Possible ecological impacts caused by GFP transgenic zebrafish, danio rerio

POSSIBLE ECOLOGICAL IMPACTS CAUSED BY GFP TRANSGENIC ZEBRAFISH, DANIO RERIO SEAH WEE KHEE B.Sc (Hons) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2006 For my beloved mother & father ACKNOWLEDGEMENTS Firstly, I am grateful to my Honours project supervisors, Associate Professor Li Daiqin and Associate Professor Gong Zhiyuan, for encouraging me to embark on this interesting project. I am deeply indebted to Associate Professor Li Daiqin for his patience and contributions towards the completion of this thesis. He has been a wonderful and inspiring supervisor for my past research projects, constantly encouraging me throughout the last seven years. Thanks also go to Associate Professor Gong Zhiyuan, for patiently educating me on the molecular aspects of the project, and providing me with priceless suggestions on my experiments. Special appreciation goes out to Associate Professor R. R. Jackson from University of Canterbury and Dr. Simon Pollard from Canterbury Museum, for their invaluable and creative sessions of discussion while being a guest of the Behavioural Ecology & Sociobiology Laboratory in National University of Singapore. My appreciation is also due to Professor W. M. Muir, of the Department of Animal Sciences, Purdue University, for assistance on the details of population modeling and his expert advice on transgenic organisms. Special mention is due to Brandon Brown for advising me in the initial stages of population modeling, and completing preliminary graphs using the BioQuest program biota. Thanks are also due to Wu Yi Lian, Ng Kok Chye, Ng Quee Kee, Subhas Balan and Goh Poi Moi, Department of Biological Sciences, National University of Singapore, for their help and provision of equipment during the course of this research project. My heartfelt thanks go to everyone past and present of the Behavioural Ecology & Sociobiology Laboratory for their relentless support and contributions towards this project in so many ways than one. I would like to thank the National University of Singapore (NUS) for providing me with a Research Scholarship and NUS Academic Research Fund grants (R-154-000-104112 & R-154-000-104-107) to carry out this work, and for providing this project with a research grant to see through the completion of this endeavor. To Grandma, Dad and Mum, I am especially indebted to them for their unwavering love, support and understanding throughout these years. To you, I will be forever grateful. And lastly, Chris, I thank you for everything you are, and have done for me. No amount of thanks will suffice. I TABLE OF CONTENTS Acknowledgements I Table of Contents II List of Tables VI List of Figures VIII Summary CHAPTER GENERAL INTRODUCTION 1.1. An Era Created via Transgenic Techniques 1.2. Are Transgenic Organisms Potential Ecological Hazards? 1.3. Fitness Affected by Genotypic and Phenotypic Alterations 1.3.1. Behavioural changes 1.3.2. Physical changes 1.3.3. Natural and sexual selection pressures 1.4. Assessment of Ecological Risk of Transgenic Organisms 1.5. Research Objectives CHAPTER THE EFFECT OF GFP TRANSGENE ON FEMALE MATE PREFERENCES OF ZEBRAFISH 11 2.1. Introduction 11 2.2. Materials and Methods 13 2.2.1. Experimental subjects 13 2.2.2. Rearing conditions 14 2.2.3. Female mate choice 14 2.2.4. Role of visual cues in female mating preference 17 2.2.5. Manipulated video-recording or mate choice tests 22 II 2.2.5.1. Preparation of video playbacks 22 2.2.5.2. Mate choice experiments 26 2.3. Results 27 2.3.1. Female mate choice 27 2.3.2. The role of male colour in female mating preference 30 2.3.3. Role of visual cues in female mate choice tests 33 2.4. Discussion 38 CHAPTER EFFECTS OF GFP TRANSGENE ON REPRODUCTIVE FITNESS OF ZEBRAFISH 42 3.1. Introduction 42 3.2. Materials and Methods 43 3.2.1. General rearing procedures 43 3.2.2. Pre-spawn fitness measurements 44 3.2.2.1. Gonado-somatic index (GSI) 44 3.2.2.2. Gamete size 45 3.2.3. Post-spawn fitness measurements 45 3.2.3.1. Fecundity 46 3.2.3.2. Fertility 46 3.2.3.3. Hatching rate and developmental rate 46 3.2.4. Larval development 47 3.2.5. Fitness parameters of the second generation 47 3.3. Results 48 3.3.1. Pre-spawn fitness measurements 48 3.3.1.1. Gonado-somatic index (GSI) 48 3.3.1.2. Gamete size 49 III 3.3.2. Post-spawn fitness measurements 