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Molecular Markers of Ecotoxicological Interest in the Rainbowfish

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Molecular Markers of Ecotoxicological Interest in the Rainbowfish Molecular markers of ecotoxicological interest in the rainbowfish Melanotaenia fluviatilis A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Pattareeya Ponza B.Sc, M.Sc School of Applied Sciences Science, Engineering and Technology Portfolio RMIT University August 2006 Summary The Crimson-spotted rainbowfish ( Melanotaenia fluviatilis ) from the Murray-Darling basin of Australia is a common indicator species in Australian ecotoxicology. Biochemical changes have been investigated in this species, but not molecular markers of ecotoxicological interest. In this study genes of M. fluviatilis were isolated using a cDNA library and sequences analysed. Of 345 randomly selected clones, 94 shared similarity with 26 different genes in other organisms in public databases. Amongst these, reproductive genes coding for vitellogenin, retinol binding protein, sialyltransferase and zona pellucida protein were considered of interest in ecotoxicology. The vitellogenin gene was selected for study as it has been widely used as a molecular marker of exposure to 17 β-estradiol (E 2) in teleosts. Gene expression was examined via northern blot, RT-PCR and Real-Time PCR relative to the housekeeping gene (18S rRNA). The expression of vitellogenin mRNA was observed at 12 hours post-exposure, peaked at 48 hours according to northern blot analysis; and cleared within 4 days, partly consistent with RT- PCR. However, Real-time PCR yielded an inconclusive result, probably due to differences between pooled and individual samples. Vitellogenin in blood plasma was confirmed by western blot, found to be significantly increased and retained in the plasma in fish treated with E 2 compared to controls. It was concluded that vitellogenin mRNA is a molecular marker of exposure to 17 β-estradiol in the rainbowfish, and could potentially be used as a marker of exposure to environmental estrogenic chemicals. Further investigations of the expression of genes in the cDNA library, could establish other molecular markers of ecotoxicological interest in M. fluviatilis . i Declaration I declare that this thesis contains my original work. Information from published or unpublished sources has been clearly acknowledged within the thesis. None of the work contained in this thesis has been submitted to qualify for any other academic award. The content of the thesis is a result of the work, which has been carried out during the enrolled period of the program. I declare that this thesis is less than 100,000 words in length. PATTAREEYA PONZA 22 August 2006 ii List of Abbreviations Abbreviations frequently used: µg Microgram µL Microlitre cDNA Complementary DNA CuSO 4 Copper (II) Sulphate DNase An enzyme to eliminate DNA dNTP Deoxyribonucleoside triphosphate DTT Dithiothreitol EC 50 The statistical estimate of the toxicant concentration, for acute toxicity testing, that has a specified adverse effect (such as immobilization, change in respiration rate, or loss of equilibrium) on 50 percent of test organisms after a defined period of exposure EDCs Endocrine Disruptive Chemicals g Gram GAPDH Glyceraldehyde 3-phosphate dehydrogenase kb Kilo basepairs kDa Kilodaltons L Litre MgCl 2 Magnesium Chloride mL Millilitre mM Millimolar or millimole per litre mRNA Messenger RNA iii MS-222 An anaesthetic ng Nanogram nM Nanomolar or nanomole per litre pg Picogram ppm Part per million ppt Part per thousand RNase An enzyme to eliminate RNA rpm Revolution per minute RT-PCR Reverse-Transcription Polymerase Chain Reaction Vtg Vitellogenin iv Acknowledgement I would like to thank a number of people who have involved in associating my thesis. • My supervisors, Associate Professor Dayanthi Nugegoda and Associate professor Trevor Stevenson for their guidance and encouragement in this project. • The Department of Science and Technology for financial support through the Royal Thai Government Scholarship throughout the study. • My colleagues (past and present) in the Plant Biotechnology lab and Ecotoxicology lab, RMIT University: Dr. Beata Skiba, Dr. Chitra Raghavan, Ajay Pundalkirad Niranjane, Deanne Britt, Priyadharshini Madhou, Brook Clinton, Belinda Denton, Tristan Coram and Kathryn Hassell, Kylie White for their valuable friendship, assistant and encouragement. I would also like to thank all my friends in the Department of Biotechnology and Environmental Biology for their support. • Churches members in Bundoora Presbyterian Church, Victoria, Australia and in Thailand for their faithfully prayers for me and my family. • My parents and my brothers for their supports in many ways. I would like to thank especially my parents, who have taught me to trust God in every situation in life. • My daughter, Modhana for bringing me joy and hope and my husband, Supat Ponza for his encouragements. • Most of all, I would like thank God for He has strengthen me and has provided all my needs. v Table of Contents Summary…………………………………………………………………………………………...i Declaration………………………………………………………………………………………...ii List of Abbriviations……………………………………………………………………………...iii Acknowledgements……………………………………………………………………………….v Table of Contents…………………………………………………………………………………vi List of Tables…………………………………………………………………………………......xii List of Figures…………………………………………………………………………………...xiii Chapter 1 Review of Literature on biomarkers of exposure to environmental chemicals..... 1 1.1 Effects of pollutants on biota............................................................................................. 1 1.2 Biomarkers studied in aquatic organisms.......................................................................... 3 1.2.1 Metallothionein........................................................................................................ 9 1.2.2 Stress proteins ........................................................................................................ 12 1.2.3 Enzymes related to the neural system as biomarkers............................................. 12 1.2.4 Cytochrome P450s................................................................................................. 15 1.2.5 Reproductive proteins ............................................................................................ 16 1.3 Endocrine disruptive chemicals (EDCs) ......................................................................... 19 1.3.1 The mechanisms and general structures: ............................................................... 19 1.3.2 Route of endocrine disrupting chemicals contamination....................................... 20 1.3.3 Biomarkers of exposure to EDCs .......................................................................... 24 1.4 Limitation of using these proteins as biomarkers............................................................ 27 1.5 Molecular markers........................................................................................................... 29 1.5.1 Selective genes as biomarkers for ecotoxicology purposes................................... 29 1.5.2 Advances in molecular markers............................................................................. 31 vi 1.5.3 Molecular markers in fish in an ecotoxicological context.................................... 32 1.6 The Rainbowfish ............................................................................................................. 35 1.6.1 Fish taxonomy........................................................................................................ 35 1.6.2 The habitat and distribution of Crimson-spotted rainbowfish. .............................. 35 1.6.3 Utilization of the Rainbowfish as a bioindicator of exposure to toxicants............ 37 1.6.4 Studies of the genome of Melanotaenia ................................................................ 42 1.6.5 Approaches to study the genome of a non-model organism.................................. 42 1.7 General objective............................................................................................................. 43 1.8 The specific objectives .................................................................................................... 43 Chapter 2 General material and methods................................................................................. 44 2.1 Fish preparation and maintenance ...................................................................................... 44 2.1.1 Source of the fish and husbandry................................................................................ 44 2.1.2 Food............................................................................................................................. 44 2.1.3 Water quality............................................................................................................... 44 2.2 Exposure of 17-estradiol..................................................................................................... 45 2.2.1 Determination of doses................................................................................................ 45 2.2.2 Preparation of 17 β-estradiol........................................................................................ 46 2.2.3 Intraperitoneal injection .............................................................................................. 46 2.2.4 Sample collection ........................................................................................................ 47 Chapter 3 Construction of cDNA library.................................................................................
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