references - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/60984/16/16_references.pdf ·...
TRANSCRIPT
REFERENCES
1. Abraham, E. P. 1977. In Biologically active substances exploration andexploitation (John Wiley and Sons, New Yark). p-2.
2. Abraham, T. J., Palaniappan, R and Dhevendaran, K. 1999. Simple taxonomic keyfor the identifying marine luminous bacteria. Indian I Mar. Sci. 29: 35-38.
3. Abraham, T. J. 2004. Antibacterial marine bacterium deters luminous vibriosis inshrimp larvae. NAGA, World Fish Center, 27(3): 28-31.
4. Aceret, T. L., Coll, J. C., Uchio, Y and Sammarco, P. W. 1998. Antimicrobialactivity of the diterpenes flexibilide and sinulariolide derived from SinulariaJiexibilis Quoy and Gaimard 1833 (Coelenterata: Alcyonacea, Octocorallia),Comparative Biochemistry and Physiology C: Pharmacol. Toxicol. Endocrinol.120: 121-126.
5. Agarwal, M. S and Bowden, B. F. 2005. Marine sponge Dysidea herbacearevisited: brominated diphenyl ether. Marine Drugs, 3: 9-14.
6. Ahmed, M., Rahman, M. T., Alimuzzaman, M and Shilpi, J. A. 2001. Analgesicsesquiterpene dilactone from Mikania cordata. Fitoterapia, 72: 919-921.
7. Alisop, A. E. 1998. New antibiotic discovery novel screens, novel targets andimpact of microbial genomics. Curr. Opin. Microbiol. 1: 530-534.
8. Amador, M. L., Jimeno, J., Ares, P. L., Furies, C. H and Hidalgo, M. 2000.Progress in the development and acquisition of anticancer agents from marinesources. Ann. Oncol. 14(11): 1607-1615.
9. Amann, R. I., Ludwig, W and Schleifer, K. H. 1995. Phylogenetic identificationand in situ detection of individual microbial cells without cultivation. Microbiol.
Rev. 59: 143-169.
10. Amin, V. M and Olson, N. F. 1968. Influence of catalase activity on resistance ofcoagulase-positive staphylococci to hydrogen peroxide. Appi. Microbio. 16 (2):
267-270.
11. Anand, P. T and Edward, J. K. P. 2002. Antimicrobial activity in the tissue extractsof five species of cowries, Cypraea spp. (Mollusca: Gastropoda) and an ascidian,Didemnum psammathodes (Tunicata: Didemnidae). Indian I Mar. Sci. 31: 239-242.
203
12. Anand, P. T. 2002. Screening for bioactive metabolites producing marine bacteriafrom Tuticorin coastal waters, Southeast coast of India. Ph. D Thesis submitted toManonmaniam Sundaranar University, Tirunelveli. Pp. 1-171
13. Anand, T. P., Rajaganapathi, T and Edward, J. K. P. 1997. Antibacterial activity ofmarine molluscs from Porto Novo region. I. I Mar. Sci. 26: 206-208.
14. Anderson, R. J., Wolfe,. M. S and Faulkner, D. J. 1974. Autotoxic antibioticproduction by a marine chromobacterium. .1. Mar. Biol. 27: 281-285.
15. Andrews, G. P and Martin, S. E. 1979. Catalase activity during the recovery ofheat-stressed Staphylococcus aureus MF-3 1. Appi. Environ. Microbiol. 38 (3):
390-394.
16. Anjaneyulu, A. S. R., Murthy, M. V. R. K., Gown, P. M., Venugopal, M. J. R. Vand Laatsch, H. 2000. A rare prostglangin from the soft coral, Sarcophyton
crassocaule of the Indian Ocean. J. Nat. Prod. 63: 1425-1426.
17. Asakura, W., Matsumoto, K., Ohta, H and Watanabe, H. 1993. Effects of alpha 2-adrenergic drugs on REM sleep deprivation-induced increase in swimmingactivity. Pharmacol. Behav. 46: 111-115.
18. Austin, B and Intosh, D, M. 1988. Natural antibacterial compounds form thesurface of the rainbow trout, Salmo gairdneri Richardson. I Fish. Dis. 11: 257-275
19. Baird, R. M and Lee, W. H. 1995. Media used in the detection and enumeration ofStaphylococcus aureus. mt. J. Food. Microbiol. 26: 15-24.
20. Baker, J. T and Duke, C. C. 1974. Precursors of Tyrian Purple. Food- Drugs from
the Sea Proceedings, pp.345-353.
21. Bakus, G. J. 1987. Coral Reefs and Marine Bioactive Substances. Sci. Rep. (India)
24 (6): 327-334.
22. Balogh, J. C and Anderson J. L. 1992. Environmental impacts of turf grasspesticides. In: Balogh, J. C. Walker, W. J. (Ed). Environmental issues, Lewis
publishers, pp.221 -354.
23. Bandurraga, M. M and Fenical, W. 1985. Isolation of the Muricins: evidence ofchemical adaptation in the marine octocoral Muricea fruiticosa (Gargonacea).
Tedrahedron, 41: 1057-1065.
24. Banerji, A. 1992. Biotechnical potential of nature products. Bioelectrochemistry
and Bioenergetics, 27: 105-113.
25. Bangkok Post, 2000. Consumer protection-calls for less farm use of chemicals,concern rose over food contamination, April, p-7.
204
26. Barbieri, E., Barry, K., Child, A and Waiñwright, N. 1997. Antimicrobial activityin the microbial community of the accessory nidamental gland and egg cases ofLoligo pealei (Cephalopoda: Lolignidae). Biol. Bullet. 193: 275-276.
27. Barja, J. L., Lemos, M. L and Toranzo, A. E. 1989. Purification andcharacterization of an antibacterial substance produced by a marine Alteromonas
sp. Antimicrobial. Agent. Chemother. 33 (10): 1674-1679.
28. Becerro, M. A., Goetz, V. J and Scheuer, P. J. 2001. Chemical defenses of thesacoglossan mollusc Elysia rufescens and its host alga Bryopsis sp. J. Chem. Ecol.
27: 2287-2299.
29. Becerro, M. A., Lopez, N. I., Turon, X and Uniz, M. J. 1994. Antimicrobialactivity and surface bacterial film in marine sponges. J Exp. Mar. Biol. Ecol. 179:
195-205.
30. Belarbi, E. H., Gomez, A. C., Chisti, Y., Camacho, F. G and Grima, E. M. 2003.Producing drugs from marine sponges. Biotechnol. Adv. 21: 585-598.
31. Benkendorff, K, Davis, A. R and Bremer, J. B. 2001a. Chemical defense in the eggmasses of benthic invertebrates: An assessment of antibacterial activity in 39mollusks and 4 polychaetes. I Invert. Pathol. 78: 109-118.
32. Benkendorff, K., Bremner, J. B and Davis, A. R. 2001b. Indole derivatives fromthe egg masses of muricid molluscs. Molecules, 6: 70-78.
33. Benkendroff, K., Davis, A and Be=er, J. B. 2001c. Indole derivatives from theEgg masses of muricid molluscs. Molecules, 6: 79-8 1.
34. Bennett, J. W and Bentley, R. 1989. What's in a Name? - Microbial secondarymetabolism. Adv. App!. Microbiol. 14: 1-27.
35. Benny, A., Murugan, A and Ayyakannu, K. 1995. Extraction of heparine likesibstances from the Muricid Gastropods Chicoreus ramosus and Chicoreus
virgineus. Phuk. Mar. Biol. Cent. Spi. Pub. 15: 197-198.
36. Bergmann, W and Burke, D. C. 1955. Contributions to study of marine products,39: the nucleosides of sponges, III; spongothymidine and spongouridine. .1. Org.
Chem. 20: 1501-1507.
37. Bergmann, W. and Feeney, R. J. 1951. Contributions to the study of marineproducts, 32: the nucleosides of sponges. I Org. Chem. 16: 981-987.
38. Bergmann, W., Lieblich, L., Cohen, E and Ganchrow, J. R. 1985. Influences ofintake of sweet solutions on the analgesic effect of all low dose of morphine inrandomly bred rats. Behav. Neural. Biol. 44: 347-353.
205
39. Berrue, F., Thomas, 0. P., Laville, R., Prado, R., Golebiowski, J., Fernandezc, Rand Arnadea, P. 2007. The marine sponge Plakortis zyggompha: a source oforiginal bioactive polyketides. Tetrahedron, 63:2328-2334.
40. Betina V. 1983. The Chemistry and Biology of Antibiotics. Amsterdam(Netherland) Elsevier. P-13.
41. Blunt, J. W., Copp, B. R., Munro, M. H. G., Northcote, P. T and Prinsep, M. R.2005. Marine natural products. Nat. Prod. Rep. 22: 15-61.
42. Bolinches, J., Lemos, M. L and Barja, J. L. 1988. Population dynamics ofheterotrophin bacterial communities associated with Fucus vericulosus and Ulvarigida inanestuary. Microbial. Ecol. 15:25-34.
43. Boyd, K. G., Sprag, M and Burgess, J. G. 1999. Screening of marine bacteria forthe production of microbial repellents using a spectrophotometric chemotaxisassay. Mar. Biotechnol. 1: 359-363.
44. Brecknell, D. J., Collett, L. A., Coleman, D. M. T., Garson, M. J and Jones, D. D.2000. New Non-contiguous polypropionates from marine molluscs: A comment ontheir natural product status. Tetrahedron, 56: 2497-2502.
45. Broderip, W.J.1832. Description of the Cirrhipeda, Conchifera and Mollusca in thecollection formed by the Officers of H.M.S. Adventure and Beagle employedbetween the years 1826 and 1830 in surveying the southern coasts of SouthAmerica, including the Straits of Magellan and the coast of Tierre del Fuego. Zool.Jb. 5: 332-349.
46. Broussallis, A. M., Ferraro, G. E., Martino., V. S., Pinzon, R., Coussiom, J. D andAlvearez, J. C. 1999. Argentine plants as potential source of insecticidalcompounds. Ethnopharmacol. 67: 219-223.
47. Bucker, E. R and Martin, S. E. 1982. Effect of free-radical scavengers onenumeration of thermally stressed cells of Staphylococcus aureus MF-3 1. Appi.Environ. Microbial. 43 (5): 1020-1025.
48. Burgess, J. G., Jorden, E. M., Brefu, M., Mearns-Spragg, A and Boyd, K. G. 1999.Microbial antagonism: a negleted avenue of natural products research. I Bacteriol.70: 27-32.
49. Burkholder, P. R., Pfister, R. M and Leitz, F. P. 1966. Production of pyrroleantibiotic by a marine bacterium. Appl. Microbiol. 14: 649-653.
50. Burnell, J. N., Doyle, J. R., Woodward, J. R and Llewellyn, L. E. 2000. Rapidthroughput biomolecula screening for marine derived C4 plant-specific herbicides,Proc 5th IBMC, 2000. Australia, p-24.
206
51. Busta, F. F and Jezeski, J. J. 1963. Effect of sodium chloride concentration in anagar medium on growth of heat-shocked Staphylococcus aureus. Appl. Microbiol.11: 404-407.
52. Calabrese, J. P and Bissonnette, G. K. 1990. Improved detection of acid minewater stressed coliforms bacteria on media containing catalase and sodiumpyruvate. Can. I Microbiol. 36: 544-550.
53. Capon, R. J., MacLeod, J. K and Scammells, P.J. 1986. The Trikentrins: novelindoles from the sponge Trikentrion flabellforme. Tetrahedron, 42 (23): 6545-6650.
54. Canesi, L., Gallo, G., Gavioli, M and Pruzzo, C. 2002. Bacteria hemocyteinteractions and phagocytosis in marine bivalves. Microsc. Res. Tech. 57: 469-476.
55. Cardozo, K. H. M., Guaratini, T. Barros, M. P., Falcao, V. R., Tonon, A. P.,Lopes, N. P., Campos, S, Torres, M. A., Souza, A. 0., Colepicolo, P and Ernani,P. 2006. Metabolites from algae with economical impact. Comparat. Biochem.Physiol. Part C. Press. Pp. 11-39
56. Carte, B. K. 1996. Biomedical Potential of Marine Natural Products. Bioscience,46 (4): 271-286.
57. Chang, G. W., K. Joyce, J. K. Fuji, and R. A. Lum. 1993. Coliforms in OceanWaters: New Methodology for Resuscitating Seawater Injured E. coli. Proceedingsof the 66th Annual Conference of the Water Environment Foundation. Anaheim,CA. Pp.78-83.
58. Charlet, M., Chernysh, S., Philippe, H., Hetru, C., Hoffmann, J. A and Bulet, P.1996. Innate immunity. Isolation of several cystein-rich antimicrobial peptide fromthe blood of mollusc, Mytilus edulis. I Biol. Chem. 271: 21808-21813.
59. Chellaram C., Gnanambal, K. M. E and Edward, J. K. P. 2005 a. Isolation andscreening of mucus associated bacteria of the gastropod, Drupa margariticola forantagonistic activity. I Mar. Biol. Ass. India. 47 (2): 154-159.
60. Chellaram C., Gnanambal. K. M. E and Edward. J. K. P. 2004 b. Antibacterialactivity of the winged oyster, Pteria chinensis (Pterioida: Pteridae). Indian I Mar.
Sd. 33 (4): 369 -372.
61. Chellaram, C., Gnanambal, K. M. E and Jamila Patterson, 2005 b. Insecticidal andherbicidal activity of a gorgonid associated winged oyster, Pteria chinensis. In:Proc. Nat. Sem. Reef Ecosystem Remediation. SDMRI Research Publication, 9:
178-181.
207
62. Chellaram, C., Patterson, J and Edward, J.K.P. 2006. Distribution of Red-banddisease and effect of boring sponges on solitary corals in Tuticorin coastal waters,Gulf of Mannar, Southeastern coast of India. Proc. Of 10th mt. Symp. Pp.133-137.
63. Chelossi, E., Milanese, M., Milano, A., Pronzanto, R and Riccardi, G. 2004.Characterization and antimicrobial activity of epibiotic bacteria from Petrosia
JIciformis, (Porifera: Demospongiae). I Exp. Mar. Biol. Ecol. 309: 21-33.
64. Choudhury, S., Pattnaik, P., Sree, A., Bapuji, M and Mukherjee, S. C. 2003.Antibacterial activity of sponge extracts against fish pathogens. Aquacul. Res. 34(12): 1075-1077.
65. Chrsitophersen, C. 1983. Marine indoles. In: Marine natural products, chemicaland biological perspectives, Vol. 5. Paul J. Scheuer (Ed). Academic Press Inc. NewYork. Pp.259-285.
66. Chu, F. L. E. 1988. Humoral defense factor in Marine bivalves. Am. Fish. Soc. Sec.Pubi. Pp. 18178-18188.
67. Cimino, G., Crispino, A., Gavagnin, M., Zubia, E., Trivellone, E. 1992.Biosynthesis in opisthobranch molluscs: General outline in the light of recent useof stable isotopes. I Nat. Prod. 56: 1642-1646.
68. Cimino, G., Gavagnin, M., Sodano, G., Puliti, R., Mattia, C. A and Mazzarella, L.1988. Verrucosin-A and -B, ichthyotoxic diterpenoic acid glycerides with a newcarbon skeleton from the dorid nudibranch Doris verrucosa. Tetrahedron, 44:2301-2310.
69. Cimino, G., Crispino, A.., Gavagnin, M., Zubia, E., Trivellone, E. 1993.Biosynthesis in opisthobranch molluscs: General outline in the light of recent useof stable isotopes. I Nat. Prod 56: 1642-1646.
70. Collier, H. 0. J., Dinneen, L. C., Johnson, C. A and Schneider C. 1968. Theabdominal constriction response and its suppression of analgesic drugs in mouse.Br. J. Pharmacol. 32: 295 -310
71. Costerton, J. W and Geesey, G. G. 1979. Which populations of aquatic bacteriashould we enumerate? In: Costerton J. W, Clowell, R. R (Eds). Native aquaticbacteria: enumeration, activity and ecology. American Society for Testing andMaterials, Philadelphia, pp.7-18.
72. Coulson, F. R and O'Donnell, S. R. 2000. The effects of contignasterol (IZP-94,005) on allergen-induced plasma protein exudation in the tracheobronchialairways of sensitized guinea-pigs in vivo. Inflam. Res. 49: 123-127.
208
73. Coval, S. J and Scheuer, P. J. 1985. An intriguing C16-alkadienone-substituted 2-pyridine from a marine mollusc. I Org. Chem. 50: 3024-3025.
74. Cragg, G. M., Newman, D. J and Snader, K. M. 1997 a. Natural products in drugdiscovery. I Nat. Prod. 60: 52-60.
75. Cragg, G. M., Newman, D. J and Weiss, R. B. 1997 b. Coral reefs, forests andthermal vents: the worldwide exploration of nature for novel antitumour agents.Semin. Oncol. 24: 156-163.
76. Graziani, E. I., Andersen, R. J., Krug, P. J and Faulkner, D. J. 1996. Stable isotopeincorporation evidence for the de novo biosynthesis of terpenoic acid glycerides bydorid nudibranchs. Tetrahedron, 52: 6869-6878.
77. Crombie. L. 1990. Natural models for the design of insect control compounds: themammerins, In: Recent advances in the chemistry of insect control II, The RoyalSociety of Chemistry Special Publication, (Crombie.L,Edn), Cambridge, England,pp.23-Si
78. Czechowicz, S. M., Santos, 0 and Zottola, E. A. 1996. Recovery of thermally-stressed Escherichia co/i 0157:H7 by media supplemented with pyruvate. mt. iFood. Microbiol. 33: 275-284.
79. Dana, J. D. 1846. Zoophytes. United States Exploring Expedition 1838-1842,Philadelphia, 7: 1-120.
80. Davidson, B.S. 1995. New dimensions in natural products research: culturedmarine microorganisms. Curr. Opin. Biotechnol. 6: 284-291.
81. Davis-Coleman, M. T and Faulkner, D. J. 1991. New diterpenoic acid glyceridesfrom the Antarctic nudibranch Austrodoris kerguelensis. Tetrahedron, 47: 9743-9750.
82. De Silva E. D. 1980. Manoalide, an antibiotic sesterterpenoid from the marinesponge Luffariella variabilis. Tetrahedron Lett. 2:1611-1614.
83. De Varries, D. J and Bearts, P. M. 1995. Fishing for drugs from the sea: Status andstrategies. TiPS. 16: 275-279.
84. Del Val, A. G., Basilio, G. P., Cabello, A., Suay, J. G., Vicente, F., Rio, E. P.,Reina, G. G and Pelaez, F. 2001. Screening of antimicrobial activities in red, greenand brown macroalgae from Gran Canaria (Canary Islands, Spain). Integ.
Microbiol. 4: 35-40.
Offirt
85. Diaz-Marrero, A. R., Dorta, E., Cueto, M., Rovirosa, J., San- Martin, A., Loyola,A and Darias, J. 2003. Labdane diterpenes with a new oxidation pattern from themarine Pulmonate Trim usculus peruvianus. Tetrahedron, 59: 4805-4809.
86. Di Salvo, L and Gundersen, K. 1971. Regenerative functions and microbialecology of coral reefs. An assay for microbial population. Can. J Microbiol. 17:1081-1089.
87. Dobler, W. I., Beil, W., Lang, S., Meiners, M and Laatsch, H. 2002. Integratedapproach to explore the potential of marine microorganisms for the production ofbioactive metabolites. Adv. Biochem. Eng. Biotechnol. 74: 207-238.
88. Dobretsov, S and Qian, P. 2004. The role of epibiotic bacteria from the surface ofthe soft coral, Dendronephthya sp. in the inhibition of larval settlement. I Exp.Mar. Biol. Ecol. 299: 35-50.
89. Dopazo, C. P., Lemos, M. L., Lodeiros, C., Bolinches, J., Barja, J. L and Toranzo,A. E. 1988. Inhibitory activity of antibiotic producing marine bacteria against thefish pathogens. J. Appi. Bacteriol. 65: 97-101.
90. Dorow, E. 1993. Present practices of controlling desert locust outbreaks. In: Newstrategies for locust control. Ed: Tembold, H, ATSAF. Bonn. 89: 7-8.
91. Douillet, P. A and Langdon, C. J. 1994. Use of a probiotic for the culture of larvaeof the Pacific oyster, (Crassostrea gigas Thunberg), Aquaculture, 119: 25-40.
92. Duke, S. 0., Menn, J. J and Plimmer, J. R. 1993. Challenges of pest control withenhanced toxicological and environmental safety, an overview. In Pest control withEnhanced Environmental safety; ACS Symp. Ser. p-524; Duke, S. 0., Menn, J. Jand Plimmer, J. R., Eds: Am. Chem. Soc.: Washington, DC. Pp. 1-13.
93. Edward, J. K. P and Murugan, A. 2000. Screening of cephalopods for bioactivity.Phuket. Mar. Biol. Cent. Pubi. 21: 253-256.
94. Edward, J. K. P., Jamila, P., Venkatesh, M., Mathews, G., Chellaram, C andWilhelmsson, D. 2004. A field guide to study to stony corals (scleractinia) oftuticorin in Gulf of Mannar, southeast coast of India. SDMRI Spl Res. Pubi. 4: 1-
80.
95. Einhellig, F. A., Leather, G. R and Hobbs, L. L. 1985. Use of Lemna minor L. as abioassay in allelopathy. I Chem. Ecol. 11: p-65.
96. Ellis and Solander, D. 1786. The natural history of many curious and uncommonZoophytes from various parts of the globe. London. P-206.
210
97. Ely, R., Supriya, T and Naik, C. G. 2004. Antimicrobial activity of marineorganisms collected off the coast of South East India. I Exp. Biol. 309: 121-27.
98. Elzen, G. W. 2001. Lethal and sublethal effects of insecticide residues on Onusinsidiosus (Hemipter: Anthocoridae) and Geocoris punctipes (Hemiptera:Lygaeidae). I. Econ. Entomol. 94: 55-59.
99. Emori, T. C and Gaynes, R. 1993. An overview of nosocomial infections,including the role of microbiology laboratory. Clinical Microbiol. Rev. 6: 428-442.
100. Esper, E. J. C. 1794. Die Pflanzenthiere in Abbildungen nach der Natur mit Farbenerleuchtet nebst Beschreibungen. Zweyter Theil. Mit hundert und sechtilluminirten Kupfertafeln. Pp. 1-303.
101. European Science Foundation (ESF). Marine Board Feasibility Study GroupReport, 2001. A European Strategy for marine biotechnology. Paper. 4. 1-26.
102. Fattorusso, E., Parapini, S., Campagnuolo, C., Basilico, N., Scafati, O.T andTaramelli, D. 2002. Activity against Plasmodium faciparum of cylcioperoxidecompounds obtrained from the sponge, Plakortis simplex. I Antimicrobial.Chemother. 50: 883-88.
103. Faulkner, D. J. 1977. Interesting aspects of marine natural products chemistry.Tetrahedron Letters, 33 (28): 1421-43.
104. Faulkner, D. J and Ghiselin, M. T. 1983. Chemical defense and evolutionaryecology of dorid nudibranchs and some other opisthobranch gastropods. Mar.Ecol.Prog. Ser. 13: 295-301.
105. Faulkner, D. J. 1988. Feedinf deterents in molluscs. Pages 29-36 in Fautin D. G,ed. Biomedical importance of marine organisms. San Francisco (CA): CaliforniaAcadamy of sciences.
106. Faulkner, D. J. 1992. Biomedical use for natural marine chemicals. Oceanus, 35:29-35.
107. Faulkner, D. J. 1995. Chemical Riches from the Ocean. Chem. Brit. Pp.680-684.
108. Faulkner, D. J. 2000 a. Highlights of marine natural products chemistry (1972-1999). Nat. Prod. Rep. 17: 1-6.
109. Faulkner, D. J. 2000 b. Marine Pharmacology. Antonie Leeuwenhoek, 77 (2): 135-145.
110. Faulkner, D. J. 2003. Marine Natural Products. Nat. Prod. Rep. 17: 7-55
211
111. Fenical, W. 1981. Investigation of benthic marine macro algae as a resource fornew pharmaceuticals and agricultural chemicals. Proceedings of Joint US-ChinaPhycological symposium, Oingdao, China. Pp. 32-35
112. Fenical, W. 1993. Chemical Studies of Marine Bacteria: Developing a NewResource. Chemical. Rev. 93 (5): 1673-1683.
113. Fenical, W. 1997. New pharmaceuticals from marine organisms. Trend.Biotechnol. 15: 339-341.
114. Fenical, W., Sleeper, H. L., Paul, V. J., Stallard, M. 0 and Sun, H. H. 1979.Defensive chemistry of Navanax and related opisthobranch molluscs. Pure. Appi.Chem. 51: 1865-1874.
115. Fernandez, L. F., Reys, F and Sanchez-puelles, J. M. 2002. The marine pharmacy:new antitumoral compounds from sea. Pharmaceutics News. 9: 495-501.
116. Finney, D. J. 1971. A statistical treatment of the sigmoid response curve. In: Probitanalysis, (31d Ed), Cambridge University Press, London, pp.333-336.
117. Flowers, R. S., Martin, S. E., Brewer, D. G and Ordal, Z. J. 1977. Catalase andenumeration of stressed Staphylococcus aureus cells. App!. Environ. Microbiol. 33(5): 1112-1117.
