Molecular analysis of the breeding biology of the asian arowana (scleropages formosus)

Molecular Analysis of the Breeding Biology of the Asian Arowana (Scleropages formosus) by Chang Kuok Weai Alex Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biological Science National University of Singapore 2010 Supervisor: Associate Professor Laszlo Orban PhD committee members : Emeritus Professor Lam Toong Jin Dr Hong Yan Dr Gregory Jedd Associate Prof Laszlo Orban 1|Page Acknowledgements Throughout my project, Prof. Laszlo Orban, my supervisor, was generous with his time and knowledge and on target with his counsel and instilling in me a real appreciation for the scientific method. . I gratefully acknowledge the essential contributions of my Supervisory Committee, Emeritus Professor Toong Jin Lam (chair), Dr Yan Hong and Dr Gregory Jedd, and I also thank Professor Lam for chairing my Examination Committee. I commend the administration of the Temasek Life Sciences Laboratory (TLL) and Qian Hu Fish Farm for facilitating the educational pursuits of their employees I also thank Yap Kim Choon and his staff at Qian Hu Fish Farm believing in me and providing me such a important platform to work on this wonderful species and I am grateful to Kenny Yap for providing invaluable early assistance. I am grateful to my colleagues, Woei Chang Liew, Hsiao Yuen Kwan, Rajini Srineevasan, Felicia Feng, Dr. Xingang Wang, Dr. Richard Bartfai for help and collaborative work; and to Chin Heng Goh, Dr. Patrick Gilligan, and Dr. Gen Hua Yue, who quickly and patiently replied to questions relating to material herein. I thank the contribution of several attachment students, Wee Kee, Qi Feng, Zi Jie, Say Aik, Serene, and Daniel who brightened up the sky every time they were around. In addition, I fondly acknowledge the help and support of numerous TLL colleagues and PIs. Also, special thanks to Dr Robert Brooks, who patiently guided me through the advanced analysis of some of my results (in understanding mating systems. I also thank Aaron Chuah and Graham Wright, who provided computing advice and code, Prof Rudolf Meier and Prof Tan Heok Hui, who helped me in understanding the interesting phylogenetic studies. Lastly, I thank my Dad for getting me hooked on fishes and fisheries science early on. 2|Page Dedication To my family, especially my dad and mum, my wife Cynthia, my son Andre Jacob and friends, for their unwavering kindness, patience, and support. 3|Page TABLE OF CONTENT ABSTRACT . 11 List of Tables . 12 List of Figures 13 List of Abbreviations . 23 INTRODUCTION . 27 1.1 Taxonomy of bonytongues 27 1.2 The general biology of Asian arowana . 31 1.3 The Asian arowana has at least six main colour strains 34 1.4 Sex and strain identification of teleosts using classical tools 42 1.5 Mating system and parental care in fishes 46 1.6 Molecular approaches in fish biology and aquaculture research 50 1.7 The aims of my research . 62 MATERIALS AND METHODS . 63 2.1 The origin of fish studied 63 2.2 Arowana breeding and holding facilities in QH 64 2.3 Field Observations . 64 4|Page 2.4 Sample collection 65 2.5 DNA isolation . 66 2.6 Isolation and genotyping of microsatellites 68 2.7 Detection of steroid hormone levels using an Enzyme Immunosorbent Assay kit …………………………………………………………………………… .69 2.8 Bradford total soluble protein (TSP) Assay 69 2.9 Amplified Fragment Length Polymorphism (AFLP) 70 2.10 Fluorescent Motif Enhanced Polymorphisms (FluoMEP) 72 2.11 Determining single nucleotide polymorphisms in the mitogenome to confirm the gender of the mouthbrooder . 72 2.12 Computational and statistical analysis . 74 2.12.1 Microsatellite genotyping 74 2.12.2 Analysis of AFLP-based phylogenetic relationship between the colour strains …………………………………………………………………………75 2.12.3 Analysis of kinship between the brooders for understanding egg thievery event …………………………………………………………………………75 2.12.4 Study of the genetic similarity among the colour strains . 76 2.13 Morphometric measurements 76 5|Page 2.13.1 External morphometric differences 76 2.13.2 Internal morphometric differences – moulding/roughness index and touch-based examination . 77 2.13.3 Morphometry of the eggs and larvae . 79 RESULTS 80 3.1 The reproduction biology of Asian arowana is very different from that of most teleosts . 80 3.1.1 3.2 Observation of mating - temporal and spatial data 80 Differences in the productivity and survival rate of colour strains and dependence of breeding events on environmental factors . 84 3.2.1 There were significant differences in offspring number per brooder and their survival between several colour strains . 