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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ItemTissue-associated and vertically transmitted bacterial symbiont in the coral Pocillopora acuta(OXFORD UNIV PRESS, 2024-01-08)Coral microhabitats are colonized by a myriad of microorganisms, including diverse bacteria which are essential for host functioning and survival. However, the location, transmission, and functions of individual bacterial species living inside the coral tissues remain poorly studied. Here, we show that a previously undescribed bacterial symbiont of the coral Pocillopora acuta forms cell-associated microbial aggregates (CAMAs) within the mesenterial filaments. CAMAs were found in both adults and larval offspring, suggesting vertical transmission. In situ laser capture microdissection of CAMAs followed by 16S rRNA gene amplicon sequencing and shotgun metagenomics produced a near complete metagenome-assembled genome. We subsequently cultured the CAMA bacteria from Pocillopora acuta colonies, and sequenced and assembled their genomes. Phylogenetic analyses showed that the CAMA bacteria belong to an undescribed Endozoicomonadaceae genus and species, which we propose to name Candidatus Sororendozoicomonas aggregata gen. nov sp. nov. Metabolic pathway reconstruction from its genome sequence suggests this species can synthesize most amino acids, several B vitamins, and antioxidants, and participate in carbon cycling and prey digestion, which may be beneficial to its coral hosts. This study provides detailed insights into a new member of the widespread Endozoicomonadaceae family, thereby improving our understanding of coral holobiont functioning. Vertically transmitted, tissue-associated bacteria, such as Sororendozoicomonas aggregata may be key candidates for the development of microbiome manipulation approaches with long-term positive effects on the coral host.
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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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ItemNo Preview AvailableFunctional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts(AMER SOC MICROBIOLOGY, 2023-12-21)Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.
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ItemNo Preview AvailableGenomic exploration of coral-associated bacteria: identifying probiotic candidates to increase coral bleaching resilience in Galaxea fascicularis(BMC, 2023-08-19)BACKGROUND: Reef-building corals are acutely threatened by ocean warming, calling for active interventions to reduce coral bleaching and mortality. Corals associate with a wide diversity of bacteria which can influence coral health, but knowledge of specific functions that may be beneficial for corals under thermal stress is scant. Under the oxidative stress theory of coral bleaching, bacteria that scavenge reactive oxygen (ROS) or nitrogen species (RNS) are expected to enhance coral thermal resilience. Further, bacterial carbon export might substitute the carbon supply from algal photosymbionts, enhance thermal resilience and facilitate bleaching recovery. To identify probiotic bacterial candidates, we sequenced the genomes of 82 pure-cultured bacteria that were isolated from the emerging coral model Galaxea fascicularis. RESULTS: Genomic analyses showed bacterial isolates were affiliated with 37 genera. Isolates such as Ruegeria, Muricauda and Roseovarius were found to encode genes for the synthesis of the antioxidants mannitol, glutathione, dimethylsulfide, dimethylsulfoniopropionate, zeaxanthin and/or β-carotene. Genes involved in RNS-scavenging were found in many G. fascicularis-associated bacteria, which represents a novel finding for several genera (including Pseudophaeobacter). Transporters that are suggested to export carbon (semiSWEET) were detected in seven isolates, including Pseudovibrio and Roseibium. Further, a range of bacterial strains, including strains of Roseibium and Roseovarius, revealed genomic features that may enhance colonisation and association of bacteria with the coral host, such as secretion systems and eukaryote-like repeat proteins. CONCLUSIONS: Our work provides an in-depth genomic analysis of the functional potential of G. fascicularis-associated bacteria and identifies novel combinations of traits that may enhance the coral's ability to withstand coral bleaching. Identifying and characterising bacteria that are beneficial for corals is critical for the development of effective probiotics that boost coral climate resilience. Video Abstract.
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ItemNo Preview AvailableAssessing the contribution of bacteria to the heat tolerance of experimentally evolved coral photosymbionts(WILEY, 2023-12)Coral reefs are extremely vulnerable to ocean warming, which triggers coral bleaching-the loss of endosymbiotic microalgae (Symbiodiniaceae) from coral tissues, often leading to death. To enhance coral climate resilience, the symbiont, Cladocopium proliferum was experimentally evolved for >10 years under elevated temperatures resulting in increased heat tolerance. Bacterial 16S rRNA gene metabarcoding showed the composition of intra- and extracellular bacterial communities of heat-evolved strains was significantly different from that of wild-type strains, suggesting bacteria responded to elevated temperatures, and may even play a role in C. proliferum thermal tolerance. To assess whether microbiome transplantation could enhance heat tolerance of the sensitive wild-type C. proliferum, we transplanted bacterial communities from heat-evolved to the wild-type strain and subjected it to acute heat stress. Microbiome transplantation resulted in the incorporation of only 30 low-abundance strains into the microbiome of wild-type cultures, while the relative abundance of 14 pre-existing strains doubled in inoculated versus uninoculated samples. Inoculation with either wild-type or heat-evolved bacterial communities boosted C. proliferum growth, although no difference in heat tolerance was observed between the two inoculation treatments. This study provides evidence that Symbiodiniaceae-associated bacterial communities respond to heat selection and may contribute to coral adaptation to climate change.
