Transcriptomic analysis of thermally stressed Symbiodinium reveals differential expression of stress and metabolism genes

Endosymbioses between dinoflagellate algae (Symbiodinium sp.) and scleractinian coral species form the foundation of coral reef ecosystems. The coral symbiosis is highly susceptible to elevated temperatures, resulting in coral bleaching, where the algal symbiont is released from host cells. This experiment aimed to determine the transcriptional changes in cultured Symbiodinium, to better understand the response of cellular mechanisms under future temperature conditions. Cultures were exposed to elevated temperatures (average 31°C) or control conditions (24.5°C) for a period of 28 days. Whole transcriptome sequencing of Symbiodinium cells on days 4, 19, and 28 were used to identify differentially expressed genes under thermal stress. A large number of genes representing 37.01% of the transcriptome (∼23,654 unique genes, FDR < 0.05) with differential expression were detected at no less than one of the time points. Consistent with previous studies of Symbiodinium gene expression, fold changes across the transcriptome were low, with 92.49% differentially expressed genes at ≤2-fold change. The transcriptional response included differential expression of genes encoding stress response components such as the antioxidant network and molecular chaperones, cellular components such as core photosynthesis machinery, integral light-harvesting protein complexes and enzymes such as fatty acid desaturases. Differential expression of genes encoding glyoxylate cycle enzymes were also found, representing the first report of this in Symbiodinium. As photosynthate transfer from Symbiodinium to coral hosts provides up to 90% of a coral’s daily energy requirements, the implications of altered metabolic processes from exposure to thermal stress found in this study on coral-Symbiodinium associations are unknown and should be considered when assessing the stability of the symbiotic relationship under future climate conditions

Integral light-harvesting complex expression in Symbiodinium within the coral Acropora aspera under thermal stress

Coral reef success is largely dependent on the symbiosis between coral hosts and dinoflagellate symbionts belonging to the genus Symbiodinium. Elevated temperatures can result in the expulsion of Symbiodinium or loss of their photosynthetic pigments and is known as coral bleaching. It has been postulated that the expression of light-harvesting protein complexes (LHCs), which bind chlorophylls (chl) and carotenoids, are important in photobleaching. This study explored the effect a sixteen-day thermal stress (increasing daily from 25–34 °C) on integral LHC (chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC)) gene expression in Symbiodinium within the coral Acropora aspera. Thermal stress leads to a decrease in Symbiodinium photosynthetic efficiency by day eight, while symbiont density was significantly lower on day sixteen. Over this time period, the gene expression of five Symbiodinium acpPC genes was quantified. Three acpPC genes exhibited up-regulated expression when corals were exposed to temperatures above 31.5 °C (acpPCSym_1:1, day sixteen; acpPCSym_15, day twelve; and acpPCSym_18, day ten and day sixteen). In contrast, the expression of acpPCSym_5:1 and acpPCSym_10:1 was unchanged throughout the experiment. Interestingly, the three acpPC genes with increased expression cluster together in a phylogenetic analysis of light-harvesting complexes

Exposure to elevated sea-surface temperatures below the bleaching threshold impairs coral recovery and regeneration following injury

Elevated sea surface temperatures (SSTs) are linked to an increase in the frequency and severity of bleaching events due to temperatures exceeding corals' upper thermal limits. The temperatures at which a breakdown of the coral-Syrnbiodinium endosymbiosis (coral bleaching) occurs are referred to as the upper thermal limits for the coral species. This breakdown of the endosymbiosis results in a reduction of corals' nutritional uptake, growth, and tissue integrity. Periods of elevated sea surface temperature, thermal stress and coral bleaching are also linked to increased disease susceptibility and an increased frequency of storms which cause injury and physical damage to corals. Herein we aimed to determine the capacity of corals to regenerate and recover from injuries (removal of apical tips) sustained during periods of elevated sea surface temperatures which result in coral stress responses, but which do not result in coral bleaching (i.e., sub-bleaching thermal stress events). In this study, exposure of the species Acropora aspera to an elevated SST of 32 degrees C (2 degrees C below the bleaching threshold, 34 degrees C) was found to result in reduced fluorescence of green fluorescent protein (GFP), reduced skeletal calcification and a lack of branch regrowth at the site of injury, compared to corals maintained under ambient SST conditions (26 degrees C). Corals maintained under normal, ambient, sea surface temperatures expressed high GFP fluorescence at the injury site, unclerwent a rapid regeneration of the coral branch apical tip within 12 days of sustaining injury, and showed extensive regrowth of the coral skeleton. Taken together, our results have demonstrated that periods of sustained increased sea surface temperatures, below the corals' bleaching threshold but above long-term summertime averages, impair coral recover/from damage, regardless of the onset or occurrence of coral bleaching

