A genome-centric view of the role of the Acropora kenti microbiome in coral health and resilience.

Microbial diversity has been extensively explored in reef-building corals. However, the functional roles of coral-associated microorganisms remain poorly elucidated. Here, we recover 191 bacterial and 10 archaeal metagenome-assembled genomes (MAGs) from the coral Acropora kenti (formerly A. tenuis) and adjacent seawater, to identify microbial functions and metabolic interactions within the holobiont. We show that 82 MAGs were specific to the A. kenti holobiont, including members of the Pseudomonadota, Bacteroidota, and Desulfobacterota. A. kenti-specific MAGs displayed significant differences in their genomic features and functional potential relative to seawater-specific MAGs, with a higher prevalence of genes involved in host immune system evasion, nitrogen and carbon fixation, and synthesis of five essential B-vitamins. We find a diversity of A. kenti-specific MAGs encode the biosynthesis of essential amino acids, such as tryptophan, histidine, and lysine, which cannot be de novo synthesised by the host or Symbiodiniaceae. Across a water quality gradient spanning 2° of latitude, A. kenti microbial community composition is correlated to increased temperature and dissolved inorganic nitrogen, with corresponding enrichment in molecular chaperones, nitrate reductases, and a heat-shock protein. We reveal mechanisms of A. kenti-microbiome-symbiosis on the Great Barrier Reef, highlighting the interactions underpinning the health of this keystone holobiont.

Validation of key sponge symbiont pathways using genome-centric metatranscriptomics.

The sponge microbiome underpins host function through provision and recycling of essential nutrients in a nutrient poor environment. Genomic data suggest that carbohydrate degradation, carbon fixation, nitrogen metabolism, sulphur metabolism and supplementation of B-vitamins are central microbial functions. However, validation beyond the genomic potential of sponge symbiont pathways is rarely explored. To evaluate metagenomic predictions, we sequenced the metagenomes and metatranscriptomes of three common coral reef sponges: Ircinia ramosa, Ircinia microconulosa and Phyllospongia foliascens. Multiple carbohydrate active enzymes were expressed by Poribacteria, Bacteroidota and Cyanobacteria symbionts, suggesting these lineages have a central role in assimilating dissolved organic matter. Expression of entire pathways for carbon fixation and multiple sulphur compound transformations were observed in all sponges. Gene expression for anaerobic nitrogen metabolism (denitrification and nitrate reduction) were more common than aerobic metabolism (nitrification), where only the I. ramosa microbiome expressed the nitrification pathway. Finally, while expression of the biosynthetic pathways for B-vitamins was common, the expression of additional transporter genes was far more limited. Overall, we highlight consistencies and disparities between metagenomic and metatranscriptomic results when inferring microbial activity, while uncovering new microbial taxa that contribute to the health of their sponge host via nutrient exchange.

Genomic signatures in the coral holobiont reveal host adaptations driven by Holocene climate change and reef specific symbionts.

Genetic signatures caused by demographic and adaptive processes during past climatic shifts can inform predictions of species' responses to anthropogenic climate change. To identify these signatures in Acropora tenuis, a reef-building coral threatened by global warming, we first assembled the genome from long reads and then used shallow whole-genome resequencing of 150 colonies from the central inshore Great Barrier Reef to inform population genomic analyses. We identify population structure in the host that reflects a Pleistocene split, whereas photosymbiont differences between reefs most likely reflect contemporary (Holocene) conditions. Signatures of selection in the host were associated with genes linked to diverse processes including osmotic regulation, skeletal development, and the establishment and maintenance of symbiosis. Our results suggest that adaptation to post-glacial climate change in A. tenuis has involved selection on many genes, while differences in symbiont specificity between reefs appear to be unrelated to host population structure.

Connectivity over a disease risk gradient enables recovery of rainforest frogs.