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ItemNo Preview AvailableColocalization and potential interactions of Endozoicomonas and chlamydiae in microbial aggregates of the coral Pocillopora acuta(AMER ASSOC ADVANCEMENT SCIENCE, 2023-05-19)Corals are associated with a variety of bacteria, which occur in the surface mucus layer, gastrovascular cavity, skeleton, and tissues. Some tissue-associated bacteria form clusters, termed cell-associated microbial aggregates (CAMAs), which are poorly studied. Here, we provide a comprehensive characterization of CAMAs in the coral Pocillopora acuta. Combining imaging techniques, laser capture microdissection, and amplicon and metagenome sequencing, we show that (i) CAMAs are located in the tentacle tips and may be intracellular; (ii) CAMAs contain Endozoicomonas (Gammaproteobacteria) and Simkania (Chlamydiota) bacteria; (iii) Endozoicomonas may provide vitamins to its host and use secretion systems and/or pili for colonization and aggregation; (iv) Endozoicomonas and Simkania occur in distinct, but adjacent, CAMAs; and (v) Simkania may receive acetate and heme from neighboring Endozoicomonas. Our study provides detailed insight into coral endosymbionts, thereby improving our understanding of coral physiology and health and providing important knowledge for coral reef conservation in the climate change era.
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ItemNo Preview AvailableAdvancing coral microbiome manipulation to build long-term climate resilience(CSIRO PUBLISHING, 2023)Coral reefs house one-third of all marine species and are of high cultural and socioeconomic importance. However, coral reefs are under dire threat from climate change and other anthropogenic stressors. Climate change is causing coral bleaching, the breakdown of the symbiosis between the coral host and its algal symbionts, often resulting in coral mortality and the deterioration of these valuable ecosystems. While it is essential to counteract the root causes of climate change, it remains urgent to develop coral restoration and conservation methods that will buy time for coral reefs. The manipulation of the bacterial microbiome that is associated with corals has been suggested as one intervention to improve coral climate resilience. Early coral microbiome-manipulation studies, which are aimed at enhancing bleaching tolerance, have shown promising results, but the inoculated bacteria did generally not persist within the coral microbiome. Here, we highlight the importance of long-term incorporation of bacterial inocula into the microbiome of target corals, as repeated inoculations will be too costly and not feasible on large reef systems like the Great Barrier Reef. Therefore, coral microbiome-manipulation studies need to prioritise approaches that can provide sustained coral climate resilience.
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ItemNo Preview AvailableComparing the Role of ROS and RNS in the Thermal Stress Response of Two Cnidarian Models, Exaiptasia diaphana and Galaxea fascicularis(MDPI, 2023-05-06)Coral reefs are threatened by climate change, because it causes increasingly frequent and severe summer heatwaves, resulting in mass coral bleaching and mortality. Coral bleaching is believed to be driven by an excess production of reactive oxygen (ROS) and nitrogen species (RNS), yet their relative roles during thermal stress remain understudied. Here, we measured ROS and RNS net production, as well as activities of key enzymes involved in ROS scavenging (superoxide dismutase and catalase) and RNS synthesis (nitric oxide synthase) and linked these metrics to physiological measurements of cnidarian holobiont health during thermal stress. We did this for both an established cnidarian model, the sea anemone Exaiptasia diaphana, and an emerging scleractinian model, the coral Galaxea fascicularis, both from the Great Barrier Reef (GBR). Increased ROS production was observed during thermal stress in both species, but it was more apparent in G. fascicularis, which also showed higher levels of physiological stress. RNS did not change in thermally stressed G. fascicularis and decreased in E. diaphana. Our findings in combination with variable ROS levels in previous studies on GBR-sourced E. diaphana suggest G. fascicularis is a more suitable model to study the cellular mechanisms of coral bleaching.
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ItemFluorescence lifetime imaging microscopy (FLIM): a non-traditional approach to study host-microbial symbioses(CSIRO PUBLISHING, 2022)Corals and their photosynthetic endosymbiotic algae (Symbiodiniaceae) produce a strong autofluorescent signal that spans the visible to near-infrared (NIR) spectrum. However, this broad-spectrum emission hinders the use of fluorescence in situ hybridisation (FISH) for the study of bacterial heterogeneity within the different niches of corals and Symbiodiniaceae, because FISH fluorophores also fluoresce within the visible to NIR spectrum. A solution to this impediment is to use fluorescence lifetime imaging microscopy (FLIM). The ‘lifetime’ property of fluorophores is a feature that enables sample (e.g. coral/Symbiodiniaceae) autofluorescence to be distinguished from FISH-labelled bacteria. In this manner, the location of bacteria around and within Symbiodiniaceae can be quantified along with their identity and spatial distribution. Furthermore, the ‘lifetime’ of the host and associated microbe cellular autofluorescence can be analysed in terms of endogenous fluorophore composition (e.g. metabolic co-factors, aromatic amino acids) and serves as information for symbiotic versus parasitic host-microbe association.