118. Fontana, A., Ciavatta, M. L., D'Souza, L., Mob, Naik, C., G, Parameshvaran, P.,5, Wahidulla, S and Cimino, G. 2001. Selected chemo-ecological studies of marineopisthobranchs from India coasts. I Indian. Inst. Sci. 81: 403-4 15.
119. Fontana, A., Tramice, A., Cutignano, A., d'Ippolito, D., Renzulli, L and G.Cimino, G. 2003. Studies of the Biogenesis of Verrucosins, toxic diterpenoidglycerides of the Mediterranean Mollusc Doris verrucosaEur. I Org. Chem.
Pp.3 104-3 108.
120. Franzen, H. 1993. Need for the development of new strategies for locust control.In: New strategies for locust control. Ed: Rembold, H, ATSAF, Bonn, pp.9-13
121. Fukuyama, Y., Iwatsuki, C., Kodama, M., Ochi, M and Kataoka, K. 1998.Antimicrobial indolequinones from the mid-intestinal gland of the muricidgastropod, Drupellafragum. Tetrahedron, 54: 10007-10016.
122. Fukuyawa, A and Masamune, T. 1981. Laurepinnacin and isolaurepinnacin: newacetylenic cyclic ethers from the marine red alga, Laurencia pinnata Yamada.Tetrahedron Letters, 22: 4081-4084.
123. Gamache, D. A., Povlishock, Jand Ellis, E. F. 1986. Carrageenan induced braininflammation. I Neurosurg. 65: 679-685.
212
124. Garson, M. J. 1989. Biosynthetic-studies on marine natural-products. Nat. Prod.Rep. 6: p-143.
125. Garson, M. J. 1996. 'Is Marine Bioprospecting Sustainable?' Search, 27(4): 114-116.
126. Gast, G. J., Weigman, S., Wieringa, E., Duyl, F. C and Bak, R. P. M. 1998.Bacteria in coral ref water types: removal of cells, stimulation of growth andmineralization. Mar. Ecol. Prog. Series. 167: 37-45.
127. Gauthier M. J. 1976. Modification of the bacterial respiration by a polyanionic
antibiotic produced by a marine Alteromonas. Antimicrobial. Agent. Chemother. 9:349-354.
128. Gavagnin, M., Carbone, M., Mollo, E and Cimino, G. 2003. Further chemical
studies on the Antarctic nudibranch Austrodoris kerguelenensis: new terpenoidacylglycerols and revision of the previous stereochemistry Tetrahedron, 59 (29):5579-55 83.
129. Gavagnin, M., de Napoli, A., Cimino, G., Iken, K., Avila, C and Garcia, F. J.1999. Absolute configuration of diterpenoid diacylglycerols from the Antarctic
nudibranch Austrodoris kerguelenensis, Tetrahedron: Asymmetry, 10: 2647-50.
130. Gavagnin, M., Spinella, A., Cimino, G and Sodano, G. 1990. Stereochemistry ofichthyotoxic diacyiglycerols from opisthobranch molluscs. Tetrahedron Letter, 31:6093-6094.
13 1. Gavagnin, M., Trivellone, E., Castelluccio, F., Cimino, G., Cattaneo-Vietti, R.
1995. Glyceryl ester of a new halimane diterpenoic acid from the skin of theAntarctic nudibranch Austrodoris kerguelenensis Tetrahedron Letter, 36
(40):7319-7322.
132. Gavagnin, M., Ungur, N., Castelluccio, F., Cimino, G. 1997. Novel verrucosinsfrom the skin of mediterranean nudibranch Doris verrucosa tetrahedron, 53: 1491-1504.
133. Gavagnin, M., Ungur, N., Castelluccio, F., Muniain, C., Cimino, G. 1999. Newminor diterpenoid diacylglycerols from the skin of the nudibranch Anisodoris
fontaini. J. Nat. Prod. 62: 269-274.
134. GESAMP. 1997. Towards safe and effective use of chemicals in coastalaquaculture. Reports and Studies. Rome, FAO. 65: 40-46
135. Giannini, C., Zampella, A., Debitus, C., Menou, J. L., Roussakis, C and D'Auria,M. V. 1999. Isolation and structural elucidation of the Crellastatins I-M: Cytotoxic
213
Bis-Steroids derivatives from the Vanuatu marine sponge Crella sp. Tetrahedron,55: 13749-13756.
136. Glombitza, K. W and Knöss, W. 1992. Phioroglucinol compounds of naturalorigin. Phytochemistry, 31: p-279.
137. Gmelin, J. F. 1791. Caroli a Linné, systema naturae. Tom. I. Pars VI. Pp.3821-3910.
138. Gnanambal, K. M. E., Chellaram, C and Jamila Patterson. 2005 a. Isolation ofantagonistic marine bacteria from the surface of the gorgonian corals at Tuticorin,Southeast coast of India. Indian I Mar. Sci. 34 (3): 316-319.
139. Gnanambal, K. M. E., Chellaram, C and Edward, J. K. P. 2005 b. Inscticidalactivity of the whole body extracts of Turbo intercostalis (Archaegastropoda:Turbnidae) and Cerithidea cingulata (Caenogastropoda: Potamiddidae). Nat.Symp. RETFER. Pp. 132-141.
140. Gnanambal, K. M. E., Chellaram, C. and Jamila Patterson, 2005 c. Antibacterialactivity of whole body extracts of Trochus radiatus (Mollusca: Gastropoda). In:Proc.Nat. Sem. Reef Ecosystem Remediation. SDMRI Research Publication, 9:182-186
141. Goh, N. K. C., Sim, T. S and Chou, L. M. 1995. Bioactivity of Gorgonians (Sub-Class-Octocorallia) in Singapore: Preliminary studies. Marine Research, 4 (1): 33-46.
142. Gonzalez, J. B., Fernandez, F. J and Tomasini, A. 2003. Microbial secondarymetabolites production and strain improvement. 1 .1 Biotechnol. 2: 322-333.
143. Gorbach, S. L. 2001. Editorial: Antimicrobial use in animal feed- Time to stop",The New England Journal of Medicine, 345 (16): 1-3.
144. Gorshkova, N. M., Lanova, E. P., Sergeev, A. F., Zhukova, N.V., Alexeeva, Y.,Wright, J. P., Nicolau, D. V., Mikhilov, V. V and Christen, R. 2003. Marinobacterexcellens sp. Nov., isolated from sediments of the Sea of Japan. J. System.
Evolution. Microbiol. 53: 2073-2078.
145. Goud, T. V., Srinivasulu, M., Reddy, V. L. N., Reddy, A. V., Rao, T. P., Kumar,D. S., Murty, U. S and Venkateswarlu, Y. 2003. Two new bromotyrosine derivedmetabolites from the sponge Psammaplysilla purpurea. Chem. Pharmacol. Bullet.
51 (8): 990-993.
146. Grabley, S and Thiericke, R. 1999. Bioactive agents and natural sources: trends indiscovery and application. Adv. Biochem. Eng. Biotechnol, 64: 101-154.
214
147. Gray, J. E. 1847. An account of Pablo, a sea worm eaten in the. Navigator Islands.Proc. Zool. Soc. London 15: 17-18.
148. Gregory, J. W. 1900. The corals. Jurassic fauna of Cutch. Palaeontologica. Indica.Ser. 9 (2): 1-195.
149. Grode, S. H., James, T. B and Cardellina, J. H. 1983. Molecular structures ofBriantheins, new insecticidal diterpenes from Briareum polyanthes. J Org. Chem.48:5203 -5207.
150. Grossart, H. P., Schlingloff, A., Bernhard, M., Simon, M and Brinkhoff, T. 2004.Antagonistic activity of bacteria isolated from organic aggregates of the GermanWadden Sea. FEMS. Microbial. Ecol. 47: 387-396.
151. Groweiss, A., Shmeuli, U., Zadock, E., Kashman, Y and Neeman, I. 1974.Sarcophine, a new epxy cembranoid from amrine origin. Tetrahedron, 30: 2817-2824.
152. Gudbjarnason, S. 1999. Bioactive marine natural products. Rit. Fiskideildar. 16:107-110.
153. Gustafson, K., Andersen, R. J., Chen, M. H. M., Clardy, J and Hochbowski, J. E.1984. Observations on the toxicity and metabolic relationships of polygodial, thechemical defense of the nudibranch Dendrodoris limbata. Tetrahedron Letter, 25:11-14.
154. Gustafson, K and Andersen, R. J. 1985. Chemical studies of British Columbianudibranchs. Tetrahedron, 41: 1101-1108.
155. Hamann, M. T., Scheuer, P. J. and Kahalalide, F. 1993. A bioactive depsipeptidefrom the sacoglossan mollusk Elysia rufescebs. I Am. Chem. Soc. 115: 5825-5826.
156. Hao, E., Fromount, J., Jardine, D and Karuso, P. 2001. Natural products fromsponges of the genus Agelas- on the tail of a [2+2]-Photoadditon enzyme.Molecules, 6: 130-141.
157. Harborne, J. B and Williams, C. A. 1976. Occurrence of sulfated flavones andcaffeic acid esters in the members of Fluviales. Biochem. Syst. Ecol. 4: 37-44
158. Harmon, S. M and Kautter, D. A. 1976. Beneficial effect of catalase treatment ongrowth of Clostridium perfringens. Appi. Environ. Microbiol. 32 (3): 409-416.
159. Harrison, P. G. 1982. Control of microbial growth and of amphipod grazing bywater soluble compounds from the leaves of Zostera marina. Mar. Biol. 67: 225-230
215
160. Harvey, A. 2000. Strategies for discovering drugs from previously unexplorednatural products. Drug Discovery Today, 5 (7): 294-300.
161. Heinments, F. 1953. Reactivation of ultraviolet inactivated Escherichia coli by
pyruvate. Environ. Microbiol. April, pp.455-457.
162. Hellio, C., Pons, A. M., Beaupoil, C., Bourgougnon, N and Gal, Y. L. 2002. 1Antimicrobial. Agent. 20: 214-219.
163. Hocklowski, J. E and D. J. Faulkner. 1983. Antibiotics from marine pulmonates,Siphonaria diemenensis, Tetrahedron Letters, 24: 1917-1920.
164. Hoffman, P. 5., Krieg, N. R and Smibert, R. M. 1979. Studies of themicroaerophilic nature of Campylobacter fetus sub sp. Jeijuni. I. Pysiologicalaspects of enhanced aerotolerance. Can. J. Microbiol. 25: 1-7.
165. Hornsey, L. S and Hide, D. 1974. The production of antimicrobial compounds byBritish marine algae. Brit. I Phycol. 9: 353-361.
166. Hubert, F., Knaap, V. D. W., Noel. T and Roch, P. 1996. Cytonic and antibacterialproperties of Mytilus galloprovincialis, Ostrea edulis and Crassostrea gigas(bivalve, molluscs) haemolymph. Aquatic. Living. Res. 9: 115-124.
167. Huppatz, J. L, 1990. Essential amino acid biosynthesis provides multiple targetsfor selective herbicides. In: Casida J. E (Ed). Pesticides and alternatives. Elsevier,New York, pp.563-572.
168. Hylleberg, J and Kilburn, R. N. 2002. Annotated inventory of molluscs from theGulf of Mannar and vicinity. In: J. Hylleberg and Nateewathana, A, Editors.Zoogeography and inventory of marine molluscs encountered in Southern India.PMBC, Thailand. Phuket. Mar. Biol. Cent. Pub!. 29: p- 1970.
169. lijima, R., Kisugi, J and Yamazaki, M. 2003. A novel antimicrobial peptide fromthe sea hare Dolabe!la auricularia. Dev. Comp. Immunol. 27: 305-311.
170. Ilagedone, M. R., Barreson, B. J and Scheuer, P. J. 1999. Bioactive naturalproducts. Helvetica. Chimica. Acta. 62: 2484-2486.
171. Ireland, C. M., Roll, D. D., Molinski, T. F., McKee, T. C., Zabriske, T. M andSwersey J. C. 1988. Uniqueness of the marine environment: categories of marinenatural products from invertebrates. In Fautin D. G. Ed. Biomedical importance ofmarine organisms. San Francisco (CA): California Academy of Sciences, pp.4 i-S7
172. Ireland, C and Faulkner, D. J. 1978. The defensive secretion of the opisthobranchmollusc Onchidella binneyi. Bioorg. Chem. 7: 125-13 1.
216
173. Ireland, C., Copp, B., Foster, M., McDonald, L., Radisky, D and Sersey, I. 1993.Biomedical potential of marine natural products. Mar. Biotechnol. 1: 1-43.
174. Isnansetyo, A., Cui, L., Hiramatsu, K and Kamei, Y. 2003. Antibacterial activity of2, 4- diacetylphloroglucinol produced by Pseudomonas sp. AMSN isolated from amarine alga, against vancomycin-resistant Staphylococcus aureus. mt .1Antimicrobial. Agent. 22: 545-547.
175. Iwashima, M., Terada, I., Iguchi, K and Yamori, T. 2002. New biologically activemarine sesquiterpenoid and steroid from the Okinawan sponge of the GenusAxinyssa. Chem. Pharmacol. Bullet. 50 (9): 1286-1289.
176. Izac, R. R., Bandurruga, M. M., Wasylyk, J. M., Dunn, F. W and Fenical, W. 1982.Germacrene derivatives from diverse marine soft-corals (Octacorallia).Tetrahedron, 38: p-301.
177. Jacobs, R. S., Culver, P., Langdon, R., Brien, T. 0 and White, S. 1985. Somepharmacological observation on marine natural products. Tetrahedron, 41 (6): 981-984.
178. Jaimun, R and Coe, E. L. 1987. Bromoperoxidase from the marine snail Murextrunculus. Comp. Biochem. Physiol. 88 B: 917-922.
179. Jayanth, K., Jeyasekaran, G and Shakila, J. 2002. Isolation of marine bacteria,antagonistic to human pathogens. I J. Mar. Sci. 31 (1): 39-44.
180. Jayaseeli, A., Anand, T. P and Murugan, A. 2001. Antibacterial activity of 4bivalves from Gulf Mannar. Phuket. Mar. Biol. Cent. Pubi. 25: 215-217.
181. Jenkins, C. L., Da Harris, R. L. N and Mc Fadden, H. G. 1987. 3,3-Dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate, a new specific inhibitor ofphosphoenolpyruvate (PEP) carboxylase. Biochem. Int.14: 219-226.
182. Jensen, P. R and Fenical, W. 1994. Strategies for the discovery of secondarymetabolites from marine bacteria: Ecological Prospective. Ann. Rev. Microbiol.48:.559-584.
183. Jensen, P. R., Jenkins, K. M., Porter, D and Fenical, W. 1998. Evidence that a newantibiotic flavone glycoside chemically defends the seagrass Thalassia testudinum
against zoosporic fungi. Appl.Environ. Microbiol. 64 (4): 1490-1496.
184. Jensen, P. R., Mincer, T. J and Fenical, W. 2003. The true potential of the marinemicroorganism. Feature, January, pp. 17-19.
185. Jeong, S. J., Inagaki, M., Higuchi, R., Miyamoto, T., Ono, M., Kuwano, M andSoest, W. M. V. 2003. 1,3 Dimethylisoguaninium, and Antiangiogenic Purine
217
analog from the sponge Amphimedon paraviridis. Chem. Pharmacol. Bullet. 51(6):731-733.
186. Jeyasekaran, G., Jayanth, K and Jeya Shakila, R. 2002. Isolation of marinebacteria, antagonistic to human pathogens. Indian J. Mar. Sci. 31: 39-44.
187. Jimeno, J. M. 2002. A clinical armamentarium of marine derived anti-cancercompounds. Anticancer Drugs, 13 (1): 515-519.
188. Joffe, S and Thomas, R. 1989. Phytochemicals: a renewable global resource.AgBiotech News Information, 1: 696-700
189. Kabaru, J. M and Gichia, L. 2001. Insecticidal activity of extracts derived fromdifferent parts of the mangrove tree Rhizophora muncronata (Rhizophoraceae)LAM. Against three arthropods, Afri. J. Sci. Technol. 2 (2): 44-49.
190. Kagoo E. L and Ayyakarmu, K. 1992. Bioactive compounds from Chicoreusramosus- antibacterial activity-in vivo, Phuket. Mar. Biol. Cent. Spi. Publ. 11:147-150.
191. Kamel, H. N., Fronczek, F. R., Fisher, N. H and Slattery, M. 2004. A novelmetabolite from the hybrid soft coral Sinularia maxima and Sinularia polydaclyla:a biosynthetically mixed skeleton lining cembrane and africanane terpenoids.Tetrahedron Letters, 45: 1995-97.
192. Kamiya, H., Muramoto, K., Goto, R., Sakai, M., Endo, Y and Yamazaki, M. 1989.Purification and characterization of an antibacterial and antineoplastic proteinsecretion of a sea hare, Aplysiajuliana. Toxicon, 27: 1269-1277.
193. Kanarek, R. B., White, E. S., Biegen, M. T and Marks-Kaufman, R. 1991. Dietaryinfluences on morhine-induced analgesia in rats, Pharm. Biochem. Behav. 38 (3):681-684.
194. Kang,H. S., Chung, H. Y., Jung, J. H., Son, B. W and Choi, J. S. 2003. A newphiorotannin from the Brown Alga Ecklonia stolonfera. Chem. Pharmacol. Bullet.51(8): 1012-1014.
195. Karuso, P and Scheuer, P. J. 2002. Natural Products from three Nudibranchs:Nembrotha kubaryana, Hypselodoris infucata and Chromodoris petechialis.
Molecules, 7: 1-6.
196. Kelly, M. S. and McKenzie, D. J. 1992. The quantification of sub-cuticularbacteria in enhinoderms. In: L. Scalera-Liaci, C. Canicatti (Eds), EchinodermResearch 1991. Rotterdam: Balkema, pp.225-228.
218
197. Kelman, D., Kashman, Y., Rosenberg, E., Ilan, M., lirach, I and Loya, Y. 2001.Antimicrobial activity of the reef sponge Amphimedon viridis from the Red Sea:evidence for selective toxicity. Aquacul. Microbial. Ecol. 24: 9-16.
198. Kernan, M. R and Faulkner, D. J. 1987. Halichondramide, an antifungal macrolidefrom the sponge, Halichondria sp. Tetrahedron Letters, 28: 2809-2812.
199. Kerr, R. G and Kerr, S. S. 1999. Marine natural products as therapeutic agents.Exp. Opin. Therapy. Pattern. 9: 1207-1222.
200. Kijjoa, A and Sawangwong, P. 2004. Drugs and Cosmetics from the Sea. MarineDrugs, 2: 73-82.
201. Koh, E. G. L. 1997. Do scieractinian corals engage in chemical warfare againstmicrobes? I Chem. Ecol. 23: 379-398.
202. Kokke, W. C. M. C., Epstein, S., Look, S. A., Rau, G. H., Fenical, W and Djerassi,C. 1984. On the origin of terpenes in symbiotic associations between marineinvertebrates and algae (Zooxanthellae). I. Biol. Chem. 259 (13): 8168-8173.
203. Korkmaz, S and Wahistrom, G. 1997. The EEG burst suppression threshold test forthe determination of CNS sensitivity to intravenous anesthetics in rats. BrainResearch Protocol, 1: 378-384.
204. Koster, R., Anderson, M and De Beer, E. J. 1959. Acetic acid for analgesicscreening. Fed. Proc. 18: 412-145
205. Kreig, N. R and Hoffman, P. S. 1986. Microaerophily and oxygen toxicity. AnnuRev. Microbiol, 40: 107-130.
206. Krug, P. J., Boyd, K. G., Faulkner, D. J. 1995. Isolation and synthesis of tanyolidesA and B, metabolites of the nudibranch Sclerodoris tanya. Tetrahedron, 51:11063-11074
207. Kulkarni, S. K., Mehta, A. K and Kunchady, J. 1986. Anti-inflammatory actionsof clonidine, guanfacine and B-HT 920 against various inflammagen-induced acutepaw edemas in rats. Arch. mt. Pharmacodyn. 279: 324-334.
208. Kumar, S. S. 2003. Studies on the cowries (Mollusca: Gastropoda: Cypraeidea) ofGulf of Mannar, Southeast coast of India, Ph. D Thesis submitted toManonmaniam Sundaranar University, Tirunelveli. Pp. 1-186.
209. Lamarck, J. B. P. 1816. Histoire naturelle des animaux sans vertèbres. Paris. 2: p-
568.
210. Lech, M. A. 2004. A shot in the arm. Nature, 431: 892-893.
719
211. Lei, J and Zhou, J. 2002. A Marine Natural Product Database. J. Chem. Informt.Comp. Sci. 42: 742-748.
212. Lemos, M. L., Toranzo, A. E and Barja, J. L. 1985. Antibiotic activity of epiphyticbacteria isolated from inter-tidal seaweeds. Microbial. Ecol. 11: 149-163.
213. Levy, S. B. 1998. The challenge of antibiotic resistance. Scintfic Am. March,pp.46-53.
214. Lewis, J. B and Price, W. S. 1975. Feeding mechanisms and feeding strategies ofAtlantic reef corals. Zool. Land, 176: 527-544.
215. Li, C. P., Goldin, A and Hartwell, J. L. 1974. Antineoplastic substrances from thesea: A review. Cancer Chemother. Rep. 4: p-97.
216. Lin, Y. C., Huang, Y. L., Khalil, A. T., Chen, M. H and Shen, Y. C. 2005.Juncenolides F and G, Two new Briarane Diterpenoids from Taiwanese GorgonianJunceellajuncea. Chem. Pharmacol. Bullet. 53 (1): 128-130.
217. Loukaci A., Bultel-Ponce V., Longeon A., Guyot M., 2000. New lipids from thetunicate Cystodytes cf. dellechiajei, as PLA2 inhibitors. I Nat. Prod. 63: 799-802
218. Lyewellyn, L. E and Burnell, J. N. 2000. Marine organisms as sources of C4-Weed-Specific herbicides. Biopesticides Outlook, April. Pp.64-67.
219. Look, S. A., Fenical, W., Jacobs, R. S and Clardy, J. 1986. The Pseudopterosins:Anti-Inflammatory and Analgesic Natural Products from the Sea WhipPseudopterogorgia elisabethae Proc. Nat. Acad. Sci. U. S. A. 83 (7): 6238-6240.
220. Long, R. A and Azam, F. 2001. Antagonistic interatctions among marine pelagicbacteria. Appi. Environ. Microbiol. 67: 4975-4983.
221. Lozano, M. B., Farias, F. G., Acosta, B. G., Gasca, A. G and Zavala, J. R. B. 1998.Variation of antimicrobial activity of the spong Aplysina fistularis (Pallas, 1766)and its relation to associated fauna. I Exp. Mar. Biol. Ecol. 223: 1-18.
222. Lynch, W. E. 2004. Duckweed and Watermeal: Prevention and Control, The OhioState University extension Fact sheet. 14: 1-4.
223. Lyndon, E. L. 2000. Marine organisms as sources of C 4-weed-specific herbicides.In: Biopesticides, Pesticide Outlook, (ed, Copping, L). Pp.64-67
224. Ma, D., Hu, Y., Wang, J., Ye, S and Li, A. 2005. Effects of antibacterials use inaquaculture on biogeochemistry processes in marine sediment. Sci. Total. Environ.
367 (1): 273-277.
220
225. Maeda, M. 1994. Biocontrol of the larvae rearing biotope in aquaculture. Bulletinof the Natural Research Institute of Aquaculture, Supplement 1: 71-74.
226. Maeda, M and Liao, C. 1992. Effect of bacterial population on the growth of theprawn larvae, Penaeus monodon. Bullet. Nat. Res. Inst. Aquacul. 21: 25-29.
227. Mandal, S. K., Boominathan, R., Parimaladevi, B., Dewanjee, S and Mandal, C.2005. Analgesic activity of methanol extract of Eupatorium adenophorum Sreng.Leaves. Indian J. Exp. Biol. 43: 662-663.
228. Maoka, T., Fujiwara, Y., Hashimoto, K and Akimoto, N. 2005. Structure of newcarotenoids with a 3,4 Dihydroxy B end Group from the Oyster Crassostrea gigas.Chem. Pharmacol. Bullet. 53 (9): 1207-1209.
229. Marenzeller, E. V. 1886. Uber die Sarcophytum benannten Alcyo- niiden. Zoo!.Jahrb. 1 (2): 341-368.
230. MarinLit. 2001. A marine literature dtabase produced and maintained by theDepartment of Chemistry, University of Canterbury, New Zeland. VersionSeptember.
231. MarinLit, Version September 2003. A marine literature database produced andmaintained by the Department of Chemistry, University of Canterbury, NewZealand. Version September.
232. Martin, G., Matson, J. A and Weinheimer, A. J. 1979. 13C-NMR studies of marinenatural products II. Total assignment of the 13 C-NMR spectrum of asperdiol.Tetrahedron Letters, 24: 2195-98.
233. Martin, S. E., Flowers, R. S and Ordal, Z. J. 1976. Catalase: Its effects onmicrobial enumeration. Appl. Environ. Microbiol. 32 (5): 731-734.