Chytridiomycosis has been a key driver of global frog declines and extinctions, particularly for high-altitude populations across Australia and the Americas. While recent evidence shows some species are recovering, the extent of such recoveries and the mechanisms underpinning them remain poorly resolved. We surveyed the historical latitudinal and elevational range of four Australian rainforest frogs that disappeared from upland sites between 1989 and 1994 to establish their contemporary distribution and elevational limits, and investigate factors affecting population recovery. Five rainforest streams were surveyed from mountain-base to summit (30 sites in total), with swabs collected from the target species (Litoria dayi, L. nannotis, L. rheocola, and L. serrata) to determine their infection status, and data loggers deployed to measure microclimatic variation across the elevational gradient. Infection probability increased with elevation and canopy cover as it was tightly and inversely correlated with stream-side air temperature. Occupancy patterns suggest varying responses to this disease threat gradient. Two species, L. rheocola and L. serrata, were found over their full historical elevational range (≥1,000 m above sea level [asl]), while L. dayi was not detected above 400 m (formerly known up to 900 m asl) and L. nannotis was not detected above 800 m (formerly known up to 1,200 m asl). Site occupancy probability was negatively related to predicted infection prevalence for L. dayi, L. nannotis, and L. rheocola, but not L. serrata, which appears to now tolerate high fungal burdens. This study highlights the importance of environmental refuges and connectivity across disease risk gradients for the persistence and natural recovery of frogs susceptible to chytridiomycosis. Likewise, in documenting both interspecific variation in recovery rates and intraspecific differences between sites, this study suggests interactions between disease threats and host selection exist that could be manipulated. For example, translocations may be warranted where connectivity is poor or the increase in disease risk is too steep to allow recolonization, combined with assisted selection or use of founders from populations that have already undergone natural selection.

Characterization of a sponge microbiome using an integrative genome-centric approach.

Marine sponges often host diverse and species-specific communities of microorganisms that are critical for host health. Previous functional genomic investigations of the sponge microbiome have focused primarily on specific symbiont lineages, which frequently make up only a small fraction of the overall community. Here, we undertook genome-centric analysis of the symbiont community in the model species Ircinia ramosa and analyzed 259 unique, high-quality metagenome-assembled genomes (MAGs) that comprised 74% of the I. ramosa microbiome. Addition of these MAGs to genome trees containing all publicly available microbial sponge symbionts increased phylogenetic diversity by 32% within the archaea and 41% within the bacteria. Metabolic reconstruction of the MAGs showed extensive redundancy across taxa for pathways involved in carbon fixation, B-vitamin synthesis, taurine metabolism, sulfite oxidation, and most steps of nitrogen metabolism. Through the acquisition of all major taxa present within the I. ramosa microbiome, we were able to analyze the functional potential of a sponge-associated microbial community in unprecedented detail. Critical functions, such as carbon fixation, which had previously only been assigned to a restricted set of sponge-associated organisms, were actually spread across diverse symbiont taxa, whereas other essential pathways, such as ammonia oxidation, were confined to specific keystone taxa.

A genomic view of the reef-building coral Porites lutea and its microbial symbionts.

Corals and the reef ecosystems that they support are in global decline due to increasing anthropogenic pressures such as climate change1. However, effective reef conservation strategies are hampered by a limited mechanistic understanding of coral biology and the functional roles of the diverse microbial communities that underpin coral health2,3. Here, we present an integrated genomic characterization of the coral species Porites lutea and its microbial partners. High-quality genomes were recovered from P. lutea, as well as a metagenome-assembled Cladocopium C15 (the dinoflagellate symbiont) and 52 bacterial and archaeal populations. Comparative genomic analysis revealed that many of the bacterial and archaeal genomes encode motifs that may be involved in maintaining association with the coral host and in supplying fixed carbon, B-vitamins and amino acids to their eukaryotic partners. Furthermore, mechanisms for ammonia, urea, nitrate, dimethylsulfoniopropionate and taurine transformation were identified that interlink members of the holobiont and may be important for nutrient acquisition and retention in oligotrophic waters. Our findings demonstrate the critical and diverse roles that microorganisms play within the coral holobiont and underscore the need to consider all of the components of the holobiont if we are to effectively inform reef conservation strategies.

Increased Numbers of Culturable Inhibitory Bacterial Taxa May Mitigate the Effects of Batrachochytrium dendrobatidis in Australian Wet Tropics Frogs.

Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. For example, antibiotic production by community members can control the pathogen Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd caused declines and disappearances of frogs in the Australian Wet Tropics; the severity of its effects varied among species and sites. Some species have since recolonized despite enzootic Bd within their populations. This variation in history among species and sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in protecting frogs against Bd infection. We collected cutaneous swab samples from three species of frogs at two upland and two lowland sites in the Wet Tropics, and used in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa were associated with frog species, site, or intensity of infection. We present preliminary results showing that the upper limit of Bd infection intensity was negatively correlated with number of inhibitory OTUs present per frog indicating that increased numbers of Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd-inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and statistically significant clustering of individual frogs with similar Bd-inhibitory signatures when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be particularly important to frogs at this site and may have played a role in their ability to recolonize following population declines. Our findings suggest that the use of multi-taxon Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective than single-taxon alternatives.

