World Journal of Fish and Marine Sciences 5 (1): 104-109, 2013 ISSN 2078-4589 © IDOSI Publications, 2013 DOI: 10.5829/idosi.wjfms.2013.05.01.66144

Feeding, Anatomy and Digestive Enzymes of False guamensis

K.V.R. Murty, A. Shameem and K. Umadevi

Department of Marine Living Resources Andhra University, Visakhapatnam 530 003, A.P., India

Abstract: Very little information has been available in the literature on the feeding habits, anatomy and histology of digestive system of siphonariid . The present study revealed Siphonaria guamensis feeds on the crustose red alga Hildenbrandia prototypus browsing on the rocks by rasping action of . The anatomy of digestive system of Siphonaria guamensis is similar with that of the other siphonariid limpets but the length of gut and colon are shorter than the patellogastropod limpets like Cellana radiata, vulgata, barbadensis and of Acmaea. The salivary glands are the main source of the enzyme system of Siphonaria guamensis. They contained enzymes which can act on carbohydrates, proteins and polysaccharides. The enzyme which can act on proteins was found only in salivary glands and not detected in any other part of the digestive system. No lypolytic activity was seen in any part of the digestive system of the .

Key words: False Limpet Feeding Anatomy Digestive Enzymes

INTRODUCTION tridentatum and C. minimum, where he described the morphology and histology of the digestive system at Little work has been done on the feeding, digestion length. anatomy and histology of the digestive organs of limpets Very little information has been available in the with an exception of patella vulgata (Davies and Fleure literature on the feeding methods, anatomy and histology [1], Graham [2], Stone and Morton [3], Fretter and Graham of the digestive system of siphonariid limpets. Dall [15] [4], Levvy and McAllan [5]). Ward [6] made some described the alimentary system of Siphonaria tristensis. observations on feeding, digestion and histology of the Cottrell [16] gave a detailed description of the morphology digestive tract in the key hole limpet Fissurella of the digestive system in Siphonaria obiliquata. barbadensis. Owen [7, 8] has studied the feeding and Hubendick [17] described the various organ systems of digestion in molluscs in a detailed way. Rao [9] studied the siphonariid limpets to study their phylogenic the feeding digestion and histology of the digestive tract relationship, but very little information was given about of Cellana radiata. Petchimuthu [10] studied the the digestive system of the species of siphonaria. Voss digestive system of Bursa spinosa. Thapanand and [18] gave a brief account of the food of Siphonaria Chunhabundit [11] studied the feeding habits top shell pectinata. Marcus and Marcus [19] briefly described the Trochus maculates, Thilaga et al. [12] studied the alimentary tract of Siphonaria hispida. Abe [20, 21], digestive enzymes and gut microflora of top shell Ohgushi [22], Hirano and Inaba [23] recorded the time of Trochus niloticus. feeding movements in Siphonaria japonica and S. atra. Among , is the least touched Levings and Garrity [24] studied the grazing patterns in group for its anatomy of the digestive system. Morton Siphonaria gigas from the Rocky pacific coast of Panama. [13] studied the functional morphology of Otina otis, This study describes the anatomy of digestive a primitive pulmonate mollusc. Morton [14] also studied system, feeding and digestive enzymes present in the the functional morphology of four British primitive digestive system of S. guamensis to compare with the pulmonates belonging to the family Ellobiidae viz., other species of Siphonaria with different feeding habits Leucophytia bidentata, Ovatella myosotis, Carychium and living in different parts of the world, studied by the

