School of BioSciences - Research Publications
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ItemA targeted approach to enrich host-associated bacteria for metagenomic sequencing.(Oxford University Press (OUP), 2024)Multicellular eukaryotic organisms are hosts to communities of bacteria that reside on or inside their tissues. Often the eukaryotic members of the system contribute to high proportions of metagenomic sequencing reads, making it challenging to achieve sufficient sequencing depth to evaluate bacterial ecology. Stony corals are one such complex community; however, separation of bacterial from eukaryotic (primarily coral and algal symbiont) cells has so far not been successful. Using a combination of hybridization chain reaction fluorescence in situ hybridization and fluorescence activated cell sorting (HCR-FISH + FACS), we sorted two populations of bacteria from five genotypes of the coral Acropora loripes, targeting (i) Endozoicomonas spp, and (ii) all other bacteria. NovaSeq sequencing resulted in 67-91 M reads per sample, 55%-90% of which were identified as bacterial. Most reads were taxonomically assigned to the key coral-associated family, Endozoicomonadaceae, with Vibrionaceae also abundant. Endozoicomonadaceae were 5x more abundant in the 'Endozoicomonas' population, highlighting the success of the dual-labelling approach. This method effectively enriched coral samples for bacteria with <1% contamination from host and algal symbionts. The application of this method will allow researchers to decipher the functional potential of coral-associated bacteria. This method can also be adapted to accommodate other host-associated communities.
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ItemDNA from non-viable bacteria biases diversity estimates in the corals Acropora loripes and Pocillopora acuta(BMC, 2023-12-08)BACKGROUND: Nucleic acid-based analytical methods have greatly expanded our understanding of global prokaryotic diversity, yet standard metabarcoding methods provide no information on the most fundamental physiological state of bacteria, viability. Scleractinian corals harbour a complex microbiome in which bacterial symbionts play critical roles in maintaining health and functioning of the holobiont. However, the coral holobiont contains both dead and living bacteria. The former can be the result of corals feeding on bacteria, rapid swings from hyper- to hypoxic conditions in the coral tissue, the presence of antimicrobial compounds in coral mucus, and an abundance of lytic bacteriophages. RESULTS: By combining propidium monoazide (PMA) treatment with high-throughput sequencing on six coral species (Acropora loripes, A. millepora, A. kenti, Platygyra daedalea, Pocillopora acuta, and Porites lutea) we were able to obtain information on bacterial communities with little noise from non-viable microbial DNA. Metabarcoding of the 16S rRNA gene showed significantly higher community evenness (85%) and species diversity (31%) in untreated compared with PMA-treated tissue for A. loripes only. While PMA-treated coral did not differ significantly from untreated samples in terms of observed number of ASVs, > 30% of ASVs were identified in untreated samples only, suggesting that they originated from cell-free/non-viable DNA. Further, the bacterial community structure was significantly different between PMA-treated and untreated samples for A. loripes and P. acuta indicating that DNA from non-viable microbes can bias community composition data in coral species with low bacterial diversity. CONCLUSIONS: Our study is highly relevant to microbiome studies on coral and other host organisms as it delivers a solution to excluding non-viable DNA in a complex community. These results provide novel insights into the dynamic nature of host-associated microbiomes and underline the importance of applying versatile tools in the analysis of metabarcoding or next-generation sequencing data sets.