234. Martin, S. A., Negrete, R and Rovirosa, J. 1991. Insecticide and acaricide activitiesof polyhalogenated monoterpenes from Chilean Plocamium cartilagineum.Phytochemistry, 30: 2165-2169.
235. Maruta, T., Saito, T., Inagaki, M., Shibata, 0 and Higuchi, R. 2005. Biologicallyactive Glycosides from Asteroidea, 41. Isolation and structure determination ofGlucocerebrosides from the star fish Linckia laevigata. Chem. Pharmacol. Bullet.
53 (10): 1255-1258.
236. Marwick, J. D., Wright, P. C and Burgess, J. G. 1999. Bioprocess intensificationfor production of novel marine bacterial antibiotics through bioreactor operationand design. Mar. Biotechnol. 1: 495-507.
221
237. Matsunaga, S., Fusetani, N and Hashimoto, K. 1986. Kabiramide C, a novel
antifungal macrolide from nudibranch egg masses. J Am. Chem. Soc. 108: 847-49.
238. Matsunagas, S., Okada, Y., Fusetani, N and Van Soest, R. W. 2000. Anantimicrobial C 14 acetylenic acid from a marine sponge Oceanapia species. I Nat.Prod. 63: 690-691.
239. May, W. 1898. Alcyonaceen von Ost-Spitzbergen, nach der Ausbeute der HerrenProf. Dr. W. Kukenthal und Dr. A. Walter in Jabre 1889. Zoo!. Jahre. (Syst.) 11:35-404.
240. Mayer, A. M. S and Gustafson, K. R. 2003. Marine pharmacology in 2000:Antitumour and cytotoxic compounds. J. Cancer. 105: 291-299.
241. Mayer, A. M. S and Gustafson, K. R. 2006. Marine pharmacology in 2003-2004:Anti-tumour and cytotoxic compounds. Eur. I Can. 42: 2241-2270
242. Mayer, A. M. S and Hamann, M. T. 2000. Marine Pharmacology in 1999;compounds with antibacterial, anticoagulant, antifungal, anti-helmintic, anti-inflammatory, anti-platelet, anti-protozoal and antiviral activities affecting the
cardiovascular, endocrine, immune and nervous systems and other miscellaneousmechanisms of action. Comp. Biochem. Physiol. 132: 315-339.
243. Mayer, A. M. S and Hamann, M. T. 2002. Marine pharmacology in 1999:compounds with antibacterial, anticoagulant, antifungal, anthelmintic, anti-inflammatory, Antiplatelet, antiprotozoal and antiviral activities affecting the
cardiovascular, endocrine, immune and nervous systems and other miscellaneousmechanisms of action. Comp. Biochem. Physiol. Part. C, 132: 325-339.
244. Mayer, A. M. S and Lehmann, V. K. B. 2001. Marine pharmacology in 1999:antitumour and cytotoxic compounds. Anticancer Research, 21: 2489-2500.
245. Mayer, A. M. S., Jacobson, P. B., Fenical, W., Jacobs, R. S and Glaser K. B. 1998.
Pharmacological characterization of the pseudopterosins: novel anti- inflammatorynatural products isolated from the Caribbean soft coral, Pseudopterogorgiaelisabethae. Life Science, 62 (26): 401-407.
246. Mayers, R. A., Cruz, L. J., River, J. E and Olivera, B. M. 1993. Conus peptides as
chemical probes for receptors and ion channels. Chem. Rev. 93: 1923-1936.
247. McDonald, L. A., Capson, T. L., Krishnamurthy, G., Ding, W., Ellestad, G. A.,Bernan, V. S., Maiese, W. M., Lassota, P., Discafani, C., Karmer, R. A andIreland, C. M. 1996. Namenamicin, a new enediyne antitumour antibiotic from the
marine ascidian Polysyncrato lithostrotum. J. Am. Chem. Soc. 118: 10898-10899.
222
248. McDonald, L. C., Hackney, C. R and Ray, B. 1983. Enhanced recovery of injuredEscherichia co/i by compounds that degrade hydrogen peroxide or block itsformation. Appi. Environ. Microbiol. 45 (2): 360-365.
249. McLachlan, J and Craigi, J. S.1964. Antialgal activity of some simple phenols. JPhycol. 2: 133-35.
250. McLaughlin, J. L., Chang, C. J and Smith, D. L. 1991. "Bench-top" bioassays forthe discovery of bioactive natural products: In: Studies in Natural ProductsChemistry, Vol. 9, (ed). Atta-ur-Rabman, Elsevier, Amsterdam, pp.383-407.
251. McMillan, C, Zapata, 0 and Escobar, L. 1980. Sulphated phenolic compounds inseagrasses. Aquatic Botany, 8: 267-278.
252. Meikle, P., Richards, G. N and Yellowlees, D. 1988. Structural investigations onthe mucus from six species of coral. Mar. Biol. 99: 187-193.
253. Melnikov, N. N. 1971. In: Chemistry of pesticides. Gunther, F. A, Gunther, J. D,Eds, Springer-Verlag: New York, p-197.
254. Mendiola, J., Hemandeza, H., Sariegoa, I., Rojasa, L., Oterob, A., Ramirez, A.,Chavezb, M. D. A., Payroic, J. A and Hernandezd, A. 2007. Antimalarial activityfrom three ascidians: an exploration of different marine invertebrate phyla.Transactions of the Royal Society of Tropical Medicine and Hygiene 100: 909-916
255. Mitta, G., Hubert, F., Noel, T and Roch, P. 1999. Myticin, a novel custeine-richantimicrobial peptide isolated form haemocytes and plasma of the mussel Mytilusgalloprovincialis. Eur. J. Biochem. 265: 71-78.
256. Monks, N. R., Lerner, C., Henriques, A. T., Farias, F. M., Schapoval, E. E. S.,Suyenaga, E. S., da Rocha, A. B., Schwartsmann, G and Mothes, B. 2002.Anticancer, antichemotactic and antimicrobial activities of marine spongescollected off the coast of Santa Catarina, Southern Brazil. I Exp. Mar. Biol. Ecol.281: 1-12.
257. Moore, B. S. 1999. Biosynthesis of marine natural products: microorganisms andmacroalgae. Nat. Prod. Rep. 16: 653-674.
258. Moreau, J., Pesando, D and Caram, B. 1984. Antifungal and antibacterial screeningof Dictyotales from the French Mediterranean coast. Hydrobiologia, 116: 521-524
259. Morris S A., De Silva E. D and Anderson R. J. 1990. Chromodorane diterpenesfrom the tropical dorid nudibranch, Chromodoris cavae. Can. I Chem. 69: 768-771.
223
260. Munro, M. H. G., Blunt, J. W., Lake, R. J., Litaudon, M. and Battershill, C. N.1994. In Sponges in time and space. Balakëma. Pp.473-484.
261. Munro, M. H. F., Luibrand, R. T and Blunt, J. W. 1987. The search for antiviraland anticancer compounds from marine organisms. In: Schuer, P. J. (Ed.). Bioorg.Mar. Chem. 1: 93-176.
262. Muralidhar, P., Kumar, M. M., Krishna, N and Rao, C. B. 2005. New sphingolipidsand a sterol from a Lobophytum Species of the Indian Ocean. Chem. Pharmacol.Bullet. 53 (2):168-171.
263. Nair, S and Simidu, U. 1987. Distribution and significance of heterotrophic marinewith antibacterial activity. Appl. Environ. Microbiol. 53 (12): 2957-2962.
264. Nair, S., Chandramohan, D. and Bharathi, L. 1992. Differential sensitivity ofpigmented and non-pigmented marine bacteria to metals and antibiotics. WaterResearch, 26 (4): 431-34.
265. Nakamura, H., Kishi, Y., Shimomura, 0., Morse, D and Hastings, J. W. 1989.Structure of dinoflagellate luciferin and is enzymatic and nonenzymaticairoxidation products. I Am. Chem. Soc. 111: 7607-7611.
266. Narahashi T. 1988. Drugs acting on calcium channels. Handbook Exp. Phramacol.83:255-274
267. Narkowicz, C. K., Blackman, A. J., Lacey, E., Gill. J. H and Heiland, K. 2002.Convolutindole A and Convolutamine H, New nematocidal brominated alkaloidsfrom the marine Bryozoan Amathia convolute. J. Nat. Prod. 65:938-941.
268. Nathan, P., Law, E. J and Murphy, D. F. 1978. A laboratory for the selection oftopical antimicrobial agents. Burns. 4: 177-178.
269. Nicolaou, K. C., Delft, F. V., Ohshima, T., Vourloumis, D., Xu, J., Hosokawa, S,Pfefferkorn, J., Kim, S and Li, T. 1997. Total synthesis of Eluetherobin. Angew.Chem. mt. Ed. Engl. 36: 2520-2524.
270. Nilsen, I. W., Overbo, K., Sandsdalen, E., Sandaker, E., Sletten, K and Myrnes, B.1999. Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity. FEBS Lett, 464: 153-58.
271. O'Grady, F., Lambert, H. P., Finch, R. G and Greenwood, D. 1997. Antibiotic andChemotheraphy: anti-infective agents and their usen in therapy. 7th Edition.Churchill Livingstone, New York. Pp.469-484.
272. Oiwa, R. 1992. Antibacterial agents. In: The search for bioactive compounds frommicroorganisms (ed.) Omura, S., Springer Verlag. New York. Pp. 1-29
273. Okada, A., Watanabe, K., Umeda, K and Miyakado, M. 1991. Calyculin E and F,novel insecticidal metabolites from the marine sponge, Discoderma sp. Agr. Biol.Chem. 55: 2765-2771.
274. Okaichi, T and Hashimoto, Y. 1962. The structure of Nereis toxin. Agri. Biol.Chem. 26: 224-227.
275. Okuda, R. K and Scheuer P. J. 1985. Latrunculin A, ichthyotoxic constituents ofthe nudibranch Chromodoris elisabethina. Expienia, 41: 1355-56.
276. Okuda, S. 1992. Herbicides: The search for bioactive compounds from marinemicroorganisms, (ed) Omura. 5, Springer-Verlag, New York. Pp.224-236.
277. Olivera, B. M., Gray, W. R., Zeikus, R., McIntosh, J. M., Varga, J., Revier, J., deSantos, W and Cruz, L. J. 1985. Peptide neurotoxins from fish-hunting cone snails.Science, 230: 1338-1343.
278.01son, J. B., Lord, C. C and Mc Carthy, P. J. 2000. Improved recoverability ofmicrobial colonies from marine sponge samples. Microbial Ecol. 40: 139-147.
279. Omura, S., Tanaka, Y., Kanaya, I., Shinose, M and Takahashi, Y. 1990.Pthoxazolin. A specific inhibitor of cellulose biosynthesis produced by a strain ofStreptomyces sp. I Antibiot. 43: 1034-1036.
280. Padget, P. J., Cover, W and Krieg, N. R. 1982. The microaerophilic Spirillumvolutans: Cultivation on complex liquid and solid media. Appi. Environ. Mrobiol.43: 469-477.
281. Padmakumar, K and Ayyakannu, K. 1997: Seasonal variation of antibacterial andantifungal activities of the extracts of marine algae from Southern coast of India.Marine Botany, 40: 507-515
282. Pastor, P. G., De Rosa, S., De Giulio, A., Paya, M and Alcaraz, M. J. 1999.Modulation of acute and chronic inflammatory processes by cacospongionolide, B,a novel inhibitor of human synovial phospholipase A 2. Brit. I Pharmacol. 126:
301-311.
283. Patil, A. D., Freyer, A. J., Carte, B., Taylor, P. B., Johnson, R. K and Faulkner, D.J. 2002. Haploscleridamine, a novel tryptamine derived alkaloid from a sponge ofthe Order Haploscierida: an Inhibitor of Cathepsin K. I Nat. Prod. 65: 628-629.
284. Patil, R., Jeyasekaran, G., Shanmugam, S. A and Shakila, R. J. 2001. Control ofbacterial pathogens, associated with fish diseases, by antagonistic marineactinomycetes isolated from marine sediments. Indian I Mar. Sci. 30 (4): 264-
267.
285. Paul, J. H., De Flaun, M. F and Jeffery, W. H. 1986. Elevated levels of microbialactivity in the coral surface monolayer. Mar. Ecol. Prog. Ser. 33: 29-40.
286. Peng, J., Shen, X., Sayed, E., Dunbar, D. C., Perry, T. L., Wilkins, S. P., Hamann,M. T., Bobzin, S., Huesing, J., Camp, R., Prinsen, M., Krupa, D and Wideman, M,A. 2003. Marine natural products as prototype agrochemical agents. J Agri. Food.Chem. 51(8): 2246-2253.
287. Perez, M. H and Rabanal, R. M. 2002. Evaluation of the anti-inflammatory andanalgesic activity of Sidertis canariesis var. pannosa in mice. J Ethnopharmacol.81: 43-47.
288. Peters, L., Konig, G. M., Wright, A. D., Pukall, R., Stackebrandt, E., Eberl, L andRiedel, K. 2003. Secondary metabolites of Flustra foliacea and their influence onbacteria. Appi. Environ. Microbiol. 69 (6): 3469-3475.
289. Pettit. G. R. 1991. The bryostatins. Pages 153-195 in Herz, W., Kirby, G. W andTamm, C, eds. Progress in the chemistry of organic natural products. 57: Berlin(Germany): Springer-Verlag.
290. Phadke, J. D and Anderson, L. A. 1988. Ethnopharmacology and WesternMedicine. J. Ethnopharmacol. 25: p-61
291. Piementel, D. 1986. Studies on integrated pest management. In: Piementel, D (ed).Some aspects of integrated pest management. Cornell University Press, Ithaca.Pp.29-68.
292. Pomponi, S. A. 1999. The bioprecess-technological potential of the sea. IBiotechnol. 70: 5-13.
293. Posadas, I., Giannini, C., Debitus, C., Paya, M and D'Auria, M.V. 2000.Dysidotronic acid, a new and selective human phospholipase A (2) inhibitor fromthe sponge Dysidea sp. Tetrahedron Letters, 41: 3257-3260.
294. Potts, B. C. M and Faulkner, D. J. 1992. Phospholipase A2 inhibitors from marineorganism. J. Nat. Prod. 55: 1701-1717.
295. Powell, R. G., Bajaj, R and Mc Laughlin, J. L. 1987. Bioactive stilbenes of Scirpus
niaritimus. .1 Nat. Prod. 50: 293-296.
296. Premanathan, M., Kathiresan, K and Nakashima, H. 1999. Mnagrove Halophytes:A source of antiviral substances. South Pacific Study, 19 (1-2): 49-57.
297. Proksch, S. 1994. Defensive roles for secondary metabolites from marine spongesand sponge-feeding nudibranches. Toxicon, 32 (6): 639-55.
226
298. Proksch, P., Edrada, R. A and Ebel, R. 2002. Drugs from the seas - current statusand microbiological implications. Appl. Microbial. Biotechnol. 59: 125-134.
299. Prota, G. 1980. Nitrogenous pigments in marine invertebrates. In: Marine NaturalProducts: Chemical and Biological Perspectives, Vol. 3. Paul J. Scheuer (Ed).Academic Press, New York: Pp. 141-178.
300. Radhika, P., Rao, P. R., Archana, J and Rao, N. K. 2005. Anti-inflammatoryactivity of a new Shingosine dervateve and Cembrenoid Diterpene(Lobohedleolide) isolated from marine soft corals of Sinulaia crassa Tixier-Durivault and Lobophytum species of Andaman and Nicobar Island. Biol.Pharmacol. Bullet. 28(7): 1311-1313.
301. Radhika, P., Rao, P. R and Laatsch, H. 2004. Chemical constituents of a marinesoft corals of the genus Lobophytum. Chem. Pharmacol. Bullet. 52 (11): 1345-1348.
302. Radhika P., Subrahmanyam C., Ellaiah P., Adinarayana K. 2003. Indian IMicrobiol. 43: 207-209.
303. Radwana, M. A. A and El-Sherbiny, M. 2007. Synthesis and antitumor activity ofindolylpyrimidines: Marine natural product meridianin D analogues. Bioorg. Med.Chem. 15:1206-1211
304. Rahman, A. U., Choudhary, M. I., Hayat, S., Khan, A. M and Ahmed, A. 2001.Two new aurones from marine brown alga Spatoglossum variabile. Chem.Pharmacol. Bullet. 49: 105-107.
305. Rajaganapathi, J. 1996. Studies on antibacterial activity of five marine molluscs.M.Sc. thesis. Annamalai University, Parangipettai. India, p-43
306. Rajaganapathi, J., Kathiresan, K and Sing, T. P. 2000. Purification Anti-HIVprotein from purple fluid of the sea hare Bursatella leachii de Blainville. J. Mar.Biotechnol. 4: 447-453.
307. Ramasamy, S. M and Murugan, A. 2003. Antifouling property of and possiblechemical defense strategies in the starfish Pentaceraster affinis from Tuticorin,Southeast coast of India, Proceedings of the National Seminar on MarineBiodiversity as a source of food and Medicine, SDMRI Research Publication, 3:129-132
308. Ramasamy, S. M. 2003. Screening for antifouling natural products from marinemolluscs off Tuticorin coast, southeast coast of India. Ph.D thesis, Submitted to theManonmaniam Sundaranar University. Pp. 1-219.
227
309. Ramkumar, R., Kumar, S. S and Edward, J. K. P. 2005. Antibacterial activity ofthe epidermal layer of coral associated fishes of Tuticorin coast. Indian J. Fish.
52(1): 119-124.
310. Ravinder, K., Reddy, A. V., Raju, T. V and Venkateswarlu, Y. 2005. A newdibromotyrosine derived metabolite from the sponge Psammaiplysilla purpurea.
ARKIVOC. Pp.51-55.
311. Ray, S. B., Verma, D and Wadhwa, S. 2005. Acute analgesic effect of loperamideas compared t morphine after intrathecal administration in rat. Indian I Exp. Biol.43: 425-429.
312. Redshaw, C. J. 1995. Ecotoxicology risk assessment of chemicals used inaquaculture: a regulatory viewpoint. Aquacul. Res. 26: 629-637.
313. Rembold, H. 1984. Secondary plant metabolites in insect control with specialreference to azadirachtins, Adv. Invert. Reprod. 3: 481-491.
314. Rhodes, G. 2000. Distribution of oxytetracycline resistance plasmids betweenaeromonads in hospital and aquaculture environments: Implications of TN 721 indissemination of the tetracycline resistance determinants Tetracycline A, Appi.Environ. Microbiol. 66 (9): 3883-3890.
315. Rifai, S., Fassouane, A., Abbouyi, A. E., Wardani, A., Kijjoa, A and Soest, R. V.2005. Screening of antimicrobial activity of marine sponge extracts. I deMycologie. Medicale. 15: 33-38.
316. Riguera, R. 1997. Isolating bioactive compound from marine organisms. I Mar.
Biotechnol. 5: 187-193.
317. Rinehart, K. L., Kishore, V., Bible. K. C., Sakai, R., Sullins, D. Wand Li, K. M.1988. Didemnins and Tunichlorin: Novel natural products from the marineTunicate Trididemnum solidum. I Nat. Prod. 51: 1-21.
318. Rinehart, K. L. 2000. Antitumour compounds from Tunicates. Med. Res. Rev. 20:1-27.
319. Ritchie, K. R and Smith, G. W. 1995. Preferential carbon utilization by surfacebacterial communities from water mass, normal, and white-band diseasedAcroporal cervicornis. Mol. Mar. Biol. Biotech. 4 (4): 345-352.
320. Rosa, S. D., Milone, A., Kujumgiev, A., Stefanov, K., Nechev, I and Popov, P.2000. Metabolites from a marine bacterium Pseudomonas! Alteromonas associatedwith the sponge Dysideafragilis. Comp. Biochem. physiol. 126: 391-396.
321. Rosenfeld, W. D and Zobell, C. E. 1947. Antibiotic production by the marinemicroorganisms. I Bacteriol. 54: 393-398.
322. Roesener, J. A and Scheuer, P. J. 1986. Ulapualide A and B, extraordinaryantitumour macrolides from nudibranch egg masses. I Am. Soc. 108: 846-847.
323. Sanduja, R., Weinheimer, A. G., Euler, K. L and Alam, M. 1985. Unusualoccurrence of fulvoplumierin, anantibacterial pigment, in the marine molluscNerita albicilla. I Nat. Prod. 48: 335-36.
324. Santalova, E. A., Makarieva, T. N., Gorshkova, I. A., Dmitrenok, A. S., Krasokhin,V. B and Stonik, V. A. 2004. Sterols from six marine sponges. Biochem. System.Ecol. 32:153-67.
325. Santavy, D. L. 1995. The diversity of microorganisms associated with marineinvertebrates and their roles in the maintenance of ecosystem. In: Allosp, D.,Colwell, R. R and Hawksworth D. L (Eds) Microbial diversity and ecosystemfunction. CAB, International, Wallingford. Pp.211-219.
326. Sato, S., Kuramotob, M and Ono, N. 2006. Ircinamine B, bioactive alkaloid frommarine sponge Daclylia sp. Tetrahedron Lett. 47: 7871-7873
327. Satyamurti, S. J. 1952 a. The mollusca of Krusadai Island, (In the Gulf of Mannar).Amphineura and Gastropods: p-45 and 158.
328. Sayed, K. A. E., Dunbar, D. C., Perry, T. L., Wilkins, P. S and Hamann, T. M.1997. Marine natural products as Prototype insecticidal agents. I Agri. Food.Chem. 45: 2735-2739.
329. Schmitz, A. 2000. Occurrence antimicrobial resistance in fish-pathogenic andenvironmental bacteria associated with four Danish rainbow trout farms. Appi.Environ. Microbiol. 66(11): 4908-4915.
330. Schmitz, F. J., Prasad, R. S., Gopichand, Y., Hussain, M. B., Van der helm, D andSchmidt, P. 1981. Acanthifolicin, a new episulfide-containing marine spongePandarus canthifolium. J. Am. Chem. Soc. 103: 2467-2469.
331. Schmitz, F. J., Bowden, B. F and Toth, S. I. 1993. Antitumor and cytotoxiccompounds from marine organisms, Mar. Biotechnol. 1: 197-208.
332. Seiderer, L. J., Newell, R. C., Schultes, K., Robb, F. T and Turely, C. M. 1987.Novel bacteriolytic activity associated with the style microflora of the musselMytilus edulis (L). J. Exp. Mar. Biol. Ecol. 110: 213-224.
333. Selvam, M. R. 2002. Studies on the biodiversity of seaweeds in Hare Island and itspotential pesticidal activity against storage pests- Tribolium castaneum and
')70
Sitophilus oryzae- M. Sc dissertation, Manonmaniam Sundaranar University,Tirunelveli. Pp.1-38.
334. Selvin, J., Joseph, S., Asha, K. R. T., Manjusha, W. A., Sangeetha, V. S.,Jayaseema, D. M., Antony, M. C and Vinitha, A. J. 2004. Antibacterial potential ofantagonistic Streptomyces Sp. isolated from marine sponge Dendrilla nigra.FEMS, Microbial. Ecol. 50:117-122.
335. Sertie, J. A. A., Woisky, R. G., Wiezel, G and Rodrigues, M. 2005.Pharmacological assay of Cordia verbenacea V: oral and topical anti-inflammatoryactivity, analgesic effect and fetus toxicity of a crude leaf extract. Phytomedicine,12: 338-344.
336. Shajini, J. 2005. Insecticidal properties and Artemia toxicity studies of the deep-sea gastropod, Nassarius suturalis, Southeast coast of India. M. Sc dissertation,Bharathidasan University, Tiruchirapalli. Pp. 1-39.
337. Sharma, s., Jam, N. K and Kulkarni, S. K. 2005. Possible analgesic and anti-inflammatory interactions of aspartame with opioids and NSAIDs. Indian J Exp.Biol. 43: 498-502.
338. Shaw, P. D., McClure, W. 0., Van Blaricom, G. V., Sims, J., Fenical, W and Rude,J. 1976. Antimicrobial activities from marine organisms. In: Webber, H. H andRuggieri, G. D (Eds), Food and Drugs from the Sea, Proceedings, Washington:Mar. Technol. Soc. Pp.429-433.
339. Shiba, T and Taga, N. 1980. Heterotrophic bacteria attached to sea weeds. I Exp.Mar. Biol. Ecol. 47: 251-258.
340. Shin, J and Fenical, W. 1991. Fucosides, A-D: anti-inflammatory diterpenoidglycosides of new structural classes from the Caribbean gorgonian, Euniea fusca.I Org. Chem. 56: 3153-3158.
341. Shoji, N., Umeyama, A and Arihara, S. 1993. A novel norditerpenoid from theOkinawan soft coral Sinularia sp. I Nat. Prod. 56: 1651-1653.
342. Sobecky, P. A., Mincer, T. J., Chang, M.C., Toukdarian, A and Helinski, D. R.1998. Isolation of broad-host-range replicons from marine sediment bacteria. Appi.
Environ. Microbiol. 64:2822-2830.
