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Coral reef science is a fast-growing field propelled by the need to better understand coral health and resilience to devise strategies to slow reef loss resulting from environmental stresses. Key to coral resilience are the symbiotic interactions established within a complex holobiont, i.e. the multipartite assemblages comprising the coral host organism, endosymbiotic dinoflagellates, bacteria, archaea, fungi, and viruses. Tara Pacific is an ambitious project built upon the experience of previous Tara Oceans expeditions, and leveraging state-of-the-art sequencing technologies and analyses to dissect the biodiversity and biocomplexity of the coral holobiont screened across most archipelagos spread throughout the entire Pacific Ocean. Here we detail the Tara Pacific workflow for multi-omics data generation, from sample handling to nucleotide sequence data generation and deposition. This unique multidimensional framework also includes a large amount of concomitant metadata collected side-by-side that provide new assessments of coral reef biodiversity including micro-biodiversity and shape future investigations of coral reef dynamics and their fate in the Anthropocene., Scientific Data, 10 (1), ISSN:2052-4463

The ability to collect samples suitable for RNA extraction in remote field settings is typically hampered by the need for immediate processing or freezing of samples in liquid nitrogen for preservation of nucleic acid integrity. To this end, a range of RNA preservation buffers are available, compatible with ambient temperature transport and subsequent RNA extraction. Here we tested two commonly available RNA storage buffers, RNAlaterfrom Thermo Fisher Scientific and DNA/RNA Shield from Zymo Research, with subsequent RNA extraction using the RNeasy Mini Kit from Qiagen. We assessed three common coral genera (Acropora,Pocillopora,Porites) reported to yield different levels of RNA quality and quantity, partially due to varying levels of mucus that co-purifies with RNA. All samples were collected in the field and preserved at site (on the boat) with storage at ambient temperature, followed by +4°C and -20°C storage, before subsequent RNA extraction. We found DNA/RNA Shield in combination with the RNeasy Mini Kit to yield non-degraded high quality total RNA. The lytic activity of the DNA/RNA Shield buffer facilitates the separation of the coral tissue from the skeleton. At the same time, minimizing the amount of storage buffer is important to obtain sufficiently concentrated RNA for direct use in downstream applications (e.g., RNA-Seq). In summary, we developed a protocol for coral RNA preservation and RNA extraction suitable for application in remote field settings that yield sufficiently concentrated, high quality RNA for direct use in downstream molecular applications.&nbsp

Summary The ability to collect samples suitable for DNA extraction in remote field settings without the necessity of freezing is a common need for molecular ecologists that has been addressed by the so-called DESS buffer, a solution containing DMSO, EDTA, and saturated NaCl(Dawson, Raskoff, & Jacobs, 1998; Seutin, White, & Boag, 1991). DESS is commonly used as a storage buffer for coral samples(Pinzón et al., 2013; Voolstra et al., 2021; Ziegler et al., 2017) and has also been shown to faithfully preserve DNA for bacterial community analyses(Gray, Pratte, & Kellogg, 2013; Lee, Adams, & Klassen, 2019). What is lesser known is that DESS buffer has been shown to preserve nematode morphology, comparable to formalin-based fixatives(Yoder et al., 2006)and, as such, preserves cell integrity. This makes it suitable for metagenomics applications, which typically rely on differentially lysis of eukaryotic (host) and prokaryotic (microbiome) cells to effectively deplete the eukaryotic host DNA. Protocols for effective depletion of coral host DNA and enrichment of bacterial DNA coverage for application in metagenome shotgun sequencing studies to our knowledge do not exist(Neave, Michell, Apprill, & Voolstra, 2017; Robbins et al., 2019). To address this, we here developed a protocol for coral DNA preservation and DNA extraction suitable for host- (e.g., sWGS), marker gene- (e.g., ITS2, 16S), and metagenomics-based (e.g., microbiome) sequencing approaches. The protocol employs the DNA- and cell morphology-/integrity-preserving properties of DESS buffer, compatible with application in remote field settings and evading the need for cooling/freezing of samples. Sprayed-off tissue from coral fragments is then used for DNA extraction using two distinct extraction kits (the Qiagen DNeasy Blood & Tissue Kit and the Qiagen QIAamp DNA Microbiome Kit) for use in host-, marker gene-, and metagenomics-based sequencing approaches. The protocol was successfully applied to coral samples fromAcroporasp.,Pocilloporasp., andPoritessp.. A detailed study on performance, efficacy, and benchmarking of the here-developed protocol in comparison to a range of other protocols is in preparation and is anticipated to be available shortly. We hope the here-developed protocol applies broadly and will be used widely.&nbsp

Figure S2 Family-level stacked bar plots for bacterial community composition of healthy and insulted skin samples of black-tip reef sharks (Carcharhinus melanopterus) collected at different sites in the Amirante Islands, Seychelles. Each bar represents an individual sample. a) Samples from the skin around the gill area. b) Samples from the skin on the back of the shark. (DOCX 350 kb)

Table S3 Details on collection date, location, health state, and sex of individual blacktip reef sharks collected in 2017 around the Amirante Islands, Seychelles. Shark identifiers printed in bold indicate individuals with observed skin insult. (DOCX 19 kb)

