulata could be using a toxic secretion of some sort from the hypobranchial gland to paralyze the barnacles they prey upon. (A toxin from the salivary glands is not excluded, but some preliminary tests of salivery gland extracts showed no toxic Sleder (14) affect on barnacles.) Laboratory observations support this hyphothesis. Often, after removing a feeding Acanthina from a gaping barnacle, no bore holes or other indications of physical damage could be observed around the opercular plates. To substantiate this observation, 10 Acanthina (with spines on) were placed in an aquaria with rocks and Chthamalus spp..10 other Acanthina with spines filed off were placed in an iden¬ tical environment. Qualitative observations a day later showed the snails without their spines caused more damage to the bar¬ nacles than the control snails. Several barnacles being preyed upon by spineless Acanthina showed damaged opercular plates and possible indications of bore holes next to the opercular opening. Damage to this extent was not seen on the control barnacles being preyed upon by normal Acanthina. These ob¬ servations suggest that the spine is used in some way for a more efficient predatory encounter with barnacles. Looking at the anatomy of Acanthina, some observations can be made as to how the spine might be related to the poison secreted by the hypobranchial gland. Near the siphon on the lip of the mantle fold, is a slightly protruding tongue of tissue corresponding in position to the spine. This slight protrusion is located near the anterior end of the hypobranchial gland. Although no ciliary tract was found connecting the hypo¬ branchial gland with the spine area, it is possible that a hypobranchial gland secretion could flow over the top of the Sleder (15) mantle cavity and drip out near the spine. The spine may be used to pry open or to hold open the scutes (or the corres¬ ponding mantle flap lip) of the barnacle as poison is dripped in, or it may be used as an applicator, applying the toxin to the soft tissues or to the operculum opening of the barnacle, The barnacles Chthamalus spp. and Balanus glandula pro- vide relatively small amounts of food to a predator. From this point of view, it would seem desirable for the gastropods to have developed a relatively fast and effortless alternative to drilling as a means for opening barnacles. The absence of drill marks, and the obvious paralysis of the prey, and the experiments performed, all suggest that Acanthina does have such a means, and that it involves the hypobranchial gland and the spine. SUMMARY 1) Vertical distribution of Acanthina punctulata near Hopkins Marine Station in Pacific Grove, California, ranges from 2 to 5 feet above MLLW, with the majority of snails in the Gigartina papillata - Endocladia muricata zone, 2.5 to 4 feet above O tide mark. Near Hopkins Marine Station, Acanthina punctulata were distributed on 4 major substrates; rocks bearing barnacles, sandy bottomed tidepools, in and among A. elegantissima beds Sleder (16) and amoung the red algae, Gigartina papillata and Endocladia muricata. 3) Activity and feeding of Acanthina punctulata at Mussel Point corresponds to a tidal cycle. At low tide, under con¬ ditions of exposure, Acanthina seeks the moisture of tidepools and A. elegantissima beds and remains relatively motionless. As the tide rises, activity increases and the snails crawl up onto the rocks and barnacles now washed or covered by the in¬ coming tide. Feeding occurs according to the same pattern. The number of snails seen feeding increases as the tide rises and decreases and the tide falls. Of those snails feeding at low tide, a large proportion of them are feeding on prey caught during the previous high tide. 4) The field diet of Acanthina punctulata at Mussel Point includes the barnacles, Chthamalus spp. and Balanus glandula and the gastropods, Littorina planaxis and Littorina scutulata and Tegula funebralis. 