Reef invertebrate viromics: diversity, host specificity and functional capacity.

Recent metagenomic analyses have revealed a high diversity of viruses in the pelagic ocean and uncovered clear habitat-specific viral distribution patterns. Conversely, similar insights into the composition, host specificity and function of viruses associated with marine organisms have been limited by challenges associated with sampling and computational analysis. Here, we performed targeted viromic analysis of six coral reef invertebrate species and their surrounding seawater to deliver taxonomic and functional profiles of viruses associated with reef organisms. Sponges and corals' host species-specific viral assemblages with low sequence identity to known viral genomes. While core viral genes involved in capsid formation, tail structure and infection mechanisms were observed across all reef samples, auxiliary genes including those involved in herbicide resistance and viral pathogenesis pathways such as host immune suppression were differentially enriched in reef hosts. Utilising a novel OTU based assessment, we also show a prevalence of dsDNA viruses belonging to the Mimiviridae, Caudovirales and Phycodnaviridae in reef environments and further highlight the abundance of ssDNA viruses belonging to the Circoviridae, Parvoviridae, Bidnaviridae and Microviridae in reef invertebrates. These insights into coral reef viruses provide an important framework for future research into how viruses contribute to the health and evolution of reef organisms.

Life history linked to immune investment in developing amphibians.

The broad diversity of amphibian developmental strategies has been shaped, in part, by pathogen pressure, yet trade-offs between the rate of larval development and immune investment remain poorly understood. The expression of antimicrobial peptides (AMPs) in skin secretions is a crucial defense against emerging amphibian pathogens and can also indirectly affect host defense by influencing the composition of skin microbiota. We examined the constitutive or induced expression of AMPs in 17 species at multiple life-history stages. We found that AMP defenses in tadpoles of species with short larval periods (fast pace of life) were reduced in comparison with species that overwinter as tadpoles and grow to a large size. A complete set of defensive peptides emerged soon after metamorphosis. These findings support the hypothesis that species with a slow pace of life invest energy in AMP production to resist potential pathogens encountered during the long larval period, whereas species with a fast pace of life trade this investment in defense for more rapid growth and development.

Endemicity of chytridiomycosis features pathogen overdispersion.

Pathogens can be critical drivers of the abundance and distribution of wild animal populations. The presence of an overdispersed pathogen load distribution between hosts (where few hosts harbour heavy parasite burdens and light infections are common) can have an important stabilizing effect on host-pathogen dynamics where infection intensity determines pathogenicity. This may potentially lead to endemicity of an introduced pathogen rather than extirpation of the host and/or pathogen. Overdispersed pathogen load distributions have rarely been considered in wild animal populations as an important component of the infection dynamics of microparasites such as bacteria, viruses, protozoa and fungi. Here we examined the abundance, distribution and transmission of the model fungal pathogen Batrachochytrium dendrobatidis (Bd, cause of amphibian chytridiomycosis) between wild-caught Litoria rheocola (common mist frogs) to investigate the effects of an overdispersed pathogen load distribution on the host population in the wild. We quantified host survival, infection incidence and recovery probabilities relative to infectious burden, and compared the results of models where pathogen overdispersion either was or was not considered an important feature of host-pathogen dynamics. We found the distribution of Bd load between hosts to be highly overdispersed. We found that host survival was related to infection burden and that accounting for pathogen overdispersion allowed us to better understand infection dynamics and their implications for disease control. In addition, we found that the pattern of host infections and recoveries varied markedly with season whereby (i) infections established more in winter, consistent with temperature-dependent effects on fungal growth, and (ii) recoveries (loss of infection) occurred frequently in the field throughout the year but were less likely in winter. Our results suggest that pathogen overdispersion is an important feature of endemic chytridiomycosis and that intensity of infection determines disease impact. These findings have important implications for our understanding of chytridiomycosis dynamics and the application of management strategies for disease mitigation. We recommend quantifying individual infectious burdens rather than infection state where possible in microparasitic diseases.

Integrated approach to understanding the onset and pathogenesis of black band disease in corals.