Coral bleaching is now the main driver of reef degradation. The common notion is that most corals bleach and suffer mortality at just 1-2°C above their mean summer maximum temperatures, but some species and genotypes resist or recover better than others. Here we conducted a series of 18-hour short-term heat stress assays side-by-side with a long-term heat stress experiment to assess the ability of both approaches to resolve putative differences in coral thermotolerance and provide a measure of in situ reef temperature thresholds. Using a suite of measures (photosynthetic performance, coral whitening, chlorophyll a, host protein, algal symbiont counts, and algal type association), we assessed bleaching sensitivity/resilience of Stylophora pistillata colonies from the exposed and protected sides of a near-shore coral reef in the central Red Sea. As suggested by the differential mortality during a previous bleaching event, coral colonies from the protected site exhibited less impacted physiological performance in comparison to their exposed site counterparts, and these differences were resolved using both experimental setups. Notably, the long-term experiment provided better resolution with regard to the different measures collected, but at the price of portability, cost, and duration of the experiment. Variability in resilience to ocean warming is critical to reef persistence, yet we lack standardized diagnostics to rapidly assess bleaching severity or resilience across different corals and locations. Using a newly developed portable experimental system termed CBASS (the Coral Bleaching Automated Stress System), we demonstrate that mobile, short-term heat stress assays can resolve fine-scale differences in coral thermotolerance across reef sites. Based on our results, photosynthetic efficiency measured by non-invasive PAM fluorometry provides a rapid and representative proxy of coral resilience. Our system holds the potential to be employed for large-scale determination of in situ bleaching temperature thresholds across reef sites and species. Such data can then be used to identify resistant genotypes (and reefs) for downstream experimental examination and to complement existing remote-sensing approaches.

Figure S1 Representative rarefaction curves for bacterial community sequencing efforts of healthy and insulted skin samples of black-tip reef sharks (Carcharhinus melanopterus) collected at five sites in the Amirante Islands, Seychelles. To facilitate presentation, one representative sample of a healthy and insulted skin sample is provided for each of the five sites, except for the site Benjamin, where no visibly healthy sharks could be sampled. a) Samples from skin around the gills, b) samples from skin on the back. Plateauing curves suggest adequate sequencing effort. (DOCX 704 kb)

Global warming is resulting in unprecedented levels of coral mortality due to mass bleaching events and, more recently, marine heatwaves, where rapid increases in seawater temperature cause mortality within days. Here, we compare the response of a ubiquitous scleractinian coral, Stylophora pistillata, from the northern Red Sea to acute (7 h) and chronic (7–11 d) thermal stress events that include temperature treatments of 27°C (i.e., the local maximum monthly mean), 29.5°C, 32°C, and 34.5°C, and assess recovery of the corals following exposure. Overall, S. pistillata exhibited remarkably similar responses to acute and chronic thermal stress, responding primarily to the temperature treatment rather than duration or heating rate. Additionally, corals displayed an exceptionally high thermal tolerance, maintaining their physiological performance and suffering little to no loss of algal symbionts or chlorophyll a up to 32°C, before the host suffered from rapid tissue necrosis and mortality at 34.5°C. While there was some variability in physiological response metrics, photosynthetic efficiency measurements (i.e., maximum quantum yield Fv/Fm) accurately reflected the overall physiological response patterns, with these measurements used to produce the Fv/Fm effective dose (ED50) metric as a proxy for the thermal tolerance of corals. This approach produced similar ED50 values for the acute and chronic experiments (34.47°C vs. 33.81°C), highlighting the potential for acute thermal assays with measurements of Fv/Fm as a systematic and standardized approach to quantitively compare the upper thermal limits of reef‐building corals using a portable experimental system., Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, National Science Foundation http://dx.doi.org/10.13039/100000001, U.S. Israeli BiNational Science foundation, Universität Konstanz http://dx.doi.org/10.13039/501100010583

Global warming is resulting in unprecedented levels of coral mortality due to mass bleaching events and, more recently, marine heatwaves, where rapid increases in seawater temperature cause mortality within days. Here, we compare the response of a ubiquitous scleractinian coral, Stylophora pistillata, from the northern Red Sea to acute (7 h) and chronic (7–11 d) thermal stress events that include temperature treatments of 27°C (i.e., the local maximum monthly mean), 29.5°C, 32°C, and 34.5°C, and assess recovery of the corals following exposure. Overall, S. pistillata exhibited remarkably similar responses to acute and chronic thermal stress, responding primarily to the temperature treatment rather than duration or heating rate. Additionally, corals displayed an exceptionally high thermal tolerance, maintaining their physiological performance and suffering little to no loss of algal symbionts or chlorophyll a up to 32°C, before the host suffered from rapid tissue necrosis and mortality at 34.5°C. While there was some variability in physiological response metrics, photosynthetic efficiency measurements (i.e., maximum quantum yield Fv/Fm) accurately reflected the overall physiological response patterns, with these measurements used to produce the Fv/Fm effective dose (ED50) metric as a proxy for the thermal tolerance of corals. This approach produced similar ED50 values for the acute and chronic experiments (34.47°C vs. 33.81°C), highlighting the potential for acute thermal assays with measurements of Fv/Fm as a systematic and standardized approach to quantitively compare the upper thermal limits of reef‐building corals using a portable experimental system., Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, National Science Foundation http://dx.doi.org/10.13039/100000001, U.S. Israeli BiNational Science foundation, Universität Konstanz http://dx.doi.org/10.13039/501100010583