Microscopic marine invertebrates are reservoirs for cryptic and diverse protists and fungi.

BACKGROUND: Microbial symbioses in marine invertebrates are commonplace. However, characterizations of invertebrate microbiomes are vastly outnumbered by those of vertebrates. Protists and fungi run the gamut of symbiosis, yet eukaryotic microbiome sequencing is rarely undertaken, with much of the focus on bacteria. To explore the importance of microscopic marine invertebrates as potential symbiont reservoirs, we used a phylogenetic-focused approach to analyze the host-associated eukaryotic microbiomes of 220 animal specimens spanning nine different animal phyla. RESULTS: Our data expanded the traditional host range of several microbial taxa and identified numerous undescribed lineages. A lack of comparable reference sequences resulted in several cryptic clades within the Apicomplexa and Ciliophora and emphasized the potential for microbial invertebrates to harbor novel protistan and fungal diversity. CONCLUSIONS: Microscopic marine invertebrates, spanning a wide range of animal phyla, host various protist and fungal sequences and may therefore serve as a useful resource in the detection and characterization of undescribed symbioses. Video Abstract.

Ranking the biases: The choice of OTUs vs. ASVs in 16S rRNA amplicon data analysis has stronger effects on diversity measures than rarefaction and OTU identity threshold.

Advances in the analysis of amplicon sequence datasets have introduced a methodological shift in how research teams investigate microbial biodiversity, away from sequence identity-based clustering (producing Operational Taxonomic Units, OTUs) to denoising methods (producing amplicon sequence variants, ASVs). While denoising methods have several inherent properties that make them desirable compared to clustering-based methods, questions remain as to the influence that these pipelines have on the ecological patterns being assessed, especially when compared to other methodological choices made when processing data (e.g. rarefaction) and computing diversity indices. We compared the respective influences of two widely used methods, namely DADA2 (a denoising method) vs. Mothur (a clustering method) on 16S rRNA gene amplicon datasets (hypervariable region v4), and compared such effects to the rarefaction of the community table and OTU identity threshold (97% vs. 99%) on the ecological signals detected. We used a dataset comprising freshwater invertebrate (three Unionidae species) gut and environmental (sediment, seston) communities sampled in six rivers in the southeastern USA. We ranked the respective effects of each methodological choice on alpha and beta diversity, and taxonomic composition. The choice of the pipeline significantly influenced alpha and beta diversities and changed the ecological signal detected, especially on presence/absence indices such as the richness index and unweighted Unifrac. Interestingly, the discrepancy between OTU and ASV-based diversity metrics could be attenuated by the use of rarefaction. The identification of major classes and genera also revealed significant discrepancies across pipelines. Compared to the pipeline's effect, OTU threshold and rarefaction had a minimal impact on all measurements.

Utilization of Agro-Industrial Byproducts for Bacteriocin Production Using Enterococcus spp. Strains Isolated from Patagonian Marine Invertebrates.

This work's objective was to determine the antagonist activity of 11 Enterococcus spp. using industrial food wastes as a culture medium. The strains were isolated from invertebrates collected on the Argentinian Patagonia coast and selected by their high antibacterial activity. Phenotypic and genotypic techniques allowed identifying five E. hirae strains, five E. faecium strains, and one E. mundtii strain. The cell-free supernatants displayed inhibitory activity against most of the Gram-positive bacteria tested and Vibrio anguilarum. PCR amplification techniques detected the encoding genes of enterocin P in ten strains, mundtiicin KS in seven strains, enterocin B in six strains, hiracin JM79 in five strains, and enterocin A in three strains. The strains did not show gelatinase or hemolytic activities and were sensitive to gentamicin, kanamycin, streptomycin, tylosine, tetracycline, chloramphenicol and vancomycin. Cheese whey and hot trub derived from beer brewing were used alone or in combination to assay enterocin production. In all cases, the highest inhibitory activities were achieved when mixtures of both byproducts were used as growth medium. The results suggest that the selected strains can produce high levels of enterocins in a low-cost media composed of a mix of cheese whey and hot trub without additional supplementation with carbon or nitrogen sources.

Zophobihabitans entericus gen. nov., sp. nov., a new member of the family Orbaceae isolated from the gut of a superworm Zophobas morio.

