THE DIGESTIVE CARBOHYDRASES IN THE A COMPARISON OF VULGARE (STIMPSON, 1857), TERRESTRIAL ISOPOD, ARMADILLIDIUN AND IN THE MARINE ISOPOD, IDOTHEA RESECATA (LATREILLE, 1804) Marian Matoba Biology 175H June 1973 Hopkins Marine Station Stanford University running title: Carbohydrases in Armadillidium vulgare and Idothea resecata Carbohydrases in Armadillidium vulgare and Idothea resecata ABSTRACT Crude extracts of the hepatopancreas from Armadillidium vulgare and Idothea resecata were investigated for hydrolytic ac- tivity using the following substrates: soluble starch, maltose, lactose, melibiose, cellobiose, sucrose, and sodium alginate. Ac¬ tivity with respect to starch, maltose, and sucrose was demon- strated. No activity for lactose, melibiose, cellobiose, or sodium alginate was detected. Carbohydrases in Armadillidium vul reseca INTRODUCTION The organisms belonging to the order Isopoda show a wide range of habitat extending from the marine to the terrestrial. Marine and terrestrial isopods differ with respect to diet. Complex car- bohydrases are conspicuous components of these diets. One might expect, then, that two different species of isopods, one marine and the other terrestrial, would possess different digestive en¬ zymes for the hydrolysis of these complex materials. A study of some of the carbohydrases belonging to the marine isopod, Idothea resecata, and the terrestrial isopod, Armadillidium vulgare, was carried out. These two species were chosen because of their re¬ lative abundance in the intertidal regions near Monterey, Calif- ornia and in the neighboring grasslands. Both species appear to be omnivorous. Previous studies on the carbohydrases of these animals have shown the presence of amylase, maltase, and a 8-glu¬ cosidase hydrolyzing arbutin and salacin in Armadillidium vulgare (Newcomer, 1952). Enzyme assays for the detection of amylase, maltase, sucrase, lactase, melibiase, cellobiase, and alginase re¬ sulted in the findings reported here. MATERIALS AND METHODS The hepatopancreas of Idothea resecata and Armadillidium vul- gare were obtained by dissections and homogenized. Distilled wa- Carbohydrases in Armadillidium vulgare and Idothea resecata ter was added to the homogenized tissue, and the resulting solu- tion was centrifuged for 60 minutes at 20,000 rpm in a Sorvall 32-B refrigerated centrifuge. The supernatant was removed and in- cubated at room temperature (22°- 24°C) with equal volumes of the following substrates in distilled water: soluble starch, sodium alginate, melibiose, cellobiose, maltose, sucrose, and lactose. Toluene was added to all of these solutions to prevent the growth of bacteria. The concentrations of the solutions were such that approximately one hepatopancreas, 25 ug of maltose, lactose, su¬ crose, melibiose, or cellobiose, or 50 ug of starch or sodium al¬ ginate were used per assay. After periods of 1, 2, 4, 8, and 12 hours, 2 ml samples of the incubating solutions were tested in du¬ plicate for reducing sugars. Enzyme and substrate controls were also tested at the end of the 12 hour period. Enzyme activity was expressed as percent hydrolysis, with the maximum or 100% hydroly- sis taken as a net increase of approximately 12.5 ug for the di¬ saccharides and approximately 50 ug for the polysaccharides starch and sodium alginate. Although this is an approximate value which does not take into account the addition of water molecules intro¬ duced through hydrolysis, it seemed accurate enough for purposes of expressing these enzyme activities. The assays were carried out at a ph between 4 and 5. Enzyme activity was detected by the reducing sugar method of Somogyi (1952) and Nelson (1944) using a Klett-Summerson Photoelec- Carbohydrases in Armadillidium vulgare and Idothea resecata tric Colorimeter and a red filter. The protein content of the en¬ zyme solution was determined by the Lowry method (1957). RESULTS The protein content of the hepatopancreatic extracts was 390 jg and 830 pg per assay for Armadillidium vulgare and Idothea re secata, respectively. Figure 1 shows the time course of hydrolysis of starch by he¬ patopancreatic extracts from the two animals. After 12 hours, the extracts from Idothea resecata produced hydrolysis of 74 of the theoretical maximum expected with this substrate. In the same in- terval, the Armadillidium vulgare extract produced 59% hydrolysis. Figure 2 shows the time course of hydrolysis of maltose by he¬ patopancreatic extracts. After 12 hours, the extract from Idothea resecata produced hydrolysis of 57% of the theoretical