16 9. ADDITIONAL INFORMATION, IF ANY, CONCERNING AUTHORS, ADDRESS, TITLE, OR CITATION DATA Jobe, dlon H. A ST PLEASE TYPE ABSTRACT DOUBLE SPACED BELOW JOBE, ALAN H. (Hopkins Marine Sta,, Stanford Univ., Pacific Grove, Calif., USA). A study of morphological variation in the limpet Acmaea pelta (Mollusca: Gastropoda: Prosobranchia). The Veliger Populations of Acmaea pelta (Eschscholtz, 1833) on the Monterey Peninsula, California exibit variation in shell shape and color. Field studies showed possible correlations of these variables with algal association, substrate, and exposure. Shell shape seems to be dependent upon substrate and exposure. Shell color may be related to diet, but preliminary investigations of gut carbohydrases failed to reveal any distinctive guantitative acwes differences in the amylase, fucoidinase and alginase, found in the A. pelta forms assayed. PLEASE DO NOT TYPE BELOW THIS LINE A Study of Morphological Variation in the Limpet Aomaea pelta (Mollusca: Gastropoda: Prosobranchia) by Alan Jobe Hopkins Marine Station of Stanford University, Pacific Grove, Calif. 93950 % Acmaea pelta (Eschscholtz, 1833) is a low intertidal species of limpet that is widely distributed on the American west coast. A great variation in shell appearance has been noted-Light,, 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, reported 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 A. 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 species using standard radula length and teeth determinations, as well as the classification by jaw shape proposed by Walker (1966). These various morphologic forms are described below in terms of color, shell shape, and length-to-height K (Se fesnste 95 Alan Jobe ratios as measured with a vernier caliper. Norohologic Formsien Brown form: The typical large brown A. pelta is rized by a convex slope from the high peaked shell charate apex to the periphery and is often infected with the fungus 1+oral common to shells of diberg animals of the Pacific Coast (Boner, 1936). These brown-colored specimens are generally over 30 mmn in length, and have a length to height ratio of 2.3 as seen in Table 1. Illustrations of the brown shells are in Fig. 1, Group A. tia): Two separate populations of the Black form (Pely small black A. pelta were observed, on the mussel beds and on and under Pelvåtia fastigata. Those found associated with Pelwötia (Fig. 1, Group c) had a straight to concave slope from the apex to edge and a characteristically cleaner shell chan any other A. pelta form. The shell shape was flatter than the brown 4. pelta, and had a uniform lengöh-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 the mussel beds (see below). Black form (Mussel beds): A. pelta was found on the mussel bed areas, generally on the mussels, Nytilus tella forianus, and on the goosenecked barnacles, cali These were generally black with length-to-heigh polymerus. 16 Alan Jobe 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 (Fig. 1, 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 Pelvétia, and the shell periphery is smooth. Green form: The third group of A. pelta, intermediate in size between the black A. pelta on Pelvétia and the large brown A. pelta form, are shown in Fig. 1, 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. Kabitat. A survey of the intertidal zone of the Monterey Peningila revealed fifferences in the density of the k. pelta populationge. Mussel Point and Pescadero Point had rich populations while Point Pinos was sparcely populated. The reason for this uneven distribution was not investigated. Brown form: This form of A. pelta occupies a high inshore intertidal position from to above 16 feet. The brown Alen Jobo orgrnisms wore found predominatoly on more barren rocks and wore oxposod to more sun and orid conditions then tho other typos. Thoy woro gonerally found on vortical rathor then orizontel faces. The broun limpots wore found in an apparontly rendom association with onerusting algae and amrä. oth, hieh elvei ähgel films as woll as m e Tastigat Tine, and other algao. Occasionally Rhodoglos Tu L ono was found on bare rock close to sand with no macroscopio algoo ovidont. a o Polv tia): At low tido 253 of this form Ltoe Polvitia. An obvious scar was on tho holdfast or stipo of un was fond undor those limpots, indiesting that this algaomay bo a peimary food sourco. Tho