Quantitative distribution of chitinolytic bacteria from the hepatopancreas, stomach and intestine of a

commercially important marine prawn Penaeus indicus was studied. Twenlyfive representative bacteria

were isolated from colonies forming well defined clear zones on chitin agar medium and were identified.

Eighteen of the isolates were members of the genus Vibrio and the rest were members of the genus

Pseudomonas

Banik, S

Ninawe, A S

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CHITINOL YTIC BACTERIA IN PENAEUS INDICUS A. NINAWE* a nd S. BANIK'* C~llfral Marine Fi.\'i1eri('s Research 111:)'{ilule, P.O. Box AD. 1704. Cochiti 682031. India ABSTRACT Qua nt itat ive d ist r ibutio n o f chitino lyt ic bacter ia from the hep alopa ncreas. stomach and intestine of a co mmercially important ma rint: prawn Pel1lJeliS indin f.\" was slUdicd . Twenlyti"e representa tive bacteria were isolated from colo nies fo rm ing well d efined clea r zones on chitin agar med ium and were id entified . Eighteen of the isolates were members of the gen us Vihriu and the rest were members of the genus Pseudomonas. Distribution of chitin has been reported in the exoskeleton of crustacean (ZoBell , 1946), mollusca, coelenterata, protozoa and in cell walls of certain molds and fila-mentous yeasts (Nabel, 1939) and principally among the Arthropoda-crabs and insects ( Karlson. 1963). Abundance of chitin in marine and es tuarine environment is well known and chitinolytic role of marine bacteria has been suggested (Aaronson, 1970). The chitinolytic enzyme fo und in the diges tive conte nts and glands or in the intes tinal mucosa in several species of verte brata is reponed to be indigenous to the system instead of being of bacterial origin (Jeuniaux, 196 1) . Chitinolytic activi ty has also been reported in the diges tive tract of Lateolahrax japonicus (Okutani, 1966) a nd Mughil cephalus (H amid er al., 1979), and bacteria were also found in their alimentary tracts. So, study was undertaken to identify the bacteria tfuly involved in chitinolytic activity in the diges tive tracts. viz. hepato pancreas, stomach and intestine of a com-mercially importa nt marine prawn Penaeus indiclis collected from Narakkal Prawn Culture Laboratory (N PCL). Cochin . India . • A. :'\inawc, Depart mt:nt of Sciencc and Techno logy, :\ew i\:lchra uli Road, New Del hi 1100 16, Ind ia . .. D r. S, Ba ni k, J ute Tech no logical Research L.a bo ratory. 12. Regen l Park. tv1 icrobio log) Div is io n. Calcutta 700 040. India. J. Aqua. Trop .. 2 (1987) 89-92 © Oxford & IBH Publishing Co. P vc Ltd. 90 MATERIALS AN D METHODS 5 nos. health y and mature specimens of prawn Penaeus indicus were collected from the NPCL ponds, Cochin, India. The pra wns were immedia tely washed with 1% HgCb_ solution for surface sterilization . After repeated washing with sterile distilled water their hepatopancreas. stomach and intestine were dissected fro m the alimen-tary tract and put into 100 ml sterile blank saline solution (0.85%) separately for serial dilution. Aseptic condition was maintained throughout the experiment. For enumeration of chitina lytic bacteria , po ur plating was carried out on mineral agar medium (Aaronson , 1970) co ntaining 2. 5% NaCI with chitin precipita te a s the sole so urce of carbon ~nd energy_ Separately sterilized chitin preci pitate was supple-mented to the melted mineral agar medium until it became turbid. pH of the mineral medium was adjusted to 7.0 . Precipitation of chitin has bee n discussed by Tracey (1955). Enumeration of tota l aerobic heterotrophic bacteria was carried out by using ZoBell's 2216 E agar medium and chitinolytic bacteria were studied (Tracey, 1955) from 10' and 10', and 10' a nd 10' dilutions respectively, following pour plate tech-nique. The plates were incubated at JO° C for three days in case of tota l heterotrophic bacteria and for seven days in case of chitinolytic bacteria. Clear zone forming colonies were counted only in case of chitinolytic bacteria . Best incubation tempera-ture were determined by incubating slant cultures at temperatures of 25°, 30° and 37° C. Poor g rowth was observed at 25° and 37° C but satisfactory growth was noticed at 30° C. Table I. :-':umber of chitinolytic bac teria a nd tota l viable aerobic heterotrophic bacteria Prawn speci men Region o f Nu mber x IO ~ g % of chitinolYlic number isolation bacteria Total bi:lcteria chitino lytic bacteria S 170 18 10.58 P, H 124 22 17.74 46 6 13.04 S 164 27 16.46 P, H 184 37 20.10 120 I I 9 .16 S 167 15 8.98 P, H 192 27 14.06 98 8 8. 16 S 122 12 9.83 P, H 168 24 14.28 64 5 7.8 1 S 122 21 17. 2 1 P, H 193 37 19.17 87 17 19.54 S- S tomach. H- Hepat opancrcas. I-I ntestine . 