Background and Significance
Coral reef ecosystems are in jeopardy of collapse due to anthropogenic global change. Tropical coral reefs protect coastlines from storms and erosion, while providing economic prosperity to local communities through food supply and tourism, establishing their value at $375 billion, annually (Mumby and Steneck, 2008). Furthermore, tropical coral reefs support an estimated 25% of known marine species (Thurber et al., 2017). Ultimately, the success of reef is dependent on the coral phenotype, dictated by the holobiont, composed of the microbiome, photosymbionts, and coral genome (Bosch and McFall-Ngai, 2011).
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Symbiodiniaceae is a eukaryotic family of dinoflagellates with 22 known species existing as obligate symbionts, facultative symbionts, or free-living plankton (Stat et al., 2008). Symbiodiniaceae evolved ~165 mya (LaJeunesse et al., 2018), corresponding with the emergence of successful reef-building corals (Simpson et al., 2011). Genetic and physiological evidence has revealed interesting divergences between the dinoflagellate genera. Symbiotic evolution involves adaptations to the metabolism of host and symbiont, possibly exemplified by the high concentration of DNA acquired through horizontal gene transfer (HGT) and frequency of genes acquired from bacteria seen in Symbiodiniaceae (Aranda et al., 2016). This is evidenced by the comparison of gene density and metabolism among the different species of Symbiodiniaceae (Liu et al., 2018; Stat et al., 2008).
During ambient environmental conditions, algal cells donate ~95% of their photosynthetic carbon to the host (Falkowski et al. 1984) in exchange for limiting nutrients and inorganic waste from the coral polyp (Muscatine and Porter, 1977). However, environmental stress (warmer temperatures and/or high radiation) disrupts this symbiotic relationship, as symbionts experience photo-oxidative stress and severely reduce their photosynthetic products (Hoegh-Guldberg, 1999), resulting in the coral animals expelling their symbionts. This is the phenomenon known as coral bleaching. During bleaching conditions, a reduction of photosynthetic pigments and the production of damaging reactive oxygen species (ROS) (Lesser and Farrell, 2004) have been observed and proposed as the mechanism behind coral bleaching. Additionally, it has been hypothesized that warmer temperatures may exacerbate the incline of ROS concentration by inhibiting carbon fixation enzymes, RuBisCO (Jones et al. 1998) and carbonic anhydrase (Leggat et al., 2002). Moreover, it has long been accepted that the specific species of symbiont supplies varying influence on their host’s bleaching tolerance, producing broad inconsistencies in bleaching rate among individual colonies. Overall, the factors controlling the bleaching mechanism in coral animals remains unknown, with a major microbiome player largely unexplored-viruses.
The coral reef virome first attracted attention 15 years ago, despite the incredible overlap between conditions that induce bleaching and induce infection. Generally, this has been due to a lack of standardization among omics methods, leading to variation in requirements for specific steps that enrich for/eliminate viral genomic sequences, such as 29 DNA polymerase (Thurber et al., 2017). It has since been revealed that viruses not only infect Symbiodiniaceae, but are affected by heat stress. This was demonstrated by Levin et al. (2017), who isolated dinoflagellates from bleached (sensitive) and unbleached (resistant) Acropora tenuis and found that transcripts of a positive-sense single-stranded RNA virus (+ssRNAV) had heightened expression levels in the resistant population and low expression levels in the sensitive population. They also discovered that nucleocytoplasmic large double-stranded DNA virus (NCLDVs) transcripts were high in populations, but NCLDV transcripts involved in DNA manipulation were only thermally induced in the sensitive population. Further study of symbiont-virus interactions are needed in order to truly elucidate the bleaching mechanism.
- Characterize the photosymbiont-viral components of the coral holobiont in response to acute and chronic stressors: Studies have observed varying compositions of the coral holobiont from one population to another (Bourne et al., 2016). These shifts are sometimes defined by a reduction of diversity and the species of photosymbiont present. Two years ago, in Hawaii, there was a major bleaching event, during which bleach resistant and bleach sensitive coral animals of the same species, were found right next to each other. It is hypothesized that a) there will be a shift to a thermotolerant photosymbiont species in the resistant coral populations, b) multiple viruses infecting photosymbionts will be present, overwhelmingly lysogenic in resistant populations and lytic in sensitive populations, and c) there will be overlap in the sequences obtained for viruses and photosymbionts, indicating transposition has contributed to photosymbiont evolution.