romaining 753 were attachod to tse rock substratum under the Po vêtin. Tho Pol 2512 bods providod moisture and shelter from the sun. In moro oxposod proas as Pescadoro Point, whero Polvêtia is not found in large beds, the black A. Selta wore less numerous and randonly distributed. Noro again thoy wero to bo found in locations offering sholtoe and moisture. (7 lack forn 90. s): A. Mlth in tho mussel beds be woro sot associated with conspicuous algao. This form could be found oxpocod to the sun at low tido, as opposed to tho Po i form which was novor found oxposod to dioct sunlicht. Tho mussol beds providod a damp, sholterod onvåron mont that is subjectod to splash oxcomt at very low tide. C Alan Jobo Geoon forn Tho middle sizod groen A. pelta had a Polviti habitat virtually idontieal to tho black e form. Wiguro 2 shows tho porcent distribution of thoso difforent ol t pos of A. n as relatod to position and habitst in the intortidal. Laboratory Studios "ho d intortidal distribution, alone with Graig's (1786) obsorvations on fooding habits suggested that,since tho differont forms woro ecting different algse, possibly thero was ad foronce in digestive enzymes in tho A. po forms. The digestivo enzyme potentinl of the different forms of A. pelta was assayod by moasuroment of roducing sucar rolonsed from purified polysaccharides (Wolson, 10h4). Tho polysaccharides studied woro fucoidin, laminarin, and alginic acid from broun alcal sourcos, agar and K-carrageenin Trom rod algao,and starch. Sinco somo of those carbohydrates contain sulfated groups, particularly fucoidin, sodiu hydroxide was substitutod for barium hydroxide (Wolson, 104) to avoid possiblo precipitation of tho sulfatod sucar fragmonts produced by onzymatic hydrolysis. A 10% exbract of tho ontire digestive system including osophagus, buccal selivary glands, and hind gut was propared from animals of tho black and broum morphologie forms. Tho tissues woro chilled on ico and homogenizod in ice cold homogenizing medium. C C Alen Jobo Wach 1 ml of tissuo was homogonized in a modiu containing o aa us stroptomein solution (5 m/l) to inhibit onctorial grouth, ! ml of a saturated solution of ovomucoid isolated by tho mothod of Frodricg and Doutsch (1949), and 7 ml of buffor (0.21 Tris buffer at pü 7.2 and 0.2 acotste buffor at oH 3.5). Ovomucoid was usod as a tryosin inhibitor bocause of probloms oncountorod with protoinasos in tho onzyme extract, ouho ovomcoid inerensod activity approximatoly two-fold. Ono half ml of onaymo oxtract, one half ml of a O.53 solution of purifiod polyseccharide ond one and ono half for was incubatod at 2090 for ono hour. Tho amount or bu of polyseccharido in a ono nl aliquot was eqnivalent to appromimatoly 200 micrograms of roducing aucar on complote hydrolysis. Tho colorimetrie doterminations woro mado with a Riott-Sumorson photo-oloctrie colorimeter uaing a green rülter, and values were corrocted for roducine sugar prosent in onzymo and substrato controls. Tho corroctod values are exprossod in torms of mierograms of glucose por hour per ml aliquet of rosction modium, and aro oquivalent to 0.02 ml of gut iquo. Rosults Markod amylaso activity, paeticularly at pR S.5,was domonstratod in oxtracts from larco broum A. pouma and the 11. 2og Alan Jobe black form found on Pelvétia. Table 2 gives the quantitative results. Higher act ivity was obsorved in extracts i 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. Disoussion The field work demonstrated a differonce in shell shape of the A. pelta found in dillerent habitats in the intertidal. t appears that shell growth and the resulting shell shape is robably a response to the substrate upon which the animal ves. The high peaked convex shell of the black variet ound in the mussel beds may be an adaption to attachment to a substrate such as is presented in the mussel beds where large flat surfaces are at a minimum. The flatter A. pelta under Pelvétia have an excess of flat but rough rock surface to grow and move on. The wavy shell periphery is an indication of an adaption to this environment. This seems to be a reaction 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 (Hewatt, 1910). A. pelta havatentallyp is ertian high, almost center peaked limpet. This is true when exposed and of reasonable size, as in the brow 26 O Alan Jobo form and the larger soocimons from tho mussol bods. Sinco no large