CHARACTERISATION OF DUPLICATED HAEMOGLOBIN GENES IN BIVALVES Mathilde Klein Bachelor of Medical Laboratory Science, QUT Submitted in fulfilment of the requirements for the degree of Master of Applied Science (Research) School of Biomedical Sciences, IHBI Faculty of Health Queensland University of Technology 2017 Keywords Arcoida, Bivalves, Gene duplication, Genomics, Globins, Haemoglobin, Limoida, Molluscs, Transcriptome Keywords i Abstract Haemoglobins (Hbs) are found in virtually all phyla and are some of the most investigated proteins in biomedical sciences. These proteins exhibit an extraordinary diversity of form and function in invertebrate lineages. This provides a unique opportunity to explore the origin and evolution of Hbs yet little is known about their distribution, function and evolution in invertebrate lineages. To explore further the functions and evolution of those Hbs, recent transcriptome data for the Arcid bivalve Anadara trapezia is investigated here. This species shows the presence of duplicated Hb encoding genes suggesting that gene duplication may have been more extensive than previously thought in bivalves. This study tests the hypothesis that these duplicated genes show patterns of tissue specific expression and evidence of neofunctionalisation. This is shown here for at least three Hb encoding genes present in A. trapezia with strong tissue specific expression in haemolymph compared to other tissues. Furthermore, the expression of these genes remains unaffected by prolonged air exposure suggesting that neofunctionalisation may confer an evolutionary advantage to this bivalve. As well as the unique Hbs found in the bivalve order Arcoida, Hbs are also found in three other bivalve orders: Carditoida, Solemyoida and Veneroida. These four orders that possess Hbs provide compelling evidence for the independent evolution of these proteins in multiple bivalve lineages. To expand data on the distribution of Hbs in bivalves, a transcriptome sequence for Ctenoides ales in the order Limoida was generated in this project. Interrogation of the transcriptome shows the presence of at least three globin-like encoding genes including two Hb-like encoding genes providing preliminary evidence for another independent origin of Hb in a bivalve lineage. Overall, this study provides novel insights into the function, evolution and distribution of Hbs in bivalves by investigating two distantly related species. Results of this study are consistent with current theories that Hb diversity in bivalves is a result of repeated rounds of gene duplication providing the raw material for evolution. Investigation of hypoxic resistance also reinforces that greater expression of Hbs in haemolymph confers a physiological advantage suggesting that Hb would evolve more often in some lineages during adaptation to unfavourable environment conditions, particularly Abstract ii hypoxia and prolonged air exposure. The finding of Hb-like encoding genes in another bivalve lineage also supports the evolution of this gene family through independent evolution and gene duplication, and gives insight into the distribution of globin genes in bivalves which is still poorly understood. The investigation of Hb genes in these bivalves also contributes to further understand the role of Hbs and provides potential novel insights for resistance in hypoxic environments, disease control and resistance to pollution in aquaculture. Abstract iii Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii List of Figures ......................................................................................................................... vi List of Tables .......................................................................................................................... xi List of Abbreviations ............................................................................................................. xii Statement of Original Authorship ......................................................................................... xiii Acknowledgements ............................................................................................................... xiv Chapter 1: Introduction ............................................................................................ 1 1.1 Functional and structural diversity within the globin superfamily .................................1 1.1.1 Recently discovered globin genes ....................................................................................... 2 1.1.2 Myoglobins and haemoglobins ........................................................................................... 2 1.2 Evolution of haemoglobins .............................................................................................4 1.2.1 Gene duplication ............................................................................................................... 5 1.2.2 Divergent evolution of duplicated genes .......................................................................... 8 1.2.3 Convergent evolution ........................................................................................................ 9 1.2.4 Invertebrate haemoglobins .............................................................................................. 10 1.3 Bivalve haemoglobins .......................................................................................................12 1.3.1 Haemoglobins in the family Arcidae, Pteriomorphia subclass.......................................... 16 1.4 Aims ..................................................................................................................................19 1.5 Thesis Outline ...............................................................................................................20 Chapter 2: Tissue specificity and neofunctionalisation of haemoglobin genes in Anadara trapezia ....................................................................................................... 22 2.1 Background .......................................................................................................................22 2.2 Material and Methods .......................................................................................................23 2.2.1 Sample acquisition and tissue dissection .......................................................................... 23 2.2.2 RNA extraction and cDNA synthesis ............................................................................... 24 2.2.3 RT–PCR (Real Time PCR) ............................................................................................... 25 2.2.4 Candidate gene validation ................................................................................................. 26 2.2.5 RT-qPCR (Real Time quantitative PCR) for quantification of gene expression .............. 26 2.2.6 RT-qPCR data analysis ..................................................................................................... 27 2.3 Results ...............................................................................................................................28 2.3.1 Haemolymph analysis ....................................................................................................... 28 2.3.2 RT-PCR ............................................................................................................................ 30 2.3.3 Candidate gene validation ................................................................................................. 31 2.3.4 RT-qPCR for quantification of gene expression ............................................................... 31 2.4 Discussion .........................................................................................................................35 2.4.1 Haemolymph characteristics ............................................................................................. 35 2.4.2 Tissue specific expression and neofunctionalisation ......................................................... 36 2.5 Conclusion ........................................................................................................................37 Chapter 3: Functional annotation of the Ctenoides ales transcriptome .............. 39 3.1 Background .......................................................................................................................39 Table of contents iv 3.2 Materials and Methods ......................................................................................................41 3.2.1 Sample collection .............................................................................................................. 41 3.2.2 RNA extraction, library preparation and sequencing ........................................................ 41 3.2.3 Transcriptome assembly and validation ............................................................................ 44 3.2.4 Functional annotation of transcripts and mapping ............................................................ 44 3.2.5 Comparative transcriptomics ............................................................................................ 46 3.2.6 Candidate genes identification .........................................................................................