Coral larvae for restoration and research: a large-scale method for rearing Acropora millepora larvae, inducing settlement, and establishing symbiosis

Here we describe an efficient and effective technique for rearing sexually-derived coral propagules from spawning through larval settlement and symbiont uptake with minimal impact on natural coral populations. We sought to maximize larval survival while minimizing expense and daily husbandry maintenance by experimentally determining optimized conditions and protocols for gamete fertilization, larval cultivation, induction of larval settlement by crustose coralline algae, and inoculation of newly settled juveniles with their dinoflagellate symbiont Symbiodinium. Larval rearing densities at or below 0.2 larvae mL -1 were found to maximize larval survival and settlement success in culture tanks while minimizing maintenance effort. Induction of larval settlement via the addition of a ground mixture of diverse crustose coralline algae (CCA) is recommended, given the challenging nature of in situ CCA identification and our finding that non settlement-inducing CCA assemblages do not inhibit larval settlement if suitable assemblages are present. Although order of magnitude differences in infectivity were found between common Great Barrier Reef Symbiodinium clades C and D, no significant differences in Symbiodinium uptake were observed between laboratory-cultured and wild-harvested symbionts in each case. The technique presented here for Acropora millepora can be adapted for research and restoration efforts in a wide range of broadcast spawning coral species

Coral larvae for restoration and research: a large-scale method for rearing Acropora millepora larvae, inducing settlement, and establishing symbiosis

Here we describe an efficient and effective technique for rearing sexually-derived coral propagules from spawning through larval settlement and symbiont uptake with minimal impact on natural coral populations. We sought to maximize larval survival while minimizing expense and daily husbandry maintenance by experimentally determining optimized conditions and protocols for gamete fertilization, larval cultivation, induction of larval settlement by crustose coralline algae, and inoculation of newly settled juveniles with their dinoflagellate symbiont Symbiodinium. Larval rearing densities at or below 0.2 larvae mL -1 were found to maximize larval survival and settlement success in culture tanks while minimizing maintenance effort. Induction of larval settlement via the addition of a ground mixture of diverse crustose coralline algae (CCA) is recommended, given the challenging nature of in situ CCA identification and our finding that non settlement-inducing CCA assemblages do not inhibit larval settlement if suitable assemblages are present. Although order of magnitude differences in infectivity were found between common Great Barrier Reef Symbiodinium clades C and D, no significant differences in Symbiodinium uptake were observed between laboratory-cultured and wild-harvested symbionts in each case. The technique presented here for Acropora millepora can be adapted for research and restoration efforts in a wide range of broadcast spawning coral species

Coral larvae for restoration and research: A large-scale method for rearing Acropora millepora larvae, inducing settlement, and establishing symbiosis

© 2017 Pollock et al. Here we describe an efficient and effective technique for rearing sexually-derived coral propagules from spawning through larval settlement and symbiont uptake with minimal impact on natural coral populations. We sought to maximize larval survival while minimizing expense and daily husbandry maintenance by experimentally determining optimized conditions and protocols for gamete fertilization, larval cultivation, induction of larval settlement by crustose coralline algae, and inoculation of newly settled juveniles with their dinoflagellate symbiont Symbiodinium. Larval rearing densities at or below 0.2 larvae mL-1 were found to maximize larval survival and settlement success in culture tanks while minimizing maintenance effort. Induction of larval settlement via the addition of a ground mixture of diverse crustose coralline algae (CCA) is recommended, given the challenging nature of in situ CCA identification and our finding that non settlement-inducing CCA assemblages do not inhibit larval settlement if suitable assemblages are present. Although order of magnitude differences in infectivity were found between common Great Barrier Reef Symbiodinium clades C and D, no significant differences in Symbiodinium uptake were observed between laboratory-cultured and wild-harvested symbionts in each case. The technique presented here for Acropora millepora can be adapted for research and restoration efforts in a wide range of broadcast spawning coral species

Metatranscriptomics of Host/Coral-associated Bacterial Communities: Enrichment of bacterial mRNA for metatranscriptomics through sequential polyA(+)-mRNA and rRNA subtractions