343. Soriente, A., Sodano, G., Reed, K. C., Todd, C. 1993. Chemical ecology and originof defensive compounds in the Antarctic nudibranch, Austrodoris kerguelenensis
(Opisthobranchia: Gastropoda). Nat. Prod. Lett. 3: 31-35
344. Sorokin, Y. I. 1973. Tropical role of bacteria in the ecosystem of the coral reef.Nature, 242: 425-417.
Tm
345. Speck, M. L., Ray, B and read, Jr. R. B. 1975. Repair and enumeration of injuredcoli forms by a plating procedure. App!. Microbiol. 29 (4): 549-550.
346. Spragg, M. A., Boyd, K. G., Hubble, M. 0 and Burgess, J. G. 1997. Antibioticsfrom surface-associated marine bacteria. Proc. 4th Underwater Sci. Symp. Societyfor Underwater Technology, London, ISBN 0906940311. Pp. 147-157.
347. Stallard, M. 0 and Faulkner, D. J. 1974. Marine natural products from molluscs. JComp. Biochem. Physiol. 49: 25-32.
348. Steiner, B. M., Wong G. H. W., Sutrave, P and Graves, S. 1984. Oxygen toxicity in
Treponema pallidurn: deoxyribonucleic acid single stranded breakage induced bylow doses of hydrogen peroxide. Can. I Microbiol. 30: 1467-1476.
349. Strahl. E. D., Dobson, W. E and Lun die, L. L. 2002. Isolation and screening ofbrittle star associated bacteria for antibacterial activity. Curr. Microbiol. 44: 450-459.
350. Sukma, M., Chaichantipyuth, C., Murakami, Y., Tohds, M., Matsumoto, K and
Watanable, H. 2002. CNS inhibitory effects of barakol, a constituent of Cassiasiamia Lamak. I. Ethnopharmacol. 83: 87-94.
351. Sung, P. J., Gwo, H. H., Fan, T. Y., Li, J. J., Dong, J., Han, C. C., Wu, S. L andFang, L. S. 2004. Natural product chemistry of gorgonian corals of the genusJunceella. Biochem. System. Ecol. 32:185-96.
352. Sureshkumar, S., John, J. A. C and Ravikumar, S. 2002. Antimicrobial activity of
acetone extracts of seaweeds against human pathogens. Seaweed ResearchUtilization, 24 (1): 111-116.
353. Sutton, D. C and Garrick, R. 1993. Bacterial disease of cultured giant clamTridacna gigas larvae. Dist. Aquacul. Organ. 16: 47-53.
354. Szolcsanyi, J., Helyes, Z., Oroszi, G., Nemeth, J and Printer, E. 1988. Release ofsomatostatin and its role in the mediation of the anti-inflammation. Braz. I
Pharmacol. 123: 936-942.
355. Targett, N. M. 1988. Allelo chemicals in marine organisms chemical fouling andantifouling strategies. In: Marine Biodeterioration. Thomson, M. F., Saiojini, R and
Nagabhushanam, R (Eds). Oxford and IBH Publisher co., Ltd. New Delhi. Pp.609-
617.
356. Targett, N. M., Bishop, S. S. McConnel., 0. J and Yoder, J. A. 1983. Antifouling
agent benthic diatom, Nacicula salinicola. Homarine form the gorgonians
Leptogorgia virgulata and L. setacea and analogs. I Chem. Ecol. 9: 817-830.
231
357. Tasdmir, D., Mangalindan, G. C., Concepcion, G. P., Haper, M. K and Ireland, C.M. 2001. 3,7-Dimethyguanine, a new purine from a Philippine sponge Zyzyafuliginosa. Chem. Pharmacol. Bullet. 49 (12): 1628-1630.
358. Teske, A.., Brinkhoff, T., Muyzer, G, Moser, D. P., Rethmeier, J and Jannasch, H.W. 2000. Diversity of thiosulfate-oxidizing bacteria from marine sediments andhydrothermal vents. Appi. Environ. Microbial. 66: 3125-3133.
359. Thakur, N. L., Thakur, A. N and Muller, W. W. 2005. Marine natural products indrug discovery. Nat. Prod. Radiance. 4 (6): 471-477.
360. Theiler, R. F., Cook, J. C and Hager, L. P. 1970. Halohydrocarbon synthesis bybromoperoxidase. Science, 202: 1094-1096.
361. Thorlacius, H., Volimar, B., Seyfert, U. T., Vestweber, D and Menger, M.D. 2000.The polysaccharide fucoidan inhibits microvascular thrombus formationindependently from P- and L- selectin function in vivo. Eur. I Clinical. Invest. 30:804-810.
362. Trisyono, A., Puttler, B and Chippenale, G. M. 2000. Effect of ecdysone agonists,methoxy-fenoxide and tebufenozide on the lady beetle, Coleomegilla maculate.Entomol. Ext. Appl. 94:103-105.
363.Tsao, R., Romanchuk, F, E., Peterson, C. J and Coats, J. R. 2002. Plant growthregulatory effect and insecticidal activity of the extracts of the tree of heaven(Ailanthus altissima L.). BMC. Ecol. 2 (1): 1-6.
364.Tsukamoto, S., Matsunaga, S., Fusetani, N and van Soest, R. W. 1998.Acanthosterol sulfates A-J: ten new antifungal steroidal sulfates from a marinesponge Acanthodendrilla sp. I Nat. Prod. .61: 1374-1378.
365. Turner, D. M. 1996. Natural product source material use in the pharmaceuticalindustry: the Glaxo Expience. .1. Ethnopharmacol. 51: 39-44.
366. Turner, R. A. 1965. Screening methods in pharmacology. Academic press, NewYork, Academic Press. Pp. 158-233.
367. Turner, S. J. 1994. The biology and population outbreaks of the corallivorousgastropod Drupa on Indo-Pacific reefs. Oceanogr. Mar. Biol. Annu. Rev. 32: 461-
530.
368. Ungur, N., Gavagnin, M., Fontana, A and Cimino, G. 2000. Synthetic Studies onnatural diterpenoid glyceryl esters. Tetrahedron, 56: 2503-2512
232
369. Vairappan, C. S. 2003. Bacterial dynamics associated with algal antibacterialsubstances during post harvest desiccation process of Sargassum stolonifolium.Indian I Mar. Sci. 41: 837-845.
370. Vernon. V and Vandiver, Jr. 2005. Biology and control of duckweed withherbicides. http://edis.ifas.ufl.edu/AG
371. Vanitha, J., Prakash, S., Bhima, B. V and Lazarus, S. 2003. Antibacterial action ofseaweeds against human upper respiratory tract pathogens. Seaweed ResearchUtilization, 25 (1): 181-187.
372. Verschuere, L., Rombant, G., Sorgeloos, P and Verstraete, W. 2000. Probioticbacteria as biological principles from the red alga, Laurencia nipponica Yamada.Agri. Biol. Chem. 3: 2513-2515.
373. Vetter, W., Gaul, S., Olbrich, D and Gaus, C. 2007. Monobromo and higherbrominated congeners of the marine halogenated natural product 2, 3, 30, 4, 40, 5,5 0-heptachioro- 10-methyl-i ,20-bipyrrole (Qi). Chemosphere, 66: 2011-2018
374. Wagenen, B. C., Larsen, R, Cardinella, J. H. II., Randazzo, D., Lidert, Z andSwithenbank, C. 1998. Ulosantoin, a potent insecticide from the sponge, Ulosaruetzleri. I Org. Chem. 58: 335-337.
375. Wagner, D. I., Beil, W., Lang, S., Meiners, M and Laatsch, H. 2002. Integratedapproach to explore the potential of marine micoorganisms for the production ofbioactive metabolites. Adv. Biochem. Eng. Biotechnol. 74: 207-238.
376. Walker, R. P and Faulkner, D. J. 1981. Chlorinated acetylenes from the nudibranchDialula sandiegensis. J. Org. Chem. 46: 1475-78.
377. Ward, D. M., Weller, R and Bateson, M. M. 1990. 16s rRNA sequences revealnumerous uncultured microorganisms in a natural community. Nature, 345: 63-65.
378. Ware, G. W. 1982. Pesticides: Theory and application. Thompson publication,Fresno, California. p-308
379. Watanabe, K, Umeda, K and Miyakado, M. 1989. Isolation and identification ofthree insecticidal principles from the red alga, Laurencia nipponica. Agri. Biol.
Chem. 53: 2513-2515.
380. Watanabe, K., Sekine, M and Iguchi, K. 2003. Isolation of three marineprostanoids, Possible Biosynthetic Intermediates for Clavulones, from theOkinawan soft coral, Clavularia viridis. Chem. Pharmacol. Bullet. 51 (8): 909-
913.
381. Webber, J. T. 1994. Epidemic cholera in Ecuador: Multi-drug resistance andtransmission by water and seafood. Epidemiology and Infection, 112: 1-11.
382. Whittaker, V. P. 1960. Pharmacologically active choline esters in marinegastropods. Annals of the New York Academy Science, 90: 695-705.
383. Wilkinson, C. R and Fay, P. 1979. Nitrogen fixation in coral reef sponges withsymbiotic cyanobacteria. Nature, 279: 527-529.
384. Wratten, S. J., Wolfe, M. S., Andersen, R. J and Faulkner, D. J. 1977. Antibioticmetabolites from a marine Pseudomonad. Antimicrobial. Agent. Chemotherapy. 11:411-414.
385. Wright, A. E. 1998. Isolation of marine natural products. In: R. P. J. Cannell,editor. Methods in biotechnology, Vol. 4. Natural product isolation. New Jersey:Humana press Inc. Pp-305-408.
386. Xue, S., Zhang, H. T., Wu, C. P., Zhang, W and Yuan, Q . 2004. Study onbioactivity of extracts from marine sponges in Chinese Sea. J Exp. Mar. Ecol.298: 71-78.
387. Ya-Ching-Shen, Chang, Y. T., Wang, S. S., Pan, Y. L., Yu, K. L.L and Lin, C.2002. J. Tixicology: Toxin Reviews, 22:533-45
388. Yamada, K., Okija, M., Kigoshi, H and Suenaga, K. 2000. Cytotocxic substancesfrom Opisthobranch Molluscs: In Drugs from the Sea; Fusetani, N., ed; Karger, A.G. Basel. Pp-50-73.
389. Yamazaki, M. 1993. Antitumor and antimicrobial glycoprotein from sea hares.Comp. Biochem. Physiol. C. 195:141-46.
390. Yemitan, 0. K and Adeyemi, 0. 2005. CNS depressant activity of Lecaniodiscuscupanioides. Fitoterapia, 76: 412-418.
391. Yousuf, A. M. H., Ali, B. H., Bashir, A. K., Tanira, M. 0. M and Blunden, G. 2002a. Central nervous system activity of Leucas inflates. Benth. In mice.Phytomedicine, 9: 501-507.
392. Yousuf, A. M. H., Bashir, A. K., Ali, B. H., Tanira, M. 0. M and Blunden, G. 2002b. Some effects of Saliva aegyptiaca L. on the central nervous system in mice. .J
Ethnopharmacol. 81: 121-127.
393. Zabriskie, M. T., Kiocke, J. A., Ireland, C. M., Marcus, A. H., Molinski, T. F.,Faulkner, D. J., Xu, C and Clardy, J. C. 1986. Jaspamide, a modified peptideisolated from a Jaspis sponge, with insecticidal and antifungal activity. I Am.
Chem. Soc. 108: 3123-3124.
Y11A
394. Zalmer, H., Bachmann, E., Hutter, R and Nuesch, J. 1962. Sideramine, eisenhaltigeWachstumsfaktoren aus Mikroorganismen. Pathol. Microbiol. 25: 708-736.
395. Zapata, 0 and McMillan, C. 1979. Phenolic acids in seagrasses. Aquatic Botany, 7:307-317.
396. Zhang, W., Liu, W. K and Che, C. T. 2003. Polyhydroxylated Steroids and otherconstituents of the soft coral, Nephthea chabroli. Chem. Pharmacol. Bullet. 51 (8):1009-1011.
397. Zobell, C. E. 1941. Studies on marine bacteria I. The cultural requirements ofheterotrophic aerobes. J Mar. Res. 4: 42-475.
398. Zobell, C. E. 1942. Bacteria of the marine world. The Scientific Monthly, 38: 35-49.
399. Zobell, C. E. 1946. Marine microbiology. A monograph on hydrobacterialogy.New. Phytologist, 45 (2): 286-287.
400. Zollinger, M., Kelter,G., Fiebigb, H. H and Lindela, T. 2007. Antitumor activityof the marine natural product dibromophakelistatin in vitro. Bioorg. Med Chem.Lett. 17: 346-349
401. Zubia, E., Gavagnin, M., Crispino, A., Martinez, E., Ortea, J., Cimino, G. 1993.Diasteroisomeric ichthyotoxic acyiglycerols from the dorsum of twogeographically distinct populations of Archidoris nudibranchs. Experiencia, 49:268-271.
Indian Journal of Marine SciencesVol. 33(4). l)ccenihcr 2004. pp. 369-372
Short Communication
Antibacterial activity of the winged oyster Pteria chinensis (Pterioida: Pteridae)
C. Chellaram, K. Mary Elizabeth Gnanambal & J.K. Patterson Edward
Suganthi Devadason Marine Research Institute, 44, Beach Road, Tuticorin-628 001, Tamil Nadu, IndiaLE-mail *coralcliella@rediffniail corn]
Received 22 December 2003, revised 13 Jul y 2004
The whole body extracts of the winged oyster. Pteria cisinensis obtained with different solvents were assayed forantibacterial activity using agar well diffusion technique against human and fish pathogens. The acetone and chloroformcrude extracts exhibited broad antibacterial activity. Highest activity was exhibited against Kiebsiella pneumoniae (5 mm)
and Staph ylococcus epidenoidrs (5 mm) by the crude extract of acetone and against Salmonella paratyphi B (5 mm) by thechloroform extract. Similarly, the crude extract of chloroform was found to inhibit 8 out of 10 fish pathogens tested. Thecolunin-purihecl acetone fractions showed higher activity against Klebsiehla pneulnoniae (5 mm), Sreptococcus pneumonicle(4 in ii), Scrratia o,c,r(cvcen.s (4 in iii) and /'rotcns ,,i irobilis (4 nun). The Ml C 01 the 1 00% acetone fraction was found to belower for the patliogcns, S. ,,iarcescens (100 gig) and I'. ,n,rabil,s (ISO ig) and hence 100% acctonatcd fraction of theextract of P. cln,iensis can be considered as potent antimicrobial compound against these pathogens.
IKey words: Marine natural products, antibacterial activity, Pieria choiensis, molluscs]IIPC Code: Iiit.Cl'. A0 IN 63/021
The marine environment is a huge source for yet to bediscovered bioactive natural products. Apart from thefood that is derived from the marine environ, a widevariety of bioactive substances are being isolated andcharacterized, several with great promise for thetreatment of human and fish diseases. For the past twodecades, pharmaceutical industry has been relativelysuccessful in overcoming problems due to singleresistant determinants; however the advent of multipleresistant mechanism has severely limited the use ofmany major classes of antimicrobial compounds. Thedemand for effective and non-toxic antibacterialtherapeutics has become even greater with theincreased incidence of bacterial infections.Aquaculture has been the world's fast growing foodproduction system for the past decade. Oil sameline, the impact of diseases in aquaculture isenormous. Therefore, there is a vital interest indiscovering new antimicrobial compounds with fewerenvironmental and toxicological risks to which thereis no resistance developed by the pathogens.
Molluscs in the oceans are a common sight and arevirtually untapped resource for the discovery of novelcompounds. Many studies have reported thebioactivity of the molluscs like ApI ysia sp.', Phyllidae
sp. 2 , bivalves 3 gastropods4 and their egg masses 5.
Bioactive metabolites from molluscs such as sea
hare6, Chroinodoris sp.7 , Onhidella sp. 8 , were isolatedand structurally elucidated. The winged oyster, Pieriachinensis, is usually found attached to gorgonids andthis bivalve was screened for antibacterial activityusing the whole body tissue extracts obtained withdifferent solvents.
Live specimens of oyster, Pieria chinensis(Pterioida: Pteridae) were collected at a depth of 6 m inTuticorin coastal waters (]at, 8°45 and long. 78°13'E).They were immediately brought to the laboratory andtheir soft bodies were removed by breaking the shells.The meat was cut into small pieces, washed thoroughlywith distilled water and air-dried. The air-dried meat ofapproximately 3 g was immersed separately in solventslike acetone, ethyl acetate, methanol, chloroform,butanol and toluene and cold steeped overnight at -18°C. The extracts from each solvent were filteredseparately for three times using Whatman No.1 filterpaper. The filtrate was poured iii previously weighedpetriplates; evaporated to dryness in rotary evaporatorand the dried extract was used for all the experiments.To test the antibacterial effect of the extracts, 12 humanpathogens [Salmonella paratyphi B, Pseudonzonasaeruginosa (ATCC 29336). Citrobacter sp, (ATCC25405), Kiebsiella pneuinoniae (ATCC 10031),Staphylococcus epidermidis (ATCC 12228), S. aureus(ATCC 29737), Slmigella dysentriae (ATCC 13313),
2
32
1.52
2.563
2.54354
1.52.54
1.5
4
43
0.5
2.5
22.5
0.5
2
54
32
1.55
1.5
45
42
22
3
44
24.5
2
4
2
2
4
42
24
3
1 1
370
INDIAN J. MAR. SC!. VOL. 33, No.4, DECEMBER 2004
Sireplococcus piieu,iioFiiae (ATCC 6301), Vi/,riocholerae (ATCC 15748), Esc/ieric/iia coli (ATCC25922), Bacillus subtilis (ATCC 6633) andEtiterohacter aerogenes (ATCC 13048)] and 10 fishpathogens [Vibrio para/iaemol','ticus (ATCC 17802),V. iIi1?liCILV (ATCC 33653), V. logei (shrimp isolate),Serraiia iitarcesceiis (MTCC 97), V. /lart'evi (shrimpisolate), V. vulnijicus (ATCC 27562), Proteus inirabilis(MTCC 1429), V. ordalli (fish isolate), Aeroinonasiiydrop/iila (ATCC 7966) and Micrococcus sp, (fishisolate)] were used as test strains. The strains wereobtained from Christian Medical College (CMC),Vehlore. All the test organisms were cultured inTry ptone Soya Broth (TSB) and the 18-24 h oldcultures were used for the experiments. Theantibacterial activity of the samples was assayed by thestandard Nathan's Agar Well Diffusion (NAWD)technique9 against the test strains on Tryptone SoyaAgar (TSA) in petridishes with drilled wells of 6 mmdiameter. A constant amount of 0.7 mg of theextract/50 jil (Dimethyl Sulfoxide) DMSO was loadedonto each well. The well at the center served as thecontrol (without the extract). After 22-24 h ofincubation at room temperature, the susceptibility of
the test organisms was determined by measuring tradius of the zone of inhibition around each welPartial purification of the extract was carried otfollowing the method outlined by Wright' () . After initi;screening, the extract obtained with acetone wfractionated using normal phase silica gel colunichromatography employing a step gradient solvetsystem from low to high polarity. The step gradieiprotocol used was: 100% acetone; 80% acetone an20% lieptane; 60% acetone and 40% heptane; 40acetone and 60% heptane; 20% acetone and 80heptane; 100% heptane; 80% heptane and 20methanol; 60% heptane and 40% methanol; 40heptane and 60% methanol; 20% heptane and 80methanol and finally 100% methanol. The fractiotithus obtained were once again evaporateconcentrated and assayed for antibacterial activitMinimal inhibitory concentration (MIC) wrdetermined by serially diluting the column purifieextracts in DMSO so that concentrations of 100, 12150, 175, 200, 225, 250, 275, and 300 ,sg I 50 pDMSO were loaded into each well for individwpathogenic strains that were found to be highisusceptible.
Table i—Antibacterial activity of Pieria cliinensis against A) human pathogens and B) fish pathogens
Pathogens Zone of inhibition (nini
A
EA M C
B
1'A)Human pathogens
Salmonella paratvphi BPseudoinonas aeruginosaCitrobacter sp.Kiebsiella pneu/noniaeStaplylococcus epiderm idisS. aureusSliiella dyseniriaeStreptococcus pneulnanic'eVil,rio cliolerneEsciterici, in coliBacillus subtilisEnterobacier acrogenes
B)Fish pathogens
Vibrio parahaemolyticusV. !?11F?IiCUS
V. logeiSerratia marcescensV. harveyiV. vulnijicusProteus ujirabilisV. ordallifleron,onas liydrophilaMicrococcus sp.
A=Acetone: EA= Ethyl acetate; M= Methanol; C=Chloroform; B=Butanol T=Toluene
SHORT COMMUNICATION
371
Out of the 6 solvents used, the extract obtainedfrom acetone and chloroform exhibited higher activityand that obtained from acetone and chloroformexhibited higher activity and that obtained fromtoluene and butanol showed mild activities (Table 1A). Of the 12 human pathogens tested, the acetoneand chloroform extracts were able to inhibit all thepathogens exhibiting broad spectral antibacterialactivity. Highest activity was exhibited againstKiebsiella pnewnoii iae (5 mm) and Staphylococcusepidertnidis (5 mm) by the extract of acetone andagainst Salmonella paratyphi B (5 mm) by the extractof chloroform. In the case of fish pathogens, acetoneextract was found to have a broad spectral activityinhibiting all the test strains used and the extract ofchloroform inhibited 8 pathogens (Table I B).Extracts obtained from ethyl acetate, methanol,butanol and toluene exhibited mild activities.Fractions obtained by column chromatography of theacetone phase of the tissue extracts exhibited broad
spectral activity for both human and fish pathogenswhen eluted with 100% acetone (Table 2). Slightlylesser activity was shown by 80% acetone and 20%heptane fractions followed by 60% acetone and 40%heptane fractions (Table 2). Higher degree ofinhibition was exhibited against Klebsiellapneumoniae (5 mm), Streptococcus pneu/noniae(4 mm) and Serratia inarcescens (4 mm) and Proteusinirabilis (4 mm) by the column fractions of 100%acetonated phase. Fractions in the methanolic phaseshowed little inhibition. Table 3 shows that theminimal inhibitory concentration (MIC) of the 100%acetone fraction was found to be lower for thepathogens Serratia tuarcescens (100 g) and Proteus,nirahilis (150 tg).
Pteria chinensis is sessile organism and the exactmechanism by which this organism acquires bioactivesubstances is not known. In the present investigation,higher degree of inhibition was confined to acetonephases indicating the substance involved in producing
Table 2—Antibacterial activity column purified fractions of Pieria chinensis in acetone, heptane and methanol
Pathogens Zone of inhibition (mm)
A) Human pathogens A
Klebsiella pneuinoiiiae 5Staphylococcus epidernidis 3Streptococcus pFzeulnonlae 4Vibrjo cholerae 3Escherichia co/i 3Enterobacter aeroç'enes 3
B) Fish pathogens
Vj/,rjo pora/znentulytuuv 3Serratia lniz,cescens 4V. harveyt 3Proteus ,njrabilis 4A ero,nonas hydrophila 2
A=Acetone, H=1-leptane: M=Methanol
3 2 2 - -2 2 2 - -3 2.5 - - -2 3 - - -2 2 2 - -
80:20 60:40 40:60 20:80 H 80:20 60:40 40:60 20:80 M- - - - - - - 0.5 I 0.5
2.5 2 - - - - - 1 1.5 1.53 2 - - - - 1 12 2 - - - - 1.5 1.5 1.53 2 2 - - - - I I 1
2.5 2 - - - - - - - -
- - I 1.5- - I (.5- I 1.5 1.5- - 0.5 -
Table 3—Minimal inhibitory concentration (MIC) of (lie acetone fractions of Pteria chinensis
Minimal Inhibitory Concentration (MIC) in jsgAcetone: heptane
100:0 80:20 60:40
40:60
200 225 250
250200 250 275
225
250 275 300
275
100 150 175
200150 175 200
200
200 250 275
300
Pathogens
(A) Human pathogens
Kiebsiella pneumoniaeStaphylococcus epidennidisStreptococcus pneumoniae
B) Fish pathogens
Serratia ,00rcescensProteus inirabilisAero,nonas hydrophila
372 INDIAN J. MAR. SCI. VOL. 33, No.4, DECEMBER 2004
the antibacterial effect could be a medium-polarcompound. But, the hypobranchial glands ofChicoreus virgineus and egg capsules of Rapanarapiforinis extracted with polar solvents like ethanoland methanol also have been reported to show widespectral antibacterial activities 5. Cypraea errones wasreported to have antibacterial activity at the non-polarend of the step gradient by the column-purifiedfractions TM . Lesser degree of inhibition by the column-fractionated extracts in comparison to the crude couldbe opined that the active compound may havedegraded or modified during the fractionationprocess 12. in the present experiment, the minimalinhibitory concentration (MIC) of the fraction of100% acetone was found to be lower for Serratiainarcescens and Proteus ,nirabilis and so the extractof this particular fraction is now under the process offractionation and purification, which can be possiblyused as antimicrobial compounds against thesepathogens.
References1 Stallard M 0 & Faulkner D J, Marine natural products from
molluscs, J. Camp. Bioche,n Physiol. 49 (1974) 25-32.
2 Ilagedone M R, Barreson B J & Scheuer P J, Bioactivenatural products. Helvetica Chimica Ada, 62 (1999) 2484-2486.
3 Jayaseeli A A. Prem Anand T & Murugan A, Antibacterialactivity of 4 bivalves from Gulf of Mannar. PI,uket Mar.
Biol. Cent. SpI. ['uN. 25 (2001) 215-217.