- Use transcriptomics to identify photosymbiont response to infection: To date, there is little to no data concerning how infection affects photosymbionts, in either ambient conditions or bleaching conditions. The choice of Symbiodinium microadriaticum (thermosensitive obligate symbiont), Cladocopium goreaui (thermotolerant facultative symbiont), and Fugacium kawagutii (free-living plankton)to explore photosymbiont response to infection is motivated by: i) their completely sequenced genomes (Aranda et al., 2016) ii) their variable responses to thermal stressors (Jones et al., 2010), and iii) their variable response to living freely and living as a symbiont. Transcriptomic analysis for infection of all three species living as free cells and symbionts, in bleached and ambient conditions, will provide insight as to what pathways in symbionts are activated/repressed during infection, as well as how viruses contribute to bleaching mechanisms. It is predicted that a) viruses will largely be lysogenic under ambient conditions, with very little algae death observed, and shift to lytic upon bleaching conditions, b) viruses will induce different and/or fewer differentially expressed genes in thermotolerant symbionts compared to thermosensitive or free-living algae, c) viruses will induce different and/or lower differentially expressed genes in plankton compared to algae in a host, and d) the majority of genes affected by infection will be involved in photosynthesis and carbon fixation.
Aim 1: Genomic DNA will be extracted from the sensitive and resistant coral samples using the Qiagen AllPrep DNA/RNA/miRNA Universal Kit, following liquid nitrogen induced cell lysis. PCR will be used to amplify the entire internal transcribed spacer 2 region (ITS2) for symbiont eukaryotic identification. The amplicons will be used to prepare libraries for sequencing. PCR will then be used to attach forward and reverse primers to unique duel-index barcodes, primer pads, and the Illumina forward and reverse flow cell adaptors. The quality of the libraries will be quantified using an Agilent Bioanalyzer and pooled. Pooled libraries will be sequenced on the Illumina HiSeq 2500. Operational taxonomic units (OTUs) will be clustered and GenBank will be used as the reference database, following concatenated alignment, Hierarchical Likelihood Ratio Tests, and Akaike Information Criterion, as outlined by Stat et al. (2008). The generated sequences will also be run through various viral databases to find genes of possible viral origin.
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Viral particles from the samples will be isolated and purified for viral RNA extractions, followed by sequencing on Illumina HiSeq2500, described by Correa et al. (2013). To ensure there is no contamination from coral, symbionts, or bacteria, PCR of 18S and 16S rRNA will be performed. Bioinformatic analyses, outlined by Correa et al. (2013) will be performed using the generated unassembled raw reads. BLASTx and tBLASTx searches of the resulting sequences will be run through MG-RAST database, various viral databases, and Gen Bank. The viral sequences will also be compared to ITS2 sequences from the photosymbionts. Additionally, the frequency of lysogenic and lytic viruses will be estimated, using methods adapted from Nguyen-Kim et al. (2015).
Aim 2: S. microadriaticum and C. will be isolated from multiple colonies of Montipora capitata in Kaneohe Bay, Hawaii. F. kawagutii will be obtained from Bigelow National Center for Marine Algae and Microbiota. All three species will be cultured using f/2 media enriched with antibiotics, as described by Stat et al. (2008). I) Purified viral particles will be added to all three cultures under ambient conditions (27 °C). II) Half of each culture will then be added to a synthetic host (Stat et al., 2008) at ambient conditions. III and IV) The other half of the initial infected cultures and the cultures in the synthetic host will incubated at 32 °C, matching bleaching conditions. Samples will be taken for omics analysis from I, II, III, and IV at set amount of time (TBD) after virus addition or bleaching initiation. Cell counts for each sample will be measured using flow cytometry at various time points (TBD) to track cell death caused by infection. The bleaching rate will be measured in the symbiotic host, as well as photosynthetic, carbon fixation, and carbon export rates in every sample under all conditions.
Total RNA will be collected using Qiagen AllPrep DNA/RNA/miRNA Universal Kit and flash frozen with liquid nitrogen from all three cultures from each condition. RNA quality will be visualized using the Agilent Bioanalyzer and cDNA libraries will be prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina) and sequenced on an Illumina HiSeq 2500 instrument. All RNA-seq reads will be trimmed using CLC Genomic Workbench and aligned, before using HTSeq to annotate the contigs (Anders et al., 2015). To identify significantly differentially expressed (DE) genes of each species and condition for nuclear genes, edgeR package will be utilized (Robinson et al., 2010). Genes will be annotated with the KEGG metabolic pathway tool (Kanehisa et al., 2017) and DE overlap of genes between conditions/species will be determined using Venny 2.027 (Oliveros, 2007).
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