84 3.3 The early development of Asian arowana larvae is a slow process 87 3.3.1 Fertilised eggs 88 3.3.2 Juveniles . 89 3.4 Sexing the brooders with classical and molecular tools 91 3.4.1 External morphometric measurements on sexually mature adults showed significant differences between the two sexes . 91 6|Page 3.4.2 Internal morphometry – The surface of the buccal cavity transformed in mouthbrooding males . 92 3.4.3 3.5 Hormonal measurements from mucus . 95 Identification of the sex of the mouthbrooding parent in the MG variety of Asian arowana 96 3.6 Genotyping data reveals complex relationships between the brooders in the ponds …………………………………………………………………………… .99 3.7 Change in breeding pattern following the loss of a male in pond WH001 105 3.8 No signs of possible inbreeding or incompatibility avoidance were observed …………………………………………………………………………….106 3.9 The Asian arowanas display an unusual phenomenon of egg thievery . 107 3.10 Transient morphological modification of the surface of buccal cavity in Asian arowana during mouthbrooding . 111 3.11 Most colour variants of Asian arowana can be differentiated from the others using molecular analysis . 113 3.11.1 Differentiation of the colour strains using microsatellite-based genotypes . 113 3.11.2 FluoMEP was able to differentiate between two commercially important colour strains 115 7|Page 3.11.3 The microsatellite-based phylogenetic tree of the colour variants of Asian arowana was congruent with geographical reconstruction of prehistoric events in South-East Asia 117 3.11.4 Bayesian clustering analysis allowed for differentiation of most colour strains ……………………………………………………………………… 119 3.12 Pairwise comparison of the FST value indicated that the colour strains are likely to be one species 121 DISCUSSION 123 4.1 Microsatellites allow for accurate parentage analysis and reveal breeding relationships of Asian arowana 123 4.2 The Asian arowana male protects its eggs by transient morphological modification of the surface of its buccal cavity during mouthbrooding 130 4.3 The advantages of being able to sex the adult Asian arowanas 132 4.4 The change in the breeding relationships in a pond after the death of a highly productive male indicates the presence of a complex hierarchical breeding system …………………………………………………………………………….138 4.5 Our data not show inbreeding or incompatibility avoidance, indicating unique mate choice and strategy 139 4.6 Egg thievery: Why Asian arowanas steal each other‟s eggs? 141 8|Page 4.7 Asian arowanas provide another example for a positive correlation between egg size and parental care 144 4.8 Genetic confirmation of a paternal care in the Malaysian golden variety of Asian arowana 145 4.9 Strong positive effect of a mating strategy involving multiple mates, number of reproductive events (broods) and lack of difference between the sexes . 146 4.10 Genetic analysis detects distinct differences between Asian arowana strains ………………………………………………………………………….150 4.11 The divergence of the different colour strains seems to be consistent with the change of the land mass configuration of South East Asia 152 4.12 The different colour strains are likely to be geographically isolated populations and not different species . 154 POSSIBILITIES FOR THE FUTURE 156 5.1 Selective breeding program – production of the first hybrids in preparation for linkage mapping . 156 REFERENCE . 158 SUPPLEMENTARY TABLES . 178 9|Page ABSTRACT The dragonfish or Asian arowana (Scleropages formosus Müller & Schlegel, 1844) is one of the few living, „near-basal‟ teleosts belonging to the family Osteoglossidae. This CITES-protected species possesses a fascinating collection of biologically interesting characters that could be important for the study of basal vertebrate breeding biology, mating behavior and mate preference. We report here, to the best of our knowledge, the most detailed documentation of the breeding behavior, including mate choice and observed mating strategy, of S. formosus. For the analysis of mate choice, we created the “genetic mating map” for three ponds containing the total of more than 60 brooders over a yr period, using 12 highly polymorphic microsatellites. Our data indicated that there were no multiple paternities, only single paternity in the 100 clutches of offspring sampled. The interspawning interval ranged from months to 17 months. Parentage assignment, together with identification of the maternal and paternal genotypes using mitochondrial haplotyping, demonstrated that Asian arowana practiced both polygamy and monogamy. 