Synergistic response to climate stressors in coral is associated with genotypic variation in baseline expression.

As environments are rapidly reshaped due to climate change, phenotypic plasticity plays an important role in the ability of organisms to persist and is considered an especially important acclimatization mechanism for long-lived sessile organisms such as reef-building corals. Often, this ability of a single genotype to display multiple phenotypes depending on the environment is modulated by changes in gene expression, which can vary in response to environmental changes via two mechanisms: baseline expression and expression plasticity. We used transcriptome-wide expression profiling of eleven genotypes of common-gardened Acropora cervicornis to explore genotypic variation in the expression response to thermal and acidification stress, both individually and in combination. We show that the combination of these two stressors elicits a synergistic gene expression response, and that both baseline expression and expression plasticity in response to stress show genotypic variation. Additionally, we demonstrate that frontloading of a large module of coexpressed genes is associated with greater retention of algal symbionts under combined stress. These results illustrate that variation in the gene expression response of individuals to climate change stressors can persist even when individuals have shared environmental histories, affecting their performance under future climate change scenarios.

A targeted approach to enrich host-associated bacteria for metagenomic sequencing.

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.

Horizon scanning the application of probiotics for wildlife.

The provision of probiotics benefits the health of a wide range of organisms, from humans to animals and plants. Probiotics can enhance stress resilience of endangered organisms, many of which are critically threatened by anthropogenic impacts. The use of so-called 'probiotics for wildlife' is a nascent application, and the field needs to reflect on standards for its development, testing, validation, risk assessment, and deployment. Here, we identify the main challenges of this emerging intervention and provide a roadmap to validate the effectiveness of wildlife probiotics. We cover the essential use of inert negative controls in trials and the investigation of the probiotic mechanisms of action. We also suggest alternative microbial therapies that could be tested in parallel with the probiotic application. Our recommendations align approaches used for humans, aquaculture, and plants to the emerging concept and use of probiotics for wildlife.

DNA from non-viable bacteria biases diversity estimates in the corals Acropora loripes and Pocillopora acuta.

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.

Gut microbiota in the short-beaked echidna (Tachyglossus Aculeatus) shows stability across gestation.

Indigenous gut microbial communities (microbiota) play critical roles in health and may be especially important for the mother and fetus during pregnancy. Monotremes, such as the short-beaked echidna, have evolved to lay and incubate an egg, which hatches in their pouch where the young feeds. Since both feces and eggs pass through the cloaca, the fecal microbiota of female echidnas provides an opportunity for vertical transmission of microbes to their offspring. Here, we characterize the gut/fecal microbiome of female short-beaked echidnas and gain a better understanding of the changes that may occur in their microbiome as they go through pregnancy. Fecal samples from four female and five male echidnas were obtained from the Currumbin Wildlife Sanctuary in Queensland and sequenced to evaluate bacterial community structure. We identified 25 core bacteria, most of which were present in male and female samples. Genera such as Fusobacterium, Bacteroides, Escherichia-Shigella, and Lactobacillus were consistently abundant, regardless of sex or gestation stage, accounting for 58.00% and 56.14% of reads in male and female samples, respectively. The echidna microbiome remained stable across the different gestation stages, though there was a significant difference in microbiota composition between male and female echidnas. This study is the first to describe the microbiome composition of short-beaked echidnas across reproductive phases and allows the opportunity for this novel information to be used as a metric of health to aid in the detection of diseases triggered by microbiota dysbiosis.

Effects of Ocean Warming on the Underexplored Members of the Coral Microbiome.

The climate crisis is one of the most significant threats to marine ecosystems. It is leading to severe increases in sea surface temperatures and in the frequency and magnitude of marine heatwaves. These changing conditions are directly impacting coral reef ecosystems, which are among the most biodiverse ecosystems on Earth. Coral-associated symbionts are particularly affected because summer heatwaves cause coral bleaching-the loss of endosymbiotic microalgae (Symbiodiniaceae) from coral tissues, leading to coral starvation and death. Coral-associated Symbiodiniaceae and bacteria have been extensively studied in the context of climate change, especially in terms of community diversity and dynamics. However, data on other microorganisms and their response to climate change are scarce. Here, we review current knowledge on how increasing temperatures affect understudied coral-associated microorganisms such as archaea, fungi, viruses, and protists other than Symbiodiniaceae, as well as microbe-microbe interactions. We show that the coral-microbe symbiosis equilibrium is at risk under current and predicted future climate change and argue that coral reef conservation initiatives should include microbe-focused approaches.

Exploring microbiome engineering as a strategy for improved thermal tolerance in Exaiptasia diaphana.

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.

Heritable variation and lack of tradeoffs suggest adaptive capacity in Acropora cervicornis despite negative synergism under climate change scenarios.

Knowledge of multi-stressor interactions and the potential for tradeoffs among tolerance traits is essential for developing intervention strategies for the conservation and restoration of reef ecosystems in a changing climate. Thermal extremes and acidification are two major co-occurring stresses predicted to limit the recovery of vital Caribbean reef-building corals. Here, we conducted an aquarium-based experiment to quantify the effects of increased water temperatures and pCO2 individually and in concert on 12 genotypes of the endangered branching coral Acropora cervicornis, currently being reared and outplanted for large-scale coral restoration. Quantification of 12 host, symbiont and holobiont traits throughout the two-month-long experiment showed several synergistic negative effects, where the combined stress treatment often caused a greater reduction in physiological function than the individual stressors alone. However, we found significant genetic variation for most traits and positive trait correlations among treatments indicating an apparent lack of tradeoffs, suggesting that adaptive evolution will not be constrained. Our results suggest that it may be possible to incorporate climate-resistant coral genotypes into restoration and selective breeding programmes, potentially accelerating adaptation.

Development of a free radical scavenging bacterial consortium to mitigate oxidative stress in cnidarians.

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.

Microplastic pollution alters forest soil microbiome.

The impact of microplastic pollution on terrestrial biota is an emerging research area, and this is particularly so for soil biota. In this study, we addressed this knowledge gap by examining the impact of aged low-density polyethylene (LDPE) and polyester fibres (i.e. polyethylene terephthalate, PET) on a forest microbiome composition and activity. We also measured the corresponding physicochemical changes in the soil. We observed that bacteria community composition diverged in PET and LDPE treated soils from that of the control by day 42. These changes occurred at 0.2% and 0.4% (w/w) of PET and at 3% LDPE. Additionally, soil respiration was 8-fold higher in soil that received 3% LDPE compared to other treatments and control. There were no clear patterns linking these biological changes to physicochemical changes measured. Taken together, we concluded that microplastics aging in the environment may have evolutionary consequences for forest soil microbiome and there is immediate implication for climate change if the observed increase in soil respiration is reproducible in multiple ecosystems.