BREEDING BEHAVIOUR, EMBRYONIC DEVELOPMENT AND BARCODING OF THE ORNAMENTAL LOACHES (COBITIDAE: CYPRINIFORMES) OF TERAI REGION OF WEST BENGAL, INDIA. Thesis Submitted to the University of North Bengal For the award of Doctor of Philosophy in Zoology By Arpita Dey (M.Sc.) Supervisor Dr. Sudip Barat Professor Department of Zoology University of North Bengal Co-Supervisor Dr. (Mrs.) Debapriya Sarkar Professor in Fishery Unit Uttar Banga Krishi Viswavidyalaya Pundibari, Cooch Behar AQUACULTURE AND LIMNOLOGY RESEARCH UNIT DEPARTMENT OF ZOOLOGY UNIVERSITY OF NORTH BENGAL DISTRICT DARJEELING WEST BENGAL, 734013 INDIA 2017 DECLARATION I declare that the thesis entitled “Breeding Behaviour, Embryonic Development and Barcoding of the Ornamental loaches (Cobitidae: Cypriniformes) of Terai region of West Bengal, India” has been prepared by me under the guidance of Dr. Sudip Barat, Professor, Department of Zoology, University of North Bengal and co-guidance of Dr. (Mrs.) Debapriya Sarkar, Professor in Fishery Unit, Uttar Banga Krishi Viswavidyalaya. Thesis has been verified for plagiarism through software ‘URKUND’. No part of the thesis has formed the basis for the award of any Degree or Fellowship previously. Date: (Arpita Dey) Aquaculture and Limnology Research Unit Department of Zoology University of North Bengal Dedicated Dedicated to my dear parents Mr. Amar Chandra Dey and Mrs. Dipti Dey ACKNOWLEDGEMENT At the onset, it is my privilege to express deep sense of gratitude and indebtedness to my Supervisor of the present research work, Dr. Sudip Barat, Professor, Department of Zoology, University of North Bengal for his unequivocal motivation, benevolent guidance, affectionate encouragement and constant supervision during the entire period of the investigation and preparation of manuscript and thesis writing. He always encouraged me to work on various aspects of Aquaculture and share developing new ideas in the research field. He always welcomed me to any problem on Aquaculture and Limnology based research work. I wish to express my deep sense of gratitude and indebtedness to my Co-Supervisor Dr. (Mrs.) Debapriya Sarkar, Professor in Fishery Unit, Uttar Banga Krishi Viswavidyalaya, Pundibari, Cooch Behar, West Bengal, for her valuable guidance during the period of research. She always encouraged me in my research work so that I could reach the research goal. It is also my pleasure to express heartiest gratitude to Prof. Somnath Ghosh, Honourable Vice-Chancellor, University of North Bengal; Dr. Laxmikanta Padhi, Registrar, Dean and other officials of the University for extending academic and administrative facilities to me. It is also a glorifying pleasure for me to express my thanks to Dr. Chirantan Chattopadhya, Honourable Vice-Chancellor, Uttar Banga Krishi Viswavidyalaya for their useful suggestion and cooperation. My deep sense of gratitude is extended to my respected teacher Prof. Apurba Kumar Chowdhury, Plant Pathology, Uttar Banga Krishi Viswavidyalaya for necessary academic facilities. It is my great pleasure to express my sincere thanks to Dr. Min Bahadur, Head, Department of Zoology, University of North Bengal. My Ph.D. thesis would never have been completed without his help. Whenever I faced any problem regarding my research work, I used to seek his advice and he always gave me good suggestions. i I sincerely express my thanks to the respected Faculty Members Prof. Joydeb Pal, Prof. Ananda Mukhopadhya, Prof. Tapas Kumar Chaudhuri, Prof. Soumen Bhattacharjee, Dr. Dhiraj Saha and Mr. Tilak Saha, Department of Zoology, University of North Bengal for their constant encouragement, cooperation and helpful suggestions. I am grateful to Mr. Kripan Sarkar, owner of the Rainbow Ornamental Fish Farm, Raninagar, Jalpaiguri, West Bengal. He was my practical teacher in the field of ornamental fish breeding and culture. I extend my thanks to his wife also Mrs. Anna Sarkar, for helping me in the research work especially during the night in the field laboratory to monitor the breeding programme. I would like to offer my thanks to the Dr J. K. Jena, DDG (Fisheries Science), Indian Council of Agricultural Research (ICAR), New Delhi, for providing the necessary facilities and suggestions. I am thankful to Dr. Mahender Singh, Principal Scientist, Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh, India for extending permission to use the vast laboratory facilities with regard to DNA barcoding of my experimental fishes. I deem it a unique opportunity and express my profound sense of gratitude and ingenuous regards to Dr. Niloy Ray from Hoogly Mohsin College, Chinsura, West Bengal, Mr. Uday Udit from CIFA, Bhubaneswar, Odisha, India, Mr. Debdut Debnath, Mr. Sukanto Debnath and Mr. Subhojit Chakraborty for their support and encouragement. I want to express my heartiest thanks to Mr. Archisman Barat, a Ph.D. research Scholar, Central University of South Bihar, Patna for extending his unique idea on my research and related topics. I sincerely express my thanks to Mr. Prasun Mazumder and Mr. Shibnath