Characterization of the carbonic anhydrase gene family and other key osmoregulatory genes in Australian freshwater crayfish (genus Cherax) Muhammad Yousuf Ali BSc (Fisheries), MSc (Animal Breeding & Genetics) Principal Supervisor: Dr. Peter J Prentis (EEBS, QUT) Associate Supervisors: Dr. Ana Pavasovic (School of Biomedical Sciences, QUT Professor Peter B Mather (EEBS, QUT) School of Earth, Environmental and Biological Sciences Science and Engineering Faculty Queensland University of Technology Brisbane, Australia A thesis submitted in fulfilment of requirements for the degree of Doctor of Philosophy 2016 Key Words Acid-base, carbonic anhydrase (CA), cytoplasmic carbonic anhydrase (CAC), membrane- associated glycosyl-phosphatidylinositol-linked carbonic anhydrase (CAg), Cherax, Cherax quadricarinatus (Cq); Cherax destructor (Cd), Cherax cainii (Cc); crayfish, expression, genomics, gills, glycosyl-phosphatidylinositol-linked (GPI-linked), H+-ATPase (HAT); marron, Na+/K+-ATPase (NKA); osmoregulation, osmoregulatory genes, pH balance, physiology, quantitative real-time polymerase chain reaction (qRT-PCR), redclaw, transcriptome, V-type H+-ATPase, V-type: vacuolar-type, yabby Page ii Abstract (Each chapter contains its own abstract. It is a general abstract of the study.) Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) are an abundant group of economically important freshwater crayfish and have been developed for aquaculture production in many countries of the world including Australia, Mexico, Ecuador, Uruguay, Argentina and China. They are endemic to Australia, and possess distinct natural distributions that span a diverse range of aquatic environments. Redclaw crayfish occurs naturally in rivers across northern Australia with an abrupt environmental pH gradient of 6.2-8.2; Yabby across southern and central Australia with a natural pH range 6-9; and Marron in south-west Western Australia with a natural pH range 7-8.25. These species have the capacity to acclimate to wide fluctuations in environmental parameters including pH and salinity. The unique physiological characteristics and their economic significance have made these three species an important group for physiological research on freshwater crustaceans. Despite this, genetic information for these crayfish is still largely limited to basic population genetic data on natural variation in wild stocks. To date, so far as we know, no studies have attempted to identify or characterise specific candidate genes that have a role in systemic acid-base balance or salinity regulation in freshwater crayfish. Thus, genomic and molecular studies aimed at characterising the major genes (or variants of the genes) involved in maintaining systemic pH and salinity balance are essential because it will provide a first step toward improving our understanding of their response to environmental changes. Page iii As crayfish are farmed in a wide range of culture conditions, optimisation of water quality parameters, particularly pH and salinity, are crucial for their maximum growth performance. Previous reports have shown that fluctuations in pH and salinity in culture environments can have a negative impact on growth and physiology of crayfish. It is also evident that physiological processes involved in osmoregulation and systemic acid-base balance are undertaken by a range of enzymes encoded by transport-related genes. Having considered this, we have sequenced, identified and characterised most major candidate genes involved in systemic pH balance and osmoregulation including: Carbonic anhydrase, Na+/K+-ATPase (NKA), V-type-H+-ATPase (HAT) and Na+/K+/2Cl- cotransporter (NKCC). We also shed light on the expression and evolution of some major genes that have roles in pH balance or salinity regulation. In our first study (Chapter 2), we deeply sequenced the whole gill transcriptome from C. quadricarinatus and generated 36,128 assembled contigs with an average length of 800bp. Approximately 40% of contigs received significant BLAST hits and 22% were assigned gene ontology terms. Full length sequences were identified and fully annotated for three CA isoforms (cytoplasmic, membrane-associated and β-CA), Na+/K+-ATPase, V-type H+-ATPase, sarcoplasmic Ca+-ATPase, Arginine kinase and Calreticulin. In order to understand potential roles of the candidate genes in response to environmental pH variation, gene expression analysis for CA, V-type H+-ATPase and Arginine kinase were undertaken at 24 h after exposure to different pH conditions (pH 6, 7 and 8). The expression results from one sampling point suggested that cytoplasmic carbonic anhydrase might be involved in pH balance, as the gene was differentially expressed across the three pH treatments, while the other genes were not. Page iv To confirm