INVESTIGATING GENETIC, GENE EXPRESSION AND PROTEOMIC CHANGES OVER TEMPERATURE GRADIENTS IN INTERTIDAL NERITA SPECIES Shorash Amin Bachelor of Biomedical Science (1A Honours) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biomedical Sciences Faculty of Health Queensland University of Technology 2018 Investigating genetic, gene expression and proteomic changes over temperature gradients in intertidal Nerita species i Keywords De novo assembly; digital gene expression; genomics; heat shock protein; Ion torrent; transcriptome; Nerita albicilla; Nerita melanotragus; molluscs; proteome; RNAseq; thermal stress; Nerita melanotragus, Illumina. ii Investigating genetic, gene expression and proteomic changes over temperature gradients in intertidal Nerita species Abstract A key area of research in physiological genomics is understanding the gene expression and proteomic responses of specific species to abiotic change in their habitat. In order to investigate these responses, an appropriate group of organisms is required that is distributed across an environmental gradient. One such group of organisms that meet this requirement are class Gastropoda, which are distributed globally in a range of different environments. This highly speciose group are important socially, economically and ecologically. Species from this taxonomic group form a large component of intertidal zone fauna in many areas, globally. The intertidal zone is amongst the harshest of environments on Earth, with constant changes in temperature, pH, sea level and UV exposure. Furthermore, species inhabiting these areas are periodically submerged due to the tidal cycle. The intertidal zone can be further subdivided into the spray, upper, mid and lower intertidal sub zones. Abiotic stresses also vary across these habitats as does the level of submergence. As a consequence, species have adapted to sub zones within the intertidal area resulting in a strong zonation of organisms among sub zones. It is suggested that zonation of species is determined in a large part by their physiological limits, with many intertidal species living near their upper thermal limits. This may suggest that organisms within these areas are under extreme physiological stress. Some intertidal organisms possess physiological defense mechanisms which allow them to cope when faced with external stressors. This is of particular importance for organisms inhabiting the intertidal zone as they are presented with continuous and varying levels of stress. Although some species have adapted to these stressors through standing genetic variation, others respond through gene expression or protein abundance changes to these specific stressors. In order to gain further understanding of how environmental stress affects intertidal grastropod species, studies are required to determine how environmental stress influences the gene repertoire, gene expression patterns and protein abundance in these species. Nerita melanotragus and N. albicilla are widespread intertidal gastropod species, distributed across a number of temporally and spatially fluctuating environmental gradients, including abrupt changes in temperature over a tidal cycle. These species differ in their ecology, as N. Investigating genetic, gene expression and proteomic changes over temperature gradients in intertidal Nerita species iii melanotragus is a mid-littoral species, while N. albicilla is a low-littoral species. Colonisation of different areas in the intertidal zone means that N. melanotragus (mid- littoral) is likely to sustain longer periods of temperature stress than N. albicilla (low- littoral), but N. albicilla is likely to experience more acute temperature stress when exposed to high temperatures. Consequently, these two species present an interesting case to examine differences in the gene repertoire, gene expression and protein abundance patterns of intertidal gastropod species in response to the same temperature stress conditions. Therefore the aim of this project was to investigate and compare the physiological response of two closely related intertidal marine snails from the genus Nerita to temperature stress using a combination of RNAseq and proteomic experiments. To achieve this, I undertook three separate but inter-related experiments. The first experiment was to optimize the assembly of next generation sequencing (NGS) data and determine which assembler produced the best quality assemblies for gastropod species. The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer physiological questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in a non-model gastropod species, an RNAseq dataset was generated for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall, the BLAST and annotation success of the assemblies was not high with only 15-19% of contigs assigned a putative function. The second experiment examined differences in the expressed gene families of N. melanotragus and N. albicilla under normal temperature conditions. Deep transcriptome sequencing was undertaken for both species to determine differences in the expressed gene compliment under natural living conditions. Samples were collected from King’s Beach, Caloundra, Australia and RNA was extracted and iv Investigating genetic, gene expression and proteomic changes over temperature gradients in intertidal Nerita species sequenced on an Illumina Hiseq 2500. A total of 6.2 Gbp and 7.4 Gbp of sequence data was produced for N. melanotragus and N. albicilla respectively. Ortholog analysis of the two Nerita species with Crassostrea gigas and Lottia gigantea revealed a total of 6,457 orthologs in common. The two Nerita species shared a total of 8,501 orthologs, with 3,618 unique orthologs in N. melanotragus and 2,280 in Nerita albicilla. Gene set enrichment of the common genes between the Nerita species revealed two over-represented terms (activation of protein kinase B and positive regulation of guanylate cyclase activity), however, neither is directly related to stress. Overall a larger number of stress transcripts were found to be expressed in N. albicilla under normal conditions when compared to N. melanotragus. The third experiment examined differences in the gene expression and protein abundance patterns in response to the three temperature stress conditions in N. melanotragus and N. albicilla. For the RNAseq component of this experiment, nine individual samples from each of N. melanotragus and N. albicilla were randomly allocated into three treatments (14 °C, 22 °C and 31 °C) with three replicates in each treatment. Five temperature treatments (14 °C, 22 °C, 31 °C, 38 °C and 45 °C) were used for the proteomic experiment with five replicate individuals in each treatment for each species. Treatment animals were euthanized, RNA/protein extracted and each individual was sequenced on an Illumina Hiseq 2500 or run on a TripleTOF 5600+ mass spectrometer (Sciex) coupled with an Ekspert nanoLC 400 system (Eksigent). The two species had highly divergent patterns of gene expression and protein abundance under specific treatment conditions. Few differentially expressed genes/proteins (~22 and 109 respectively, ) were observed in Nerita albicilla, and these were dominated by molecular chaperones. More differentially expressed genes but fewer proteins (~131 and 80 respectively) were observed in N. melanotragus, but no dominant class of genes was observed in either datatset. Little overlap existed between differentially expressed transcripts and differentially abundant proteins in either species. The molecular data generated in these experiments is the largest produced for the genus Nerita. Overall the data generated from these three experiments has enabled us to determine that N. albicilla, the lower intertidal species iniates a thermal stress response at much lower temperatures than N. melanotragus. This supports the idea that low-littoral species undergo thermal stress at lower temperatures than mid- Investigating genetic, gene expression and proteomic changes over temperature gradients in intertidal Nerita species v littoral species. Using a genome wide approach, this study has established that lower intertidal species may be far more susceptible to future climate change due to a more acute temperature stress response. vi Table of Contents KEYWORDS ......................................................................................................................................... II ABSTRACT .......................................................................................................................................... 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