Emerging infectious diseases are contributing to global declines in coral reef ecosystems, highlighting a growing need for aetiological knowledge to develop effective management strategies. In this review, we focus on black band disease (BBD), one of the most virulent diseases and the only polymicrobial disease so far known to affect corals. A multipartite microbial consortium dominated by Cyanobacteria, but also including sulfur-cycling bacteria, other bacterial groups and members of the Archaea and Eukarya, forms a sulfide-rich anaerobic mat that migrates across the surface of coral colonies, killing the underlying tissues. The polymicrobial nature of the disease challenges classic aetiological approaches to unravelling disease causation. Here, we synthesize current knowledge on the range of pathogens forming the microbial consortium with recent studies on the transmission, biogeochemistry and environmental drivers of BBD to develop a conceptual model of BBD pathogenesis. The model illustrates how the development of BBD virulence factors is linked to a cascade of microbial community shifts and associated functional roles that progressively develop the microbial consortium from comparatively benign cyanobacterial patches to virulent BBD lesions. This review showcases how an approach that integrates multiple key aspects of the disease provides insights essential to elucidating the aetiology of BBD.

Cool temperatures reduce antifungal activity of symbiotic bacteria of threatened amphibians--implications for disease management and patterns of decline.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a widespread disease of amphibians responsible for population declines and extinctions. Some bacteria from amphibians' skins produce antimicrobial substances active against Bd. Supplementing populations of these cutaneous antifungal bacteria might help manage chytridiomycosis in wild amphibians. However, the activity of protective bacteria may depend upon environmental conditions. Biocontrol of Bd in nature thus requires knowledge of how environmental conditions affect their anti-Bd activity. For example, Bd-driven amphibian declines have often occurred at temperatures below Bd's optimum range. It is possible these declines occurred due to reduced anti-Bd activity of bacterial symbionts at cool temperatures. Better understanding of the effects of temperature on chytridiomycosis development could also improve risk evaluation for amphibian populations yet to encounter Bd. We characterized, at a range of temperatures approximating natural seasonal variation, the anti-Bd activity of bacterial symbionts from the skins of three species of rainforest tree frogs (Litoria nannotis, Litoria rheocola, and Litoria serrata). All three species declined during chytridiomycosis outbreaks in the late 1980s and early 1990s and have subsequently recovered to differing extents. We collected anti-Bd bacterial symbionts from frogs and cultured the bacteria at constant temperatures from 8 °C to 33 °C. Using a spectrophotometric assay, we monitored Bd growth in cell-free supernatants (CFSs) from each temperature treatment. CFSs from 11 of 24 bacteria showed reduced anti-Bd activity in vitro when they were produced at cool temperatures similar to those encountered by the host species during population declines. Reduced anti-Bd activity of metabolites produced at low temperatures may, therefore, partially explain the association between Bd-driven declines and cool temperatures. We show that to avoid inconsistent antifungal activity, bacteria evaluated for use in chytridiomycosis biocontrol should be tested over a range of environmental temperatures spanning those likely to be encountered in the field.

Serum metabolomics indicate altered cellular energy metabolism in children with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a multi-system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis. AIM: We hypothesized that metabolomics using serum from children would differentiate CF from non-CF lung disease subjects and would provide insight into metabolism in CF. METHODS: Serum collected from children with CF (n = 31) and 31 age and gender matched children with other lung diseases was used for metabolomic profiling by gas- and liquid-chromatography. Relative concentration of metabolites was compared between the groups using partial least square discriminant analyses (PLS-DA) and linear modeling. RESULTS: A clear separation of the two groups was seen in PLS-DA. Linear model found that among the 459 detected metabolites 92 differed between CF and non-CF. These included known biochemicals in lipid metabolism, oxidants, and markers consistent with abnormalities in bile acid processing. Bacterial metabolites were identified and differed between the groups indicating intestinal dysbiosis in CF. As a novel finding several pathways were markedly different in CF, which jointly point towards decreased activity in the ß-oxidation of fatty acids. These pathways include low ketone bodies, low medium chain carnitines, elevated di-carboxylic acids and decreased 2-hydroxybutyrate from amino acid metabolism in CF compared to non-CF. CONCLUSION: Serum metabolomics discriminated CF from non-CF and show altered cellular energy metabolism in CF potentially reflecting mitochondrial dysfunction. Future studies are indicated to examine their relation to the underlying CF defect and their use as biomarkers for disease severity or for cystic fibrosis transmembrane regulator (CFTR) function in an era of CFTR modifying drugs.

