Common aquarium antiseptics do not cause long-term shifts in coral microbiota but may impact coral growth rates

IntroductionThough bacterial pathogens and parasites can compromise coral health, coral microbiome research increasingly suggests a beneficial role for bacterial species living in coral tissue and mucus. Recent studies suggest the application of targeted antibiotic treatments, while inhibiting the growth of harmful bacteria, may have unintended and persistent impacts on coral health. Land-based coral nurseries use antiseptic treatments such as Lugol’s solution and KoralMD™ dip to reduce infectious agents as part of restoration best practices. These antiseptic treatments often halt tissue loss, but the short- or long-term effects of these treatments on the coral microbiome is unknown.MethodsWe conducted a controlled tank experiment to assess the effects of these broad-spectrum treatments on coral growth rates and microbial communities when used as a prophylactic measure on healthy corals. Sixty individuals from each of two genotypes of the coral species Acropora palmata and Orbicella faveolata were treated with either Lugol’s solution or KoralMD™. Coral tissue, mucus, and skeleton were sampled pre-treatment, during treatment, and 1 and 2 months after treatment to assess microbiome shifts and recovery. The impact of the two treatments on coral growth was assessed using surface area measurements from 3D imagery.ResultsAlthough we found that A. palmata treated with Lugol’s solution had significantly reduced growth rates compared with untreated controls, impacts of antiseptic treatment were otherwise limited and microbiomes were not significantly different by treatment either immediately after application or 2 months thereafter. DiscussionStudy of the effects of these widely-used interventions may have significant repercussions on management and propagation strategies for corals reared in land-based nurseries. Furthermore, our findings indicate that antiseptic treatments can be applied to mitigate coral health issues without long-term harmful effects or significant microbiome shifts

Impacts of Environmental Change on a Newly-characterized Symbiosis between a Novel Bacterial Parasite, Genus Aquarickettsia, and the Coral Acropora cervicornis

Coral reef ecosystems continue to be significantly altered by disease epizootics, but why some host populations remain resistant while others succumb to outbreaks remains unknown. Research across diverse animal and plant host systems has revealed that disease severity is strongly influenced by host genetics and by environmental influences on both host health and pathogen virulence. Further, microbiome imbalances (referred to as dysbiosis) characterized by the loss of commensal species and an increase in opportunists are often associated with negative host health consequences including increased disease susceptibility. These changes in microbiome ecological state may both be driven by and exacerbate stress from environmental change. Microbiome dysbiosis has repeatedly been found to occur in response to both global and local environmental stressors. The dual stressors of rising ocean temperatures and increased nutrient pollution have influenced both disease severity and prevalence by leading to increases in pathogen virulence and by reducing host immune capacity. Further, it has been suggested that environmental stress may trigger normally-commensal bacterial species to invade new host niches. These species may then outcompete beneficial microbial species, resulting in microbiome dysbiosis. This dissertation examines how two environmental extremes cause dramatic swings in the population of a dominant bacterial species in an essential reef-building coral. A bacterial species from the order Rickettsiales has been found to proliferate under nutrient‐enriched conditions to upwards of 80% of the microbiome of the coral genus Acropora. Though this bacterial species appears to require nutritional supplementation from its host, it does not appear to be pathogenic on its own, as it is tolerated at high abundances under normal environmental regimes. Nutrient enrichment, however, leads to increased abundance of this parasitic species, which is accompanied by reduced coral growth rates. Due to its dependency on nutrition from the host and its algal symbiont, this parasite is lost with thermal stress, causing total collapse of microbial community structure and allowing an influx of opportunistic bacterial species which subsequently lead to disease. We used both marker gene and concatenated gene phylogenetic trees to classify the phylogeny of the dominant parasite of A. cervicornis, and found that this species did not cluster closely with any named taxon in the order Rickettsiales. Sequences from this newly-identified species formed a distinct clade with other unclassified 16S rRNA sequences from diverse freshwater and marine hosts, with sufficient divergence to classify this clade as a new genus, which we named “Candidatus Aquarickettsia.” We assembled the genome sequence of “Ca. A. rohweri” from a sample of A. cervicornis, and found a reduced genome characteristic of an obligate symbiont. We posit, based on genome annotation, that A. rohweri requires host supplementation of most amino acids and possesses the capacity to import host ATP in exchange for ADP. This species also encodes numerous predicted two-component systems that are potentially involved in sensing extracellular conditions, and may play a role in the ability of A. rohweri to respond to environmental cues. To examine the distribution of this putative parasite across Acropora cervicornis genotypes currently used for restoration purposes, we classified microbiomes of 16 genotypes known to exhibit varying disease susceptibility. We found that disease-susceptible coral genotypes were characterized by a near-total dominance of A. rohweri, representing, on average, almost 90% of the detectable members of their microbial communities. In contrast, disease-resistant genotypes harbored A. rohweri populations comprising less than 5% of the microbiome, and exhibited significantly higher community evenness, with no single sequence variant exceeding 11% of the microbial community. We posit that the high abundance of parasitic A. rohweri infection in susceptible genotypes likely imposes a considerable burden on the host immune system, while the comparatively high microbiome diversity of other genotypes contributes to disease resistance by providing the host with a large arsenal of antimicrobial defenses. Further, we found that a thermal stress event homogenized microbiomes across all genotypes due to a significant decrease in populations of A. rohweri in susceptible genotypes. We hypothesized that this loss was related to the expulsion of the algal symbiont Symbiodiniaceae with thermal stress-induced bleaching, as A. rohweri may rely on Symbiodiniaceae for amino acid supplementation. Additionally, heat stress led to a sudden expansion of minor community members in disease-susceptible genotypes, with members of the order Alteromonadales and other potentially opportunistic genera significantly increasing in these samples, while these taxa did not increase significantly in disease-resistant genotypes. A nutrient enrichment experiment was conducted to assess the magnitude of change in abundance of Aquarickettsia in genotypes of Acropora cervicornis that under homeostatic conditions, naturally vary in abundances of Aquarickettsia. We determined that a disease-susceptible genotype of A. cervicornis (with high starting abundance of Aquarickettsia) exhibited significant declines in Simpson’s index of alpha diversity over the course of nutrient enrichment, and increases in the absolute abundance of Aquarickettsia (as assessed by qPCR) across all treatments. Differential abundance analysis, however, indicated that only treatments containing phosphate induced a shift in Aquarickettsia abundance relative to other taxa, suggesting that qPCR results may have been indicative of an increase in absolute abundance of all taxa. Corals of this genotype also exhibited suppressed growth rates in response to treatments containing phosphate. In contrast, a disease-resistant genotype of A. cervicornis (with low starting abundance of Aquarickettsia) were found to be dominated by an unclassified ASV belonging to Campylobacterales. Microbiomes of this coral genotype did not exhibit significant shifts in Simpson’s diversity in response to nutrient enrichment. While Aquarickettsia was found to respond to nutrient enrichment in these genotypes, relative abundances of this parasite remained very low (< 0.5%) at all timepoints. Although overall community structure did not change over the course of the experiment, individual taxa were found to respond to nutrient enrichment, including an increase in Ruegeria and Myxococcales, which have been identified as possible commensal or beneficial species, and a decrease in the dominant taxon from Campylobacterales. As microbial community composition and structure appeared to be stable in response to nutrient stress, but proportions of individual taxa shifted slightly, we hypothesize that these microbiomes represent an intermediate state between robust and flexible classes of coral microbiomes previously proposed in other studies. In this dissertation, I demonstrate that a dominant microbial community member of an essential reef-building coral species is highly responsive to changes in the environment. Shifts in the genus Aquarickettsia in response to thermal stress and nutrient enrichment lead to changes in community diversity and dispersion, which are associated with changes to host health and stress resilience. In contrast, coral genotypes with low abundance of this parasite exhibit more robust microbial community structure that is more resistant to dysbiosis. This body of work underscores the key role this bacterial taxon plays in the health of its host and presents new data to support a causative role of Aquarickettsia in acroporid disease susceptibility