4 Emerson Kagoo L & Ayyakannu K, Bioactivc compfrom Chicoreus raolosus- antibacterial activity-inPhuket Mar. Biol. Cent. Spi. PubI. 11(1992)147-150.
5 Prem Anand T, Rajaganapathy J & Pattersoti Edward
Antibacterial activity of marine molluscs from Portregion, Indian J. Mar. Sci. 26(1997)206-208.
6
Schmitz F J, Bowden B F & Toth S I, Antitumocytotoxic compounds from marine organisms,Biolechnol, 1(1993)197-208.
7 Morris S A, De Silva E D & Anderson R J, Chromod
diterpenes from the tropical dorid nudibranch, Chronucavae, Can. J. Chem, 69(1990)768-771.
8
Ireland C, Copp B, Foster M, Mc Donald L, RadiskySwersey J, Biomedical potential of marine natural proMar. Biotechnol, 1 (1993) 1-43.
9 Nathan P E, Law J & Murphy D F, A laboratory nieth
the selection of topical antimicrobial agents, Burns, 4177-178.
10 Wright . A E, Isolation of marine natural products, in Min biotechnology, Vol.4: Natural products isolation,by R J P Canell, (Humana Press Inc., New Jersey, I1998. pp. 365-408.
11 Prem Anand T & Patterson Edward J K, Antimicactivity in the tissue extracts of five species of coCypraea spp. (Mollusca: Gastropoda) and an ascDideinnum psanunathodes ( Tunicata: Didemnidae), IncMar. Sci, 31 (2002) 239-242.
12 Cannell R J P. How to approach the isolation of a rproduct, in Methods in biotechnology, Vol. 4: Nproducts isolation, edited by R. J. P. Cannell, (HumanaInc., New Jersey, USA) 1998, pp. 1-51.
I. Mar Biol. Ass. India, 47 (2) : 154 - 159, Jul. - Dec., 2005
Isolation and screening of mucus-associated bacteria of the gastropod, Drupd
margariticola for antagonistic activity
C. Chellaram*, K. Mary Elizabeth Gnanambal and J. K. Patterson Edward
Su'anihi Dei'adason Marine Research Institute, 44, Beach Road, Tuticorin - 628 001. Tamil Nadu, India
Email: coralc/iella@red(ffivai/. coat
Abstract
The mucus-associated bacteria of the gastropod, Drupa margariticola were screened for their ability toinhibit the human and fish pathogens. Out of the two hundred and eighty live bacterial strains isolated. 23%(65) were found to he pigmented. 71% (202) were identified as Gram-negative. A hi g her percentage of non-pigmented (77%) and Gram-negative (7)%) strains were observed in the present investigation. 16 1X , (46) ofthe isolates was found to have antagonistic activity against both human and fish pathogens tested and 63 ofthe Gram-negative strains (29) were found to he antibiotic producers. Antagonistic activit y was found to beexhibited by pigmented strains too. A higher degree of inhibition was conferred b y 3 of the isolates (D 1 . D
and D) a gainst both human and fish pathogens. These strains exhibited full or complete degree of inhibi-
tion a gainst E.cI,cru1zw colt.
Key words: Gastropod. Drupa ,,ia,i,'arii,'cola mucus-associated bacteria and antagonistic activity.
Introduction
Bacterial infections were considered won in the fate
1960's but now antimicrobial resistance threatens to turn
hack. Resistance is spreading rapidly particularly where
antibiotics are heavily used (Lech. 2004). The erroneous
use of' antibiotics both for therapeutic and aquaculture
Purposes has resulted in the advent of multiple drug
resistance of the pathogenic bacterial strains. Hence theneed of the hour is the search for novel antibiotics with
lesser side effects. The ultimate source- the ocean, is a
unique resource that provides a diverse of' natural prod-
ucts primarily t'rom bacteria and cyanobacterta and inver-tebrates such as corals, sponges, tunicates, bryozoans andmolluscs. Mans- marine chemicals often possess quite
novel structures which lead to pronounced biological
activity and novel pharmacology (Let and Zhou, (2001).A number of discovery efforts have yielded several
hioacttve metabolites which have been successfully devel-
oped by the pharmaceutical industry (Kong ci al, 1994:Faulkner, 2001: Rosenfeld and Zohell, 1947; Fenical and
Jensen; 1993 and Fenical, 1993). The study of marinebacteria has also led to the realization that microorganism
from specific sy mbiotic relationship with marine organ-
isms which may he responsible for the production of
some bio-active compounds (Kobayashi and Ishihashi,
1993). The marine microorganisms have had a major
impact on the development of medical science and these
Journal of Marine Biological A .vsoria! I 0/i Of India (200_)
bacteria form highly specific s y mbiotic relationships with
marine plants and animals, (Fenical, 1993). There are anumber of works reporting the occurrence of bacteriaassociated with marine fauna, in particular, the gastropods
(Kharlamenko ci al. (2001), Distel, (1998). Belkin, ci al.(1986), Stein ci al. (1988) and Windoffer and Giere.
(1997). Recent studies have shown that these antibacterialcompounds are not only inhibiting the human pathogens
but also fish pathogens (Strahl ci (11., 2002).
Drupa ,nar'arincola is one of the comnsoriest gas-
tropods inhabiting the reefs and may he seen adhering thecorals rocks in large numbers. Bacteria associated to themucus of this organism are being less explored as poten-tial sources of antagonistic compounds. This paper de-
scribes the isolation of the bacteria associated with the
mucus of D. mnargariucola and testing the isolated strains
for their ability to inhibit the growth of selected human
and fish pathogens in m'iiro.
Materials and methods
Live D. mnargariticola (Stcnoglossa: Muricidae) were
collected from the intertidal region of the Tuticorin port
area (Lat. 8"45'N and Long. 7S"13'E). The animals were
immediately transported to the laboratory and placed in
the aquaria containin g natural sediment and sea water. The
animals were washed twice in sterile seawater and the
Isolation and screening of mucus-associated bacteria of gastropod
shells were removed aseptically. The mucus was thencollected from the animals in sterile glass tubes. Approxi-mately I mL of the mucus was seriall y diluted and platedon two different media: I. Zobell Marine 2216E Agar(ZMA) and 2. Cetrimide agar (supplemented with 3.5 and1.75% Sodium chloride) using pour plating method. The
plates were then incubated at room temperature for 3-4days. Morphologically different colonies were selectedrandomly. The number of Gram positive and negativecolonies as well as pigmented and non-pigmented colo-nies was noted. Axenic cultures were obtained by streak-
ing and re-streaking on ZMA plates and subsequentlystored as ZMA stab cultures at 4°C.
Screening of the isolates for antimicrobial activity
Pigmented strains (65)
Non-pigmented strains (220)
Fi g I. Percentage of pigmented and non-picmeni.strains isolated from the mucus of D. 'nor çaruirola
The mucus-associated isolates were applied as singlestreaks on pie-poured Yeast Pcptonc Extract (YPE) (75%
sea water) agar plates and incubated at 2S°C for 48 hrs.The human pathogens (Bacillus ru/ni/is. B. cercus. Es-cliericli ia co/i Salmonella tvph iniuriumn. Kiebsie/lapnewnoniae. Staph ylococcus epidermniclis. S. aureus andSinge//a (IVselitriae) were obtained from Christian Medi-cal College. Vcl lore. The fish pathogens (Prnu'us mnira hi/is.Serrana niarcescens, Aeroinonas formica,is, A.hvdrop/ii/a. Vibrio /iarvevii, V viminijicus. Vcamnpbe/ii andV logei) were collected f rom Fisheries Colle g c. Tuticorin.The test strains were grown on YPE (75% sea water)plates for three or more transfers before usin g them in thescreening assays. All isolates were tested for the produc-tion of antibacterial metabolites using the cross streakingmethod (Strahi et al., 2002). All the test strains wereapplied as single streaks perpendicular to the marine bacteriawithout touching it. After cross streaking with test bac-teria, the plates were incubated for an additional 24 h at28°C and observed for the inhibition of test bacterialgrowth. A zone inhibition is defined as an area on the teststreak of reduced growth or lack of growth comparedwith the control plates (streaked with non-producer strains).partial inhibition as significantly reduced over 3-9 mm (P)but not completely inhibited and lack of growth over 10-19mm of the streak was defined as moderate inhibition(M). If the test organisms did not grow, it was scored ascomplete inhibition (C) which was about 20-25 mm.
Results
Isolation of mucus-associated bacteria
Two hundred and eighty five bacterial strains wereisolated from the mucus of D. mnargariticola (232 colo-nies from Zobell marine agar and 48 colonies fromCetrimide agar were isolated). Out of 285 bacterial strainsisolated, 23% (65) were found to be pigmented, 71%(202) were identified as Gram-negative and 29% as Gram-
(;ruii [x)sitiw strains9% (83)
71'
Gram negative strains (202)
Fig 2. Percentage of Gram positive and Grarn-negatmvstrains isolated from the mucus of D. inargarmtuola
positive strains (Figs. 1 & 2). The pigmented bacteriacolonies were observed to he yellow, red, brown, orangcand black in colour. A higher percentage of non-pigmcntcd (77%) and Gram-negative (71%) strains wercobserved in the present investigation.
Antibacterial activity of the marine bacteria
Out of the total bacterial strains (285). 16% (46) ofthe isolates were found to have antagonistic activhv againstboth human and fish pathogens tested (Fig-3). 70% of thenon-pigmented strains (32) and 63% of the Gram-nega-tive strains (29) were found to be antagonistic (Figs. 4 &5). Results of the screening of the isolates against humanand fish pathogens are presented in Figures. 6 and 7respectively. A higher degree of inhibition was conferredby 3 of the isolates (D,, D 30 and D, 17) as shown inFigures 8, 9 and 10 against both human and fish patho-gens. The strains D 30 and D,37 exhibited full or completedegree of inhibition against Escheric/ua co/i. Strain D5was able to elicit a moderate antagonism to seven of thehuman and fish pathogens tested. It was observed thatall antagonistic strains were able to inhibit at least five ofthe pathogens tested.
Discussion
In the present study the non-pigmented strains (77%)were higher in numbers than the pigmented ones (23%).
Journal of Marine Biological Association of India (2005)
Pigmented strains (14)
O %
70
Non-pigmented strains (32)
Fig 4. Percentage of pigmented and non- pigmented strainsshowing activity
30
25
20
15
10
5
0
63 c7c
Gram negative strains (29)
156 C. Cliellarcun.
50
70
60
50
40
30
20
10
0
Producer strains (46)16%
84%Non-producer strains (239)
Fig 3. Percentage of producer and non- producer strainsisolated from the mucus of D. inarç'arl!ico/a
c
i.;\5o -S ,
\e 1\O
OP•M El N
Fig. 6. Percentage of producer strains showing partial,erate and nil inhibition against human bacterial pathc
positive strains (17).,
\O
S -7% ,<:'°
-
C, op .M cN
Fig. 7. Percentage of producer strains showing partmoderate and nil inhibition against fish bacterial pal,pens
Fig 5. Percentage of Gram-positive and Gram —negativestrains showing activity
This observation is on par with the findings of Jcyasckaran
et al.(2002), who have reported that pigmented bacterialflora was lower by about 2-3 log counts than the totalculturable bacterial flora observed in the marine samplesfrom seawater, sediments, sea plants and bivalves. Thefinding that the percentage of Gram-positive strains wasfound to be lower (29) than the Gram-negative strains(71) agrees with one of the earlier works which reportsthat the bacteria present in seawater are mainly Gram-
negative rods (Fenical, 1993). Another study revealed I
the bacterial strains isolated from various regimens of
marine environ showed that 82.8% were Gram-negat(Strahl ei al. 2002). There are a number of repcpertaining to the study of mucus-associated marine bteria (Di Salvo, 1971., Ducklow and Mitchell, 19Rublec etal., 1980; Paul ci al., 1986 and Coffroth,199A smaller percentage (16%) of the bacterial isolates vantagonistic compound producers. This agreed with Ireport of Nair and Simidu (1987) who stated that 8.
out of the 45 epiphytic bacteria isolated from differmarine samples displayed anti staphylococcus activity.the 16% (46) of the producer strains isolated in
Journal of Marine Biological Association of India (2005)
Fig. 10. Strain No. 237 antagonistic to (a) human and (b) fish pathogens
isolation and screening of mucus-associated bacteria of gastropod
157
Fig. 8. Strain No. 15 antagonistic to (a) human and (b) fish pathogens
Fig. 9. Strain No : 130 antagonistic to (a) human and (b) fish pathogens
(B.S- Bacillus subrilis, B.0 - B. cereus, E.0 - Escherichiaco/i, S. T - Salmonella typhimurium, K. P- Kiebsiella
pneumoniae, S. E - Staphylococcus epidermidis, S. A- S.
Journal of Marine Biological Association of India (2005)
aureus, S. D - Shigella dysentriae R M - Proteus mirabilis,S. M - Serratia ,narcescens, A. F - Aeromonas form icaflS,A. H - A. hydrophila, V. H - '7brio harveyii, V. V - Vvulnificus, V. C. - V campbelli, V. L- V iogei)
158
C. Chellarain et
present study, 70% (32) were non-pigmented which con-tradicts the findings of Rosenfeld and Zobell (1947) whoreported that most of the antibiotic-producing marine bac-teria are pigmented. However, a smaller percentage of
antagonistic compound producers (30%) were found tobe pigmented which may be due to the reason that pig-ments have been associated with antibacterial activity asis the case for cyanobacteria (L.emos et al., 1985). Pig-nented bacteria are also known to be potential antibioticproducers as reported by Shiba and Taga (1980). One ofthe findings of the present study is that Gram-negative
strains (29) showed comparatively higher antagonismagainst the test strains than the Gram-positive ones (17)which deviate from the works of Fenical (1993) whoreported that most of the Gram-negative bacteria from themarine samples have chemically proven to be unproduc-tive. In the present study, among the producer strains.D, D 30 . D, 17 were able to inhibit a minimum of II ofthe pathogens tested with (at least a partial inhibition)zones of 3-9 mm. Similar zones of inhibition by marineantagonistic bacteria were reported by earlier workers(Rosenfeld and Zobell (1947), Patil et al. (2001) andChelossi ci al. (2004).
Marine microbes have a higher possibility of yieldingnatural products with unprecedented and interesting hio-activity. The antagonistic marine bacteria isolated from themucus of D. nzargariticola may produce antibacterialcompounds with novel structures which can be exploredto generate pronounced biological activity in the future.
Acknowledgements
The authors are thankful to the authorities ofTuticorinPort Trust for research permission.
References
Belkin, S. D., C. Nelson and H. W. Jannasch. 1986. Sym-biotic assimilation of CO in two hydrothermal ventanimals, the mussel, B. 'lheriiiop/iil:i.r and the tubeworm, R. pacl:vpiila. Dial. Bull., 170:110-121.
Chelossi, E., M. Milanese, A, Milano, R. Pronzanto and G.Riccardi. 2004. Characterization and antimicrobialactivity of epibiotic bacteria from Petrosia ficiformi.',
(Porifera: Demospongiae). J. Exp. Mar. Biol. Eral.,
309: 21-33.
Coffroth, M. A. 1990. Mucous sheet formation on poritidcorals: an evaluation of coral mucus as a nutrientsource on reefs. Mar. Biol., 105: 39-49.
Di Salvo, L. H. 1971. Regenerative function and microbialecology of coral reefs: labelled bacteria in a coral reefmicrocosm. I. Exp. Mar. Biol. Ecol.. 7: 123-136.
Distel, D. L. 1998. Evolution of chemoautotrophic endo-symbioses in bivalves. Bioscience., 48: 277-286.
Journal of Marine Biological Association of India (2005)
Ducklow, H. W and R. Mitchell. 1979. Bacterial poplation and adaptations in the mucus layers on livitcorals. Limnol. Oceanogr., 24: 715-725.
Faulkner, D. J. 2001. Marine natural products. Nat. Pro
Rep., 18: 1-49.
Fenical, W. 1993. Chemical studies of marine bacteriDeveloping a new resource. Chem. Rei, 93: 167:1683.
W and P. Jensen. 1993. Marine microorganisms:new biomedical resource, Mar. Bioteclinol., 1: 41457.
Jeyasekaran, G., K. Jayanth, and R. Jeya Shakila. 200Isolation of marine bacteria, antagonistic to humapathogens. Indian J. mar. Sri., 31: 39-44.
Kharlamenko, V. I., S. I. Kiyashko, V.I. Vyshkvartze2001. Identification of food sources of invertebratefrom the seagrass, Zo.rtera marina community usincarbon and sulfur stable isotope ratio and fatty adanalyses, Mar. Ecol. Prog. Ser., 220: 103-117.
Kobayashi. J and M. lshibashi.1993. Bioactivc metaboliteof symbiotic marine microorganisms. Cizemn. Rev., 931753-69.
Kong, G. M., A. D. Wright, D. Sticher, C. K. Angerhofciand J.M. Pczuto. 1994. Biological activities of seicctcd marine natural products. Plow. Med., 6: 53237.
Lech, M. 2004. A shot in the arm. Nature, 431: 892-893
Lei, J and J. Zhou. 2001. A marine natural produciDatabase. J. Cheat. lnf Camp. Sri., 42: 742-748.
Lemos, M. L., A. Toranzo and J.L. Burjo. 1985. Antibioticactivity of epibiotic bacteria isolated from inter-tidalseaweeds: Mirrob. Eral., 11: 149-164.
Paul, R., G. Jcyasckaran, S. A. Shanmugam and R. JcyaShakila. 2001. Control of bacterial pathogens associ-ated with fish diseases by antagonistic marine Aciino-mycctes isolated from marine sediments. Indian I.
,,zar. Sri., 30: 264-267.
Paul, J. H., M. I. Dc Flaun and W. H. Jeffrey. 1986.Elevated levels of microbial activity in the coral sur-face microlayer. Mar. Eral. Proc. Ser., 33: 29-40.
Rosenfeld, W. D and C. Zobcll. 1947. Antibiotic produc-tion by marine microorganisms. J. Bacterial., 54:
394-98.
Rublee, P. A., H. R. Lasker, M. Gottfried and M. R.Roman. 1980. Production and bacterial colonizationof mucus from the soft coral, Briarium a.vbesti,iumn.
Bull. Mar. Sri., 30: 888-893.
Shiba. I and N. Taga. 1980. Heterotrophic bacteriaattached to sea weeds. J. Exp. Mar Biol. Eral., 47:
251-258.
Isolation and screening of mucus-associated bacteria of gastropod
159
Stein, J. L., S. C. Can'. R. R. Hessler, S. Ohta, R. D.Vetter,J. J. Childress and H. Felbeck.I988. Chemoautotrophicsymbiosis in a hydrothermal vent gastropod. Biol.
Bull., 174: 373-378.
Strahi. E. D., W. E. Dobson, and Jr. L. L Lun die. 2002.Isolation and screening of Brittle star associated bac-
teria for antibacterial activity. Cur Microbiol.. 44:
450- 59.
Vindoffer, R and 0. Giere. 1997. Symbiosis of the gas-
tropod. Ifremeria nautilei (Provannidae) withendobacteria-structural analyses and ecological con-
siderations. Biol. Bull., 193: 381-392.
Accepted: 20 August 2005
Journal of Marine Biological Association of India (2005)
Indian Journal of Marine SciencesVol. 34(3), September 2005, pp. 316-319
Short Communication
Isolation of antagonistic marine bacteria from the surface of the gorgonian cora1at Tuticofin, south east coast of India
*K . Mary Elizabeth Gnanambal, C. Chellararn & Jamila PattersonSuganthi Devadason Marine Research Institute, No: 44, Beach Road, Tuticorin- 628 001, Tamil Nadu, India
*IE.n,ail: maryelzi77 @yahoo.com ]
Received 28 September 2004, revised 9 March 2005
Culturable heterotrophic bacteria present on the surface of two gorgonian corals, Subergorgia suherosa and iwiceellajuncea were isolated and the number of pigmented and non-pigmented strains was noted. The antagonistic effect of themarine isolates was tested against 8 human pathogens and 7 fish pathogens. It was observed that 82% (289) of the isolatedbacterial strains were found to be non-pigmented and 61% (214) were identified as Gram-negative. Only 13% (46) of theisolated bacteria were found to be antagonistic against both human and fish pathogens. 74% (34) of the producer strainswere found to be non-pigmented, however, mild antagonistic activity was found to be exhibited also by the pigmentedstrains. Percentage of Gram-negative strains showing activity was found to be 68% (145). All the indicator strains testedwere inhibited by at least 10 of the antagonistic marine bacteria. A higher degree of inhibition was conferred by 3 of theisolates (Go, G ill and 0113) with maximum zones of inhibition against Escherichia coli (5.5mm) by the strain G ill . Thestrains showing higher degree of inhibition against human pathogens ( G 110 , Gill and G 113 ) showed potent inhibition againstthe fish pathogens too with maximum inhibition against Proteus nzirabilis (5mm) by the strain G 110 . The symbiotic bacteriapresent on the surfaces of these gorgonian corals may yield novel metabolites.
[Key words: Bacteria, corals, Tuticorin, Subergorgia suberosa, Junceella juncea, symbiotic bacteria, aiiagonistic activity][IPC Code: mt. Cl. 7 AOIN 63/021
Interactions between epibiotic marine bacteria andtheir host organisms are known to play a significantrole in marine ecosystems but this association hasreceived little attention. The important microhabitatsfor marine bacteria are the sediments, biotia andabiotic and internal tissues of invertebrates. Marineplants and animals are well known to have developedsymbiotic relationships with Iumerous microbes.Microbes are ubiquitous in the marine environmentand not surprisingly, mucus-covered coral surfacesare often colonized by bacteria and othermicroorganisms 1,2. It appears that these microbes arenot generally detrimental to their coral hosts 3 . Theimportance of bacterial symbiosis is growing inrecognition that they may be the true producers ofmany bioactive compounds isolated from corals,sponges, ascidians and other marine in vertebra tes 4 . Inthe past 20 years, the pharmaceutical industry hasbeen successful in containing problems due to singleresistance determinants. However, the advent ofmultiple resistance mechanisms has severely limitedthe effective use of many major classes of drugs. Sosearches for new drug classes with novel mechanism
of rction are of relevance. There is a growing conceabout the use and particularly the abuse of antimicrobidrugs not only in human medicine but alsoaquaculture. Hence, the need of the hour is a search fnovel antibacterial compounds with therapeutic potentifor which the pathogens may not have developresisitance5 . The symbiotic microorganisms from Umarine environs are a rich source of new metabolitwith a wide variety of biological activities and somethem display significant practical applications 6 . Theare studies which have focused on the metabolites of tigorgonian corals Euiiicea fusca7, Lophogorgia spPseudopierogorgia elizabethae9, etc. But worlinvestigating the symbiotic bacteria on gorgonid coraas potential sources of antimicrobial drugs are tsparse. Gorgonian corals, S'ubergorgia suberosa atJunceella juncea are found relatively common in turbnear-shore environs below 5 m depths. The symbiotbacteria attached to these gorgonian corals are being lestudied as sources of potent antibacterial compounds.a preliminary attempt was undertaken to isolate Uantagonistic bacteria associated with the surfacethese two gorgonids.