Corresponding Author: K. Umadevi, Department of Marine Living Resources, Andhra University, Visakhapatnam 530 003, A.P., India. 104 World J. Fish & Marine Sci., 5 (1): 104-109, 2013 earlier investigators. Comparisons were also made with The end products of the carbohydrate digestion were the true limpet Cellana radiata which is living with tested qualitatively by using Benedict’s reagent. Siphonaria guamensis on the rocky shores of Proteolytic enzymes were qualitatively demonstrated Visakhapatnam. by the method described by Harrow et al. [26] for pepsin. To detect the presence of Photolytic enzymes, exposed MATERIALS AND METHODS photographic film of 1x1 cm. was used in the experiments. The gelatinous surface of the film will be made transparent Feeding, Digestive System and Digestive Enzymes: by the action of photolytic enzymes. In situ observations were made on the limpets present on The end products of fat digestion were qualitatively high and low water levels of the rocky shores of demonstrated by the method adopted by Ward [6] and Visakhapatnam namely at station 1(Palm Beach), station Rao [9]. Fresh and boiled milk diluted to 1: 50 was used as 2 (Waltair Point-1) and station 3 (Waltair Point-2) during a substrate. To 10 ml of this milk, one drop of 0.04 percent high tide and low tide periods to determine the time and Bromothymol blue was added as an indicator and drop of the frequency of feeding excursions. In the laboratory the 0.2 N NaOH to turn the milk pale blue. The presence of feeding mechanisms were observed by watching the lipases causes the blue solution to turn yellow. animal through the wall of the glass trough to which it In all the experiments, the enzyme-substrate mixtures was attached under a high power magnifying lens. were covered with a few drops of toluene before The feeding mechanisms were also observed by the incubation to prevent bacterial action. Boiled extracts method adopted by Graham [2] in patella vulgata, by were used as controls. The temperature during the course watching the animal lying on its back in a dish of sea of experimentation was regulated by an electrically water under a binocular microscope. The food of the controlled thermostat. The pH of the experimental media animal was established by direct observations, observing was regulated by using suitable buffers. the contents of the gut and also by observing the faecal matter. RESULTS To determine the pH in different regions of the digestive tract and in the salivary glands, the tissues were Feeding: Direct observations on the feeding habits of this ground with clean sand in distilled water separately and limpet in the field have revealed that Siphonaria centrifuged at 500 rpm for 5 minutes. The pH of the guamensis feeds on the crustose red alga Hildenbrandia supernatant solution of the extracts was determined prototypus browsing on the rocks by rasping action of directly with a digital pen pH meter. [ Eutech Instruments radula. The limpets made their feeding excursions when (OAKTON) pHTestr30 ] the tide just exposing the and still the rock is wet To study the enzymes present in the digestive tract, and also when the tide just about to inundate. the method adopted by Rao [9] was followed. The Occasionally small undigested fronds of Enteromorpha digestive tract, salivary glands and the digestive gland were separated from the digestive System, dissecting the and Chaetomorpha were observed in the gut contents. live specimens of 8 mm. shell length and were homogenized separately with distilled water and Anatomy of the Digestive System: The entire digestive centrifuged, at 3,000 rpm for 15 minutes to remove the cell tract in Siphonaria guamensis can be clearly divisible into debris. The presence of enzymes was demonstrated by a small thin tube called buccal tube (Figure 1: c), a dilated studying the action of extract on various substrates, thin walled oesophagus (Figure 1: e & f); small sac or following the procedure of Jayaraman [25] as discussed pouch like stomach (Figure 1: i), thick walled tubular below. intestine / colon (Figure 1: j) and a thin walled dilated To establish the presence of sucrolastic enzymes rectum (Figure 1: k). substrates like starch, sucrose, dextrose, filter paper, Siphonaria guamensis feeds by rasping action of the cotton wool and saw dust were used. Solutions of ground radula browsing over the crustose algae attached to the thalloid alga species Ulva and 1% agar were used to rock surface. The food tends to be more finely selected establish the presence of more complex polysaccharides. and the radula is kept constantly at work raking particles Proteolytic and lypolytic enzymes in the extracts were into buccal cavity. The mouth is a horizontal ventral slit demonstrated by using Gelatine and Casein, Milk and surrounded by thin liplike structures - the oral lappets Olive oil respectively. (Figure 1: a). The mouth leads into a short tube named as

