Physiological and Mo lecular Studies of Deey-Sea Fungi Thesis submitted for the degree of Doctor of Philosophy in Marine Sciences to the Goa University by Purnima Singh National Institute of Oceanography, Dona Paula, Goa-403004, India December 2011 T- 521 STATEMENT As per requirement, under the University Ordinance 0.19.8 (vi), I state that the present thesis titled "Physiological and Molecular Studies of Deep-Sea Fungi" is my original contribution and the same has not been submitted on any previous occasion. To the best of my knowledge, the present study is the first comprehensive work of its kind from the area mentioned. The literature related to the problem investigated has been cited. Due acknowledgements have been made whenever facilities or suggestions have been availed of. Purnima Singh CERTIFICATE This is to certify that the thesis titled "Physiological and Molecular Studies of Deep-Sea Fungi" submitted for the award of the degree of Doctor of Philosophy in the Department of Marine Sciences, Goa University, is the bona fide work of Mrs Purnima Singh. The work has been carried out under my supervision and the thesis or any part thereof has not been previously submitted for any degree or diploma in any university or institution. Place: Date: r4 29: It Of2,,,La Dr. Chandralata Raghukumar Ph.D. Supervisor, 313, Vainguinim Valley, Dona Paula, Goa, 403004. te, c.ert Ye-re-ta ha/ye 1,0/4 .4t co-+ayek-e.4 - 12- • 2,911 Acknowledgments I express my sincere thanks to Dr. Chandralata Raghukumar, my research supervisor. She encouraged me to choose this topic for my thesis and I am very grateful to her for valuable guidance and constant encouragement throughout this study. She always supported patiently and taught me experimental and writing skills. She as a person is always ready to hear and solve the problems of her students in whatever way possible. Working with her was the most memorable achievement for me. I also thank Dr. S. Raghukumar, for his valuable and thoughtful suggestions regarding my work. Both of them have influenced me a lot with their enthusiastic and positive approach. I wish to thank Dr. S. R. Shetye, Director, MO for giving me opportunity to work and providing the infrastructure facilities in the institute. I am thankful to my Co-guide Dr. S. Ghadi, Department of Biotechnology, Goa University for all his support throughout my study. I would also like to extend my appreciation to all my FRC members, Dr. G. N. Nayak, Dean, Department of Marine Science, Goa University, Dr. H. Menon, Head, Department of Marine Sciences, Goa University and VC's nominee Dr. N. Ramaiah, Scientist, NIO for all their critical comments and for their periodic assessment of my work. I would also thank Dr. D. J. Bhat, Head, Department of Botany, Goa University for helping me in the identification of fungi. I am thankful to the University Grants Commission, India for the award of Research Fellowship. I ani thankful to Dr. M.P. Tapaswi, Head Librarian, MO, for providing some important references whenever required. I am also grateful to Mr. Arun Mahale, Mr. Uchil and staff of DTP section to help me in modification of some of the figures in this thesis. I specially thank Department of Oceanography (DOD) funded PMN-EIA project for providing me opportunity to go for sampling cruises to the Central Indian Basin without which this work would not have been possible. I thank Dr. Rahul Sharma, Project Leader, PMN-EIA, for allowing me to participate in these cruises and all the support for my work. I am also thankful to all the crew members of the Russian ship Akademik Boris Petrov for their co-operation and all the sampling and lab facilities extended on board. I am thankful to Dr. Yogesh Shouche, Scientist, NCCS, Pune for extending the sequencing facility in his lab. I am also very much grateful to Pankaj Verma, NCCS, Pune for his great help and useful suggestions throughout my thesis. I am thankful to all my friends in the Marine Biotechnology and Microbial Ecology Lab, MO for all their timely help. I specially thank Dasu, who was always there to help me in all the possible ways. I thank all my friends from BOD, COD, GOD and POD for the help provided during the study. I am deeply indebted to all my family members for their support and encouragement. I am thankful to my loving husband Gaurav for his help, thoughtful suggestions and encouragement throughout my study. And last but not the least; I am thankful to God for giving me all the strength to achieve this goal. Purnima Singh A -01 Table of Contents Page no. Chapter 1 Fungi in deep-sea ecosystem: an introduction... ....... 1-22 Chapter 2 Isolation of fungi from deep-sea sediments of the Central Indian Basin (CIB) and their identification using molecular approaches 23-59 Chapter 3 Fungal Community analysis from deep-sea sediments of CIB using culture-independent approach 60-101 Chapter 4 Effect of simulated deep-sea conditions on growth and protein patterns of a few select cultures.... ...... 