Influence of host phylogeny and water physicochemistry on microbial assemblages of the fish skin microbiome.

The skin of fish contains a diverse microbiota that has symbiotic functions with the host, facilitating pathogen exclusion, immune system priming, and nutrient degradation. The composition of fish skin microbiomes varies across species and in response to a variety of stressors, however, there has been no systematic analysis across these studies to evaluate how these factors shape fish skin microbiomes. Here, we examined 1922 fish skin microbiomes from 36 studies that included 98 species and nine rearing conditions to investigate associations between fish skin microbiome, fish species, and water physiochemical factors. Proteobacteria, particularly the class Gammaproteobacteria, were present in all marine and freshwater fish skin microbiomes. Acinetobacter, Aeromonas, Ralstonia, Sphingomonas and Flavobacterium were the most abundant genera within freshwater fish skin microbiomes, and Alteromonas, Photobacterium, Pseudoalteromonas, Psychrobacter and Vibrio were the most abundant in saltwater fish. Our results show that different culturing (rearing) environments have a small but significant effect on the skin bacterial community compositions. Water temperature, pH, dissolved oxygen concentration, and salinity significantly correlated with differences in beta-diversity but not necessarily alpha-diversity. To improve study comparability on fish skin microbiomes, we provide recommendations for approaches to the analyses of sequencing data and improve study reproducibility.

Novel pelagiphage isolate Polarivirus skadi is a polar specialist that dominates SAR11-associated bacteriophage communities at high latitudes.

The SAR11 clade are the most abundant members of surface marine bacterioplankton and a critical component of global biogeochemical cycles. Similarly, pelagiphages that infect SAR11 are ubiquitous and highly abundant in the oceans. Pelagiphages are predicted to shape SAR11 community structures and increase carbon turnover throughout the oceans. Yet, ecological drivers of host and niche specificity of pelagiphage populations are poorly understood. Here we report the global distribution of a novel pelagiphage called "Polarivirus skadi", which is the sole representative of a novel genus. P. skadi was isolated from the Western English Channel using a cold-water ecotype of SAR11 as bait. P. skadi is closely related to the globally dominant pelagiphage HTVC010P. Along with other HTVC010P-type viruses, P. skadi belongs to a distinct viral family within the order Caudovirales, for which we propose the name Ubiqueviridae. Metagenomic read recruitment identified P. skadi as one of the most abundant pelagiphages on Earth. P. skadi is a polar specialist, replacing HTVC010P at high latitudes. Experimental evaluation of P. skadi host range against cold- and warm-water SAR11 ecotypes supported cold-water specialism. Relative abundance of P. skadi in marine metagenomes correlated negatively with temperature, and positively with nutrients, available oxygen, and chlorophyll concentrations. In contrast, relative abundance of HTVC010P correlated negatively with oxygen and positively with salinity, with no significant correlation to temperature. The majority of other pelagiphages were scarce in most marine provinces, with a few representatives constrained to discrete ecological niches. Our results suggest that pelagiphage populations persist within a global viral seed bank, with environmental parameters and host availability selecting for a few ecotypes that dominate ocean viromes.

Correction: Alathari et al. A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia. Viruses 2023, 15, 965.

In the original publication [...].

A Multiplexed, Tiled PCR Method for Rapid Whole-Genome Sequencing of Infectious Spleen and Kidney Necrosis Virus (ISKNV) in Tilapia.

Tilapia farming is one of the most important sectors in aquaculture worldwide and of major importance to global food security. Infectious spleen and kidney necrosis virus (ISKNV) has been identified as an agent of high morbidity and mortality, threatening tilapia aquaculture. ISKNV was detected in Lake Volta, Ghana, in September 2018 and spread rapidly, with mortality rates between 60 and 90% and losses of more than 10 tonnes of fish per day. Understanding the spread and evolution of viral pathogens is important for control strategies. Here, we developed a tiled-PCR sequencing approach for the whole-genome sequencing of ISKNV, using long read sequencing to enable field-based, real-time genomic surveillance. This work represents the first use of tiled-PCR for whole genome recovery of viruses in aquaculture, with the longest genome target (>110 kb dsDNA) to date. Our protocol was applied to field samples collected from the ISKNV outbreaks from four intensive tilapia cage culture systems across Lake Volta, between October 2018 and May 2022. Despite the low mutation rate of dsDNA viruses, 20 single nucleotide polymorphisms accumulated during the sampling period. Droplet digital PCR identified a minimum requirement of template in a sample to recover 50% of an ISKNV genome at 275 femtograms (2410 viral templates per 5 µL sequencing reaction). Overall, tiled-PCR sequencing of ISKNV provides an informative tool to assist in disease control in aquaculture.