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Mank JE, Promislow DEL, Avise JC (2005) Phylogenetic perspectives in the evolution of parental care in ray-finned fishes. Evolution 59: 1570-1578. 321. Reynolds JD, Goodwin NB, Freckleton RP (2002) Evolutionary transitions in parental care and live bearing in vertebrates. Philos T Roy Soc B 357: 269-281. 322. Volff JN (2004) Genome evolution and biodiversity in teleost fish. Heredity 94: 280-294. 323. Yue GH, Li Y, Chen F, Cho S, Lim LC, et al. (2002) Comparison of three DNA marker systems for assessing genetic diversity in Asian arowana (Scleropages formosus). Electrophoresis 23: 1025-1032. 324. Chistiakov DA, Tsigenopoulos CS, Lagnel J, Guo YM, Hellemans B, et al. (2008) A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L. Anim Genet 39: 623-634. 325. Sakamoto T, Danzmann RG, Gharbi K, Howard P, Ozaki A, et al. (2000) A microsatellite linkage map of rainbow trout (Oncorhynchus mykiss) characterized by large sex-specific differences in recombination rates. Genetics 155: 1331-1345. 326. Lin QF (2009) Molecular analysis of osteoglossid teleosts. Singapore: National University of Singapore. 327. Taylor JS, Van de Peer Y, Meyer A (2001) Genome duplication, divergent resolution and speciation. Trends Genet 17: 299-301. 328. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, et al. (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2: 225-238. 176 | P a g e SUPPLEMENTARY TABLES Supplementary table 1: Master list of QH ponds and its involvement in the different experimentation. WH001 WH002 WH003 WH004 WH005 WH006 10 11 12 13 14 15 16 17 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Legend : 10 11 12 13 14 15 16 17 Observation of mating Offspring survival between colour strains Breeding frequency following rainfall Embryonic and larval development External morphometric study showing differences between two sexes Internal morphometry of buccal cavity transformation in mouthbrooding males Hormonal measurements from mucus Identification of the sex of the mouthbrooding parent in the MG Genotyping study on relationships in the ponds Change in breeding pattern after loss of a male Inbreeding or incompatibility avoidance study Egg thievery Differentiation of the colour strains using microsatellite-based genotypes Differentiatation of two commercially important colour strains using FluoMEP Microsatellite-based phylogenetic tree of the colour variants Differentiation of colour trains using Bayesian clustering analysis Pairwise comparison of the FST value study 177 | P a g e Supplementary table 2: PAPA results from WH002 family. RESULTS Collected parents file:D:\New Folder\PAPA results\WH002\WH002 adult D0438429210811531104105106109115119.txt Offspring file:D:\New Folder\PAPA results\WH002\ospring p1 D0438429210811531104105106109115119.txt Global level of transmission error:0 Distribution of transmission error:0 Names of loci file: D:\New Folder\PAPA results\WH002\name of loci D0438429210811531104105106109115119.txt Choice of loci: D04 D42 D92 D108 D115 D31 D104 D105 D106 D109 D116 D119 Offspring DZ1 165199 000000 147165 148162 223231 204212 229233 234234 242242 209233 204214 113113 155181 Parents D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 ************************************* Offspring DZ2 199203 000000 165165 164164 223231 204212 229233 234234 242242 209233 214214 107107 189189 Parents D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 ************************************* Offspring DZ3 199199 000000 147165 164164 223223 204212 229233 000000 242242 199209 214214 113113 000000 Parents D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189 D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 Parents D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 178 | P a g e ************************************* Offspring DZ10 199203 000000 147147 162164 223239 212212 229229 234234 242242 209199 206214 107113 155189 Parents C8 199203 000000 147147 148164 239245 212204 229233 234240 242242 209199 206212 113115 181189 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 Parents D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 ************************************* Offspring EA1 199199 000000 147165 148164 223223 204212 233233 232240 242242 209199 000000 107113 000000 Parents C7 199203 000000 147165 148164 223239 212204 233229 232234 242242 209199 204212 107113 189181 D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 ************************************* Offspring