<p><strong>Summary</strong></p> <p>Metatranscriptomics is a powerful technique to study the ongoing processes in complex communities through the analysis of their gene expression profiles. As such, it is possible to assess, for example, whether there are inter- and intraspecific differences between the functioning of the microbiota of various host organisms, or how the functioning of the microbiome/microbiota may be altered under different conditions. This method has been used mostly to study free-living microbial communities (e.g. in sediment or water) and biofilms, as well as the microbiota of model organisms. However, metatranscriptomics approaches on non-model organisms from the marine environment are still relatively uncommon.</p> <p>Metatranscriptomic analysis of host tissue-associated microbial communities is namely complex as the amount of RNA originating from host cells generally greatly outnumbers the RNA originating from microbes. To avoid that the metatranscriptome sequencing data consists overwhelmingly of reads from the host rather than the microbiota and that very deep sequencing is required to offset this, it is important to remove as much host RNA (both coding polyA(+) messenger RNA and non-coding ribosomal RNA) from samples prior to sequencing library preparation. This is an additional step to the subtraction of ribosomal RNAs from microbes. As commercial kits for rRNA subtraction are not available for non-model organisms and their associated communities of prokaryotic and eukaryotic microbes, it is important to use tailormade sample-specific subtraction protocols.</p> <p>We studied the metatranscriptome of three octocoral species (<em>Corallium rubrum</em>, <em>Eunicella cavolini</em>, <em>Paramuricea clavata</em>)<em> </em>with a well-characterized microbiota composition. This required the development of a protocol to reduce rRNA and polyA(+)-mRNA from these host organisms. The protocol described here is a modification of the protocol used by Daniels <em>et al</em>. (2015), studying the metatranscriptome of a scleractinian coral, and is<em> </em>based on the protocol developed by Stewart <em>et al</em>.<em> </em>(2010). The protocol for the enrichment of bacterial mRNA for metatranscriptomics through rRNA and polyA(+)-mRNA subtraction consists of four stages: (1) the simultaneous extraction of both DNA and RNA from the same sample; (2) the development of biotin-labelled, sample-specific anti-sense rRNA probes targeting eukaryotic and prokaryotic rRNA using <em>in vitro </em>transcription; (3) the depletion of polyA(+)-mRNA from the total RNA sample using oligo(dT)<sub>25</sub>-coated beads to remove eukaryotic mRNA; followed by (4) subtractive hybridization for the removal of rRNA using the biotin-labelled anti-sense RNA probes. The RNA remaining after the polyA(+)-mRNA depletion and rRNA subtraction, will be relatively enriched in prokaryotic mRNA and is ready to use for sequencing library preparation.</p><p>The development of this protocol was supported through KAUST baseline funding to CRV, and through Chanel and the Fondation Paul Hamel to JvdW and CFP.</p&gt

Elevated seawater temperatures have a limited impact on the coral immune response following physical damage

Recurrent disturbances on coral reefs that cause injuries, like predation and storm damage, and elevated seawater temperatures reduce coral fitness and immunocompetence. An effective immune response is essential to prevent disease and enhance colony survival. To evaluate how elevated seawater temperatures affect the coral immune response following injury, fragments of Acropora aspera were exposed to ambient (27–29°C) or elevated (32–33.5°C) seawater temperatures for 8 days and subsequently experimentally injured. Expression patterns for 15 immune genes 24 h post-injury revealed that most genes involved in the Toll-like receptor pathway were unaffected by elevated seawater temperatures. Exceptions to this pattern were cFos and cJun, which were upregulated and likely played a role in repair processes, and TRAF-6 and NFκB, which were downregulated suggesting reduced immune function. Components of the complement system were upregulated (millectin, C3) or downregulated (Bf, Tx60, apextrin) in corals at high temperatures. However, corals that also sustained injury, showed normal Tx60 and apextrin expression, suggesting roles in the wounding response. Overall, basal expression levels of immune genes are sufficient to mount a response to injury in the short term, and the immune response of A. aspera following injury is not significantly affected by minor elevations in seawater temperatures

Antimicrobial and stress responses to increased temperature and bacterial pathogen challenge in the holobiont of a reef-building coral