SHORT COMMUNICATION
317
Viable heterotrophic bacteria were obtained byswabbing a small area (1 cm 2 in triplicates) of theexternal surface of live gorgonian corals, Subergorgiasuherosa (Octocoral I ia: Alcyanacea: Subergorgiidae)and Junceella juncea (Octocorallia: Alcyanacea:Ellisellidae) from Tuticorin coastal waters, south eastcoast of India, using a sterile cotton swab, which wasthen placed in 2 ml of sterile seawater and vortexed.Serial 10-fold dilutions of each solution were preparedand aliquots (0.1 ml) were plated on ZoBell MarineAgar 2216 (ZMA)'°. Plates were incubated for 7 days at27°C. The number of pigmented and non-pigmentedstrains was noted. Perceptible different morphotypeswere isolated in pure culture on ZMA. Bacterial isolateswere kept in ZMA slants at 4°C. Gram staining wasperformed for all the isolated strains. To test theantagonistic effect of the isolated bacterial strains, 8human pathogens [Bacillus cereus (ATCC 10876),B. subtilis (ATCC 6633), Escherichia coli (ATCC25922), Salmonella typhi (ATCC 6539), Klebsiellapneuinoniae (ATCC 10031), Staphylococcusepidermidis (ATCC 12228), S. aureus (ATCC 29737)and Sijigella dysenrriae (ATCC 13313)] and 7 fishpathogens [Proteus 'nirabilis (MTCC 1429), Serratia/narcescens (MTCC 97), Aero,nonas hydrophila (ATCC7966),A. forinicans (shrimp isolate), Micrococcus sp. (fishisolate), V. Iiarveyi (shrimp isolate) and V. vulnificus(ATCC 27562)] were used as test strains. All the humanpathogens and the fish pathogens, Proteus niirahilis,Serratia inarcescens, A erononas Izydroph ila,V. vulnijicus were obtained from Christian MedicalCollege [CMC, Vellore, India] and the remaining fishpathogens were isolated from the above mentionedsources. Double agar overlay method was used for theassay of antagonistic bacteria against the humanpathogens". Colonies of antagonistic bacteria weredeveloped on ZMA plates by spotting 18 h old cultureand incubating at 30°C for 40 h. All the test organismswere cultured in Tryptone Soya Broth (TSB) and the18-24 Ii old cultures were used for the experiments.About 10 tl of the culture was suspended in 8 ml of softTryptone Soya Agar (TSA) with 0.7% w/w agar waspoured immediately over the macro-colonies of theantagonistic marine bacteria on the ZMA plates. Theplates were incubated at 30°C for 24 ii. The cleared zonearound the macro-colonies of the antagonistic bacteriawas measured and the radius of zone of inhibition wasnoted (in mm). Antagonistic activity of the isolatedmarine bacteria was also tested for the fish pathogens
following the method of Gauthier ' 2 . About 1 ml of18-24 h old culture of fish pathogens in TSB was mixedwith molten TSA (supplemented with 1% NaCI) andwas poured immediately on the petriplates and allowedto harden. Marine bacteria were cultured on ZMA platesand a small amount of cell paste was scraped off anddeposited onto the surface of TSA plates seeded with thetest bacterium. After a diffusion time of 30 mm, plateswere incubated at 30°C for 24 h. A clear zone ofinhibition around the cells of marine bacteria indicatedantibacterial activity and the radius of zone of inhibitionwas noted (in mm). Some of the producer strains wereidentified up to the generic level using the biochemicalmethods outlined in Bergey's manual of systematicbacteriology1314
A total of 352 bacterial strains were isolated from thegorgonids, Subergorgia suberosa and Junceella junceaand among them 82% (288) of the bacterial strains werefound to be non-pigmented of which 61% (214) wereidentified as Gram-negative. Majority of the pigmentedcolonies were yellow, red, brown, orange and black incolour. Only 13% (47) of the isolated bacteria were foundto be antagonistic against both human and fish pathogens.About 74% (35) of the producer strains were found to benon-pigmented and only 26% (12) were pigmented.However, mild antagonistic activity was exhibited bymany pigmented strains also. In the present study, thepercentage of Gram-negative strains showing activity (32)was higher (68%) than the Gram-positive strains (32%).Screening of the marine bacterial isolates against humanpathogens (Table I) showed that all the indicator strainstested were inhibited by at least 10 of the antagonisticmarine bacteria. A higher degree of inhibition wasconferred by 3 of the isolates (G,, 0, G ill and G 113) withmaximum zones of inhibition against Escherichia coli(5.5 mm) by the strain G 113 . However, some of the strains(G89 and G) were able to display mild antagonism. Datafor the inhibitory activity of the isolated marine bacteriaagainst the fish pathogens are shown in Table 2. Thestrains showing higher degree of inhibition against humanpathogens (0 110, G, 11 and 0113) showed potent inhibitionagainst the fish pathogens also with maximum inhibitionagainst Proteus mirabilis (5 mm) by the strainHowever, strains G 12 and G 163 showed inhibition againstSerratia inarcescens with zones of 4 mm each and 0162and G 163 against Aeroinonas hydrophila. Strains G,, G2and G320 displayed only mild activities. The present studyshowed that major antibiotic producing strains belong tothe genera Vibrio (G,, , 0,,,), Pseudomonas (G,,3),Micrococcus (G89 , G9 ) and Bacillus (G ,
318
INDIAN J. MAR. SCI., VOL. 34, No.3, SEPTEMBER 2005
Producer BacilliStrains cereu
01 2
G5 2
G8 2.5
G9 2
010 2
025 --
030 1.5
032 T
041 2.5
056 2
060 1.5
067 --
089 I
090 1.5
0110 4
Gill 4
0113 5
0162 1.5
0189 --
0203 --0209
0225 1.5
0289 --
T= Trace: --=No activity
Table 1-Surface bacteria associated with gorgonids antagonistic to human pathogensZone of inhibition (values show radius of the zone of inhibition in mm)
s B. subtilis Escherichia Salmonella Kiebsiella Staphylococcu S. aureuscoli ryphi pneumoniae .c epiderinidis
2.5 2 2 2 1.5
1.52.5 2 1.5 1.5 1.5
2
3 3 3 2.5 3
32.5 2.5 2.5 2 2.5
2
2 2 1.5 2 2
1.5T -- -- 2 2.5
1.5
-- -- -- -- T
2 2 1.5 1 1-- -- 1 T -- 1.5-- 1.5 -- T
1.51.5
-- 1 1.5 -- -- 1.5-- -- 1.5 -- -- 1.54 4.5 4 3.5 4
4
4 5 4 4 4.5
45 5.5 5 4.5 5
5
1 1 -- -- ---- T -- 1 1T -- 1.5 1.5 1.5-- 1.5 1.5 1 1.5
-- -- -- T --
Shigtdyseni
2.52.5
1.51.5
4.54.54.5
1.5T
Table 2-Surface bacteria associated with gorgonids antagonistic to fish pathogensZone of inhibition (values show radius of the zone of inhibition in mm)
Pathogens Proteus Serratia Aeroinonas A. farmicans Micrococcus sp., Vibrio harveyii V. vuln(f,nirabilis inarcescens hvdrophila
01
2 2 2.5 -- -- T
1.505
1.5 1 I 1.5 -- 1.508 -- -- -- 1.5 -- T09 -- T 1 -- --010
T -- 1.5 1 1.5025
T 1.5 1.5 1 1.5 1
1.5030
5 -- 4 4 4 4.5
4032
4 4 4 4 3.5 4
4041
4 4 3.5 3.5 4 4
3.5056
1 -- 1 2 1 T060
1.5 4 2 2 2 1.5
1.5067
1.5 4 2 2 2 1.5
1.5089
1 1 1.5 1 1 -- 1.5G90
T0110
2 2 1.5 1.5 1 2
2Gill
1.5 1 1 -- T 1
1.5G113
1.5 1 1 -- 1.5 10162 -- -- -- -- T --0189
2 1.5 2 2 1.5 2G203
1.5 1.5 1.5 1.5 1.5 1.5
1.50209
I -- I T --G225 -- 1.5 -- 1.5 1.5 -- T6289
1 1 -- 1 1 1
T= Trace: --=No activi
SlIORT COMMUNICATION
A lower percentage of pigmented strains (18%) wereisolated from the surface of the gorgonian corals,Subergorgia suberosa and Junceella juncea. Thisobservation is similar to the findings of Jeyasekaran et
al.' 5 who reported that pigmented bacterial populationwas lower by about 2-3 log counts than the totalculturable bacterial population observed in the marinesamples. In the present study, of the 352 isolates, a totalof 214 accounting to about 61% of the bacterial strainswere Grain-negative which is in line with an earlierwork' which reports that the bacteria present in seawaterare mainly Gram-negative rods. A higher percentage ofantagonistic strains was found to be non-pigmented(74%) which contradicts an earlier report that states thatmost of the antibiotic-producing marine bacteria grownon marine agar are pigmented ' 6 . Present finding suggeststhat 68% of the producer strains isolated was identifiedto be Gram-negative, which is in line with the view thatmajority of the antagonistic bacteria isolated from theTuticorin coast of Tamil Nadu' 5 were found to be Gram-negative and only one strain was Gram-positive.However this observation disagrees with the findings ofFenical4 who reported that the Gram-negative rodsisolated from the marine samples have proven to bechemically unproductive. The antagonistic bacteriaisolated from the surface of the gorgonids were able toinhibit most of the test strains used for the experiment.Inhibition zones of up to 22 mm were observed for 3coral species against human pathogens 15 . It has beendocumented that bacteria associated with the soft coral,Dendronephthya sp. are suggested to produce bioactivecompounds against the attachment of bacteria onto thesurface of these organisms ' 7 . Presence of antagonisticbacteria on the surface of this soft coral is to controlbiofouling. It has been hypothesized that this organismmay control biofouling on their surfaces by regulatingthe bacterial species composition. Ducklow & Mitchel13found that some species of the marine bacterium Vihrioare adapted for living in the mucus layers of livingcorals. Actinomycetes, recognized as sources ofantimicrobial compounds have been isolated fromvarious marine invertebrates such as corals'8.
A better recovery of strains from the surface ofthese gorgonian corals with antibacterial activitysuggests that these organisms represent an ecologicalniche which harbors a largely uncharacterizedmicrobial variety. Thus, the symbiotic bacteriaattached to the surface of these gorgonid corals mayyield a vast array of new compounds with novelactivities.
ReferencesDiSalvo L H, Regenerative function and microbial ecolof coral reefs: labeled bacteria in a coral reef microcoJExp Mar Biol Ecol,7 (1971) 123-136.
2 Rublee P A, Lasker H R, Gottfried M & Roman MProduction and bacterial colonization of mucus from thecoral, Briariu,n asbes ginu,n, Bull Mar Sci, 30 (19888-893.
3 Ducklow 1-1 W & Mitchell R, Bacterial populationadaptations in the mucous layers on living corals, LinOceanogr, 24(1979)715-725.
4 Fenical W, Chemical studies of marine bacteria: Develola new resource, Citeizi Rev, 93 (1993) 1673-1683.
5 Patil R, Jeyasekaran 0, Shanmugam S A & Jeya ShakiliControl of bacterial pathogens associated with fish diseby antagonistic marine Actinomycetes isolated from masediments, Indian J Mar Sci. 30 (2001) 264-267.
6 Fenical W & Jensen P. Marine microorganisms: A I
biomedical resource, Mar Biotechnol, 1(1993)419-457.7 Shin J & Fenical W, Fucosidcs A-D: Anti-inllanima
diterpenoid glycosides of new structural classes fromCaribbean gorgonian, Eunicea fusca, J Org Chem, 56 (13153-3158.
8 Jacobs R S, Culver P. Langdon R, O'Brien T & WhitSome pharmacological observations on marine naproducts, Tetrahedron, 41(1985)981-984.
9 Roussis V. Wu Z, Fenical W, Stobel S A, VanDyne 0 1Clardy J. New anti-inflammatory pseudopterosins frommarine Octocoral. Pseudopierogorgia elizabethae, JClieni. 55 (1990) 4916-4922.
10 Chelossi E, Milanese M. Milano A, Pronzato R & Ric0, Characterization and antimicrobial activity of epibbacteria from Petrosia ficiforntis (Porifera, DemospongJ Exp Mar Biol Ecol, 309 (2004) 21-33.
11 Dopazo C P. Lemos M L, Lodeiros C, Bolinches J, Barj& Toranzo A E, Inhibitory activity of antibiotic produmarine bacteria against the fish pathogens, J Appi Bactt65 (l988)97-101.
12 Gauthier M I, Modification of the bacterial respirationpolyanionic antibiotic produced by a marine Alieronu/inrin,icrob Agents Chemother. 9 (1976) 349-354.
13 Kreig N R & Holt J G, Bergey's manual of syslerbacteriology. Vol 1 (Edo, Williams & Wilkins, Baltim1994 pp.910.
14 Kieig N R, Simeath P 11 A, Mair N S & Sharpe E M, lienmanual of systematic Bacteriology, Vol 2 (WillianiWilkins, Baltimore), 1984 pp. 911-1504.
15 Jeyasekaran G, Jayanth K & Jeya Shakila R, Isolaticmarine bacteria, antagonistic to human pathogens, mdi
Mar Sci, 31 (2002) 39-44.16 Rosenfeld W D & Zobell C E, Antibiotic production b
marine microorganisms. J Bacteriol. 54 (1947) 393-398.17 Dobretsov S & Qian P. The role of epibiotic bacteria
the surface of the soft coral, Dendronepht/mya sp. iiinhibition of larval settlement, J Exp Mar Biol Ecol,(2004) 35-50.
18 Zheng Z, Zeng W, Huang Y, Yang Z, Li J, Cai H & SDetection of anti-tumor and antimicrobial activities in IF
organisms associated Actinomycctes isolated froniTaiwan Strait, China, FEMS Microbiol Lett, 188 C
7-91.
Proceedings of 10th International Coral Reef Symposium, 133-137 (2006)
Distribution of Red-band disease and effect of boring sponges onsolitary corals in Tuticorin coastal waters, Gulf of Mannar,
Southeast coast of India
Chellaram C, Jamila Patterson and JK Patterson Edward
Suganthi Devadason Marine Research Institute, 44- Beach Road, Tuticorin— 628 001, India*jkpattisanchametin
Abstract The Gulf of Mannar (GOM) covers an areaof approximately 10,500 sq. km and includes a chain of21 islands surrounded by fringing and patchy reefs.Studies on the distribution of coral diseases and theeffect of boring sponges on corals were carried out insitu in Tuticorin coast 95 belt transects were laid toquantify the number of Red band affected colonies,unaffected colonies and boring sponge colonies in eachtransect, and the distribution of affected species andtheir intensity. During the survey, live corals werecounted along transects besides the diseased, and boringsponge affected corals. Red-band disease (RBD) wasrecorded in the mainland patch reef area whereTurbinaria sp. is dominant and the disease was notencountered around the islands. Selective species suchas T. mesenterina and T. peltata were affected and T
mesenlerina was most susceptible to RBD. About 15-
20% of T. peltata and T. mesenterina sp. in Tuticorinwaters was affected by RBD. Coral boring spongesaffected about 0.5% corals. Further studies are inprogress to identify the causative agent for the RBD.
Keywords: Gulf of Mannar, Coral survey, Red banddisease, coral boring sponge
IntroductionCorals diseases are expanding rapidly around
the world and the situation is unprecedented. Theproblem is a genuinely new and rapidly increasing inthe corals and associated reef organisms. Mortality ismajor threat to reef health around the world. Over thepast 20 years, there has been increase in possible newdiseases (Antonius, 1981, Williams and Bunkly -Williams, 1990; Santavy and Peters, 1997), which arecurrently referred to as syndromes affectinghermotyphic corals (Goreau et al. 1998; Richardson,1998). Some of these epizootics are associated withunusual stresses from natural or man-made causes (e.g.
sediment action, temperature fluctuations andpollutants (Mitchell Chet, 1975; Antonius, 1977),suggesting that external stress may lower coralresistance or stimulate the growth of pathogenicorganisms. However, most reef epizootics show littleobvious linkage to local spatial and temporal Stresses,and these may be due to the dissemination of new ornewly adapted pathogenic agents in the marineenvironment (Goreau et al. 1998). Some postulatedanthropogenic stress linked to coral reef diseaseincludes de-forestation and soil erosion. Also windor ocean transport of dust could potentially result inthe introduction of terrestrial microbes into themarine environment (Smith et al. 1996, Nagelkerkenet al. 1997; Geiser el al. 1998). Aspergillus sydowiihad been shown to be an important pathogenassociated with diseased sea fan tissue throughout theCaribbean, affecting Gorgonia yenta/ma and G.
flabellum (Cnidaria: Gorgoniidae) (Smith et al. 1996;Nagelkerken et al. 1997; Smith et al. 1998). 4.
sydowii is not a common marine fungus but it wastypically found in soil and other habitats.
Four major coral diseases (black-band,white-band type II, white plague type 11 andAspergillosis) have been described (Santavy andPeters 1997; Riëhardson 1998; Green and Bruckner2000) and the effects of disease on coral communitieshave ranged from partial mortality of a fewindividuals to community level changes (Edmunds1991; Aronson and Precht 1997). White-band diseasehas caused profound effects on coral communities inCaribbean region. Acropora palmata standing in theUS Virgin Islands have been decimated by acombination of white-band disease (Gladfelter 1982)and hurricane damage (Bythell et al. 1993).
Red band diseaseAs the name indicates, the band is a soft
microbial mat that is brick red or dark brown in color
133
and can be easily dislodged from the surface of thecoral tissue. This disease affects hard star, stag horn,and brain corals. The microbial mat movements aredifferent and the types of microbes present might bedifferent depending on the host, but little is knownabout this. Several scientists are studying thecomposition of these microbial mats. Red-band disease(RBD) has been reported to affect hard corals(Richardson, 1993) and sea fan (Santavy, 1996) inCaribbean. The dominant Cyanobacteria, in Red-banddisease found on Scleractinian corals from the Bahamaswere two species of &cillatoria (Richardson 1993).Subsequent investigation in Puerto Rico has confirmedthe red band obtained from diseased gorgonians wascomposed primarily of two species of Cyanobacteria.
Coral boring spongesBoring sponges of the family Clionidiae have
the capacity to bore into calcareous substrates such ascorals, shells of mollusks and calcareous rocks. Theyexert an important influence on the erosion andrecycling of accumulated calcium carbonate,particularly from coral reefs. Sponges destroy corals(known as boring sponges) by their ability to dissolvethe calcium carbonate (coral skeletons and bivalveshells) and cause problems to the coral growth. Up to50 -90% of calcium carbonate from coral skeleton maybe removed by sponge activity (Mc Geachy and steam1976). Studies from several reef areas, around theglobe, have been showing that bioerosion rates in coralreefs, change with water depth (Goreau and Hartman1963), the age of coral colonies (Kiene 1988), type ofreef framework (Bromley 1978) and the water energyregime (Mc Geachy and Steam 1976). They are alsoassociated with several other environmental parameterssuch as, the available nutrients (Risk and Ma Greachy1978; Hallock et al. 1993) and coral bleaching (Glynn1977).
The aim of the study was to identify species ofcorals from the Tuticorin reef area that are susceptibleto RBD and to determine its prevalence in affectedcolonies. Difference in susceptibility of coral species toRBD was investigated by comparing the number ofindividuals within a species that showed signs of RBDwhen experimentally placed to direct contact withnecrotic tissue. Also the corals affected by boringsponges were investigated.
Materials and Methods
Study areaGulf of Mannar (GOM) (Fig.1) located on the
Southeast coast of India is the first Marine BiosphereReserve inSouth and Southeast Asia, covering an areaof 10,500 sq. km. GOM has 21 uninhibited islands (Lat.
8° 47' - 9° 15' N and Long. 78° 12' - 79° 14' E),
surrounded by fringing and patchy reefs. The islandsare divided into 4 groups namely, (1) Mandapam, (2)Keezhakkarai, (3) \'embar and (4) Tuticorin. Thestudy was conducted in the Tuticorin coastal areaincluding four islands, namely Vaan, Koswari,Vilanguchalli and Kariachalli and mainland patchreef.
General approachPotential sites with dense live coral cover
were selected using a stratified random design, withinthe five sites (4 islands and mainland patch reef)during February - April 2003. Surveys wereconducted using belt transects, each covering an areaof 1 x 15m (English et al. 1997). In total, 95 transectswere laid. For disease studies, the number of coloniesin an area is usually recorded (Edmunds 1991, Kuta
Rxnenywam
IM)L ,l "1
Tt4icor*, /:.:,.3
Gut OFM)KkR
)WC44NU.U4i.M I 4.Pis6,lmd
—4 -c77 hnOcen 79 79
Fig. I. Map showing Gulf of Mannar
and Richardson 1996). A colony is defined anyautonomous part of coral with living tissue. RBD wasidentified in the field using the gross morphologicalcharacteristics, which includes a red band of necrotictissue abutting relative healthy tissues. The numbersof RBD affected and non-affected colonies in eachtransect were quantified.
Photo documentationAll transects were laid between depths of 3
and 6 m. Counts of RBD and coral boring spongeswere compared to those of healthy corals species.Stainless steel nails with numbered tags were placedat the affected edges to monitor the progression ofRBD. Colonies in mainland patch reef werephotographed from February to April 2003 using anUnderwater Canon digital camera.
ResultSelectively two species of corals were
affected byRBD and three species by boring spongesin Tuticorin coastal waters. Turbinaria mesenterina
134
and T. peltata had 502 and 149 colonies respectivelyaffected with RBD and Porites lutea, T. mesenterina
and T. peltata had 1, 14 and 9 colonies affectedrespectively with boring sponges (Figs 2 and 3).
In mainland patch reef, 95 transects were laid,in which a total of 3398 (2396 colonies of T.mesenterina and 1002 colonies of T. peltata) colonieswere counted, and 19.58% (14.78% of T. mesenterina
Fig.2. Red-Band disease in T. peltata and I
,nesenlerifla
Fig. 3. Coral boring sponge in T. mesenterina and I
peltata
and 4.38% of I peltata) colonies were found to beinfected with RBD. 95 transects were laid in all islandsand 918, 872, 665 and 1210 colonies were counted inVaan, Koswari, Velanguchalli and Kariachalli islandsrespectively, which were not found to be affected withRBD (Tab 1).
Boring SpongesTotalSite Trans -no ' s P. T. T.
luiecj mesenterifla peltala
I 95 3398 0 tO 7
2 95 918 I 2
3 95 872 0 2 I
4 95 665 0 0 0
5 1 95 1210 0 0 0
Tab I: (Site 1- Mainland patch reef; 2-Vaan; 3-Koswari; 4- Velanguchalli and 5- Kariachalli)
In the mainland patch reef, 0.5% coral
colonies (0.29% T. mesenlerina and 0.21% T. peltata),
Vaan island, 0.44% colonies (0.11% Pontes lutea,
0.22% T. mesenterina and 0.11% T. peltala), Koswari
Island 0.34% coral colonies (0.22% T. mesenterina and
0.11% T. peltata) were affected by boring sponges (Tab
2). No coral colony was observed to be affected byboring sponges in Kariachalli and Velanguchalli
islands.
RBDTotal __________
Site Transect Colonies T. mese7Ix' T. peltata
I 95 3398 502 149
2 95 918 0 0
3 95 872 0 0
4 95 665 0 0
5 95 1210 0 0
Tab 2: (Site I- Mainland patch reef, 2-Vaan; 3-Koswari; 4- Velanguchalli and 5- Kanachalli)
It was observed that 0.52±0.12 (n=) cm oftissues died in the affected area per month due toRBD. After the infection by RBD, mucus productionin corals was not present in the infected portion. Theaffected area become bleached and broken. Thecolour of RBD tissue is a characteristic feature andranged from red to orange in T. mesenrerina and T.
peltata with strong increase in pigmentation towardsthe edge. The surface depression was greatest at thedying edge or centre of the lesion.
DiscussionRed-band disease was first noticed as a
variant of Black band disease (BBD) infectingGorgonia yenta/ma near Carrie Bow Cay, Belize(Rutzler and Santavy 1983). The term RBD was usedto describe a brick red cyanobacterial mat resemblingBBD on Scieractinian corals from the Bahamas(Richardson 1993). Reports of a "brown band"infecting Acropora formosa, from the Great BarrierReef might also be RBD. It has affected 20 coralspecies in five Scleractinian families and wasreported to be different from BBD (Dinsdale 1994).
The identity of the primary cyanobacteriacomprising the "band" of RBD is unclear, withdifferent species proposed from distant locations. Thedominant cyanobacteria were two species ofOscillatoria in RBD, found on Scleractinlan coralsfrom the Bahamas (Richardson 1993). Subsequentinvestigation in Puerto Rico has confirmed the redband obtained from diseased gorgonians to becomposed primarily of two species of cyanobacteria.Each cyanobacterium has trichome morphology and16S rRNA sequences differ from each other (Santavyet al. 1996). Additional research is needed to identify
135
the pathogenic agent (s) of the disease (s) appearing asRBD on corals.
The migration of the "band" in BBD wasmuch faster than in RBD, where the movement of thered "band" occurring only during the day while theblack "band" moving at night and day times(Richardson 1993). Different migration patterns of thecyanobacteria associated with RBD were observed ascompared to P. corallyticum on Scleractinian corals(Richardson, 1993).
Out of the 7063 colonies surveyed, 651 and 24coral colonies from two Scieractinian species wereaffected with Red band disease and coral boringsponges respectively in Tuticorin coastal waters. Thiswork suggests that RBD has affected coral colonies inmainland patch reef in Tuticorin coast, whereas suchsevere impacts were not noted in corals around theislands. A high--r percentage of T. mesenterina coloniesaffected with RBD (14.78%) suggest that this coralspecies might be highly susceptible to RBD. In the caseof boring sponges, T. pellata and T. mesenterina hadhigher levels of affected colonies than Porite.s luteaboth in mainland patch reef and islands. The realcausative agent for RBD in Tuticorin coastal waters isso far unknown and further studies are in progress.
AcknowledgementsThe authors are thankful to the Ministry of
Environment and Forests, Govt. of India and Coral ReefDegradation in Indian Ocean (CORDIO), Sweden forthe financial support and Chairman, Tuticonn PortTrust and Chief Wildlife Warden, Tamil Nadu ForestDepartment for Research permissions..
ReferencesAntonius A (1981) The "band" diseases in coral reefs.