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ItemExploring microbiome engineering as a strategy for improved thermal tolerance in Exaiptasia diaphana(OXFORD UNIV PRESS, 2022-04)AIMS: Fourteen percent of all living coral, equivalent to more than all the coral on the Great Barrier Reef, has died in the past decade as a result of climate change-driven bleaching. Inspired by the 'oxidative stress theory of coral bleaching', we investigated whether a bacterial consortium designed to scavenge free radicals could integrate into the host microbiome and improve thermal tolerance of the coral model, Exaiptasia diaphana. METHODS AND RESULTS: E. diaphana anemones were inoculated with a consortium of high free radical scavenging (FRS) bacteria, a consortium of congeneric low FRS bacteria, or sterile seawater as a control, then exposed to elevated temperature. Increases in the relative abundance of Labrenzia during the first 2 weeks following the last inoculation provided evidence for temporary inoculum integration into the E. diaphana microbiome. Initial uptake of other consortium members was inconsistent, and these bacteria did not persist either in E. diaphana's microbiome over time. Given their non-integration into the host microbiome, the ability of the FRS consortium to mitigate thermal stress could not be assessed. Importantly, there were no physiological impacts (negative or positive) of the bacterial inoculations on the holobiont. CONCLUSIONS: The introduced bacteria were not maintained in the anemone microbiome over time, thus, their protective effect is unknown. Achieving long-term integration of bacteria into cnidarian microbiomes remains a research priority. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiome engineering strategies to mitigate coral bleaching may assist coral reefs in their persistence until climate change has been curbed. This study provides insights that will inform microbiome manipulation approaches in coral bleaching mitigation research.
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ItemLack of evidence for the oxidative stress theory of bleaching in the sea anemone, Exaiptasia diaphana, under elevated temperature(SPRINGER, 2022-08)Abstract To survive in nutrient-poor waters corals rely on a symbiotic association with intracellular microalgae. However, increased sea temperatures cause algal loss—known as coral bleaching—often followed by coral death. Some of the most compelling evidence in support of the ‘oxidative stress theory of coral bleaching’ comes from studies that exposed corals, cultures of their algal endosymbionts, or the coral modelExaiptasia diaphanato exogenous antioxidants during thermal stress. Here, we replicate these experiments usingE.diaphanawith the addition of the antioxidants ascorbate + catalase, catechin, or mannitol under ambient and elevated temperatures along with an antioxidant-free control. In the absence of exogenous antioxidants,E.diaphanaexposed to elevated temperatures bleached with no change in reactive oxygen species (ROS) levels associated with their microalgal cells. Ascorbate + catalase and mannitol treatments rescued the anemones from bleaching, although microalgal ROS levels increased in these antioxidant treatments under elevated temperature conditions. While bleaching was not associated with changes in net ROS for the intracellular algal symbionts, it is evident from our findings that excess ROS is connected to the bleaching phenotype as exogenous antioxidants were successful in mitigating the effects of thermal stress in cnidarians. This understanding may assist applied research that aims to reduce the impact of climate change on coral reefs.
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ItemDevelopment of a free radical scavenging bacterial consortium to mitigate oxidative stress in cnidarians(WILEY, 2021-09)Corals are colonized by symbiotic microorganisms that profoundly influence the animal's health. One noted symbiont is a single-celled alga (in the dinoflagellate family Symbiodiniaceae), which provides the coral with most of its fixed carbon. Thermal stress increases the production of reactive oxygen species (ROS) by Symbiodiniaceae during photosynthesis. ROS can both damage the algal symbiont's photosynthetic machinery and inhibit its repair, causing a positive feedback loop for the toxic accumulation of ROS. If not scavenged by the antioxidant network, excess ROS may trigger a signaling cascade ending with the coral host and algal symbiont disassociating in a process known as bleaching. We use Exaiptasia diaphana as a model for corals and constructed a consortium comprised of E. diaphana-associated bacteria capable of neutralizing ROS. We identified six strains with high free radical scavenging (FRS) ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae and Micrococcaceae. In parallel, we established a consortium of low FRS isolates consisting of genetically related strains. Bacterial whole genome sequences were used to identify key pathways that are known to influence ROS.
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ItemShort-Term Exposure to Sterile Seawater Reduces Bacterial Community Diversity in the Sea Anemone, Exaiptasia diaphana(FRONTIERS MEDIA SA, 2021-01-21)The global decline of coral reefs heightens the need to understand how corals may persist under changing environmental conditions. Restructuring of the coral-associated bacterial community, either through natural or assisted strategies, has been suggested as a means of adaptation to climate change. A low complexity microbial system would facilitate testing the efficacy of microbial restructuring strategies. We used the model organism for corals,Exaiptasia diaphana, and determined that short-term (3 weeks) exposure to filter-sterilized seawater conditions alone reduced the complexity of the microbiome. Metabarcoding of the V5–V6 region of the bacterial 16S rRNA gene revealed that alpha diversity was approximately halved in anemones reared in filter-sterilized seawater compared to controls reared in unfiltered seawater and that the composition (beta diversity) differed significantly between the two. By reducing the complexity of theE. diaphanamicrobiome, the development of a system for testing assisted strategies such as probiotics, is more feasible.