A Comparative Analysis of Microbial DNA Preparation Methods for Use With Massive and Branching Coral Growth Forms

In the last two decades, over 100 studies have investigated the structure of the coral microbiome. However, as yet there are no standardized methods applied to sample preservation and preparation, with different studies using distinct methods. There have also been several comparisons made of microbiome data generated across different studies, which have not addressed the influence of the methodology employed over each of the microbiome datasets. Here, we assess three different preservation methods; salt saturated dimethyl sulfoxide (DMSO) – EDTA, snap freezing with liquid nitrogen and 4% paraformaldehyde solution, and two different preparation methodologies; bead beating and crushing, that have been applied to study the coral microbiome. We compare the resultant bacterial assemblage data for two coral growth forms, the massive coral Goniastrea edwardsi and the branching coral Isopora palifera. We show that microbiome datasets generated from differing preservation and processing protocols are comparable in composition (presence/absence). Significant discrepancies between preservation and homogenization methods are observed in structure (relative abundance), and in the occurrence and dominance of taxa, with rare (low abundance and low occurrence) phylotypes being the most variable fraction of the microbial community. Finally, we provide evidence to support chemical preservation with DMSO as effective as snap freezing samples for generating reliable and robust microbiome datasets. In conclusion, we recommend where possible a standardized preservation and extraction method be taken up by the field to provide the best possible practices for detailed assessments of symbiotic and conserved bacterial associations

Marine heatwave hotspots in coral reef environments: physical drivers, ecophysiological outcomes and impact upon structural complexity

A changing climate is driving increasingly common and prolonged marine heatwaves (MHWs) and these extreme events have now been widely documented to severely impact marine ecosystems globally. However MHWs have rarely recently been considered when examining temperature-induced degradation of coral reef ecosystems. Here we consider extreme, localised thermal anomalies, nested within broader increases in sea surface temperature, which fulfil the definitive criteria for MHWs. These acute and intense events, referred to here as MHW hotspots, are not always well represented in the current framework used to describe coral bleaching, but do have distinct ecological outcomes, including widespread bleaching and rapid mass mortality of putatively thermally tolerant coral species. The physical drivers of these localised hotspots are discussed here, and in doing so we present a comprehensive theoretical framework that links the biological responses of the coral photo-endosymbiotic organism to extreme thermal stress and ecological changes on reefs associated after MHW hotspots. We describe how the rapid onset of high temperatures drives immediate heat-stress induced cellular damage, overwhelming mechanisms that would otherwise mitigate the impact of gradually accumulated thermal stress. The warm environment, and increased light penetration of the coral skeleton due to the loss of coral tissues, coupled with coral tissue decay support rapid microbial growth in the skeletal microenvironment, resulting in the widely unrecognised consequence of rapid decay and degeneration of the coral skeletons. This accelerated degeneration of the coral skeletonson a reef scale hinder the recovery of coral populations and increase the likelihood of phase shifts towards algal dominance. We suggest that MHW hotspots, through driving rapid heat-induced mortality, compromise reefs' structural frameworks to the detriment of long term recovery. We propose that MHW hotspots be considered as a distinct class of thermal stress events in coral reefs, and that the current framework used to describe coral bleaching and mass mortality be expanded to include these. We urge further research into how coral mortality affects bioerosion by coral endoliths