3.3.2.1. Fecundity, fertility, hatching rate and developmental rate 50 50 3.3.3. Larval development 52 3.3.4. Fitness parameters of the second generation 54 3.4. Discussion 56 CHAPTER TRANSGENE EFFECTS ON ADULT VIABILITY OF GFP TRANSGENIC ZEBRAFISH 61 4.1. Introduction 61 4.2. Materials and Methods 63 4.2.1. General rearing procedures 63 4.2.2. Associative learning 64 4.2.2.1. Cognitive to forage 64 4.2.2.1.1. Preparation 64 4.2.2.1.2. Learned experience 65 4.2.2.1.2.1. Individual tests 66 4.2.2.1.2.2. Group tests 67 4.2.2.2. Threat recognition and avoidance 67 4.2.2.2.1. Preparation 68 4.2.2.2.2. Paired tests 70 4.2.2.2.3. Associative predator recognition 76 4.2.3. Predation 77 4.3. Results 78 4.3.1. Associative learning 78 4.3.1.1. Cognitive to forage 78 4.3.1.2. Threat recognition and avoidance 81 IV 4.3.2. Predation 87 4.4. Discussion 87 4.4.1. Associative learning 88 4.4.1.1. Cognitive to forage 88 4.4.1.2. Threat recognition and avoidance 90 4.4.2. Predation 95 4.4.3. Conclusions 95 CHAPTER FITNESS COMPONENTS AND POSSIBLE ECOLOGICAL RISKS OF TRANSGENIC ZEBRFISH: MODELING FUTURE POPULATIONS 97 5.1. Introduction 97 5.2. Methods 99 5.2.1. Deterministic model 99 5.2.2. Determined parameters 102 5.3. Results 103 5.4. Discussion 105 CHAPTER GENERAL DISCUSSION 109 6.1. Transgene Effects and Its Significance 109 6.2. Natural and Sexual Selection 110 6.3. Conflicting Hypotheses 113 6.4. Last Considerations 114 References 117 Appendix 132 V LIST OF TABLES Table Number 2-1 Title Page Objectives of the various video playback manipulations. Abbreviations are as follows: WT male courtship behaviour without yellow fin colour (WTNY); WT male courtship behaviour with yellow fin colour (WTY); GFP male courtship behaviour without yellow fin colour (GFPNY); GFP male courtship behaviour with yellow fin colour (GFPY); GFP male courtship behaviour but GFP colour (green) removed, without yellow fin colour {WT (GFP)NY}; WT male courtship behaviour with GFP colour (green) added, without yellow fin colour {GFP (WT)NY}. 25 2-2 Results obtained from Dichotomous Choice test with WT and GFP transgenic zebrafish. Test for Goodness of fit determines significant differences within the experimental sets. 28 2-3 Results from the cross-chambered tests in which a test female was permitted to choose from four phenotypically similar fish under four different light environments manipulated by four different types of filters. Filter #130 (FS) is clear, permitting all light transmitted; filter #328 (MW1-) blocks green; filter #142 (MW2-) blocks yellow; filter #14 (SW-) blocks blue. Predicted female mate preference rank: MW1- = FS > MW2- = SW-. 31 2-4 Results from testing females with manipulated video images. Abbreviations follow those of Table 2-1. 34 3-1 Mean (± SE) weight of WT and GFP juveniles at the respective ages. 53 3-2 Statistical test results used to determine significant differences among mating pairs and between the two generations. 54 4-1 Summary of the items required for quantitative data collection for response to the alarm cue, Schreckstoff. Here, (4a) represents the infusion needed for Pavlovian conditioning of the zebrafish, while (4b) was utilized for determining whether there were any differences in conditioned response in the three zebrafish populations. 69 4-2 Results from the individual choice test in associative foraging behaviour for WT, GFP transgenic and non-transgenic zebrafish. 79 4-3 Multivariate statistical test results used to determine significant differences in location of the zebrafish in the experimental set-up and their clumping indexes among different treatments and different populations (WT, GFP and non-transgenic). 82 VI 4-4 Results from predator choice made after 24 h by starved WT and GFP zebrafish adults on juvenile prey choice. VII 87 LIST OF FIGURES Figure Number 1-1 Legend Page (A) Wild type (WT) and (B) GFP transgenic zebrafish. Note how the muscle cells in the transgenic zebrafish glows fluorescent green. 10 2-1 Top-view of the dichotomous choice apparatus. The phenotypically different stimulus males vie for the test female’s attention by courting her through the transparent glass partition. An opaque barrier between males prevents competition displays, but allowing the test female to view courtship behaviour of both stimulus males. 