Sarkar for helping me in my computational work. The completion of my thesis is also due to the whole hearted support of my senior research colleague Ms. Ruksa Nur. I am also happy to express my thanks to other senior colleagues Dr. Prithwiraj Jha, Dr. Manik Lal Acharjee, Dr. Soma Banerjee, Dr. Rudra Prasad Roy, Mr. Rakesh Prasad Bhagat and friend Sanjib Sarkar. I am thankful to my senior colleagues Tanmay Mukhopadhyay, Kumar Basnet, Anjali Prasad, Somit Dutta, Sutanuka Chattaraj, Uttara Dey Bhoumick, Tanmoy Dutta, ii Bappaditiya Ghosh and Swati Singh for their suggestions time to time. My thanks is extended to my friend Subhashis Paul and Ms Ankita Mukherjee. I would like to express a word of thanks to the Librarian, University of North Bengal, for extending all the necessary facilities throughout the research work. I am also thankful to the Non-teaching staff, Department of Zoology, University of North Bengal. My field study would not have been completed without the devoted help of Mr. Sunil Barman, a fisherman and Ms. Mampi Dey for helping me in harvesting of fishes from the river my field laboratory work, respectively. Most important, I would like to thank my dear parents Mr. Amar Chandra Dey and Mrs. Dipti Dey who are the pillars of strength and to whom this thesis is dedicated. They have always encouraged and inspired me. My brother, Mr. Apurba Dey, always suggested to me to “dream big” so that I could reach the main goal of my life. Lastly, I would like to thank all those well wishers who helped me knowingly or unknowingly during the period of the present study. Place: University of North Bengal Dated: Arpita Dey iii Abstract Ornamental fish production globally is a multibillion dollar industry. India‟s overall ornamental fish trade was about 1.06 million US$ during year 2009. India has recorded at least 150 commercially important ornamental fish species and trade mainly indigenous freshwater species collected from rivers. Among them, Botia loaches classified as aquarium fish due to their beautiful colouration, small size, bright bands, blotches, peaceful nature and hardiness can be reared and bred in aquarium throughout their life span. Botia almorhae (Grey), Botia dario (Hamilton-Buchanan), Botia lohachata (Chaudhuri) and Botia rostrata (Gunther) were selected for present study. They are highly demanding both as an ornamental and edible fish in the Terai region of Eastern Himalaya of West Bengal, India, a “Hot Spot” for fresh water fish biodiversity. With a view to rearing and breeding of loaches in captivity, which are Vulnerable and Endangered, their conservation and ichthyofaunal diversity of river Kaljani, Cooch Behar district a study was executed during the period August 2012 to July 2015. In the present study, for both river Kaljani and Captive study water was soft, alkaline in nature with high Dissolved Oxygen and medium productive condition. The temperature was good for growth of the fishes of Kaljani river water and Laboratory water (Captive condition). TDS, Free Carbon Dioxide and average concentrations of ammonium-nitrogen (0.017 mg L-1), nitrite-nitrogen (0.009 mg L-1), nitrate-nitrogen (0.312 mg L-1) and Phosphate-phosphorous concentration in the Kaljani river and captive condition were also within normal range. The average Gonado-somatic Index data of Botia species revealed to be Botia almorhae (11.96±10.29), Botia dario (8.34±5.4), Botia lohachata (13.86±11.50) and Botia rostrata (10.29±9.01). Among the Botia species, Botia lohachata had the highest GSI and Botia rostrata the lowest. Condition Factor or K- factor in Captive condition for iv Abstract Botia almorhae was 1.390, Botia dario was 1.788., Botia lohachata was 1.538 and Botia rostrata was 1.399. The values indicated good general condition of fish as „K‟ was greater than 1.0. Length - Weight relationship was calculated by the determination of Coefficient of Correlation (r). The Coefficient of Correlation of Botia almorhae was 0.811; Botia dario: 0.802; Botia lohachata: 0.753 and Botia rostrata: 0.936. Botia species indicated positive allometric growth and suggested that all fish grows in proportion to the length in Captive condition. Average fecundity of Botia almorhae, Botia dario, Botia lohachata and Botia rostrata were 18539, 22573, 18053 and18698 respectively and fertilization rate 90.03%, 82.09 %, 95.98 % and 67.60 % respectively Fish collected from river water were first acclimatized in aquarium and induced bred using synthetic hormone WOVA-FH at a dose of 0.025ml/fish. The latency period was 4.30 to 6.00 hours. The embryonic development studied in Botia species was divided into eight stages namely Zygote, Cleavage, Blastula, Gastrula, Segmentation, Pharyngula, Hatching and Early larval period. Complete adult stage was obtained within one year. After captive breeding of wild Botia species, F1 generation of Botia loaches were aqua-ranched into the natural environment of the river system (River Kaljani). Spawning behaviour was observed during the night or afternoon in absence of light.