A novel bacterial strain, designated IPMB12T, isolated from the gut of the superworm Zophobas morio in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, facultatively anaerobic, non-motile, coccoid or rod-shaped and formed translucent colonies. Optimal growth occurred at 25-37 °C, pH 9-10, and with 0-2 % NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain IPMB12T is affiliated with genus in the the family Orbaceae in the class Gammaproteobacteria. Strain IPMB12T was most closely related to Gilliamella mensalis LMG 29880T with a 94.6 % 16S rRNA gene sequence similarity. Strain IPMB12T showed less than 71.6 % average nucleotide identity, less than 71.5 % average amino acid identity and less than 21.2 % digital DNA-DNA hybridization identity compared to the strains of related genera within the family Orbaceae. The major fatty acids of strain IPMB12T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c), C16 : 0 and C14 : 0. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one uncharacterized phosphoaminoglycolipid and one uncharacterized aminophospholipid. The major isoprenoid quinone was Q-8. Genomic DNA G+C content of strain IPMB12T was 39.3 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain IPMB12T represents a novel species of a new genus in the family Orbaceae, for which the name Zophobihabitans entericus gen. nov., sp. nov. is proposed. The type strain is IPMB12T (=BCRC 80908T =LMG 32079T=KCTC 82347T=KACC 22323T).

Lethal and sub-lethal activity of Brevibacillus laterosporus on the mosquito Aedes albopictus and side effects on non-target water-dwelling invertebrates.

The biocidal potential of Brevibacillus laterosporus against mosquitoes of major medical importance has been widely documented, but its effects on non-target invertebrates are still poorly known. In this study, we determined the lethal and sub-lethal effects of B. laterosporus strain UNISS 18, an entomopathogenic bacterium known for its effectiveness against synanthropic Diptera, on the larvae of the Asian tiger mosquito Aedes albopictus, a vector of several pathogens to humans. Moreover, we compared the larvicidal activity with the lethal action on the invasive snail Physella acuta and on two non-target water-dwelling species: the mayfly Cloeon dipterum, and the harlequin fly Chironomus riparius. B. laterosporus exhibited significant lethal effects on all the tested species with a concentration-dependent activity. However, the susceptibility varied among species, with a higher susceptibility of Ae. albopictus (LC50 = 0.16 × 107 spores mL-1) than the other species (LC50 = 0.31, 0.33, and 0.30 × 107 spores mL-1 for C. dipterum, C. riparius, and P. acuta, respectively). While 1st instar mosquito larvae were very susceptible to the bacterial infection, no effects on preimaginal development stages and adult emergence were observed at sub-lethal spores' concentrations. Even if the efficacy of B. laterosporus against Ae. albopictus and the invasive freshwater snail P. acuta is promising for their control, the susceptibility of non-target beneficial aquatic insects, highlights the need of accurate evaluations before applying B. laterosporus for pest management in water environments.

Fungal immunity and pathogenesis in mammals versus the invertebrate model organism Galleria mellonella.

In recent decades, Galleria mellonella (Lepidoptera: Pyralidae) have emerged as a model system to explore experimental aspects of fungal pathogenesis. The benefits of the G. mellonella model include being faster, cheaper, higher throughput and easier compared with vertebrate models. Additionally, as invertebrates, their use is subject to fewer ethical and regulatory issues. However, for G. mellonella models to provide meaningful insight into fungal pathogenesis, the G. mellonella-fungal interactions must be comparable to mammalian-fungal interactions. Indeed, as discussed in the review, studies suggest that G. mellonella and mammalian immune systems share many similarities, and fungal virulence factors show conserved functions in both hosts. While the moth model has opened novel research areas, many comparisons are superficial and leave large gaps of knowledge that need to be addressed concerning specific mechanisms underlying G. mellonella-fungal interactions. Closing these gaps in understanding will strengthen G. mellonella as a model for fungal virulence in the upcoming years. In this review, we provide comprehensive comparisons between fungal pathogenesis in mammals and G. mellonella from immunological and virulence perspectives. When information on an antifungal immune component is unknown in G. mellonella, we include findings from other well-studied Lepidoptera. We hope that by outlining this information available in related species, we highlight areas of needed research and provide a framework for understanding G. mellonella immunity and fungal interactions.