105 World J. Fish & Marine Sci., 5 (1): 104-109, 2013

Table 1: pH (Hydrogen ion Concentration) in different regions of the digestive system Region pH Salivary glands 7.17 Oesophagus 7.58 Stomach 7.45 Intestine 7.55 Digestive gland 6.94

at the extreme posterior end of the body of limpet and reaches the dorsal surface at the right half of the body. When reaching about the middle of the length of the body of the limpet, it takes a sharp turn towards right, runs forward as a thin walled tube-rectum (Figure 1: k), ends as the anus (Figure 1: l) in the lower side of the respiratory orifice or siphonal opening (Figure 1: n). The stomach and colon are embedded in the thick olive green coloured Fig. 1: The course of the digestive system in Siphonaria digestive gland (Figure 1: m). The wall of the anterior and guamensis (Length-5mm, Breadth-3mm). a. Mouth; the posterior oesophagus and rectum has thrown into b. Buccal mass; c. Buccal tube; d. Buccal glands; folds and appear like vertical groves are present on the e. Anterior oesophagus; f. Nerve collor wall of the oesophagus. A pair of salivary glands (Figure g. Posterior oesophagus; h. Salivary glands; 1: h), emerges out of the anterior part of the buccal mass, I. Stomach; j. Colon; k. Rectum; l. Anus; runs down along with the buccal tube surrounding both m. Digestive gland; n. Siphonal opening; sides until the nerve collar (Figure 1: f) and inserted into o. Body wall the nerve collar. Before emerging out of the buccal mass the two small buccal tube (Figure 1: c), embedded in the cavity of the glands like structures which were opening into the buccal muscular odantophore, also called as buccal mass tube are the buccal glands (Figure 1: d). Projecting into (Figure 1: b). The buccal mass is round and red in colour the buccal cavity, behind the buccal mass is the thick in the live specimens and in the fresh specimens. muscular Odantophore with its radula. The buccal tube emerges out of the buccal mass from the The muscular odantophore called as buccal mass is middle of its dorsal surface near to the anterior end, run supported by two cartilaginous plates which are broad posteriorly, but very soon gives a dialated thin walled towards its posterior region and pointed at the anterior tube known as oesophagus. At the point where the side. The two plates are connected by muscle fibres. oesophagus leaves the buccal mass and touches the floor The entire structure is dorsoventraly flattened spread among the odantophoral muscles thus giving insertion to of the body cavity, the tube of the oesophagus is muscles when operating the radula. surrounded by a collar of nerves and muscle fibres, thus dividing the entire oesophagus into anterior Enzymes in the Digestive System: The pH in the oesophagus (Figure 1: e) and posterior oesophagus Salivary glands Oesophagus, Stomach, Digestive (Figure 1: g). The posterior oesophagus is distally slightly gland and Intestine were given in the Table 1. In constricted; behind this constriction is the true stomach. S. guamensis, the pH in different regions of the After giving the sac like stomach (Figure 1: i) the digestive system was slightly alkaline and varies from alimentary canal turns to left and runs forward in the 6.94 to 7.58. anterior direction as a narrow thick walled tube called The results of the experiments conducted on the colon (Figure 1: j), again turn to right at the region of digestive enzymes in the salivary glands, digestive tract nerve collar. Then it runs down as an ‘S’ shaped coil and the digestive gland are given in the Table 2. among the lobes of the digestive gland. Below the The salivary glands are the main source of the stomach it again turn to left side runs anteriorly and enzyme system of Siphonaria guamensis. They contained finally runs down from the extreme left side of the enzymes which can act on carbohydrates, proteins and digestive gland, turns round the distal part of the stomach polysaccharides. The enzyme which can act on proteins