EA2 165203 000000 147147 148164 223239 212212 233233 000000 242242 199209 214214 113113 155155 Parents D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189 E4 199165 000000 147165 148148 239245 212212 233229 240234 242242 209199 204214 113115 155189 ************************************* Offspring EA3 165199 000000 147147 148164 223239 212212 229229 234234 242242 209233 214214 107113 189189 Parents D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 Parents D3 199203 000000 147165 162164 223231 204212 233229 232234 242242 199233 204214 107113 155189 179 | P a g e E4 199165 000000 147165 148148 239245 212212 233229 240234 242242 209199 204214 113115 155189 ************************************* Offspring EA4 199199 000000 147165 164164 231239 212212 229229 232240 242242 209199 214214 107113 155181 Parents D1 199203 000000 147165 162164 223231 212212 229233 232240 242242 209199 204214 107113 155189 D2 165199 000000 147165 164148 223239 204212 229233 234240 242242 209209 206214 107113 189181 ************************************* Offspring CB1 199199 000000 147165 164164 223245 212212 229233 234240 242242 209233 212212 113115 189189 Parents D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189 D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189 ************************************* Offspring CB2 165199 000000 147147 162164 231239 212212 229233 232234 242242 209233 204212 113113 189181 Parents D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189 D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189 ************************************* Offspring CB3 165203 000000 147165 162162 231239 212212 233233 234240 242242 233233 204212 113113 189189 Parents D5 199165 000000 147165 162164 223239 204212 229233 232240 242242 209233 204212 113115 155189 D6 199203 000000 147165 162164 231245 212212 229233 234234 242242 233199 204212 107113 181189 ************************************* 180 | P a g e Supplementary table 3: The batch of offsprings were collected from brooder A7 but the genotyped results of 12 microsatellite marker showed that A7 was not one of the genetic parent. The table below showed the results of additional microsatellite markers used, which still showed that the genetic parents were A1 and A2. RESULTS Collected parents file:D:\New Folder\PAPA results\WH001\WH001_parents.txt Offspring file:D:\New Folder\PAPA results\WH001\WH001_offspring_p1.txt Global level of transmission error:0 Distribution of transmission error:0 Names of loci file: D:\New Folder\PAPA results\WH001\name of loci.txt Choice of loci: D04 D38 D42 D106 D108 D109 Offspring DE1 203209 189199 163175 195213 000000 214238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DE2 193209 189199 000000 195213 000000 214214 Parents A1 209215 197199 165175 203213 245253 214238 B8 193203 189197 171177 195207 225241 214214 ************************************* Offspring DE3 203209 197199 163175 195213 000000 208214 Parents 181 | P a g e A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DE4 203215 189197 163165 195203 000000 214238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DE5 203215 197197 163165 000000 000000 208238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************ Offspring DE6 193209 000000 163165 195203 241245 208238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DE7 000000 000000 163163 207213 000000 000000 Parents none ************************************* Offspring DE8 203215 189199 163165 195213 241253 214214 Parents 182 | P a g e A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DE9 193215 189199 000000 195213 225255 214238 Parents none ************************************* Offspring DE10 193215 197197 163175 203207 225253 208238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DF1 203209 189197 171175 195203 000000 208214 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* Offspring DF2 000000 000000 171175 000000 000000 208238 Parents A1 203215 183201 171171 195207 219241 208210 A1 209215 197199 165175 203213 245253 214238 ************************************* Offspring DF3 203209 000000 000000 203207 000000 208214 Parents 183 | P a g e A1 209215 197199 165175 203213 245253 214238 C3 193203 195197 177187 203207 215217 208208 ************************************* Offspring DF4 203209 189197 171175 195203 225245 208238 Parents A1 209215 197199 165175 203213 245253 214238 A2 193203 189197 163171 195207 225241 208214 ************************************* 184 | P a g e [...]