Proc 4th mt Coral Reef Sym 2:7-14Antonius A (1977) Coral mortality in reefs: a problem
for science and management. Proc Id mt CoralReef Sym 2: 618-623
Aronson RB, Precht WF (1997) Stasis, biologicaldisturbance, community structure of a Holocenecoral reef. Paicobiology 23: 236-246
Bromley RG (1978) Bioerosion of Bermuda reefs.Paleogeography, Pa!eoclimatology, Paleoecology
23 (3-4): 169-197Bythell JC, Gladfelter, EH, Bythell M (1993) Chronic
and catastrophic natural mortality of three commonCaribbean reef corals. Coral Reef 12: 143-152
Dinsdale EA (1994) Coral disease on the Great BarrierReef, J Conf on Sci Mgt Sustainability of MarHab on GBR. Richardson LL (1993) Red banddisease: A new cyanobacterial infestation of corals.Am Acad Und Sci 10th Ann Sci Div Symp: 153-160
Edmunds PJ (1991). Extent and effect of black-banddisease on a Caribbean reef. Coral Reef 10: 161-165
English S, Wilkinson C, Baker V (1997) Surveymanual for tropical marine resources. AustralianInst Mar Sci, Townsville, Australia: 390
Geiser DM, Taylor JW, Ritchie KB. Smith GW(1998) Cause of Sea fan death in the WestIndies. Nature 394: 137-138
Glatfelter \VB (1982) White-band diseases inBermuda. Nature 253:349-350
Glynn PW (1977) Bioerosion and coral reef growth:a dynamic balance. In: Birkeland, C. (ed) Lifeand Death of Coral Reefs. Chapman Hall NewYork: 69-95
Goreau IF, Hartman WD (1963) Borin g sponges ascontrolling factors in the formation andmaintenance of coral reefs. In: R F Sognnaes(ed) Mechanisms of Hard Tissue Destruction.Amer Ass for the Adv Sci Pub 75: 25-54
Goreau TJ, Cervino J, Goreau M, Hayes R. Hayes M,Richardson L, Smith G, DeMeyer K,Nagelkerken I, Garzon Ferra J, Gil D, Garrison
G, William EH, Bunkley William L Quirolo C,Patterson K, Porter J, Porter K (1998) RapidSpread of Caribbean Coral Reef Diseases. RevBio Trop 6: 157-171
Green B', Bruckner AW (2000) The significance ofcoral disease epizootiology for coral reefconservation. Biol Cons 96: 347-361.
Hallock P, Muller-Karger FE, Halas JC (1993) CoralReef decline. National Geographic Res ExpI 9(3): 358-378
Kiene WE (1988) A model of bioerosion and itseffect on the Great Bnamer Reef. Proceedings ofthe dh mt Coral Reef Sym Townsville 3: 449-454
Kuta KG, Richardson LL (1996) Abundance anddistribution of black-band disease on coral reefin the northern Florida Keys. Coral Reefs 15:
219-223Mc Geachy JK, Steam CW (1976) Boring by
macroorganisms in the coral Montastreaanularis on Barbados reefs. mi Rev derGesaniten Hydrobiologie 61: 715- 745
Mitchell R, Chet 1(1975) Bacterial attack of corals inpolluted seawater. Micro Ecol 2: 227-223
Nagelkerken 1K, Buchan GW, Smith K, Bonair P,Bush J, Garzon-Ferreira I, Pors P, Yoshioka(1997) Widespread disease in Caribbean seafans: H Patterns of infection and tissue loss. MarEcol Pmg Ser 160: 255-263
Richardson LL (1998) Coral disease: what is reallyknown? TREE 13: 438-443
136
Risk Ml, MacGeachy ilK (1978). Aspects of bioerosionof modern Caribbean reefs. Revista de BiologiaTropical 26(1): 85-105
Rutzler K, Santavy DL (1983) The black-band diseaseof Atlantic reef corals Description of thecyanophyte pathogen. PSZNI Mar Ecol 4: 301-319
Santavy DL, Peters EC (1997) Micro pests Coraldisease in the western Atlantic. Proc gh mt Coral
Reef Syrn, Panama. 1:607-612Santavy DL, Schmidt T, Wilkinson SS, Buckley DH,
Bruckner AW (1996) The Phylogency and allianceof cyanobacteria affiliated with two band disease incorals from the western Atlantic. 91h Gen MtgASM: 325.
Smith GW, Ives LD, Nagelkerken AA, Ritchie KB(1996) Aspergilliosis associated with Caribbeansea fan mortalities. Nature 382: 487
Williams EH, Bunkley-Williams L (1990) The worldwide coral reef bleaching cycle and related sourceof coral mortality. Atoll Res Bull 335:1-71
137
Proceedings of the National Seminar on Reef Ecosystem Remediation
Insecticidal and herbicidal activity of a gorgonid associatedwinged oyster, Pteria chinensis
C. Chellararn, K. Mary Elizabeth Gnanambal and Jamila Patterson*Suganthi Devadason Marine Research Institute
44, Beach Road, Tuticorin - 628 001, India*jarnjiapat®hotmail. corn
Abstract
Marine organisms processing novel bioactive substances could form a potential source ofherbicidal and insecticidal compounds. The ethyl acetate, acetone and dichloromethaneextracts of the gorgonid associated bivalve, Pteria chinensis were evaluated for herbicidal andinsecticidal activities. The acetone extract showed 100% mortality in Sitophilus oiyzae, adreadful pest of rice at 20mg/nil concentration. The extract also showed herbicidal activity invitro and effectively decayed duckweed, Lemna minor at a concentration of 500 j.ig/ml in day 7.The preliminary screening for insecticidal and herbicidal activies indicates that P. chinensiscould be a potential source of natural pesticides.
IntroductionThe marine resources possess
biologically active compounds withpotential for use as pharmaceuticals,nutritional supplements, cosmetics,agrochemicals, molecular probes,enzymes and fine chemicals. Research onbio-products is growing rapidly to meetout the demand for new chemicals.Although chemicals with increasedefficacy and safety have been developed,the fight for controlling insect pestsexists both in the field and storehouse.Also, the pesticide and agro-chemicalindustry face growing criticism over thetoxicity and residue problems associatedwith the use of chemical control agents(Huppatz, 1990). Since these chemicalsare toxic and biologically active, their usefor insect control has become a seriousconcern (Balogh and Anderson, 1992).Apart from that, the insects acquireresistance to the existing insecticides andthus the development of new insecticideis always under consideration to tacklethe problem.
The judicious application ofherbicides has been an integral part of
agriculture (Piementel, 1986). Theever-increasing needs in agriculture toprotect crops have promoted extremelyintensive research efforts all over the world.The appropriate application of herbicideshas brought about a great reduction oflabour and increase in crop yield. An idealherbicide should have potent activityagainst weeds, minimum toxicity againstliving things other than plants, highselectivity between crop plants and weedsand cause no damage to the environmentthrough residual material. In the hunt fornew agro-chemical agents, the plants,animals and microorganisms of themarine environment with their wide rangeof chemical diversity prove to be anunexplored resource. Insecticidalcompounds were isolated from red algae(Watanabe et al., 1989), corals (Grode etal., 1983) marine annelids (Okaichi andHashimoto, 1962) and many importantherbicides from marine algae (Fenical,1981). In the present study, the wingedoyster, Pteria chinensis attached togorgonids were screened for theirInsecticidal activity against a dreadfulrice pest Sitophilus oryzae, in vitroherbicidal activity against Lemna minor(duckweed).
178
SDMRI Research Publication No. 9, 178 - 181, 2005
Material and Methods
Live Pteria chiriensis (Satyamurthi,1952) (Mollusca: Bivaivia: Pteriidae)attached to gorgonids were collected froma depth of 6 meters by SCUBA diving andtransported to the laboratory in livecondition. The shells were opened andthe soft body was taken out, cut intosmall pieces and air-dried to remove thewater content. A constant amount of lOgof the air-dried meat each was used theextraction in different solvents. Theextracts of the air-dried animals wereobtained using different solvents such asethyl acetate, dichioromethane andacetone after cold steeping at -18°C. Theextracts from each solvent were filteredseparately using Whatmann No.1 filterpaper. The filtrate was poured into thepreviously weighed petri plates,evaporated to dryness and used for allthe experiments and the concentrationof extracts were noted in mg.
The insecticidal activities of theextracts of Pteria chinensis from differentsolvents were tested using the modifiedcontact bioassay method of Broussalis etal. (1999). The dried extracts werere-dissolved in their respective solventsat 1mg/nil concentration. From this 2,3, 5, 10, 20 and 40m1 were poured intoseparate petri plates in triplicates andallowed to evaporate overnight to get thesame concentration in mg/ ml of extracts.Controls with solvents alone were takenin separate petri plates and evaporatedovernight. Ten healthy adults of Sitophilu.soryzae were introduced into each petriplate and were sufficiently fed, so thatdeath due to starvation could be ruledout. 4-5 small holes were made on thelids of the petri plates to ensure thatthe test organisms do not die due tosuffocation. Number of dead insects wascounted after 24 hrs and the percentagemortality was noted. The ED50 values of
the extracts were determined by theprobit analysis (Finney, 1971).
The herbicidal activity of thecrude extracts was assayed using theduckweed, LeTnna minorL. following a benchtop bioassay described by McLaughlin(1991). L. minor (duckweed), a miniatureaquatic monocot consists of a central ovalfrond or mother frond with two attacheddaughter fronds and a filamentous root.Under normal conditions, the plantsreproduce exponentially with budding ofdaughter fronds from pouches on thesides of the mother fronds.
To assay the herbicidal activity,the dried crude extracts were re-dissolvedin their respective solvents at 1mg/miconcentration. From this solution, 1000,500, 100, 50 and 10iL were pipetted intodram vials corresponding to 1000, 500,100, 50 and loug/ ml respectively intriplicates. Controls with appropriatesolvents alone were maintainedseparately. The solvents of the controland test vials were allowed to evaporateovernight and 2mi of E-medium(consisting of KH 2 PO4 - 680mg, KNO3 -1515mg, Ca (NO 3) 2 . 4H2 0 -1180m9,MgSO4 .7H20 - 492mg, H3B03 - 286mg,MnCl2 . 4H20 - 3.62 mg, FeC12 . 6H20 -5.40mg, ZnSO4 . 7H20 - 0.22mg, CuSO4.5H20 - 0.22mg, Na2Mo04.2H 20 - 0.12mgand EDTA - 11.2mg in one liter ofdistilled water) was added into each vial.A single L. minor plant containing arosette of three fronds was introducedinto each vial and were placed in glasschamber with about 2 cm water at thebottom to maintain the moisture contentof the chamber and sealed with glassplate and the whole set up was main-tained under normal light conditions. Thefronds per vial were counted daily up to7 days and symptoms of damage to thefronds such as yellowing and decayingwere noted.
179
0E
120
100
80
60
40
20
0
2 3 5 10 20 40
0 Control
0 Tissue extract inethyl acetate
0 Tissue extract indichioromethane
E Tissue extract inacetone
Extract concentration (ing)
Fig. 1. Percentage of mortality of Sftophthis oryzaeby the tissue extracts of Pteria chfrsen.sis
Proceedings of the National Seminar on Reef Ecosystem Remediation
Results and DiscussionRice weevil, Sitophilus oryzae is a
common pest, which destroys stored rice,wheat and sorghum grains and producesa rise in humidity, which inturnquickens the propagation of molds andbacteria. The results of the insecticidalactivity of the extracts of Pteria chinensisagainst this insect are given in Fig 1. Allthe extracts were found to causemortality. But, 100% mortality wasobserved only for the insects exposedto the extracts of acetone at aconcentration of 20mg/ ml and 40mg/ml after 24hrs. The extracts of ethylacetate and dichioromethane caused only60.6 and 50.0% mortality respectivelyat a concentration of 40mg/ml. Nomortality was observed in the controlplates. The ED values for the extractsof ethyl acetate, acetone anddichiorornethane were found to be27.793, 15.321 and 25.698 mg / mlrespectively. The low ED,. values ofacetone extract indicate that it is efficientin killing the pest than the other twosolvents. The significance between theextracts such as ethyl acetate anddichioromethane, acetone and ethylacetate and acetone and dichioromethanein killing the insects were analyzedusing 't' Test and the 'V values obtained
were 0.66, 0.95 and 1.405 respectively.The t' value of the extracts of acetone andethyl acetate is significantly higher thanethyl acetate and dichioromethane,whereas the 't' values for the extractsof acetone and dichioromethane issignificantly higher than the other twosolvents. Therefore, the activity of acetoneis highly significant than the other twosolvents in killing the insects. Similarworks on S. oryzae were performedearlier and the extracts of Annonareticulata in methanol were found to beefficient in causing mortality. The authorsopined that the methanolic extract beinga contact poison for insects couldpenetrate the body wall and the trachealsystem bringing about death (Jaswanthet al., 2002).
The mode of action of the extractin the present study has not beendetermined as it is beyond the scope ofthe present study. Insecticidal activitiesof sea pen, Ptilosareus gurneyi(Hendrickson and Cardinella, 1986) andsoft corals, Lzptophyton sp., (Ochi et al.,1988) have been reported in marinenatural product research.
Einhellig et al. (1985) havereported that using Lemna assay, thesearch for biodegradable herbicides maybe extended to include naturalcompounds (allelochemicals) and this isa simple screen for such activity. Theresults of Lemna minor bioassay are givenin Table 1. It was observed that theextract from acetone was able to causeyellowing of plants on 7 th day at aconcentration of 50 jig/mi, whereas, theplants were decayed at the same time ata concentration of 500 j.tg/rnl. The extractsfrom the other two solvents did not decaythe plants but yellowing of the leaves wasnoted on 51 day at a concentration of100 jig/mi. There was luxuriant growth ofplants kept in control vials.
180
SDMRI Research Publication No. 9, 178 - 181, 2005
Table 1. Lemna minor bioassay using tissue extracts of Pteria chinensis
Days Day Day Day Day Day Day Day
Control Gil Gil GHEL1 GHEL2 GHEL2 GHEL2 GHEL2
Solvents EA A 0CM EA A DCM EA A DCM EA A 0CM EA A DCM EA A 0CM EA A 0CMpg/mi
10 Gil Gil Gil Gil Gil Gil Gil Gil Gil Gil GHEL 1 Gil Gil GHEL, Gil Gil GHEL1 Gil Gil GHEL2 Gil
50 GHGHGHGHGHGHGHGHGHGHGHEL,Gil GGHEL,G GGHEL,G G LY G
100 GH Gil Gil Gil Gil GH G G Gil G LY G LY LY LY LY LY LY LY L LY
500 Gil Gil Gil Gil G Gil G LY G LY Y G LY Y LY LY Y LY Y 0 LY
1000 G G G G LY.G LY Y G LY Y LY Y D LY Y 0 Y Y D Y
GH - Green and healthy; GH EL - Green and healthy with new extra leaf 1; GH EL2 - Green and healthy with new extra leaf2; G - Green; LY - Light yellow; Y - Yellow; D - Decayed
Reports are available on the 8. Hendrickson, R. L and J. H. Cardinella, 1986.
herbicidal activities of 9 purified algal Structure and stereochemistry of insecticidal
metabolites (Fenical, 1993). The extract diterpenes from the sea pen, Pti.losareus gurneyi.
of Pteria chinensis, screened for Tetrahedron, 42: 6565 - 6570.
insecticidal and herbicidal activities 9. Huppatz,J.L.,1990. Essential amino acid bio-synthesis provides multiple targets for selective
clearly shows that the extract of acetone herbicides. In: CasidaJ.E. (Ed.) Pesticides andpossesses some biologically active alternatives. Elsevier, New York: 563 - 572.
compounds. Work on the isolation of 10. Jaswanth,A., M. Ramanatran., V. Krisbnaraj
active compounds is in progress. and K.Ruckmani,2002. Insecticidal activity
References
of the leaf extracts of Annona reticulata. Advancesin Pharmacology and Toxicology, 3 (2) : 13-16.
1. Balogh, J.C. and J.L. Anderson, 1992. 11. McLaughlin J. L, Jr. and D. L. Smith, 1991.Environmental impacts of turf grass pesticides. "Bench - Top' Bioassays for the Discovery ofln:Balogh, J.C.Walker, W.J. (Ed.) Environ
mental issues, Lewis publishers : 221 - 354. Bioactive Natural Products: an update, In:Studies in Natural Products Chemistry, Vol. 9:
2. Brousallis,A.M., G.E.Ferraro., V.S. Martino., edited by Att-ur-Rahman, (Elsevier ScienceR. Pinzon., J.D. Coussio and J. C. Alvearez, Publisher B.V.,Amsterdam) 199 1:pp.383-409.1999. Argentine plants as potential source
of insecticidal compounds. Ethnophazmacology, 12. Ochi, M., K.Futataughi, Y.Kume., H.Kotsuki.,
67: 219 - 223. K. Asso and K.Shibata, 1988. Litophynin C,
3. Einhellig, P.A., G.R. Leather and L.L. Hobbs, a new insect growth regulatory diterpenoid
1985. Journal of Chemical Ecology, 11: 65. from a soft coral, Litophyton sp. Chemical
4. Fenical, W., 1981. Investigation of benthic Letters: 1661 - 1662.
marine macro algae as a resource for new 13. Okaichi, T and Y. Hashimoto, 1962. The
pharmaceuticals and agricultural chemicals. In: structure of Nereis toxin. Agricultural Biological
Proceedings of Joint US-China Phycological Chemistry, 26: 224 - 227.symposium, Oingdao, China. 14. Piementel, D., 1986. Studies on integrated pest
5. Fenical, W., 1993. Chemical studies of marine management. In: Piementel, D. (Ed.) Somebacteria. Chemical Review, 93: 1673 1683. aspects of integrated pest management. Cornell
6, Finney, D. J., 1971. A statistical treatment of University Press, Ithaca : 29 - 68.the sigmoid response curve. In : Probitanalysis, (3 ed), Cambridge University
15. Sathyamurthi, T., 1952. Mollusca of Kru-sadai
Islands. Vol I and H.London : 333 - 336.
7. Grode, S.H., T.B. James and J.H. Cardellina,16. Watanabe, K., K. Umeda and M. Miyakado,
1989. Isolation and identification of1983. Molecular structures of Briantheins, three insecticidal principles from the rednew insecticidal diterpenes from Briareum
polyanthes. Journal of Organic Chemistry, 48: algae, Laurencia npponica. Agricultural
5203 - 5207. Biological Chemistry, 53: 2513. - 2515.
181
Proceedings of the National Seminar on Reef Ecosystem Rernediation
Antibacterial activity of whole body extracts of Trochus radiatus(Mollusca: Gastropoda)
K. Mary Elizabeth Gnanambal, C. Chellaram and Jamila Patterson*Suganthi Devadason Marine Research Institute
44 Beach Road, Tuticorin - 628 001, India*[email protected]
Abstract
Whole body acetone, ethyl acetate and dichioromethane extracts of Trochus radiatus were screenedfor antibacterial activity against 9 human and 5 fish pathogens using agar well diffusiontechnique. All the extracts exhibited clear zones of inhibition 7 of the 9 human pathogens and 4of the 5 fish pathogens tested. The extracts of ethyl acetate: dichloromethane (1:1) showedprominence in inhibiting the growth of Enterobacter aerogenes, Staphylococcus aureus andEschertchia colr. Maximum zone of inhibition was observed against Proteus mirabths for all theextracts except that of acetone. The Minimum Inhibitory Concentration (MIC) forStaphylococcus aureus, Enterobacter aerogenes and Proteus mirabths was 0.07mg and 0.15mg for Serratia marcescens.
Introduction
The marine environment is anexceptional reservoir of bioactivenatural products, many of which exhibitstructural/ chemical features not foundin terrestrial natural products (Irelandet al., 1988). There has been a profoundincrease in the research on marineorganisms for biomedical compoundsover the last two decades, which provideda broad and better support of discoveringnew biomolecules. Of the diversifiedmarine organisms, molluscs are widelydistributed throughout the world havingmany representatives in marine,freshwater, estuarine and terrestrialecosystems. Studies on bioactivecompounds from molluscs exhibiting antitumoral, anti leukemic, antibacterial andantiviral activities have been reportedworldwide (Hochlowskj and Faulkner,1983, Faulkner and Ghiselin, 1983 andPrem Anand et al., 1997). Most of thepathogens are becoming increasinglyresistant to the major classes of theroutinely used antibiotics. There is anurgent need for the discovery of new andnovel antimicrobial drugs to effectively
combat not only the drug resistance butalso the new disease producers. So, thesearch for active drugs from alternativesources including marine environment,obviously becomes imperative. New drugclasses with novel mechanism of actionwill create effective therapy at least for aperiod of time. By far, the majority of thenatural products research has beenfocused on the shell-less snails andworks on other classes of gastropodaremain sporadic. The broad and coneshaped top shell, Trochus radiatus foundabundant and associated with live ordead corals along the Tuticorin coasthas not been studied for biomedicalpotential. So, this prosobranchiatemollusc was chosen for the presentstudy with an objective to explore theantibacterial potentiality of its whole bodyextracts.Material and Methods
Live 7)oc1wsradjatus Gmelin, 1791(Mollusca Gasfropoda Vestigas&opodaTrochidae) were collected by handpicking from the intertidal areas coveredwith dead corals in Tuticorin coastalwaters. They were immediately brought
182
SDMRI Research Publication No. 9, 182 - 186, 2005
to the laboratory and their soft bodieswere removed by breaking the shells. Thepre-weighed whole meat was washedthoroughly with sterile distilled water andcut into small pieces. Then, it wasextracted separately with solventsacetone, ethyl acetate, dichioromethaneand ethyl acetate: dichloromethane (1:1)and cold steeped overnight at -18° C. Theextracts from each-solvent were thenfiltered separately using Whatmann No.1filter paper. The filtrate was poured intopre-weighed petri plates, evaporated todryness and used for all the experiments(Wright, 1998).
The antibacterial effect of theextracts was 'assayed using 9human pathogens (Vibrio cholerae,Bacillus subtilis, Staphylococcus aureus,S. epidermidis, Escherichia coli,, Salmonellatyphimurium, Enterobacter aero genes,Kiebsiella pneumoniae and Streptococcuspneumoniae) and 5 fish pathogens(Aeromonas hydrophila, Vibrioparahaemolyticus, V. harveyi, Serratiamarcescens and Proteus mirabilis). Thehuman pathogens were obtained fromChristian Medical College (CMC), Velloreand the fish pathogens were isolated fromdiseased shrimps, fishes and prawns. Allthe test organisms were cultured inTryptone Soy Broth (TSB) and the 18-24hrs old cultures were used for theexperiment.
The antibacterial activity of thesamples was assayed by following thestandard Nathan's Agar Well Diffusion(NAWD) technique (Nathan et al., 1978).Five wells of 6 mm diameter were madeon the pre-poured Tryptone Soy Agar(TSA) plates in sterile conditions. TheseTSA plates were inoculated by swabbingthe 18-24 hrs old test bacterialsuspensions to create a confluent lawnof bacterial growth. A constant amountof 28 mg of the extract was dissolved in
2ml Dimethyl Sulfoxide (DMSO) and fromthis 50.tL DMSO containing 0.7 mg of theextract was loaded onto each well. Thewell at the center served as the control(with DMSO alone). After 22-24 hrs ofincubation at room temperature, thesusceptibility of the test organisms wasdetermined by measuring the diameterof the zone of inhibition around each wellto the nearest mm. Minimum InhibitoryConcentration (MIC) was determined byserially diluting the extracts (6mg, 3mg, -1.4mg and 0.6mg/mi) in DMSO and 50MLDMSO from each concentration containing(0.3, 0.15, 0.07 and 0.03mg) of theextracts were loaded into each well andassayed against the pathogenic strains,Staphylococcus aureus, Enterobacteraero genes, Serratia marcescens andProteus mirabilis. -
Results and Discussion
The amount of crude extractsvaried with the solvents used. The crudeacetone extract yield was 200mg! 1 O wetweight of the animal tissue and theextract of ethyl acetate, dichloromethaneand 1:1 mixture of ethyl acetate anddichioromethane yielded only 100mgfrom the same wet weight of the tissue.The results of the antibacterial activityof the whole body extracts of Trochusradiatus against 9 human pathogens aregiven in Table 1. The extracts of ethylacetate: dichioromethane (1:1) showedgood inhibitory activity againstEnterobacter aero genes, Staphylococcusaureus, Escherichia coli with zones of 4, 3 -and 3 mm respectively, whereas the zonesfor the extracts of ethyl acetate, for thesame pathogens were observed as 2.5, 2.5and 2 mm respectively (Fig. 1). Theextracts of dichioromethane and acetoneshowed higher inhibition againstS.aureus (3.5mm) and Enterobacteraerogenes (3.5mm) - respectively. In thepresent study, none of the extracts
183
Proceedings of the National Seminar on Reef Ecosystem Remediation
Table 1: Effect of whole body extracts of Trochzis radicztuson human pathogens
Zone of Inhibition (mm)Ethyl acetate Dicbloromethane Ethyl acetate: Acetone
Dichloromethane(1:1)
2 2 2 2
2 3 2.5 1.5
2.5 3.5 3 2
1 3 1
15 2 1.5 .2
215 3 4 3.5
3 2 1.5
Pathogens
Vibrio choleraeBacillus siibttlisStaphylococcus aureusS. epidermidisEscherichia coliSalmonella typhimuruimEnterobacter aerogenesKlebsiella pneumnoruaeStreptococcus pneumorUae
- No activity
Fig. 1. Inhibition of Enterobacter aerogeneS
showed activity against S. epidermzdiS
and Kiebsiella pneumoniae. Prem Anandand Edward (2002) had reported widespectral activity, of ethyl acetatephase fractions of Cypraea erronesextracts against 6 human pathogens.Benkendorff et al. (2001) studied theantibacterial activity of 39 molluscs and4 polychaeteS.