Variation in thermal performance of a widespread pathogen, the amphibian chytrid fungus Batrachochytrium dendrobatidis.

Rates of growth and reproduction of the pathogens that cause emerging infectious diseases can be affected by local environmental conditions; these conditions can thus influence the strength and nature of disease outbreaks. An understanding of these relationships is important for understanding disease ecology and developing mitigation strategies. Widespread emergence of the fungal disease chytridiomycosis has had devastating effects on amphibian populations. The causative pathogen, Batrachochytriumdendrobatidis (Bd), is sensitive to temperature, but its thermal tolerances are not well studied. We examined the thermal responses of three Bd isolates collected across a latitudinal gradient in eastern Australia. Temperature affected all aspects of Bd growth and reproduction that we measured, in ways that often differed among Bd isolates. Aspects of growth, reproduction, and their relationships to temperature that differed among isolates included upper thermal maxima for growth (26, 27, or 28 °C, depending on the isolate), relationships between zoospore production and temperature, and zoospore activity and temperature. Two isolates decreased zoospore production as temperature increased, whereas the third isolate was less fecund overall, but did not show a strong response to temperature until reaching the upper limit of its thermal tolerance. Our results show differentiation in life-history traits among isolates within Australia, suggesting that the pathogen may exhibit local adaptation. An understanding of how environmental temperatures can limit pathogens by constraining fitness will enhance our ability to assess pathogen dynamics in the field, model pathogen spread, and conduct realistic experiments on host susceptibility and disease transmission.

Mitigating amphibian chytridiomycosis with bioaugmentation: characteristics of effective probiotics and strategies for their selection and use.

Probiotic therapy through bioaugmentation is a feasible disease mitigation strategy based on growing evidence that microbes contribute to host defences of plants and animals. Amphibians are currently threatened by the rapid global spread of the pathogen, Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. Bioaugmentation of locally occurring protective bacteria on amphibians has mitigated this disease effectively in laboratory trials and one recent field trial. Areas still naïve to Bd provide an opportunity for conservationists to proactively implement probiotic strategies to prevent further amphibian declines. In areas where Bd is endemic, bioaugmentation can facilitate repatriation of susceptible amphibians currently maintained in assurance colonies. Here, we synthesise the current research in amphibian microbial ecology and bioaugmentation to identify characteristics of effective probiotics in relation to their interactions with Bd, their host, other resident microbes and the environment. To target at-risk species and amphibian communities, we develop sampling strategies and filtering protocols that result in probiotics that inhibit Bd under ecologically relevant conditions and persist on susceptible amphibians. This filtering tool can be used proactively to guide amphibian disease mitigation and can be extended to other taxa threatened by emerging infectious diseases.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay.

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Immune evasion or avoidance: fungal skin infection linked to reduced defence peptides in Australian green-eyed treefrogs, Litoria serrata.

Many parasites and pathogens suppress host immunity to maintain infection or initiate disease. On the skin of many amphibians, defensive peptides are active against the fungus Batrachochytrium dendrobatidis (Bd), the causative agent of the emerging infectious disease chytridiomycosis. We tested the hypothesis that infection with the fungus may be linked to lower levels of defensive peptides. We sampled both ambient (or constitutive) skin peptides on the ventral surface immediately upon capture, and stored skin peptides induced from granular glands by norepinephrine administration of Australian green-eyed treefrogs, Litoria serrata. Upon capture, uninfected frogs expressed an array of antimicrobial peptides on their ventral surface, whereas infected frogs had reduced skin peptide expression. Expression of ambient skin peptides differed with infection status, and antimicrobial peptides maculatin 1.1 and 2.1 were on average three times lower on infected frogs. However, the repertoire of skin peptides stored in granular glands did not differ with infection status; on average equal quantities were recovered from infected and from uninfected frogs. Our results could have at least two causes: (1) frogs with reduced peptide expression are more likely to become infected; (2) Bd infection interferes with defence peptides by inhibiting release or causing selective degradation of peptides on the skin surface. Immune evasion therefore may contribute to the pathogenesis of chytridiomycosis and a mechanistic understanding of this fungal strategy may lead to improved methods of disease control.