Suboxic DOM is bioavailable to surface prokaryotes in a simulated overturn of an oxygen minimum zone, Devil's Hole, Bermuda.

Oxygen minimum zones (OMZs) are expanding due to increased sea surface temperatures, subsequent increased oxygen demand through respiration, reduced oxygen solubility, and thermal stratification driven in part by anthropogenic climate change. Devil's Hole, Bermuda is a model ecosystem to study OMZ microbial biogeochemistry because the formation and subsequent overturn of the suboxic zone occur annually. During thermally driven stratification, suboxic conditions develop, with organic matter and nutrients accumulating at depth. In this study, the bioavailability of the accumulated dissolved organic carbon (DOC) and the microbial community response to reoxygenation of suboxic waters was assessed using a simulated overturn experiment. The surface inoculated prokaryotic community responded to the deep (formerly suboxic) 0.2 µm filtrate with cell densities increasing 2.5-fold over 6 days while removing 5 µmol L-1 of DOC. After 12 days, the surface community began to shift, and DOC quality became less diagenetically altered along with an increase in SAR202, a Chloroflexi that can degrade recalcitrant dissolved organic matter (DOM). Labile DOC production after 12 days coincided with an increase of Nitrosopumilales, a chemoautotrophic ammonia oxidizing archaea (AOA) that converts ammonia to nitrite based on the ammonia monooxygenase (amoA) gene copy number and nutrient data. In comparison, the inoculation of the deep anaerobic prokaryotic community into surface 0.2 µm filtrate demonstrated a die-off of 25.5% of the initial inoculum community followed by a 1.5-fold increase in cell densities over 6 days. Within 2 days, the prokaryotic community shifted from a Chlorobiales dominated assemblage to a surface-like heterotrophic community devoid of Chlorobiales. The DOM quality changed to less diagenetically altered material and coincided with an increase in the ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) gene number followed by an influx of labile DOM. Upon reoxygenation, the deep DOM that accumulated under suboxic conditions is bioavailable to surface prokaryotes that utilize the accumulated DOC initially before switching to a community that can both produce labile DOM via chemoautotrophy and degrade the more recalcitrant DOM.

The divergent genome of Scorpion Group 1 (SG1) intracellular bacterium from the venom glands of Vaejovis smithi (Scorpiones: Vaejovidae).

Scorpions were among the first animals on land around 430 million years ago. Like many arachnids, scorpions have evolved complex venoms used to paralyze their prey and for self-defense. Here we sequenced and analyzed the metagenomic DNA from venom glands from Vaejovis smithi scorpions. A metagenome-assembled genome (MAG) of 624,025 bp was obtained corresponding to the previously reported Scorpion Group 1 (SG1). The SG1 genome from venom glands had a low GC content (25.8%) characteristic of reduced genomes, many hypothetical genes and genes from the reported minimal set of bacterial genes. Phylogenomic reconstructions placed the uncultured SG1 distant from other reported bacteria constituting a taxonomic novelty. By PCR we detected SG1 in all tested venom glands from 30 independent individuals. Microscopically, we observed SG1 inside epithelial cells from the venom glands using FISH and its presence in scorpion embryos suggested that SG1 is transferred from mother to offspring.

Linkages Among Dissolved Organic Matter Export, Dissolved Metabolites, and Associated Microbial Community Structure Response in the Northwestern Sargasso Sea on a Seasonal Scale.