16 2-2 The courting males and females of both (A) WT and (B) GFP zebrafish, Danio rerio. Arrows point to sexual colouration detected in courting WT and GFP males. 18 2-3 Top view of the experimental set-up for the four-chambered choice test, consisting of a central chamber and four stimulus chambers. A female was left in the centre of the central chamber and allowed to select from males each placed in one of the stimulus chambers. Colours of males were manipulated by the various colour filters (see Figure 2-4). All chambers were physically separated hence only visual communication is permitted. Incomplete darkened lines across the central chamber were opaque glass panels which acted as a barrier between males at opposite ends to omit male-male interaction, yet allowing females to swim freely from one preference zone to another. These opaque panels also compartmentalized the preference zones for each individual male. 19 2-4 Reflectance spectra of the four filters used. (A) Clear filter (FS); (B) Pink filter blocking green colours (MW1-); (C) Violet filter blocking yellow colours (MW2-); (D) Yellow filter blocking blue colours (SW-). 21 2-5 Pictorial representation of the video recording of male courtship behaviour for video manipulation to determine courtship, fin colour and body colouration in female mate choice. The male and female were only permitted visual contact. 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Fluorescent male zebrafish are no match for their wild competitors. CREDIT: National University of Singapore The zebrafish Danio rerio, native to streams in southern Asia, is normally silvery-grey with dark stripes. But in the 1990s, scientists in Taiwan and Singapore genetically modified strains with genes from jellyfish and anemones, giving the fish a green or red "glow" under UV or even visible light. Originally developed to aid in the detection of water pollutants (with a switch gene added, the fish would glow whenever the target pollutant was in the water), these and similar fish have been popular in the aquarium trade in the U.S. since late last year, with the red variety marketed under the name GloFishTM. But environmentalists have expressed concern that the modified fish will escape and interbreed with wild zebrafish, particularly in their native tropical Asia. Wee-Khee Seah, Zhiyuan Gong, and Daiqin Li of the National University of Singapore made aquariums where a normal or green fluorescent zebrafish female would be confronted with the choice between a normal and a glowing green male behind glass. They found that both types of female spent more than 80% of their time with their 133 noses glued to the glass of the unaltered males' compartments, with the green males jealously courting in vain. Suspecting that the green-glowing fish might have subdued courtship behavior, they then showed the females videos of courting males after digitally doctoring the images of some of the wild males' courtships to make them look fluorescent green. Sure enough, the females always preferred wild males' courtships, whether cloaked in green or not. Finally, when forced to mate with green males, females would show their dissatisfaction by laying only half as many eggs as when paired with a wild male, the researchers found. Seah thinks the genetically-engineered fishes' lethargic courtship behavior may be the result of having too much energy drained by the glowing jellyfish protein in their muscles. Fish ethologist Adam Shohet of the University of Sussex in Brighton, U.K., agrees that the insertion of a foreign fluorescent protein may upset the fishes' finely-balanced energy budget. He's convinced that the new results show that there is "little threat posed by the popular proliferation of these fish." --MENNO SCHILTHUIZEN ‘Too Tired to be Sexy’ was based on an oral presentation delivered in the Biology in Asia International Conference, Nanyang Technological University, Singapore. The topic, Limitations in Mating Success in Green Transgenic Danio rerio, was featured in ScienceNow: American Association for the Advancement of Science on the 16th December 2004. http://sciencenow.sciencemag.org/cgi/content/full/2004/1216/2 134 [...]