5) Both field and laboratory observations on Acanthina punctulata indicate a hammering action of its tooth on the opercular opening of their barnacle prey. The obvious paralysis of the barnacle coupled with the absence of drill marks sug¬ gest the secretion of some sort of toxin through the barnacle's opercular opening. Hypobranchial gland extract has been found to have this same paralyzing affect when applied to the soft tissues or to the opercular openings of Chthamalus dalli. The spine may be used to pry open or to hold open the scutes (or the corresponding mantle flap lip) of the barnacle as poison is Sleder (17) dripped in, or it may be used as an applicator, applying the toxin to the soft tissues or to the operculum opening of the barnacle. ACKNOWLEDGEMENTS My sincere thanks to the entire staff of the Hopkins Marine Station. Thanks especially to Dr. Donald Abbott for his time, patience and encouragement. Thanks also to the Spring Class of 1979 who made it a great quarter. Sleder (18) LITERATURE CITED Abbott, D.P. and E.C. Haderlie 1979. Prosobranchia: Marine Snails, in Morris, R., D.P. Abbott, and E.C. Haderlie (eds), Intertidal Invert¬ ebrates of the California Coast. Stanford, California, Stanford University Press: (in press) Bender, J.A., K. DeRiemer, T.E. Roberts, R. Rushton, P. Booth, H.S. Moser, and F.A. Fuhrman 1974. Choline esters in the marine gastropods Nucella em¬ arginata and Acanthina spirata. Comp. Gen. Pharmacol. 5: 191-198 Bigler, Eric 1964. (spring) Attrition on the Littorina planaxis population. Unpublished Student report on file at the Hopkins Marine Station Library, 28 pgs. Glynn, P.W. 1965. Community composition, structure, and interrelation¬ ships in the marine intertidal Endocladia muricata - Bal- anus glandula association in Monterey Bay, California. Beaufortia (Zool. Mus. Amsterdam) 12 (148):1-198 Hemingway, G.T. 1973. Feeding in Acanthina spirata (Prosobranchia: Noegas- tropoda) P.H.D. Thesis, California State Univ., San Deigo Sleder (19) LITERATURE CITED (cont.) Hewatt, W.G. 1934. Ecological Studies on Selected Marine Intertidal Communities of Monterey Bay. P.H.D. Thesis, Stanford Univ. pgs. 1-150. G.E. MacGinitie and Nettie MacGinitie 1968. Natural History of Marine Animals, 2nd Ed. McGraw¬ Hill Book Co. pp 1-449. Menge, J.L. 1974. Prey selection and Foraging Period of the Predaceous Rocky Intertidal Snail, Acanthina punctulata. Oecologia (Berl.) 17,293-316. Paine, R.T. 1966. Function of labial spines, composition of diet, and size of certain marine gastropods. Veliger 9:17-24. e 9 2 9 8 SVOGTAISNT 40 SVIN 2·9-21 Lh-2h 2h-9•2 L'§-22 2n-82 12-82 2•2-8•1 1 NUASER OF ACANTHINA %o SURFACE COVERED BY: (9=285) ON SURFACE k- 6588. ROCK BEARING EARNACLES BOIOMED PS L ANTHOPLEURA ELEGANTISSIMA BESS — GIGANTINA PAPRLATA 8 RHODEGLOSSUA AFFINE ENDOCLACTA MURICATR CTHER —— ——. 6 — — — —— — — ——— — — —5 P I L . 2 —.—— A 2 MDMBER OF NEN PREV 0 5 E- N333 m 40 W 2P3 2 — L —- l kakata- ONITO mr 40 33311 2 LE 29 ESPONSE - O HENI 2 4 DEAS 5 L MANTLE CAVIT TME AFTER INTECTON (HOOES) IMECTES wm: 25 5 115 6 12 2 1 21 HPOBRANCHIAL GAND ERT —.— — 1i — ——— HYPOBRAMCIIAL GLND EXTRACT — ——— MAMIE FIAP TSS EAC (CONTR — Sleder (27) FIGURE LEGENDS Figure 1: Vertical distribution of the population of Acanthina punctulata in a 15 by 20 meter rectangular study area at Mussel Point, Pacific Grove, California. Figure 2: Distribution of the population of Acanthina pun¬ ctulata on different types of surfaces in the study site. Figure 3: Movement and feeding of 60 Acanthina punctulata over a 25 hour tidal cycle. Figure 4: Relation of number of new prey caught by Acanthina punctulata to tidal height over a 25 hour period. Figure 5: Summary of field observations during 8 high and 8 low tides. Lower portions of the graph indicate the number of individuals of Acanthina punctulata observed for high and low tides, and the conditions of emmersion or exposure. Top por¬ tions of the graph show the number of these individuals moving or feeding for a given condition of the tide. Figure 6: Feeding preferences of 70 Acanthina punctulata found feeding in the field at the Mussel Point study site. Relative abundances of prey species were subjectively determined on a scale of 0 to 4 (0-none, 4-very abundant). Figure 7: Lab results indicating the responses of Chthamalus dalli to 0.2 ml injections of hypobranchial gland or control Sleder (28) FIGURE LEGENDS (cont.) extract. Hypobranchial extract injections were given through the opercular openings in 14 barnacles, and in the soft tissue between the opercular and wall plates in 6 barnacles. Response was determined on a scale of O to 5 (healthy to dead). Among barnacles injected with hypobranchial extract through the o¬ percular plates, individuals 11 through 14 showed identical responses of 4 (very sick) over the 24 hour tidal period. Con¬ trol individuals 1 through 6 had healthy responses over 24 hours.