Deep convective mixing of dissolved and suspended organic matter from the surface to depth can represent an important export pathway of the biological carbon pump. The seasonally oligotrophic Sargasso Sea experiences annual winter convective mixing to as deep as 300 m, providing a unique model system to examine dissolved organic matter (DOM) export and its subsequent compositional transformation by microbial oxidation. We analyzed biogeochemical and microbial parameters collected from the northwestern Sargasso Sea, including bulk dissolved organic carbon (DOC), total dissolved amino acids (TDAA), dissolved metabolites, bacterial abundance and production, and bacterial community structure, to assess the fate and compositional transformation of DOM by microbes on a seasonal time-scale in 2016-2017. DOM dynamics at the Bermuda Atlantic Time-series Study site followed a general annual trend of DOC accumulation in the surface during stratified periods followed by downward flux during winter convective mixing. Changes in the amino acid concentrations and compositions provide useful indices of diagenetic alteration of DOM. TDAA concentrations and degradation indices increased in the mesopelagic zone during mixing, indicating the export of a relatively less diagenetically altered (i.e., more labile) DOM. During periods of deep mixing, a unique subset of dissolved metabolites, such as amino acids, vitamins, and benzoic acids, was produced or lost. DOM export and compositional change were accompanied by mesopelagic bacterial growth and response of specific bacterial lineages in the SAR11, SAR202, and SAR86 clades, Acidimicrobiales, and Flavobacteria, during and shortly following deep mixing. Complementary DOM biogeochemistry and microbial measurements revealed seasonal changes in DOM composition and diagenetic state, highlighting microbial alteration of the quantity and quality of DOM in the ocean.

A Novel and Ubiquitous Marine Methylophage Provides Insights into Viral-Host Coevolution and Possible Host-Range Expansion in Streamlined Marine Heterotrophic Bacteria.

The methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known and have highly streamlined genomes. OM43 represents an important microbial link between marine primary production and remineralization of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here, we characterize a novel myophage infecting OM43 called Melnitz. Melnitz was isolated independently from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range. IMPORTANCE Isolation and cultivation of viruses are the foundations on which the mechanistic understanding of virus-host interactions and parameterization of bioinformatic tools for viral ecology are based. This study isolated and characterized the first myophage known to infect the OM43 clade, expanding our knowledge of this understudied group of microbes. The nearly identical genomes of four strains of Melnitz isolated from different marine provinces and the global abundance estimations from metagenomic data suggest that this viral population is globally ubiquitous. Genome analysis revealed several unusual features in Melnitz and related genomes recovered from viromes, such as a curli operon and virally encoded tmRNA controlled by a glutamine riboswitch, neither of which are found in the host. Further phylogenetic analysis of shared genes indicates that this group of viruses infecting the gammaproteobacterial OM43 shares a recent common ancestor with viruses infecting the abundant alphaproteobacterial SAR11 clade. Host ranges are affected by compatible cell surface receptors, successful circumvention of superinfection exclusion systems, and the presence of required accessory proteins, which typically limits phages to singular narrow groups of closely related bacterial hosts. This study provides intriguing evidence that for streamlined heterotrophic bacteria, virus-host transitioning may not be necessarily restricted to phylogenetically related hosts but is a function of shared physical and biochemical properties of the cell.

Influence of short and long term processes on SAR11 communities in open ocean and coastal systems.

SAR11 bacteria dominate the surface ocean and are major players in converting fixed carbon back to atmospheric carbon dioxide. The SAR11 clade is comprised of niche-specialized ecotypes that display distinctive spatiotemporal transitions. We analyzed SAR11 ecotype seasonality in two long-term 16S rRNA amplicon time series representing different North Atlantic regimes: the Sargasso Sea (subtropical ocean-gyre; BATS) and the temperate coastal Western English Channel (WEC). Using phylogenetically resolved amplicon sequence variants (ASVs), we evaluated seasonal environmental constraints on SAR11 ecotype periodicity. Despite large differences in temperature and nutrient availability between the two sites, at both SAR11 succession was defined by summer and winter clusters of ASVs. The summer cluster was dominated by ecotype Ia.3 in both sites. Winter clusters were dominated by ecotypes Ib and IIa.A at BATS and Ia.1 and IIa.B at WEC. A 2-year weekly analysis within the WEC time series showed that the response of SAR11 communities to short-term environmental fluctuations was variable. In 2016, community shifts were abrupt and synchronized to environmental shifts. However, in 2015, changes were gradual and decoupled from environmental fluctuations, likely due to increased mixing from strong winds. We demonstrate that interannual weather variability disturb the pace of SAR11 seasonal progression.

Seasonal mixed layer depth shapes phytoplankton physiology, viral production, and accumulation in the North Atlantic.