Differential Responses of the Coral Host and Their Algal Symbiont to Thermal Stress

The success of any symbiosis under stress conditions is dependent upon the responses of both partners to that stress. The coral symbiosis is particularly susceptible to small increases of temperature above the long term summer maxima, which leads to the phenomenon known as coral bleaching, where the intracellular dinoflagellate symbionts are expelled. Here we for the first time used quantitative PCR to simultaneously examine the gene expression response of orthologs of the coral Acropora aspera and their dinoflagellate symbiont Symbiodinium. During an experimental bleaching event significant up-regulation of genes involved in stress response (HSP90 and HSP70) and carbon metabolism (glyceraldehyde-3-phosphate dehydrogenase, α-ketoglutarate dehydrogenase, glycogen synthase and glycogen phosphorylase) from the coral host were observed. In contrast in the symbiont, HSP90 expression decreased, while HSP70 levels were increased on only one day, and only the α-ketoglutarate dehydrogenase expression levels were found to increase. In addition the changes seen in expression patterns of the coral host were much larger, up to 10.5 fold, compared to the symbiont response, which in all cases was less than 2-fold. This targeted study of the expression of key metabolic and stress genes demonstrates that the response of the coral and their symbiont vary significantly, also a response in the host transcriptome was observed prior to what has previously been thought to be the temperatures at which thermal stress events occur

Recent progress in Symbiodinium transcriptomics

Dinoflagellate symbionts of the genus Symbiodinium are integral to the success of the coral holobiont (a coral host and the microbial community it harbours), however despite their importance we currently have a very limited knowledge of the genes which they possess and their genomic organisation. Analysis shows that the number of expressed sequence tags (genes that are expressed) available for Symbiodinium (7964) is less than 1/10 of those available for the scleractinian coral host (103,434). This lack of DNA sequence information limits the functional genomic studies that can be undertaken from the symbiont perspective. In addition these sequences are from only three Symbiodinium types (C3, A1, A3) and do not represent the large diversity of clades and subclades seen. Here we summarise our current understanding of the Symbiodinium genomic content with reference to our knowledge of other dinoflagellates. The genetic information of Symbiodinium is encompassed in the nuclear, plastid and mitochondrial genomes. As is the case with other dinoflagellates these three genomes are significantly different from the &quot;general&quot; phototrophic eukaryote. Firstly the nuclear genome of dinoflagellates is extremely large, utilises modified DNA bases not normally found in eukaryotes, and tandem repeat regions seem to contain the most highly expressed genes. Meanwhile the plastid genome, which normally contains between 40 and 250 genes in other eukaryotes, has been reduced to 18 genes encoded in &quot;minicircles.&quot; Finally the dinoflagellate mitochondrial genome only encodes for 2 or 3 proteins instead of the normal 40-50 in other eukaryotes. While we have some knowledge of Symbiodinium genome structure, little is known about its transcriptome. With the advent of inexpensive high throughput sequencing technologies, our understanding of the Symbiodinium genome will rapidly increase and we will begin to be able to look into the responses of these important single celled organisms

Intestinal Microbiome Richness of Coral Reef Damselfishes (Actinopterygii: Pomacentridae)