Global increases in coral disease prevalence have been linked to ocean warming through changes in coral-associated bacterial communities, pathogen virulence and immune system function. However, the interactive effects of temperature and pathogens on the coral holobiont are poorly understood. Here, we assessed three compartments of the holobiont (host, Symbiodinium and bacterial community) of the coral Montipora aequituberculata challenged with the pathogen Vibrio coralliilyticus and the commensal bacterium Oceanospirillales sp. under ambient (27°C) and elevated (29.5 and 32°C) seawater temperatures. Few visual signs of bleaching and disease development were apparent in any of the treatments, but responses were detected in the holobiont compartments. V. coralliilyticus acted synergistically and negatively impacted the photochemical efficiency of Symbiodinium at 32°C, while Oceanospirillales had no significant effect on photosynthetic efficiency. The coral, however, exhibited a minor response to the bacterial challenges, with the response towards V. coralliilyticus being significantly more pronounced, and involving the prophenoloxidase-activating system and multiple immune system-related genes. Elevated seawater temperatures did not induce shifts in the coral-associated bacterial community, but caused significant gene expression modulation in both Symbiodinium and the coral host. While Symbiodinium exhibited an antiviral response and upregulated stress response genes, M. aequituberculata showed regulation of genes involved in stress and innate immune response processes, including immune and cytokine receptor signalling, the complement system, immune cell activation and phagocytosis, as well as molecular chaperones. These observations show that M. aequituberculata is capable of maintaining a stable bacterial community under elevated seawater temperatures and thereby contributes to preventing disease development

Antimicrobial and stress responses to increased temperature and bacterial pathogen challenge in the holobiont of a reef‐building coral

Global increases in coral disease prevalence have been linked to ocean warming through changes in coral-associated bacterial communities, pathogen virulence and immune system function. However, the interactive effects of temperature and pathogens on the coral holobiont are poorly understood. Here, we assessed three compartments of the holobiont (host, Symbiodinium and bacterial community) of the coral Montipora aequituberculata challenged with the pathogen Vibrio coralliilyticus and the commensal bacterium Oceanospirillales sp. under ambient (27°C) and elevated (29.5 and 32°C) seawater temperatures. Few visual signs of bleaching and disease development were apparent in any of the treatments, but responses were detected in the holobiont compartments. V. coralliilyticus acted synergistically and negatively impacted the photochemical efficiency of Symbiodinium at 32°C, while Oceanospirillales had no significant effect on photosynthetic efficiency. The coral, however, exhibited a minor response to the bacterial challenges, with the response towards V. coralliilyticus being significantly more pronounced, and involving the prophenoloxidase-activating system and multiple immune system-related genes. Elevated seawater temperatures did not induce shifts in the coral-associated bacterial community, but caused significant gene expression modulation in both Symbiodinium and the coral host. While Symbiodinium exhibited an antiviral response and upregulated stress response genes, M. aequituberculata showed regulation of genes involved in stress and innate immune response processes, including immune and cytokine receptor signalling, the complement system, immune cell activation and phagocytosis, as well as molecular chaperones. These observations show that M. aequituberculata is capable of maintaining a stable bacterial community under elevated seawater temperatures and thereby contributes to preventing disease development

Coastal urbanization influences human pathogens and microdebris contamination in seafood

Seafood is one of the leading imported products implicated in foodborne outbreaks worldwide. Coastal marine environments are being increasingly subjected to reduced water quality from urbanization and leading to contamination of important fishery species. Given the importance of seafood exchanged as a global protein source, it is imperative to maintain seafood safety worldwide. To illustrate the potential health risks associated with urbanization in a coastal environment, we use next-generation high-throughput amplicon sequencing of the 16S ribosomal RNA gene combined with infrared spectroscopy to characterize and quantify a vast range of potential human bacterial pathogens and microdebris contaminants in seawater, sediment and an important oyster fishery along the Mergui Archipelago in Myanmar. Through the quantification of >1.25 million high-quality bacterial operational taxonomic unit (OTU) reads, we detected 5459 potential human bacterial pathogens belonging to 87 species that are commonly associated with gut microbiota and an indication of terrestrial runoff of human and agricultural waste. Oyster tissues contained 51% of all sequenced bacterial pathogens that are considered to be both detrimental and of emerging concern to human health. Using infrared spectroscopy, we examined a total of 1225 individual microdebris particles, from which we detected 78 different types of contaminant materials. The predominant microdebris contaminants recovered from oyster tissues included polymers (48%), followed by non-native minerals (20%), oils (14%) and milk supplement powders (14%). Emerging technologies provide novel insights into the impacts of coastal development on food security and risks to human and environmental health