... versus the ave k value amongst the brooders, we assume that if the k value of the thief/genetic parents is higher than the ave k, then there is a possibility of kinselection occurring in the pond where kins are helping each other in parental care The grey bar indicates the k value of the thief with the genetic father where the white 18 | P a g e bar refers to the k value of the thief with the genetic mother... work will enhance the understanding of the evolution of the breeding biology of teleost, and provide a genetic glimpse into the biology of ancient teleosts using molecular tools 32 | P a g e Figure 3: The architecture of the Asian arowana s buccal cavity and the tongue bite apparatus (TBA) TBA is thought to be a derived feature of the Osteoglossomorpha used for anchoring the prey to the buccal cavity... pairs of brooders available in the pond and calculates the ratio of the primary hypothesis that they are related (full-sibs or half-sibs) to the null hypothesis that they are unrelated k refers to the kinship coefficient where higher k value indicates higher chances of the pair being kin of each other (relatedness), where k of 1 indicates full siblings while 0 shows no relation We compared the k of the. .. http://www.youtube.com/watch?v=5ltmk6oHXg8 83 Figure 12: The breeding cycle of the Asian arowana in the farm The graph shows the total production in terms of number of batches（dotted line）and number of offspring (solid line) for the three experimental ponds in relation to the rainfall (bar) data from January 2003 to November 2006 87 Figure 13: The development of Asian arowana embryos, larvae and juveniles Panels:... direct observations of the temporal and spatial scope of the arowana breeding activities is almost impossible in the dense pond water There is no documentation of artificial fertilization of this species, probably due to the large eggs and low fecundity and lack of available specimens Such lack of basic biological information of the Asian arowana creates a bottleneck for the study of its phylogeny, behaviour,... represent breeding connections that were determined by microsatellite genotyping Each circle (○) on the line represents one breeding event that occurred between the two brooders, whereas the number beside the circle represents the number of offspring that was collected from the mouthbrooding 16 | P a g e individual The column on the left indicates the date of the collection of the offspring from the buccal... Teeth on the medial edge of entopterygoid; B) Basihyal/basibranchial toothplate 1.3 The Asian arowana has at least six main colour strains One interesting and distinct difference between the Asian arowana and its related species in the family Osteoglossidae is the presence of naturally occurring colour strains in the former Unlike the rest of the family that only have one colour form, the Asian arowana. .. similar to the MG, the only distinct difference is the distribution of the iridiophores on the scales of the fish (compare Fig 7G and 7H), the IG only has “golden” scales up to the fourth rows of scales (from bottom up), whereas the MG has the golden scales “crossing” 34 | P a g e over the dorsal side of the fish, thus, it is also known as the crossback golden (compare Fig 5D and 5C) Figure 4: The colour... presence of large tooth plates on the tongue (basihyal) and basibranchials that bite against the roof of the mouth cavity or the parasphenoid [14] (Fig 3) Asian arowana is the top predator in its natural habitat and thus, probably plays an important role in its ecosystem Asian arowanas possess a fascinating collection of interesting characters that are important for the study of breeding biology, mating... 230 Asian arowana brooders 103 Table 9: Average FST values of the different colour varieties of Asian arowana and two Australian arowana species 122 Table 10: The fecundity of some females from WH001 130 11 | P a g e List of Figures Figure 1: The phylogenetic analysis of the Osteoglossids and other teleosts by using concatenated mitochondrial protein-coding genes The data . Molecular Analysis of the Breeding Biology of the Asian Arowana (Scleropages formosus) by Chang Kuok Weai Alex Thesis submitted in partial fulfillment of the requirements for the. Figure 12: The breeding cycle of the Asian arowana in the farm. The graph shows the total production in terms of number of batches（dotted line）and number of offspring (solid line) for the three. 4.3 The advantages of being able to sex the adult Asian arowanas 132 4.4 The change in the breeding relationships in a pond after the death of a highly productive male indicates the presence of