102-132 Chapter 5 Summary 133,142 Appendix 143-147 Bibliography 148-189 Publications 190 -1/ Chapter 1 Introduction Fungi in deep-sea ecosystem 1.1 Fungi in deep-sea ecosystem: an introduction 1.1.1 Oceanic environments The ocean is the entire body of salt water, comprising — 70% of the total earth surface to an average depth of 3800 m. The water in the oceans is estimated to have a volume of 1.368* 10 9 km3, and to be equivalent to about 0.24% of the Earth's total mass. The - shelf seas 0-200 m deep, that fringe the continental landmasses, extend over about 5% of the Earth's area. At the outer edge of most shelf seas is the shelf-break where the seabed falls quite steeply to depths of around 3000 m, forming the continental slope and rise (Fig 1.1); these continental margins account for a further 13% of the Earth's area. Major oceans are Atlantic, Pacific, Indian, Arctic and Antarctic oceans. There are various types of habitats existing in the oceanic environment differing in their characteristics. Major environments include pelagic that, is entire water column and other is benthic, the near bottom water and bottom sediment surface area of the ocean. The open ocean can be divided vertically on the basis of penetration of sunlight into the following zones (Fig. 1.1): • Epipelagic zone (ocean surface up to 200 meters deep) This is the euphotic zone of the ocean, where sunlight is easily available for photosynthetic processes. This zone is characterized by maximum primary production by the existing organisms which are the main fuel source for all other pelagic and benthic life forms. This zone is inhabited by most of the phytoplankton communities which produce considerable oxygen through photosynthesis. Other organisms which feed on these phytoplanktons include various kinds of zooplanktons (pteropods, salps, foraminifers, copepods, protozoa, larval stages of many crustaceans, worms and fishes), fishes (Sardines, Tuna, sea turtles, dolphins and whales) and swimming mammals. These organisms function as consumers at higher trophic levels. Fungi in deep-sea ecosystem * Mesopelagic zone (from 200-1,000 m depth) This is the zone where faint sun light reaches and is known as twilight zone. This light is not sufficient for photosynthesis. Therefore the main option for feeding in this zone becomes restricted to detrivory or carnivory. Some of the animals inhabiting this zone include octopus, squid and other fishes. Among mesozooplankton, copepods are numerically dominant forms present, but in the upper part of this zone the larger-sized organisms are predominantly gelatinous, particularly siphonophores. These animals have to withstand elevated hydrostatic pressure and low temperature conditions. Some animals develop the lsr property of bioluminescence i.e. production of their own light to sustain in the dark ambient environment. The characteristic morphology of fishes living at mesopelagic depths is exemplified by the myctophids (Lantern fishes) and argyropelecids (hatchetfishes). They have black backs, flanks that are highly reflective and mirror-like and their undersides are lined with photophores (light organs). The silvering of the flanks is produced by regularly spaced platelets of a white pigment, guanine (Denton and Nicol, 1966). • Bathypelagic zone (from 1,000-4,000 m depth) This zone is characterized by complete darkness and extreme conditions of temperature and pressure. This zone is also known as midnight zone. In the temperate Atlantic, it also approximates to the deep oxygen minimum and the base of the permanent thermocline (Angel, 1989). This zone is inhabited by greatest variety of pelagic species. Among animals, mollusks, some squids, amphipods, octopods, jellies, crustaceans and angler fish are known to be present. Occasionally sperm whales dive in search of food to this zone. • Abyssopelagic zone (from 4,000-6,000 m depth) This zone is also known as abyss i.e. without bottom. It is characterized by complete darkness, very high pressure and freezing temperature of the surrounding water. The organisms surviving there mostly include some of the tiny squids or basket stars without back bone. Fungi in deep-sea ecosystem • Hadalpelagic zone (greater than 6,000 m deep) This zone is the deepest part of ocean and is also known as trench. The temperature and darkness are same as abyssopelagic zone but hydrostatic pressure is comparatively high. The deepest trench so far known is the mariana trench of coast Japan which is 11,000 m deep. Some of the organisms surviving in this zone include invertebrates such as star fishes and tubeworms. oceanic provinces and zones s nentic r oceanic pe'agic epipetagic A -----_ COm sublitioral disphotic mesopetagic batyat aphotic tatto at 90 bathypelagic aphotic bat ya9 4000m ben#hic Fig.