106 World J. Fish & Marine Sci., 5 (1): 104-109, 2013

Table 2: Enzymes present in the different regions of the digestive system Substrate Enzyme Salivary Glands Digestive Tract (DT) Digestive Gland (DG) Digestive system (DT + DG) Carbohydrates Starch Amylase x Sucrose Invertase x x Filter Paper Cellulase x x x x Dextrose Dextrase x Proteins Gelatine Protease xx x Fats Olive Oil Lipase x x x x Milk Lipase x x x x Agar - agar Enzyme that can digest Ulva Complex Polysaccharides x x x x Present x Absent

Table 3: Reaction of digestive enzymes on various substrates Salivary Gland extract Digestive Gland Extract ------Substrate Enzyme Period of incubation Control Experiment Control Experiment Carbohydrates Starch Amylase 3 hours - + - +++ Sucrose Invertase 3 hours - - - + Filter Paper Cellulase 24 hours - - - - Dextrose Dextrase 3 hours - +++ - ++ Cotton wool Cellulase 24 hours - - - - Saw dust Cellulase 24 hours - - - - Agar- agar Enzyme that can digest 24 hours - ++ - +++ Ulva extract Complex Polysaccharides 24 hours - - - - +++ Very prominent activity, ++ Prominent activity, + weak activity, - No activity was found only in salivary glands and not detected in above four species. A comparison of the gut of any other part of the digestive system. No lypolytic Siphonaria guamensis with Patellogastropod limpets activity was seen in any part of the digestive system of like Cellana radiata, patella vulgata, Diodora the animal. apertura, Fissurella pastula, Fissurella barbadensis and The results of the experiments carried out on the species of Acmaea revealed that the gut of these above action of 1% digestive gland extract and 1% salivary limpets is very much longer when compared to the gut of gland extract on different carbohydrate substrates were Siphonaria guamensis. given in the Table 3. Extracts of both the glands can act In Siphonaria guamensis the alimentary canal is on carbohydrates like starch and dextrose. The digestive short (not very much long). In this respect this limpet gland extract can also act on sucrose. Enzymes which can resembles the other siphonariids in which also the gut is digest cellulose were absent in both the glands but not separately divisible into fore gut, mid gut and hind enzymes that can digest polysaccharides like agar were gut. Fretter and Graham [4] pointed out that in docoglossa present in salivary gland extract and digestive gland the intestine is vastly longer and that the differences in extract. the intestinal development is due to the need for consolidation of the faecal material before it is passed into DISCUSSION the mantle cavity. The feeding habits of Siphonaria guamensis and Anatomy of Digestive System and Feeding Biology: food are similar to those of other limpets of this genus. A comparison of the gut of Siphonaria guamensis with The limpet mainly feeds on the crustose red alga that of other species like Siphonaria obliquata, Hildenbrandia prototypus. The limpet also feeds on some Siphonaria tristensis and Siphonaria hispida shows that filamentous algae like Enteromorpha intestinalis, the anatomy of the digestive system is similar in the Chaetomorpha antennina. S. guamensis feed by scraping