the results of study 1 and further investigate the role of the genes in systemic pH balance, further in-depth study was carried out that assessed the effects of pH on expression for carbonic anhydrase (cytoplasmic form CAc and membrane-associated form CAg), Na+/K+-ATPase α subunit (NKA-α) and V-type H+-ATPase subunit a (HAT-A) in gills of Redclaw crayfish. Individuals were challenged to pH 6.2, 7.2 (control) and 8.2; and sampling was done at 0h, 3h, 6h, 12h and 24h post-exposure. All genes showed significant differences in expression levels, either across pH treatments or over time. Expression levels for CAc significantly increased at low pH and decreased at high pH conditions 24 h post-transfer. Though expression levels for CAg increased at both low and high pH conditions at 6 h post exposure, but expression remained elevated only at low pH (6.2) at the end of the experiment. Expression of HAT and NKA also showed a significant increase at low pH conditions, 6 h post transfer, and their expression remained significantly elevated throughout the experiment. Overall, these results suggest that CAc, CAg, NKA and HAT may play a role in response to pH change in freshwater crayfish. In the third study (Chapter 4), we undertook deep sequencing of gill transcriptomes from two other important freshwater crayfish, C. cainii and C. destructor, and generated full length sequences of the candidate genes. The sequences from this study and those obtained from our previous study (study 1) were compared to examine patterns of molecular sequence variation; and if the genes were evolving with patterns of nucleotide variation consistent with the action of natural selection. Over 83 and 100 million high quality reads (Q ≥ 30) were assembled into 147,101 and 136,622 contigs for C. cainii and C. destructor, respectively. A total of 24630 and 23623 contigs received significant BLASTx hits and allowed the identification of multiple gill expressed candidate genes associated with pH balance and osmoregulation. The comparative molecular analysis of the candidate genes Page v showed a high level of sequence conservation across the three Cherax sp. All candidate genes showed patterns of molecular variation consistent with neutral evolution. Overall these results demonstrate a high degree of conservation in these candidate genes, despite the ecological and evolutionary divergence of the species studied. In fourth study (included in Chapter 4), we carried out a tissue-specific expression (occurrences) analysis of 80 candidate genes that have potential role in pH balance and ionic/osmotic regulation across different organs (gills, hepatopancreas, heart, kidney, liver, nerve and testes). Approximately 50% of the candidate genes were expressed in all tissue types examined. The largest number of genes was observed in nerve tissue (84%) and gills (78%). The study also revealed that most of the candidate genes that have a role in systemic acid-base balance and osmoregulation (CA, NKA, HAT, NKCC, NBC, NHE) were expressed in all tissue types. This indicates the potential for an important physiological role for these genes outside of osmoregulation in other tissue types. Overall, we generated, for the first time, full length sequences for major systemic acid-base balance and osmoregulatory genes in freshwater crayfish. We also provided the first report on the distribution of the genes across multiple tissue types. Moreover, we present the first in-depth expression study for major transport-related genes in freshwater crayfish. The study provides valuable genomic information for future genomic studies in decapod crustaceans, particularly in crayfish. Taken together this information provides much needed information to help improve crayfish aquaculture in the future. Page vi Table of Contents Table of Contents Key Words ....................................................................................................................................................... ii Abstract .......................................................................................................................................................... iii Table of Contents........................................................................................................................................... vii List of Figures ................................................................................................................................................... x List of Tables .................................................................................................................................................. xii List of Abbreviations ....................................................................................................................................