... both the transgenic and the wild type D rerio My results showed that the transgenic trait reduced the fitness of the transgenic fish in several aspects Compared with wild type zebrafish, GFP transgenic zebrafish had disadvantages in mate choice, reproduction success and viability Both wild type and GFP transgenic females showed distinctive preferences for the wild type male zebrafish over the GFP transgenic. .. done to determine whether transgenic organisms can pose any ecological risk, it is important to assess their potential dangers and not rely on hypothesized theories and models to determine their possible ecological impacts Using the zebrafish (Danio rerio) as a model organism, I aim to assess the potential ecological risk of the transgenic zebrafish by examining how insertion of GFP (Green Fluorescent... male courtship behaviour [Chapter 2.2.5] on female D rerio mate choice 2.2 Materials and Methods 2.2.1 Experimental subjects Wild type (WT) and genetically modified (GFP) Danio rerio were used Transgenic GFP zebrafish used were obtained from the eighth and ninth generation of mylz2 :gfp transgenic parental stock, which was generated by injection of a transgenic DNA construct containing a 2-kb, fast skeletal... with many artificially created novel transgenic organisms, this much celebrated pet has caused a great deal of controversy associated with potential ecological risks because the fish remains reproductively viable Using the zebrafish (Danio rerio) as a model organism, I examined the ecological impacts of GFP (Green Fluorescent Protein) transgene on fitness components by investigating the mate choice, reproductive... fibres by transgenic techniques could affect some aspects of fitness components including mating success, reproduction and viability of both GFP transgenics and wild type (WT) zebrafish, under controlled experimental conditions (Muir & Howard 1999, 2001; see Figure 1-1) The specific objectives are as follows: 1 In Chapter 2, I examined the effects of GFP transgene on mating success of both GFP transgenic. .. type and GFP transgenic zebrafish Adult viability of both the wild type and GFP transgenic zebrafish was examined via the determination of their ability to survive by effectively foraging for food and avoiding risky situations My results showed that GFP zebrafish seemed to be more limited in food acquisition and predator avoidance compared to the wild type However, adult viability of GFP transgenic. .. of female mate choice showed that the intrinsically altered courtship rituals of the GFP transgenic males resulted in the reduced female preference for GFP males Moreover, the GFP transgenic males also lacked the required optimum colouration on their flanks and fins to attract females Thus, mating success of the GFP transgenic males were significantly lower than that of the wild type ones Reproductive... risk By establishing possible predator preference for conspicuous colouration, it offered an insight as to the transgenic organism’s vulnerability, hence survivability, in the natural environment 4 In Chapter 5, I assessed the potential ecological impacts by using a deterministic model All necessary fitness metrics that were collected in this study was included in the model to determine whether the GFP. .. GFP transgenic zebrafish, or GloFish, transgene will persist when interactions between the transgenic and WT are allowed This study is expected to have general implications on potential long-term risks associated with accidental or intentional release of colourful yet conspicuous transgenic fish 9 A B Figure 1-1 (A) Wild type (WT) and (B) GFP transgenic zebrafish Note how the muscle cells in the transgenic. .. hatching rate, developmental rate and juvenile viability was compared in all possible mating permutations in a population of wild type and GFP transgenic zebrafish The collective data showed that clutches produced by at least one or both GFP transgenic parents were 1 inferior in quantity and quality as compared to those produced by both wild type parents These inferiorities included significantly lower . POSSIBLE ECOLOGICAL IMPACTS CAUSED BY GFP TRANSGENIC ZEBRAFISH, DANIO RERIO SEAH WEE KHEE B.Sc (Hons) A THESIS. the transgenic and the wild type D. rerio. My results showed that the transgenic trait reduced the fitness of the transgenic fish in several aspects. Compared with wild type zebrafish, GFP transgenic. viable. Using the zebrafish (Danio rerio) as a model organism, I examined the ecological impacts of GFP (Green Fluorescent Protein) transgene on fitness components by investigating the mate choice,