maximum ex- pected with this substrate. Armadillidium vulgare produced 65%. Figure 3 shows the time course of hydrolysis of sucrose. Af¬ ter 12 hours, the extract from Idothea resecata produced hydrolysis of 36% of the theoretical maximum expected, and Armadillidium vul- gare extract produced 23% hydrolysis. No hydrolytic enzyme activity for melibiose, cellobiose, lac¬ tose, or sodium alginate was detected by this method. Carbohydrases in Armadilli hea resecata DISCUSSION The presence of a £-fructofuranosidase or invertase is not conclusively demonstrated since it has been reported by Pigman (1948) that sucrose can be hydrolyzed by o-glucosidases as well. The percentages obtained for hydrolysis of the various substrates reflect only the activity of crude enzyme preparations. Investi¬ gation of ph and temperature optima still need to be carried out. The absence of melibiases, cellobiases, lactases, and alginases require further investigation under other assay conditions, par- ticularly since a B-glucosidase was shown to be present in Arma¬ dillidium by Newcomer (1952). Such an enzyme should have been de¬ tected through the use of cellobiose as a substrate. These assays did not show cellobiase activity. Armadillidium vulgare and Idothea resecata which live in two widely differing environments have so far failed to show any dif- ferences in the carbohydrases that they possess. This is inter¬ esting since the carbohydrates present in their diets are obvious¬ ly different. Armadillidium vulgare has been reported to eat mostly dead terrestrial plant material and dead invertebrates (Paris, 1963). The diet of Idothea resecata has not been fully characterized, but it has been reported to prefer brown algae over other kinds of marine food materials (Jones, 1971), and has been seen to feed on the brown algae, Macrocystis, in particular. An interesting point is that brown algae contain mannitol, some re- Carbohydrases in Armadillidium vulgare and Idothea resecata ducing sugars, laminarin, fucoidin, and cellulose, but starch ap¬ pears to be absent (Blinks, 1951). The possession of an enzyme ca¬ pable of hydrolyzing a substrate apparently lacking in the diet of an animal is a rather unexpected finding. It would be interesting to carry out further studies on the digestive enzymes of these i¬ sopods and their diets, and to see what sort of a correlation ex¬ ists between the two. Carbohydrases in Armadillidium vulgare and Idothea resecata LITERATURE CITED Blinks, L. R. 1951. Physiology and biochemistry of algae. In: Manual of Phycology, ed. Gilbert M. Smith, p. 263. Waltham, Mass.: Chronica Botanica Co. Jones, Laurence G. 1971. Studies on selected small herbivorous invertebrates inhabiting Macrocystis canopies and holdfasts in southern California kelp beds. In: The Biology of Giant Kelp Beds (Macrocystis) in California, ed. Wheeler J. North, p. 343. Lehre, Germany: J. Cramer. Lowry, O. H. et al. 1951. Protein measurements with Folin phenol reagent. Jour. Biol. Chem., 193: 265. Nelson, Norton. 1944. A photoelectric adaptation of the Somogyi method for the determination of glucose. Jour. Biol. Chem., 153: 375. Newcomer, W. S. 1952. The occurrence of beta-glucosidase in the digestive juice of Porcellio and Armadillidium. Anat. Rec., 113: 536. Paris, Oscar H. 1963. The ecology of Armadillidium vulgare (I¬ sopoda: Oniscoidea) in California grassland: food, enemies, and weather. Ecol. Mono., 33: 1. Pigman, William Ward and Randolph Maximillian Goepp, Jr. The Chemistry of Carbohydrates. New York: Academic Press, Inc., 1948. Carbohydrases in Armadillidiumy Idothearesecata ACKNOWLEDGEMEENTS I would like to thank the entire staff at Hopkins Marine Sta- tion for their invaluable assistance with this study, particularly Dr. John Phillips for his patient help and understanding. 8 X. N skaa 90 • 0 + + — + 2 2 — e 2 o - * 2 - o o . -ot No 1. k v- vatavaa- 0 o o 00 * k kt statatatataaa- O 4 + : . . o- Carbbhydrases in Armadillidium vulgal dothea resecata FIGURE CAPTIONS Figure 1. Time course of hydrolysis of starch by hepatopancrea¬ tic extracts of Armadillidium vulgare and Idothea re- secata. Activity is expressed as percent of theoreti- cal maximum. Figure 2. Time course of hydrolysis of maltose by hepatopancrea¬ tic extracts of Armadillidium vulgare and Idothea re- secata. Activity is expressed as percent of theoreti- cal maximum. Figure 3. Time course of hydrolysis of sucrose by hepatopancrea¬ tic extracts of Armadillidium vulgare and Idothea re- secata. Activity is expressed as percent of theore- tical maximum.