Seasonal shifts in phytoplankton accumulation and loss largely follow changes in mixed layer depth, but the impact of mixed layer depth on cell physiology remains unexplored. Here, we investigate the physiological state of phytoplankton populations associated with distinct bloom phases and mixing regimes in the North Atlantic. Stratification and deep mixing alter community physiology and viral production, effectively shaping accumulation rates. Communities in relatively deep, early-spring mixed layers are characterized by low levels of stress and high accumulation rates, while those in the recently shallowed mixed layers in late-spring have high levels of oxidative stress. Prolonged stratification into early autumn manifests in negative accumulation rates, along with pronounced signatures of compromised membranes, death-related protease activity, virus production, nutrient drawdown, and lipid markers indicative of nutrient stress. Positive accumulation renews during mixed layer deepening with transition into winter, concomitant with enhanced nutrient supply and lessened viral pressure.

Efficient dilution-to-extinction isolation of novel virus-host model systems for fastidious heterotrophic bacteria.

Microbes and their associated viruses are key drivers of biogeochemical processes in marine and soil biomes. While viruses of phototrophic cyanobacteria are well-represented in model systems, challenges of isolating marine microbial heterotrophs and their viruses have hampered experimental approaches to quantify the importance of viruses in nutrient recycling. A resurgence in cultivation efforts has improved the availability of fastidious bacteria for hypothesis testing, but this has not been matched by similar efforts to cultivate their associated bacteriophages. Here, we describe a high-throughput method for isolating important virus-host systems for fastidious heterotrophic bacteria that couples advances in culturing of hosts with sequential enrichment and isolation of associated phages. Applied to six monthly samples from the Western English Channel, we first isolated one new member of the globally dominant bacterial SAR11 clade and three new members of the methylotrophic bacterial clade OM43. We used these as bait to isolate 117 new phages, including the first known siphophage-infecting SAR11, and the first isolated phage for OM43. Genomic analyses of 13 novel viruses revealed representatives of three new viral genera, and infection assays showed that the viruses infecting SAR11 have ecotype-specific host ranges. Similar to the abundant human-associated phage ɸCrAss001, infection dynamics within the majority of isolates suggested either prevalent lysogeny or chronic infection, despite a lack of associated genes, or host phenotypic bistability with lysis putatively maintained within a susceptible subpopulation. Broader representation of important virus-host systems in culture collections and genomic databases will improve both our understanding of virus-host interactions, and accuracy of computational approaches to evaluate ecological patterns from metagenomic data.

Stable Isotope Probing Identifies Bacterioplankton Lineages Capable of Utilizing Dissolved Organic Matter Across a Range of Bioavailability.

Bacterioplankton consume about half of the dissolved organic matter (DOM) produced by phytoplankton. DOM released from phytoplankton consists of a myriad of compounds that span a range of biological reactivity from labile to recalcitrant. Linking specific bacterioplankton lineages to the incorporation of DOM compounds into biomass is important to understand microbial niche partitioning. We conducted a series of DNA-stable isotope probing (SIP) experiments using 13C-labeled substrates of varying lability including amino acids, cyanobacteria lysate, and DOM from diatom and cyanobacteria isolates concentrated on solid phase extraction PPL columns (SPE-DOM). Amendments of substrates into Sargasso Sea bacterioplankton communities were conducted to explore microbial response and DNA-SIP was used to determine which lineages of Bacteria and Archaea were responsible for uptake and incorporation. Greater increases in bacterioplankton abundance and DOC removal were observed in incubations amended with cyanobacteria-derived lysate and amino acids compared to the SPE-DOM, suggesting that the latter retained proportionally more recalcitrant DOM compounds. DOM across a range of bioavailability was utilized by diverse prokaryotic taxa with copiotrophs becoming the most abundant 13C-incorporating taxa in the amino acid treatment and oligotrophs becoming the most abundant 13C-incorporating taxa in SPE-DOM treatments. The lineages that responded to SPE-DOM amendments were also prevalent in the mesopelagic of the Sargasso Sea, suggesting that PPL extraction of phytoplankton-derived DOM isolates compounds of ecological relevance to oligotrophic heterotrophic bacterioplankton. Our study indicates that DOM quality is an important factor controlling the diversity of the microbial community response, providing insights into the roles of different bacterioplankton in resource exploitation and efficiency of marine carbon cycling.

Small phytoplankton dominate western North Atlantic biomass.