91 Twenty-five isolates of chitinolytic bacteria were taken from well defined clear 7.one produced colonies. Cultures were purified by rcpeated subcultures. Isolates of pure cultures were maintained on nutrient agar slants. Their morphological. cultural and biochemical characters were studied following Bergey's manual (Buchanan and Gibbons. 1974) for identification. , , + , + + , + , + + + + + + + + + Table 2. Di! h:rcnt ial biochemica l charach:rs 01 the i ~ola{ed bacteria :r. ;-, ::; "2 '-"::J -r. ~ + + , , + + + , + + + + , + + + , + o - :::; ; , ':JJ :I: _ , + + + + , , , , , + , + , + + , • + + + + + + > + + + + + + + + + + + + , , , + , + + + + , , + + + + + + + + + + + , + + + , , , , , , , + , + + + + + , , + + , + , + + + + + + + + + + + + + + + + + , + , + + , , , , + , + + + 4?:i ~ -~­'r. -.~ '" -+ + + + + + + + + + , + + + + , + + 2 2 2 2 I 2 2 I 2 * No isolate produced any gas like CO:: or H; S. + indicates positive: -indicates negati ve. I indicates j "ihr;u. 2 indicates Pseudomof/a.~. M.O.F. Marine oxidative fermentation. T.S .1. Triple sugar iron: V.P. Voges-Proskaucr ; M.R. Methy l red; 0 1292.4 diamino-6. 7-diisopropyl pteridine. RESULTS AND DISCUSSION The percentage of chitinolytic bacteria in total aerobic heterotrophic viable bacte-ria in the alimentary tract of the prawn Penaeus indicus ranged from 7.81 to 20.10% (Table I). Their number varied from specimen to specimen but not significantly. Okutany (1966) reported significantly higher number of chitinolytic bacteria in intestine and stomach of a fish (Loreolabraxjapollicus) than that in sea water. As the 92 prawns collected for the present study were cultured in artificial NPC L culture tanks and were rcd with artificial reed rich in chitinic ma terial (Ali, 1982), higher accumula-tion of the chitinolytic bacteria in their hepatopa ncreas, stomach and intestine than in water is most likely as reported by earlier worker (Okutany, 1966). Na turally the bactc ria might have been actively involved in digestion of chitinous material by secreti ng chitinolytic enzyme as suggested by Zo Beli and Rittenberg (193 7). The chi t inolytic bacterial population wa s found in decreasing o rder in the three regions of al imentary canal- hepatopancreas. stomach and intestine. Higher accum-ulat ion of chitinolytes in hepatopancreas followed by stomach and intes tine needs furth er attention. All the isolated bacteria were Gram negative, asporogenous motile rod s, catalase and oxidase J'Ositive and showed better growth at 30° C than at 25° C and 37°C on slant culture. Other biochemical behaviour listed in Table 2 indicate the bacteria to be members of the genus Vibrio and Pseudomonas. So, the results of the study demonstrate that chitin utilizing Vibrio and Pseudom onas species were present in the digestive tract of the marine crustaceans, viz. Penaeus indicus. These two bacteria may aid in digestion of chitinous food items in Penaeus indicus. ACKNOWLEDGEMENTS The first author gratefully acknowledges the II,dian Council of Agricultural Research (ICAR) for providing Research Fellowship. Both the authors a re grateful to the Director, Central Marine Fisheries Resea rch Institute (CMFRI) , Cochin for the facilities provided for the study. REFEREN CES Aaro nso n. S. ( 1970). Experim ellla/ Microbial Ecology. Academic Press. London. Ali , S.A. ( 1982) Relative efficiencies of pa lleet ized feeds co mpounded with d ifferent ani mal proteins a nd the erfect of protein level on the growth of the prawn. Punaeus indiclIs. Prot". !;}\mp. Coos/a/ Aquae-u/-lUre, I, 32 1 ~3 28 . Bucha na n. R.E. , and Gibbons. N.E. (eds). (1 974). Bergey's manualfor dererminulil'e bacleriology , 8th ed n. Williams and Wilkins Co .. Baltimore. Hamid, A. , Sakata , T. , and Kaki moto . D. (1 979). Microllora in the alimentary t ract of Gray mulletlV. Est imat ion of enzyme activi ties in the intestinal bacteria. Bull. lap. Soc. Sci. Fish 45. 79~ l06. Jeu niaux, C. (1961) Chitinase: An ad dition to the list of hydrolases in the d igestive tract of verteh rates. Nsture. 192. 135-136. Karlson. P. (1 963). Imroduc/ion to Modem BiochemislrY. Acad emic Press. New York a nd Lond on. Na bel. K. ( 1939) Uber d ie mem bran nicderer pi lze. besonders \"on Rhizidiomyces bivella lUr no\". spec .. Arch. Mikrobiol. 10(4),515-541. Okuta ni , K. (1966). Studies of chit inolytic systems in the d igestive tract of Lareolabrax japoniclis. Bull. lap. MiJoki. :\.lar. BioI. In.n, Kyoto University. 10, 1-47. Tracey, M .V. (1955 ) Chitinase in some Basidiomycetes . Bio("hem. J. 61,579- 586. ZoBel!. C. E. (1946). Marine J1icrobi%gy, Waltha m. Mass; Chroniea Botanica Co. ZoBe11. C. E. and Rittenberg, S.c. (1937). Contribution / rom the Scrips InstitlJle o/Oceanography. Nell ' series. 5, 275. 

Chitinolytic bacteria in Penaeus indicus

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Chitinolytic bacteria in Penaeus indicus

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