107 World J. Fish & Marine Sci., 5 (1): 104-109, 2013 the algae from the rock surface with the help of radula salivary gland extract and the digestive gland extract when the limpets are just inundated by the flooding tide indicates that the enzymes which can digest the complex and also at the time that the ebbing tide just exposing the polysaccharides were present in both the glands. limpets. Rock particles or sand grains were never This was coincided with the direct observations made on observed in the gut contents or in the faecal matter of the the limpet’s feeding habit. The limpet habitually feeds on limpet S. guamensis. No observations on the gut analysis the mats of cructose red alga Hildenbradia prototypus, of the other species of Siphonaria were available. Rao [9] which is present on the rock surface. Agar is the observed ingestion of rock particles by Cellana radiata. principal food reserve in red algae. The negative Ward [6] also observed the same in the tropical keyhole result with the Ulva extract indicates that the limpet will limpet Fissurella barbadensis. Willcox [27] suggested not feed on Ulva mats. In the direct observations on the that these rock particles help in pulverizing the food. feeding activity of the limpet in the field, when the rock In S. guamensis the salivary glands and the digestive is barren and devoid of crustose red algae, these limpets gland are the main sources of enzymes. No information on prefer to feed on some filamentous green algae like the digestive enzymes is available on the other species of Enteromorpha and Chaetomorpha. Voss [18] observed this genus. Rao [9] observed that there were no enzymes Siphonaria pectinata feeding on Ulva. present in the salivary glands of Cellana radiata. Graham The presence of amylase, invertase and dextrase in [2] in patella vulgata and Ward [6] in Fissurella the salivary glands and digestive glands of S. guamensis barbadensis also did not observe digestive enzymes in suggests that the animal mainly depends on the the salivary glands. Fretter and Graham [4] suggest that carbohydrate substrates present in the consumed algae. the salivary glands are used only for lubrication in the Presence of protease enzyme is also recorded in the diotocardians. salivary glands which may enable the digestion of No undigested food material was observed beyond proteins present in the consumed algae. The digestion of the buccal mass even in the freshly dissected limpets agar indicates that S. guamensis mainly feeds on the red which indicates that digestion apparently begins at the algae which are rich in agar resources. The restricted region where the salivary glands open into the buccal distribution of S. guamensis, to those rocks covered by tube near the buccal mass. Amylase is located in the the crutose red alga Hildenbrandia prototypus, analysis salivary glands of S. guamensis in little quantities. This of gut contents and direct observations on the feeding enzyme was located in the fore gut of patella vulgata [2] habits of S. guamensis in the field and laboratory and also in the fore gut of Cellana radiata [9]. Enzymes supports these results. which can digest proteins were located only in salivary glands in S. guamensis. Enzymes which can act on ACKNOWLEDGEMENT carbohydrates appear to be mainly located in the digestive gland than in the salivary glands. Absorptions The third author acknowledge the first two authors and intracellular digestion may occur in the digestive for their intellectual and academic guidance at the time of gland cells in Siphonaria guamensis as in the undertaking research work and preparation of paper. patellogastropod limpets like Haliotis, Acamaea, Thanks to Mr. Sk. Yaseen Baba for assisting in the Fissurella and Cellana [2, 4, 6, 9]. typographical work. There was no indication of cellulase activity in the digestive system of Siphonaria guamensis. Stone and REFERENCES Morton [3] while studying the distribution of cellulase in molluscs, observed the presence of cellulase in the 1. Davis, J.R.A. and H.J. Fleure, 1903. Patella. Liverpool digestive system of patella vulgata. The negative results Mar. Biol. Comm. Mem., 10: 1-76. for cellulase activity suggest that S. guamensis cannot 2. Graham, A., 1932. On the structure and function of digest the cell wall components. Ward [6] also observed the alimentary canal of the limpet. Trans. Roy. Soc. of no cellullase activity in Fissurella barbadensis. Edin., 57: 287-308. Meeuse [28] stated that green algae store sucrose as 3. Stone, B.A. and J.E. Morton, 1958. The distribution of a reserve substance. An enzyme which is capable of cellulase and related enzymes in . Proc. digesting sucrose was found in minute quantities in the Malac. Soc. Lond., 33: 127-141. digestive gland of Siphonaria guamensis. A strong 4. Fretter, V. and A. Graham, 1962. British Prosobranch positive result for the agar digestion with both the Molluscs. London, Ray Society.