Housing microbial symbionts: evolutionary origins and diversification of symbiotic organs in animals.

In many animal hosts, microbial symbionts are housed within specialized structures known as symbiotic organs, but the evolutionary origins of these structures have rarely been investigated. Here, I adopt an evolutionary developmental (evo-devo) approach, specifically to apply knowledge of the development of symbiotic organs to gain insights into their evolutionary origins and diversification. In particular, host genetic changes associated with evolution of symbiotic organs can be inferred from studies to identify the host genes that orchestrate the development of symbiotic organs, recognizing that microbial products may also play a key role in triggering the developmental programme in some associations. These studies may also reveal whether higher animal taxonomic groups (order, class, phylum, etc.) possess a common genetic regulatory network for symbiosis that is latent in taxa lacking symbiotic organs, and activated at the origination of symbiosis in different host lineages. In this way, apparent instances of convergent evolution of symbiotic organs may be homologous in terms of a common genetic blueprint for symbiosis. Advances in genetic technologies, including reverse genetic tools and genome editing, will facilitate the application of evo-devo approaches to investigate the evolution of symbiotic organs in animals. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Evolution of animal immunity in the light of beneficial symbioses.

Immune system processes serve as the backbone of animal defences against pathogens and thus have evolved under strong selection and coevolutionary dynamics. Most microorganisms that animals encounter, however, are not harmful, and many are actually beneficial. Selection should act on hosts to maintain these associations while preventing exploitation of within-host resources. Here, we consider how several key aspects of beneficial symbiotic associations may shape host immune system evolution. When host immunity is used to regulate symbiont populations, there should be selection to evolve and maintain targeted immune responses that recognize symbionts and suppress but not eliminate symbiont populations. Associating with protective symbionts could relax selection on the maintenance of redundant host-derived immune responses. Alternatively, symbionts could facilitate the evolution of host immune responses if symbiont-conferred protection allows for persistence of host populations that can then adapt. The trajectory of immune system evolution will likely differ based on the type of immunity involved, the symbiont transmission mode and the costs and benefits of immune system function. Overall, the expected influence of beneficial symbiosis on immunity evolution depends on how the host immune system interacts with symbionts, with some interactions leading to constraints while others possibly relax selection on immune system maintenance. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Pathogen resistance may be the principal evolutionary advantage provided by the microbiome.

To survive, plants and animals must continually defend against pathogenic microbes that would invade and disrupt their tissues. Yet they do not attempt to extirpate all microbes. Instead, they tolerate and even encourage the growth of commensal microbes, which compete with pathogens for resources and via direct inhibition. We argue that hosts have evolved to cooperate with commensals in order to enhance the pathogen resistance this competition provides. We briefly describe competition between commensals and pathogens within the host, consider how natural selection might favour hosts that tilt this competition in favour of commensals, and describe examples of extant host traits that may serve this purpose. Finally, we consider ways that this cooperative immunity may have facilitated the adaptive evolution of non-pathogen-related host traits. On the basis of these observations, we argue that pathogen resistance vies with other commensal-provided benefits for being the principal evolutionary advantage provided by the microbiome to host lineages across the tree of life. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Optimal integration between host physiology and functions of the gut microbiome.

Host-associated microbial communities have profound impacts on animal physiological function, especially nutrition and metabolism. The hypothesis of 'symmorphosis', which posits that the physiological systems of animals are regulated precisely to meet, but not exceed, their imposed functional demands, has been used to understand the integration of physiological systems across levels of biological organization. Although this idea has been criticized, it is recognized as having important heuristic value, even as a null hypothesis, and may, therefore, be a useful tool in understanding how hosts evolve in response to the function of their microbiota. Here, through a hologenomic lens, we discuss how the idea of symmorphosis may be applied to host-microbe interactions. Specifically, we consider scenarios in which host physiology may have evolved to collaborate with the microbiota to perform important functions, and, on the other hand, situations in which services have been completely outsourced to the microbiota, resulting in relaxed selection on host pathways. Following this theoretical discussion, we finally suggest strategies by which these currently speculative ideas may be explicitly tested to further our understanding of host evolution in response to their associated microbial communities. This article is part of the theme issue 'The role of the microbiome in host evolution'.