The extracts also showed anti-bacterial activity against 4 of the 5 fishpathogens tested (Table 2). In the case offish pathogens, the extracts from all thesolvents except acetone were able toinhibit the growth of Proteus mirabilis toa higher level in comparison with othertest cultures (Fig. 2). Extracts of acetonewere able to appreciably inhibit Serratza
marcescens cell growth when comparedto other solvents. Very trace inhibitoryactivity was exhibited against Vibrio
parahaemOlytiCuS culture by the extracts.The Minimum Inhibitory Concentration(MIC) of ethyl acetate, dichioromethafleand ethyl acetate: dichloromethafle (1: 1)extracts for the pathogens tested for thepathogens Staphylococcus .aureus,Enterobacter aero genes and Proteus
mirabilis (Fig. 3) was found to be 0.07mg,where as for Serratia marsescefl-S it wasabout 0. 15mg (Table 3).
Table 2 : Effect of whole body extracts of Trochus radiatus on fish pathogens
Zone of Inhibition (mm)
Pathogens Ethyl acetate Dichlorornetb.ane Ethyl acetate: AcetoneDichioromethafle
(1:1)
Aeromonas hydrophila - -Vibrio harueyi 1 1
V.parahaemolYtials 0.5 1
Serratia rnarcescens 2 2
Proteus mirabilis 2.5 3
1 0.51 0.52 2.53 2
184
SDMRI Research Publication No. 9, 182 - 186, 2005
Table 3 Values of Minimum Inhibitory Concentration (MIC) of whole body extracts of Trochus radiatus
Zone of inhibition (mm)
Pathogens Ethyl acetate Dichloromethane 1:1 ratio of ethyl
extract extract acetate and
(mg) (mg) dichloromethaneextract (mg)
0.03 0.07 0.15 0.30 0.03 0.07 0.15 0.30 0.03 0.07 0.15 0.30
Staphylococcus aureusEnterobacter aero genesSerratia marcescen.sProteus mirabilis
- 1 1.5 2 - 1 2 3 - 1 2 2.5
- 1 1.5 2 - 1 2 2.5 - 1 2 3- - 1 1.5 - - 1 1.5 - - 1 1.5
- 1 1.5 2 - 1 2 2.5 - 1 2 2.5
Fig. 2. Inhibition of Serratia marcescens
The present observation is incoincidence with that of Rajaganapathy(1996) who have reported an inhibitionzone of nil to 1.5mm for the whole bodyextracts of Cerithidea cingulata andHemifusus pugilinus against 9 humanpathogens. Sanduja et al. (1985) reportedthe occurrence of an antibacterial agent,fulvoplumierin in Nerita albicilla. Most ofthe molluscan secondary metabolites arederived form their diets (Cimino andSodeno, 1993). They concentrate themetabolites from their highly specializeddiets and incorporate into their owndefensive strategies (Faulkner, 1988).T.radiatus, being a shelled gastropod iswell protected and hence the possibilityof de nouo origin of the antibacterialactivity observed for the whole bodyextracts in the present study could beruled out. T. radiatus is an algal feeder
Fig. 3. Inhibition of Staphyloacais alLreus at 0.07 mgMinimal Inhibitory Concentration (MIC) of the extract
and hence the antibacterial compoundsof this mollusc may have originatedfrom their dietary macroalgae. This viewis in line with the reports on theantibiotic (Thieler et al., 1978), antiviral(Ehresmann et al., 1977), cytotoxic(Jacobs et al., 1985) and insecticidal(Fenical, 1981) properties of macro algae.The present observation is furthersubstantiated by Schmitz et al. (1993)who have reported that the herbivorousmollusc, Dolabella auricularia concentratesand stores selected algal metabolites.Rinehart et al. (1981) opined thatmarine molluscs might provide potentialfor isolating compounds with specificactivity against certain organisms or celltypes.
The wide spectrum antibacterialactivity exhibited by the whole bodyextracts of Trochi.ts radiatus indicates that
185
Proceedings of the National Seminar on Reef Ecosystem Remediation
it may possess biologically active observation on marine natural products.
metabolites. So, further fractionation and Tetrahedron, 41: 981 - 984.
purification would reveal the nature of the 10. Nathan, P., E. J. Law and D. F. Murphy, 1978.
compound. A laboratory method for the selection of topicalantimicrobial agents. Burns, 4: 177 - 178.
References
3. Ehresmann, D. W., E. F. Deig., M. I. Hatch.,L.H. Di Salva and N. A. Vedros, 1977. Antiviralsubstances from California marine algae.Journal of Phycology, 13: 37-40.
4. Faulkner, D. J. and M. T. Ghiselin, 1983.Chemical defense and evolutionary ecologyof dorid nudibranchs and some other opistho-branch gastropods. Marine Ecology ProgressSeries, 13: 295 -301.
5. Faulkner, D. J., 1988. Feeding deterrents inMolluscs. Biomedical importance of marineorganisms, CA, California Academy of Sciences:29-36.
6. Fenical, W., 1981. Investigation of benthicmarine algae as a resource for newpharmaceutical and agricultural chemicals.Proceedings joint US-China Phycologicalsymposium. Academia Sinica, Oingdao.
7. Hochiowski, J.E. and D. J. Faulkner, 1983.Antibiotics from marine pulmonate, S1wnanadiemensL Tetrahedron Letters, 24: 1917- 1920.
8. Ireland, C. M., D. M. Roil., T. F. Mo]inski., I. C.Miker., L M. Zabriske and J. C. Swersey, 1988.Uniqueness of the marine environment.Categories of marine natural products frominvertebrates. In: Biomedical importance ofmarine organisms. San Francisco Academy ofSciences: 41 - 57.
9. Jacobs, R. S., P. Culver., R., T. O'Brien andS.White, 1985. Some pharmacological
11 Prem. Anand, T. and J. K. Patterson Edward,2002. Antimicrobial activity in the tissueextracts of five species of Cowries, Cypraea spp.,and an ascidian, Didemnum Psaminathodes.Indian Journal of Marine Sciences, 31 (3)239-242.
Prem. Anand, T., J. Rajaganapathy and J. K.Patterson Edward, 1997. Antibacterial activityof marine molluscs from Portonovo region,Indian Journal of Marine Sciences, 26: 206-208.
13. Rajaganapathy, J., 1996. Studies on anti-bacterial activity offive marine molluscs, M.ScThesis, Annarnalai University, India, pp 43.
14 Rinehart, K. L., P. D. Shaw., L.S. Shield, J.B.Gloer, G. C. Harbour, M. E. S. Koker,D.Saniain, R. E. Schwartz, A. A. Tymiak,E.G.Swynenberg, D. A, String Fellow,J.J.Vauva, J. H. Coats, G. E. Zurenko,S.L.Kuentzel, L. H, Li, G. J. Bakus, R. C. Brasca,L. L. Craft, D. N. Young, J. L. Connot, 1981.Marine natural products as a source ofantiviral, antimicrobial and antineoplasticagents.Pure and Applied Chemistry, 53:795-817.
15. Sanduja, R., A. J, Weinhemer, K. L. Euler andM. Alam, 1985. Unusual occurrence offulvoplumierin, an antibacterial agent in themarine mollusc, Nerita albicilla, Journal ofNatural Products, 48: 335-336.
16. Schmitz, F. J., B. F. Bowden and S. I. loch,1993. Antitumor and cytotoxic compounds frommarine organisms. Marine Biotechnology, 1197-308.
17. Thieler, R. F., J. F. Suida and L. D. Hager, 1978.Drugs and food from the sea. In: Myth orreality. University of the Oklahoma Press153-169.
18. Wright, A. E., 1998. Isolation of marine naturalproducts, In: Methods in Biotechnology, Vol, 4:Naturaiproduct isolation, Cannell, R P. J. (Ed.,),Humana press Inc., New Jersey, USA: 365-408.
1. Benkendorif, K., A. Davis and J. B. Bremer,2001. Chemical defenses in the egg masses ofbenthic invertebrates. An assessment ofantibacterial activity in 39 molluscs and 4polychaetes. Journal of Invertebrate Pathology,78:109-118. 12
2. Cimino, G. and G. Sodeno, 1993. Biosynthesisof secondary metabolites in marine molluscs.Topics in Current Chemistry, 67: 78-180.
186
RETFER 04
INSECTICIDAL ACTIVITY OF THE WHOLE BODY EXTRACTSOF TURBO INTER COSTALIS (ARCHAEGASTROPODA:
TURBINIDAE) AND CERJTHIDEA CINGULATA
(CAENOGASTROPODA: POTAMIDIDAE)
K MARY ELIZABETH GNANAMBAL, C. CHELLARAMAND J.K. PATTERSON EDWARD
Suganthi Devadason Marine Research Institute44, Beach Road, Thoothukudi
ABSTRACTRice weevil, Sitophilus oryzae is a common pest, which destroys stored rice, wheat and sorghum
grains and quickens the propagation of molds and bacteria. Although chemicals with increased efficacyand safety have been developed, the fight for controlling insect pests exists both in the field and storehouse.The natural marine world can provide alternatives to the existing chemical insecticides both in the long andshort terms. Studies on bioactive compounds from molluscs exhibiting antibacterial, herbicidal andinsecticidal activities have been reported worldwide. Molluscs have always proven to be potential sourcesfor the discovery of new pesticides. The turban shell, Turbo inlercostalis and horn shell, Cerithideacing-ulata are found abundantly in Thoothukudi intertidal areas. However, efforts dedicated to screening ofnatural products for insecticides are small compared to pharmacological natural product programs. Thepresent work is undertaken to extract the whole body of these molluscs and to obtain column purifiedfractions to the insecticidal properties against S. oryzae. The acetone extracts of both the organisms werefound to cause significant mortality of the pest. It was found out that the efficiency of the column-purified100% acetone fractions of both the organisms in killing the pest was higher when compared to the crudeextracts.
INTRODUCTION
Insects, weeds and phytopathogenic organisms cause great damage toagriculture. Where pests and diseases are not systematically controlled, an estimatedone-third of a typical crop is lost (Melniknov, 1971). Although chemicals with increased
efficacy and safety have been developed, the fight for controlling insect pests exists both
in the field and in the store house. Also the pesticide and agro-chemical industry face
growing criticism over the toxicity and the residue problems associated with the use of
chemical control agents (Huppatz, 1990). Since these chemicals are toxic and
biologically active, their use for insect control has become a serious concern (Balogh and
Anderson, 1992). Apart from that, the insects acquire resistance to the existing
insecticides and thus the development of new insecticide is always under consideration
to tackle the problem. The plants, animals and microorganisms of the marine environ
132
RETFER '04
with their wide range of chemical diversity are still an unexplored resource for the
development of new agro-chemical agents (Crombie, 1990). Of the marine organisms, in
particular, the molluscs have always proven to be potential sources of marine natural
products with vast array of diversified bio-activities. There are massive evidences to
witness marine molluscs as potential supply of potent metabolites (Hocklowski andFaulkner, 1983, Hubert etal., 1996; Whittaker, 1960 and Baslow, 1977). Turban shell,Turbo intercostalis and horn shell, Cerithidea cingulata are found abundantly inThoothukudi intertidal areas. However, efforts dedicating to screening of natural
products for insecticides from these molluscs are not carriedout hitherto. So the present
work was taken up to extract the whole body of these two molluscs using different
solvents, column fractionate the active crude extracts and to test the activity against adreadful rice pest, Sit oph i/us oryzae.
MATERIALS AND METHODSLive Turbo intercostalis (Archaegastropoda: Turbinidae) and Cerithidea
cingulata (Caenogastropoda: Potamididae) were collected in the intertidal areas of the
Thoothukudi coastal waters and immediately brought to the laboratory. The shells were
opened and the soft body was taken out, cut into small pieces and air-dried to remove the
water content. The extract of the air-dried animals was obtained using different solvents
such as ethyl acetate, dichioromethane acetone, methanol, hexane and toluene after cold
steeping at -1 8°C. The extract from each solvent was filtered separately using Whatniann
No. I filter paper. The filtrate was poured in petri plates, evaporated to dryness and usedfor all the experiments.
The insecticidal activity of the extracts of T intercostalis and C. cingulata fromdifferent solvents was tested using contact bioassay by the modified method ofBroussaljs el al., (1999). 1 g of each of the dried extracts was dissolved in 1 ml of their
respective solvents and from this 500, 300, 100, 50 and 25 jiL were poured in separate
petri plates in triplicates and allowed to evaporate overnight to get the same
concentration in mg/ ml of extracts. Controls with solvents alone were taken in separate
petri plates and evaporated overnight. Ten healthy adults of Sitoph i/us oiyzae(Coleoptera: Curculionidae) were introduced into each petri plate and sufficient amount
of food was provided to the test organisms, so that the view that death due to starvation
could be ruled out. Number of dead insects was counted after 24 hrs and the percentage of
mortality was noted. The efficiency of the extracts obtained with different solvents at
133
RETFER 04
varied concentrations in killing the insects was determined statistically using Two Way
ANOVA test. Partial purification of the extract was carriedout following the method
outlined by Wright, (1998). After initial screening, the extract showing activity obtained
with acetone was fractionated using normal phase silica gel column chromatography
employing a step gradient solvent system from low to high polarity. The step gradient
protocol used was: 100% hexane, 80% hexane: 20% acetone, 60% hexane: 40% acetone,
40% hexane: 60% acetone, 20% hexane: 80% acetone, 100% acetone, 80% acetone: 20%
methanol or 60% acetone: 40% methanol; 40% acetone: 60% methanol; 20% acetone:
80% methanol and finally 100% methanol. The fractions thus obtained were once againevaporated and concentrations of 200, 100, 50, 25 and 10Pig/ ml of the extracts werepoured in separate petri plates and tested for insecticidal activity. Number of dead insects
was counted after 24 hrs and the percentage of mortality was once again noted. The LC '
0values of the crude extracts as well as the column purified fractions were determined bythe probit analysis (Finney, 1971).
RESULTS AND DISCUSSION
Out of the six solvents tested, the whole body extract of Turbo intercosta/isobtained with acetone (80±1.0) was found to cause the highest mortality to Sitophilusoryzae when compared to the solvents ethyl acetate (63.3±0.58), dichioromethane(63.3+1.53), methanol (73.3+0.58), hexane (53.3±1.53) and toluene (46.712.08) at aconcentration of 500 mg/ ml (Table 1). Similarly, the acetone extract of Cerithideacingulala was found to cause the highest mortality (76.7±0.58) at the sameconcentration. The mortality rates were observed to be lower for the extracts obtained
with other solvents. There was a significant difference between the extracts obtainedfrom different solvents for acetone extracts of both the organisms (T intercostalis and C.cingulata) in killing the insects as evidenced by statistical analysis (Table 2 and 3). Thecolumn purified 100% acetone extracts of T intercostalis and C. cingiilata were found tocause 90% (±1.0) and 93.3% (±1.15) mortalities respectively at a concentration of200jg/ ml to S. oryzae, whereas, the (80:20) acetone: methanol fractions were found to
cause 76.7% (±0.58) and 80% (±0) mortalities at the same concentrations (Table 4). TheLC,, value for the acetone extracts of intercostà/js was found to be lower 104.41 mg incomparison with other solvents like ethyl acetate (226.35), dichloromethane (280.96),methanol (126.7), hexane (495.37) and toluene (432.78). Also the LC,,, value for theextract of C. cingulata was noted to be lower for acetone (79.61 mg) than the other
134
RETFER '04
Table 1. Percentage of mortality of Silophilus oryzae by the crude extracts of Turbotercoslalis and Cerith idea cingulata
Percentage of mortality (mean D)Cone.
Marine (mg) EA DCM ACE MET HEX TOLorganisms
500 63.3+0.58 63.3±1.53 80±1.0 73.3±0.58 53.3±1.53 46.7±2.08
300 56.7±0.58 46.7+0.58 76.7±0.58 63.3+0.58 23.3±0.58 16.7±0.58
100 43.3±0.58 36.7+0.58 63.3±0.58 53.3±0.58 13.3±0.58 10+0.0
50 16.7±0.58 13.3±0.58 33.3±0.58 40±1.0 6.7±0.58 6.7±0.58
25 6.7±0.58 6.7±0.58 13.3±1.15 6.7±0.58 3.3+0.58 0
500 66.7±0.58 56.7+0.58 76.7+0.58 66.7±0.58 40.0±1.0 66.7±0.58
300 53.3±0.58 43.3±0.58 70±1.0 46.7+0.58 20±1.0 10+1.0
100 30±1.0 20±1.0 63.3±0.58 33.3±0.58 10±1.0 3.3±0.58
50 10±1.0 6.7±0.58 53.3+0.58 23.3±0.58 3.3±0.58 0
25 0 0 16.7±0.58 3.3±0.58 0 0
EA- ethyl acetate; DCM- dichloromethane; ACE-acetone; MET- methanol; HEX- hexane;TOL- toluene
135
RETFER 04
Table 2. Two-way ANOVA showing significance in variation for the percentage ofmortality of Sitophilus oryzae by the crude extracts of Cerithidea cingulata
Source of Variation SS df MS F
Between concentration (Rows) 12399.96 4
3099.99 32.20
Between solvents (Columns) 5772.238 5
1154.45 11.99
Error 1925.441 20
96.2720
Total 20097.63 29
Level ofSignificance
P<0.01
P<0.01
Table 3. Two-way ANOVA showing significance in variation for the percentage ofmortality of Siiophilus oryzae by the crude extracts of Turbo intercosta!is
Source of Variation SS df MS F Level ofSignificance
Between concentration (Rows) 12175.57 4 3043.89 42.15 P<0.01
Between solvents (Columns) 5401.399 5 1080.28 14.96 P<0.01
Error 1444.181 20 72.20
Total 19021.15 29
136
0
-4.-U
E
0Ua)
-4.-
0
IHoj cts4.-
0)
0)
Cl
00 00 00 0003
00 00 00 00 '1CA
N N N C enen 0 '0 O enen en — —
03 00 00 00 00C,
0 9 9 — — Q 0 0 —d -41 -41 -II -41 C) -+4 -41 -H 4N ('- , N en Cl o\C) \0 '0 en en ('40 en ' en
0.3 00 00 00 00 00 00C, "n In In
C' (3 C) C' C) C' C'-41 41 -41 -H C' -41 -H -41 -41Cl ô
CD
(' Cl N (' N ten " O en '0 '0 \O '0N " en
03 00 00 00 00',-l. c' " " ,,
C1) ra C) C) ._ c' d Q 9 -
a) \0 '0 '0 00 en enE N '-0
00 03 00 00C Q
t< -.4 ' —o ('- N N en N ,C
,C' '0 en 'C ,-.- 00 ' • —N N vi Vi0
a)
00 03 00 00 00 00NC) '' '1
cu oo
N r- N N (' Cl ('
'C '0 en 'CVi 03 N Vi en
00 00 00 00 00 C0C) ©
C' N ó N ô In ('- Clen en en 0 (n'0 en N- '0 en C4
00 m 00 03 00C) ' C C
C' Cl ô N- Cl ô Cl Clen ' fl kn enVi N '.0 N —
00 coC'I Cl ô Cl Cl Cl Nen ('4 en '.0 en en('4 ('4 It en —
00 en "kn
CD
CN
N
0 3 2u In
! .I SZJV)SO31)U! DJVJflUflca 03oqin vap!IjJ!1a3
0Cl
0)
E
0)
00)UCO
0)
Cl
0)
RETFER '04
solvents. It was also observed that the LC, value of 100% acetone fractions of Tinte,-costalis and C. cingulala were noted to be 29.11 and 38.16 jig/ ml respectively which
is very much lower when compared with crude extracts as shown in Table 5.
Rice weevil, Sitophilus oryzae is a common pest, which destroys stored rice,
wheat and sorghum grains. A single larva of S. oryzae is reported to consume 10 mg of
grain during its development. In the present study, highest mortality was caused to S.oryzac by the whole body acetone extracts of T intercostalis and C. cingulata. However,an earlier work of Selvam (2002) reported that ethyl acetate extract of a sea weed,
Gracilaria crassa caused 100% mortality to S. oryzae. Generally many marine-derived
diterpenes show significant insecticidal activity. Novel insecticidal briarances were
isolated from the sea fan, Plilosarcus gurneyi which showed toxicity to the larvae of thetobacco hornworm, Manduca sexta inducing 40% mortality (Hendrickson and
Cardcllina, 1986). Similarly, Ulostantin isolated from the sponge, Ulosa ruetzleri isequivalent in potency to paraoxonin inhibiting acetyl cholinesterase and hence,
considered the most potent insecticide from marine origin (Van-Wagenen et al., 1998). Inthe present study, the 100% acetone fractions of intercostalis and C cingulata showedgreater efficiency in causing mortalities in comparison to the crude extracts suggesting
that the column-purified fractions are highly potent in killing the insect. Thus it can be
implied that the compound exhibiting insecticidal activity could be of medium polar
type, however, further purification will confer the exact nature of the compound. The
lower LC,, values obtained for the acetone extracts of T intercostalis and C. cingulatasuggest that these extracts are potent in causing mortality to S. oryzae. However, the100% column-purified acetone extracts of T intercostalis and C. cingulata showed stilllower LC, values (29.11 and 38.16 jig respectively). Similar lower LC,,, (329.686 to588.717 jig) values have been reported for the extracts of marine Streptomyces sp. strainsin causing mortality to this insect (Anand, 2002). A work of Zabriskie et al., 1986 reportsthat jaspamide, a modified peptide isolated from a Jaspis sponge, showed insecticidalactivity against Heliothis virescens with a LC50 of4 ppm. The marine environment with its
chemical diversity cticidal agents. Many marine-derived structural classes have not yet
been examined for their insecticidal activity. The 100% acetone fractions of Tintercostalis and C. cingulata are now subjected to further purification to find out the
exact compound elucidating the insecticidal activities..
138
RETFER '04
REFERENCES
Anaud, P.(2002). Screening for bioactive metabolites producing marine bacteria from
Tuticorin coastal waters, South east coast of India, Ph. D Thesis, ManonmaniamSundaranar University, Tii-unelveji.
13a logh, J. C., and Anderson, J. L. (1992). Environmental impacts of turf grass pesticides. In:
Balogh, J. C. Walker, W. J. (ed) Environmental issues, Lewis publishers, 221 -354.
E3aslow, M. 11(1977). Marine pharmacology. Robert E, Kriega Publishing Company,Huntington, New York.
Brousallis, A. M., G.E. Ferraro, V.S. Martino, R. Pinzon, J.D. Coussiom and J.C.
Alvearez.(1999). Argentine plants as potential source of insecticidal
compounds. Eihn opha rmnacol , 67: 219-223.
Crombie, L.(1990). Natural models for the design of insect control compounds: the
mammerins. In: Recent advances in the chemistij' of insect control II. The RoyalSociety of Chemistry Special Publication. (Crombie, L., Edn), Cambridge,
England, pp 23-51.
Finney, D. J.(1971). A statistical treatment of the sigmoid response curve. In: Probitanalysis, (3r1 ed), Cambridge University Press, London, 333 -36.
Hendrickson, R. L., and J.H. Cardellina,.(1986). Structure and stereochemistry of
insecticidal diterpenes from the sea pen, Ptilosarcus gurneyi, Tetrahedron, 20,1707-1708.
I-Iocklovski, J. E., and D.J. Faulkner. (1983). Antibiotics from marine pulmonates,
Siphonaria diemnenensis, Tetrahedron Letters, 24:1917-1920.
Hubert, F, W. Knaap, T. Noel and P. Roch.(1996). Cytonic and antibacterial properties of
Afytilus galloprovincialis, Osirea edulis and Crassostrea gigas haemolymph,Aquatic LivingResowces, 9: 115-124.
139
RETFER 04
Huppatz, J. L.(1990). Essential amino acid biosynthesis provides multiple targets for
selective herbicides. In: Casida J.E (Ed). Pesticides and alternatives. Elsevier,New York, 563 - 572.
Melnikov, N. N.(1971). Chenistry of pesticides. Gunther, F. A., Gunther, J. D., Eds,Springer-Verlag: New York, pp 197.
Selvam, M.(2002). Studies on the biodiversity of sea weeds in Hare island and its
potential pesticidal activity against storage pests- Tribolium castaneum andSit ophilus olyzae, M.Sc., Thesis, Manonmaniam Sundaranar University,Ti run elvel i.
Van-Wagenen, B. C, R. Larsen, J.H. Cardellina, D. Randazzo, Z.C. Lidert and C.
Swithenbank(1998) Ulostantin, a potent insecticide from the sponge, Ulosaruetzleri, Journal ofOrganic chemistry, 58 : 335-337.
Whittaker, V. P.(1960). Pharmacologically active choline esters in marine gastropods,
Annals of the New York Academy Science, 90: 695-705.
Wright, A. E.(1998). Isolation of marine natural products, in Methods in biotechnology,V61.4: Natural products isolation, edited by R.J.P. Canell, (Humana Press Inc.,New Jersey, USA),pp365408.
Zabriskie, M. T, J.A. Kiocke, C.M. Ireland, A.H. Marcus, T.F. Molinski, D.J. Faulkner,
C.Xu and J.C. Clardy.(1986). Jaspamide, a modified peptide isolated from aJaspis sponge, with insecticidal and antifungal activity. Journal ofthe AmericanChemical Society, 108:3123-3124.
140