The North Atlantic phytoplankton spring bloom is the pinnacle in an annual cycle that is driven by physical, chemical, and biological seasonality. Despite its important contributions to the global carbon cycle, transitions in plankton community composition between the winter and spring have been scarcely examined in the North Atlantic. Phytoplankton composition in early winter was compared with latitudinal transects that captured the subsequent spring bloom climax. Amplicon sequence variants (ASVs), imaging flow cytometry, and flow-cytometry provided a synoptic view of phytoplankton diversity. Phytoplankton communities were not uniform across the sites studied, but rather mapped with apparent fidelity onto subpolar- and subtropical-influenced water masses of the North Atlantic. At most stations, cells < 20-µm diameter were the main contributors to phytoplankton biomass. Winter phytoplankton communities were dominated by cyanobacteria and pico-phytoeukaryotes. These transitioned to more diverse and dynamic spring communities in which pico- and nano-phytoeukaryotes, including many prasinophyte algae, dominated. Diatoms, which are often assumed to be the dominant phytoplankton in blooms, were contributors but not the major component of biomass. We show that diverse, small phytoplankton taxa are unexpectedly common in the western North Atlantic and that regional influences play a large role in modulating community transitions during the seasonal progression of blooms.

Cophylogenetic analysis suggests cospeciation between the Scorpion Mycoplasma Clade symbionts and their hosts.

Scorpions are predator arachnids of ancient origin and worldwide distribution. Two scorpion species, Vaejovis smithi and Centruroides limpidus, were found to harbor two different Mollicutes phylotypes: a Scorpion Mycoplasma Clade (SMC) and Scorpion Group 1 (SG1). Here we investigated, using a targeted gene sequencing strategy, whether these Mollicutes were present in 23 scorpion morphospecies belonging to the Vaejovidae, Carboctonidae, Euscorpiidae, Diplocentridae, and Buthidae families. Our results revealed that SMC is found in a species-specific association with Vaejovidae and Buthidae, whereas SG1 is uniquely found in Vaejovidae. SMC and SG1 co-occur only in Vaejovis smithi where 43% of the individuals host both phylotypes. A phylogenetic analysis of Mollicutes 16S rRNA showed that SMC and SG1 constitute well-delineated phylotypes. Additionally, we found that SMC and scorpion phylogenies are significantly congruent, supporting the observation that a cospeciation process may have occurred. This study highlights the phylogenetic diversity of the scorpion associated Mollicutes through different species revealing a possible cospeciation pattern.

Species-specific diversity of novel bacterial lineages and differential abundance of predicted pathways for toxic compound degradation in scorpion gut microbiota.

Scorpions are considered 'living fossils' that have conserved ancestral anatomical features and have adapted to numerous habitats. However, their gut microbiota diversity has not been studied. Here, we characterized the gut microbiota of two scorpion species, Vaejovis smithi and Centruroides limpidus. Our results indicate that scorpion gut microbiota is species-specific and that food deprivation reduces bacterial diversity. 16S rRNA gene phylogenetic analysis revealed novel bacterial lineages showing a low level of sequence identity to any known bacteria. Furthermore, these novel bacterial lineages were each restricted to a different scorpion species. Additionally, our results of the predicted metagenomic profiles revealed a core set of pathways that were highly abundant in both species, and mostly related to amino acid, carbohydrate, vitamin and cofactor metabolism. Notably, the food-deprived V. smithi shotgun metagenome matched almost completely the metabolic features of the prediction. Finally, comparisons among predicted metagenomic profiles showed that toxic compound degradation pathways were more abundant in recently captured C. limpidus scorpions. This study gives a first insight into the scorpion gut microbiota and provides a reference for future studies on the gut microbiota from other arachnid species.

Arthropod-Spiroplasma relationship in the genomic era.

The genus Spiroplasma comprises wall-less, low-GC bacteria that establish pathogenic, mutualistic and commensal symbiotic associations with arthropods and plants. This review focuses on the symbiotic relationships between Spiroplasma bacteria and arthropod hosts in the context of the available genomic sequences. Spiroplasma genomes are reduced and some contain highly repetitive plectrovirus-related sequences. Spiroplasma's diversity in viral invasion susceptibility, virulence factors, substrate utilization, genome dynamics and symbiotic associations with arthropods make this bacterial genus a biological model that provides insights about the evolutionary traits that shape bacterial symbiotic relationships with eukaryotes.

Gut and root microbiota commonalities.

Animal guts and plant roots have absorption roles for nutrient uptake and converge in harboring large, complex, and dynamic groups of microbes that participate in degradation or modification of nutrients and other substances. Gut and root bacteria regulate host gene expression, provide metabolic capabilities, essential nutrients, and protection against pathogens, and seem to share evolutionary trends.