Phylogenetic, genomic, and biogeographic characterization of anovel and ubiquitous marine invertebrate-associated Rickettsiales parasite,Candidatus Aquarickettsia rohweri, gen. nov., sp. nov

Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as “Candidatus Aquarickettsia.” Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of “Ca. Aquarickettsia” are found globally in dozens of invertebrate lineages. The coral-associated “Candidatus A. rohweri” is the first finished genome in this new clade. “Ca. A. rohweri” lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, “Ca. A. rohweri” possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that “Ca. A. rohweri” reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of “Ca. A. rohweri” in its host by providing abundant N-rich metabolites to be scavenged

Wastewater Disposal from Unconventional Oil and Gas Development Degrades Stream Quality at a West Virginia Injection Facility

The development of unconventional oil and gas (UOG) resources has rapidly increased in recent years; however, the environmental impacts and risks are poorly understood. A single well can generate millions of liters of wastewater, representing a mixture of formation brine and injected hydraulic fracturing fluids. One of the most common methods for wastewater disposal is underground injection; we are assessing potential risks of this method through an intensive, interdisciplinary study at an injection disposal facility in West Virginia. In June 2014, waters collected downstream from the site had elevated specific conductance (416 μS/cm) and Na, Cl, Ba, Br, Sr, and Li concentrations, compared to upstream, background waters (conductivity, 74 μS/cm). Elevated TDS, a marker of UOG wastewater, provided an early indication of impacts in the stream. Wastewater inputs are also evident by changes in ⁸⁷Sr/⁸⁶Sr in streamwater adjacent to the disposal facility. Sediments downstream from the facility were enriched in Ra and had high bioavailable Fe­(III) concentrations relative to upstream sediments. Microbial communities in downstream sediments had lower diversity and shifts in composition. Although the hydrologic pathways were not able to be assessed, these data provide evidence demonstrating that activities at the disposal facility are impacting a nearby stream and altering the biogeochemistry of nearby ecosystems

Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov.

Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as "Candidatus Aquarickettsia." Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of "Ca. Aquarickettsia" are found globally in dozens of invertebrate lineages. The coral-associated "Candidatus A. rohweri" is the first finished genome in this new clade. "Ca. A. rohweri" lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, "Ca. A. rohweri" possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that "Ca. A. rohweri" reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of "Ca. A. rohweri" in its host by providing abundant N-rich metabolites to be scavenged

Correction: Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov (The ISME Journal, (2019), 13, 12, (2938-2953), 10.1038/s41396-019-0482-0)

Since publication of the original article the authors noticed that an incorrect file was uploaded instead of the Supplementary Tables. The incorrect file has been replaced with the Supplementary Tables file. Furthermore the abstract has been edited slightly in both HTML and PDF versions for clarity

Correction: Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Tara Pacific 16S rRNA data analysis release

data setThis data is the result of the primary analysis of the 16S rRNA gene sequencing data collected from all islands as part of the Tara Pacific expedition. The analysis was conducted using cutadapt/snakemake/dada2 and usearch. A full README is contained within the data upload.</p

Tara Pacific V9 18S rDNA metabarcoding dataset

data setThis data is the result of the primary analysis of the V9 18S rDNA metabarcoding sequencing data from the Tara Pacific expedition. The analysis was conducted using cutadapt, vsearch, swarm and lulu. Please refer to the readme file for more details