108 World J. Fish & Marine Sci., 5 (1): 104-109, 2013

5. Levvy, G.A. and A. McAllan, 1963. B-D. Fucosidase 17. Hubendick, B., 1947. Phylogenic und Tiergeographic in the limpet Patella vulgata. Biochem. J., der . Zur Kenntnis der Phylogenic in 87: 206-209. der Ordnung Bassommatophora und des Ursprungs 6. Ward, J., 1966. Feeding, digestion and histology of Pulmonatengruppe. Zool. Bid. Fran. Uppsala, the digestive tract in the keyhole limpet Fissurella 24: 1-216. barbadensis (Gmelin). Bull. Mar. Sci., 16: 668-684. 18. Voss, N.A., 1959. Studies on the pulmonate 7. Owen, G., 1966a. Feeding. In Physiology of Mollusca, gastropod Siohonaria pectinata (Linnaeus) from the 2: 1-51. Ed. K.M. Wilbur and C.M. Yonge. London, southeast coast of Florida. Bull. Mar. Sci. Gulf and Academic Press. Carib, 9: 84-99. 8. Owen, G., 1966b. Digestion. In Physiology of 19. Marcus, E. and E. Marcus, 1960. On Siphonaria Mollusca, 2: 53-96. Ed. K.M. Wilbur and C.M. Yonge. hispida. Bole. De Zool., 23: 107-39. London, Academic Press. 20. Abe, N., 1940. The homing, spawning and other 9. Rao, M.B., 1975. Some observations on feeding, habits of a limpet, Siphonaria japonica Donovan. anatomy, histology of the digestive tract and Toh. Univ. Sci. Rep., 15: 59-95. digestive enzymes in the limpet Cellana radiata 21. Abe, N., 1941. Ecological observations on a (Born) (Gastropoda: Prosobranchia). Proc. Malac. pulmonate limpet Siphonaria atra (Quoy et Gaimard). Soc. Lond., 41: 309-320. Palo. Trop. Biol. Stat. Stud., 2: 239-278. 10. Petchimuthu, S.M., 1985. Studies on digestive 22. Ohgushi, R., 1954. Ethological studies on the system, of Bursa spinosa (Lamarck) (Bursidae: intertidal limpets. I. On the tidal rhythmic activities of Mesogatopoda: Mollusca) PhD Thesis. M.K. two species of limpets. Jap. J. Ecol., 4: 120-122. University, S. India. 23. Hirano, Y. and A. Inaba, 1980. Siphonaria (pulmonate 11. Thapanand, T. and S. Chunhabundit, 1995. Feeding limpet) survey of Japan. I. Observations on the habits of top shell Trochus maculates (Linnaeus). behavior of Siphonaria japonica during breeding J. Thai. Aquat. Sci., 1(2): 185-193. season. Publ. Seto Mar. Biol. Lab., 25: 323-34. 12. Thilaga, R.D., D. Jayaprabha, V. Sivakumar and 24. Levings, S.C. and S.D. Garrity, 1984. Grazing patterns M. Tamilselvi, 2010. Digestive enzymes, gut in Siphonaria gigas (Mollusca, Pulmonata) on the microfloral study of top shell Trochus niloticus. W. rocky Pacific coast Panama. Oecol., 64: 152-159. J. Fish. Mar. Sci., 2(6): 495-498. 25. Jayaraman, J., 1992. Laboratory Manual in 13. Morton, J.E., 1955a. The functional morphology of Biochemistry. Widely Eastern Ltd, New Delhi. Otina otis, a primitive marine pulmonate. J. Mar. Biol. 26. Harrow, B., E. Borek, A. Mazur, C.H.G. Stone and Assoc. U.K, 34: 113-150. H. Wagreich, 1960. Laboratory Manual of 14. Morton, J.E., 1955b. The functional morphology of Biochemistry. London, W.B. Saunders Co. the British Ellobiidae (Gastropoda: pulmonata) with 27. Willcox, M.A., 1905. Biology of Acmaea testudinalis special reference to the digestive and reproductive Muller. Amer. Natu., 39: 325-333. systems. Phil. Trans. Ser. B., 239: 89-160. 28. Meeuse, J.D., 1962. Storage products. In physiology 15. Dall, W.H., 1870. remarks on the Anatomy of the and Biochemistry of Algae, 289-313. R.A. Lewin, Ed. genus Siphonaria with a description of a new species. New York, Academic Pres. Amer. J. ., 6: 30. 16. Cottrell, A.J., 1911. Anatomy of Siphonaria obliquata (Sowerby), Trans. New Zea. Inst., 43: 582-594.

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