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ItemDevelopment of a free radical scavenging probiotic to mitigate coral bleaching(Cold Spring Harbor Laboratory, 2020)
ABSTRACT
Corals are colonized by symbiotic microorganisms that exert a profound influence on the animal’s health. One noted symbiont is a single-celled alga (from the family Symbiodiniaceae ), which provides the coral with most of its fixed carbon. During thermal stress, hyperactivity of photosynthesis results in a toxic accumulation of reactive oxygen species (ROS). If not scavenged by the antioxidant network, ROS may trigger a signaling cascade ending with the coral host and algal symbiont disassociating; this process is known as bleaching. Our goal was to construct a probiotic comprised of host-associated bacteria able to neutralize free radicals such as ROS. Using the coral model, the anemone Exaiptasia diaphana , and pure bacterial cultures isolated from the model animal, we identified six strains with high free radical scavenging ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae , and Micrococcaceae . In parallel, we established a “negative” probiotic consisting of genetically related strains with poor free radical scavenging capacities. From their whole genome sequences, we explored genes of interest that may contribute to their potential beneficial roles, which may help facilitate the therapeutic application of a bacterial probiotic. In particular, the occurrence of key pathways that are known to influence ROS in each of the strains has been inferred from the genomes sequences and are reported here.IMPORTANCE
Coral bleaching is tightly linked to the production of reactive oxygen species (ROS), which accumulates to a toxic level in algae-harboring host cells leading to coral-algal dissociation. Interventions targeting ROS accumulation, such as the application of exogenous antioxidants, have shown promise for maintaining the coral-algal partnership. With the feasibility of administering antioxidants directly to corals being low, we aim to develop a probiotic to neutralize toxic ROS during a thermal stress event. This probiotic can be tested with corals or a coral model to assess its efficacy in improving coral resistance to environmental stress. -
ItemExaiptasia diaphana from the great barrier reef: a valuable resource for coral symbiosis research(SPRINGER, 2020-02-06)The sea anemone, Exaiptasia diaphana, previously known as Exaiptasia pallida or Aiptasia pallida, has become increasingly popular as a model for cnidarian-microbiome symbiosis studies due to its relatively rapid growth, ability to reproduce sexually and asexually, and symbiosis with diverse prokaryotes and the same microalgal symbionts (family Symbiodiniaceae) as its coral relatives. Clonal E. diaphana strains from Hawaii, the Atlantic Ocean, and Red Sea are now established for use in research. Here, we introduce Great Barrier Reef (GBR)-sourced E. diaphana strains as additions to the model repertoire. Sequencing of the 18S rRNA gene confirmed the anemones to be E. diaphana while genome-wide single nucleotide polymorphism analysis revealed four distinct genotypes. Based on Exaiptasia-specific inter-simple sequence repeat (ISSR)-derived sequence characterized amplified region (SCAR) marker and gene loci data, these four E. diaphana genotypes are distributed across several divergent phylogenetic clades with no clear phylogeographical pattern. The GBR E. diaphana genotypes comprised three females and one male, which all host Breviolum minutum as their homologous Symbiodiniaceae endosymbiont. When acclimating to an increase in light levels from 12 to 28 μmol photons m−2 s−1, the genotypes exhibited significant variation in maximum quantum yield of Symbiodiniaceae photosystem II and Symbiodiniaceae cell density. The comparatively high levels of physiological and genetic variability among GBR anemone genotypes make these animals representative of global E. diaphana diversity and thus excellent model organisms. The addition of these GBR strains to the worldwide E. diaphana collection will contribute to cnidarian symbiosis research, particularly in relation to the climate resilience of coral reefs.