Fish gastro-intestinal system harbors diverse microbiomes that affect the host's digestion, nutrition, and immunity. Despite the great taxonomic diversity of fish, little is understood about fish microbiome and the factors that determine its structure and composition. Damselfish are important coral reef species that play pivotal roles in determining algae and coral population structures of reefs. Broadly, damselfish belong to either of two trophic guilds based on whether they are planktivorous or algae-farming. In this study, we used 16S rRNA gene sequencing to investigate the intestinal microbiome of 5 planktivorous and 5 algae-farming damselfish species (Pomacentridae) from the Great Barrier Reef. We detected Gammaproteobacteria ASVs belonging to the genus Actinobacillus in 80% of sampled individuals across the 2 trophic guilds, thus, bacteria in this genus can be considered possible core members of pomacentrid microbiomes. Algae-farming damselfish had greater bacterial alpha-diversity, a more diverse core microbiome and shared 35 ± 22 ASVs, whereas planktivorous species shared 7 ± 3 ASVs. Our data also highlight differences in microbiomes associated with both trophic guilds. For instance, algae-farming damselfish were enriched in Pasteurellaceae, whilst planktivorous damselfish in Vibrionaceae. Finally, we show shifts in bacterial community composition along the intestines. ASVs associated with the classes Bacteroidia, Clostridia, and Mollicutes bacteria were predominant in the anterior intestinal regions while Gammaproteobacteria abundance was higher in the stomach. Our results suggest that the richness of the intestinal bacterial communities of damselfish reflects host species diet and trophic guild

Understanding decay in marine calcifiers: Micro-CT analysis of skeletal structures provides insight into the impacts of a changing climate in marine ecosystems

Calcifying organisms and their exoskeletons support some of the most diverse and economically important ecosystems in our oceans. Under a changing climate, we are beginning to see alterations to the structure and properties of these exoskeletons due to ocean acidification, warming and accelerated rates of bioerosion. Our understanding has grown as a result of using micro‐computed tomography (μCT) but its applications in marine biology have not taken full advantage of the technological development in this methodology. We present a significant advancement in the use of this method to studying decalcification in a marine calcifier. We present a detailed workflow on best practice for μCT image processing and analysis of marine calcifiers, designed using coral skeletons subjected to acute, short‐term microbial bioerosion. This includes estimating subresolution microporosity and describing pore space morphological characteristics of macroporosity, in perforate and imperforate exoskeletons. These metrics are compared between control and bieroded samples, and are correlated with skeletal hardness as measured by nanoindentation. Our results suggest that using subresolution microporosity analysis improves the spatiotemporal resolution of μCT data and can detect changes not seen in macroporosity, in both perforate and imperforate skeletons. In imperforate samples, the mean size and relative number of pores in the macroporous portion of the images changed significantly where total macroporosity did not. The increased number of pores and higher microporosity are both directly related to a physical weakening of the calcareous exoskeletons of imperforate corals only. In perforate corals, increased macroporosity was accompanied by an overall widening of pore spaces though this did not correlate with sample hardness. These novel techniques complement traditional approaches and in combination demonstrate the potential for using μCT scanning to sensitively track the process of decalcification from a structural and morphological perspective. Importantly, these approaches do not necessarily rely on ultra‐high resolution (i.e. single micron) scans and so maintain the accessibility of this technology. The continued optimization of these tools for a variety of marine calcifiers will advance our understanding of the effect of climate change on marine biogenic calcified structures.Australian Research Council, Grant/Award Number: DP180103199; International Coral Reef Societ

Rebuilding relationships on coral reefs: Coral bleaching knowledge-sharing to aid adaptation planning for reef users: Bleaching emergence on reefs demonstrates the need to consider reef scale and accessibility when preparing for, and responding to, coral bleaching

Coral bleaching has impacted reefs worldwide and the predictions of near-annual bleaching from over two decades ago have now been realized. While technology currently provides the means to predict large-scale bleaching, predicting reef-scale and within-reef patterns in real-time for all reef users is limited. In 2020, heat stress across the Great Barrier Reef underpinned the region's third bleaching event in 5 years. Here we review the heterogeneous emergence of bleaching across Heron Island reef habitats and discuss the oceanographic drivers that underpinned variable bleaching emergence. We do so as a case study to highlight how reef end-user groups who engage with coral reefs in different ways require targeted guidance for how, and when, to alter their use of coral reefs in response to bleaching events. Our case study of coral bleaching emergence demonstrates how within-reef scale nowcasting of coral bleaching could aid the development of accessible and equitable bleaching response strategies on coral reefs. Also see the video abstract here: https://youtu.be/N9Tgb8N-vN0