spocimons woro found in the mussel bods and small ones woro abundant, mortality may inerease with ago, tho larger limots being unable to survivo on this aubstrato. Substrato and exposure therofore appoar to be large factors in dotermining sholl morphology. po Tho reason for tho different colors soon in A. is not cloar, but a possible correlation with diffowout algal food is plausible. Tho reported experiments do not ate that digestivo potential is tho critical sm toindi Tactor. Thoy also shou that alginic acid and fucoidin could be utilized only slouly in comparasen to utilization of tarch. Althouch extracts prepared from broun forms displayed more activity tham thoso obtainod from blach ones, this di foronce may bo simply rolated to tho onso of ng ereptor purity of tho extrachs dissecting and tho rosul proparod from the larcer broun limpots. thouch not rolated to digestivo potential, the difforonces in color of poak and poriphoral portions of shells suggests possible changes in diet denge tge lite of tie amal. on te omporimonts will bo roquirod to oxploro this rolationship. C 20 Alan Jobo Surmary Populations of Aemaoa da (Nachscholtz, 1033) oxhibit ot variation in sholl shapo and color. Fiold studies showed correlations of these variables with algal association, substrate, and oxpesuro. Shell shapo sooms to bo dopondont upon substrate and oxposure. Sholl color bo rolatod to diot, but proliminary invostimations of carbohydrases failod to revoal any distinctivo quantit att foronces in the amylaso, fucoidina: nd a t in tho A. polt forms assayed. 20 C C C c Alan Jobo Ackouledgemonts This work was mado possiblo by Grant G/006 from the Undorgraduate Rosearch Participation Program of tho Wational Scionco Foundation. Thanks to Dr, J. Thillips, my advisor, who isolated and jurified tho polysascharides agar, K-caragoonin, lamina fucoidin, and alcinic aeid for tho digestive enzym e Thacks to Miss Cathy Walker who holpe ification to tho A. positivo idon 20 C 11 Alan Jobe LITERATURE CITED Bonar, Lee 1936. An unusual ascomycete in the shells of marine animals. Univ. Calif. Publ. Botany 19: 187-194. Craig, Peter C. 1968. The activity pattern and food habits of the limpet Acmaea pelta (Mollusca: Gastropoda: Prosobranchia). The Veliger Fredricg, E., and H. F. Deutsch 1949. Studies on ovomucoid. Jour. Biol. Chem. 181: 499-510. Hewatt, Willis G. 1940. Observations on the homing limpet, Acmaea scabra Gould. Amer. Midland Naturalist 24: 205-208. Nelson, N. 1944. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: 375-380. Walker, Catherine G. 1968. Studies on the jaw, digestive system, and coelomic deri- vatives in representatives of the genus Acmaea (Mollusca: Gas- tropoda: Prosobranchia). The Veliger 208 1. Permanent address: Footnotes 12 Alan Jobe 206 0 O Alan Jobe Table Legenda Table 1: Length to height ratios of the different forms of A. pelta as related to position in the intertidal. Table 2: Enzyme activity expressed in micrograms of reduced sugar per 0.02 ml of gut tissue. Any value less than 2 micro¬ ams is considered inconclusive. C Alan Jobe Figure Legends Figure 1: A photograph that illustrates the different varieties of A. palta encountered on the Monterey Peninsula. Group A is the brown form, Group B is the green form, Group C is the black form (Eelvitia), and Group D is the black form (mussel beds). Sigure 2: Percent distribution of the three morphologic types of A. palta (each type z 1005) as related to position and habitat in the intertidal. Black A. palta (231 animals) show in solid bars, brown (123 animals) in open bars, and green (168 animals) in cross hatched bars. 04 C Form of Anirul Brown A. pelta Black Apalta Plack A. pelta Black 4. pelta Black A. polta Black A. pelta Table 1 Sizo Location Nich intertidal 30 m Under Peltia 15 m Under Pelvtia 15 ma Wussel beds 15 m 10 to 15 mm Mussel beds Mussel beds 10 r Nubor Checkod 22 12 23 Langth to Moicht ratio 2.3 2.86 2.93 2.53 2.89 3.21 Klan oto 20 Standard Deviaticn O.246 0.270 0.263 O.295 0.272 C Polysaccharide Sbarch Agar K-carrageenln Laminarin Fucoidin Alginic Acid Table 2 Plack A. pelta (Pelvitia) 7 pll 5.5 p 7.2 52ug 2ug 1ug 3ug 13 2g Bromm A. polta pl 5.5 pI 7.2 112 ug 50 ug iug 513 5ug tan Jote 216 O C C B A C f H 3ch R O Dlan Jota (Sae 4loto 21 O C —————--—————-——---- . ------------.. 34 S 3 ———--—-- m ——---——------------ — -------------------. a + — Flau Iee 22