In the beginning: egg-microbe interactions and consequences for animal hosts.

Microorganisms are associated with the eggs of many animals. For some hosts, the egg serves as the ideal environment for the vertical transmission of beneficial symbionts between generations, while some bacteria use the egg to parasitize their hosts. In a number of animal groups, egg microbiomes often perform other essential functions. The eggs of aquatic and some terrestrial animals are especially susceptible to fouling and disease since they are exposed to high densities of microorganisms. To overcome this challenge, some hosts form beneficial associations with microorganisms, directly incorporating microbes and/or microbial products on or in their eggs to inhibit pathogens and biofouling. Other functional roles for egg-associated microbiomes are hypothesized to involve oxygen and nutrient acquisition. Although some egg-associated microbiomes are correlated with increased host fitness and are essential for successful development, the mechanisms that lead to such outcomes are often not well understood. This review article will discuss different functions of egg microbiomes and how these associations have influenced the biology and evolution of animal hosts. This article is part of the theme issue 'The role of the microbiome in host evolution'.

Holobionts: Ecological communities, hybrids, or biological individuals? A metaphysical perspective on multispecies systems.

Holobionts are symbiotic assemblages composed by a macrobe host (animal or plant) plus its symbiotic microbiota. In recent years, the ontological status of holobionts has created a great amount of controversy among philosophers and biologists: are holobionts biological individuals or are they rather ecological communities of independent individuals that interact together? Chiu and Eberl have recently developed an eco-immunity account of the holobiont wherein holobionts are neither biological individuals nor ecological communities, but hybrids between a host and its microbiota. According to their account, the microbiota is not a proper part of the holobiont. Yet, it should be regarded as a set of scaffolds that support the individuality of the host. In this paper, we approach Chiu and Eberl's account from a metaphysical perspective and argue that, contrary to what the authors claim, the eco-immunity account entails that the microorganisms that compose the host's microbiota are proper parts of the holobiont. Second, we argue that by claiming that holobionts are hybrids, and therefore, not biological individuals, the authors seem to be assuming a controversial position about the ontology of hybrids, which are conventionally characterized as a type of biological individual. In doing so, our paper aligns with the contemporary tendency to incorporate metaphysical resources to shed light on current biological debates and builds on that to provide additional support to the consideration of holobionts as biological individuals from an eco-immunity perspective.

Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates.

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.

The Microbial Community of Tetrodotoxin-Bearing and Non-Tetrodotoxin-Bearing Ribbon Worms (Nemertea) from the Sea of Japan.

A potent marine toxin, tetrodotoxin (TTX), found in a great variety of marine and some terrestrial species, leaves intriguing questions about its origin and distribution in marine ecosystems. TTX-producing bacteria were found in the cultivable microflora of many TTX-bearing hosts, thereby providing strong support for the hypothesis that the toxin is of bacterial origin in these species. However, metagenomic studies of TTX-bearing animals addressing the whole microbial composition and estimating the contribution of TTX-producing bacteria to the overall toxicity of the host were not conducted. The present study is the first to characterize and compare the 16S rRNA gene data obtained from four TTX-bearing and four non-TTX-bearing species of marine ribbon worms. The statistical analysis showed that different nemertean species harbor distinct bacterial communities, while members of the same species mostly share more similar microbiomes. The bacterial species historically associated with TTX production were found in all studied samples but predominated in TTX-bearing nemertean species. This suggests that deeper knowledge of the microbiome of TTX-bearing animals is a key to understanding the origin of TTX in marine ecosystems.

First Identification of ß-Lactamases in Antibiotic-Resistant Escherichia coli, Citrobacter freundii, and Aeromonas spp. Isolated in Stream Macroinvertebrates in a Central Italian Region.

Antibiotic-resistant bacteria (ARB) are widespread in nature and represent a serious public and environmental problem. In the present study, we report for the first time the presence of bacterial ß-lactamases in two macroinvertebrate species with different feeding traits. The class A ß-lactamases, SHV-1 and TEM-1, were found in Citrobacter freundii isolated from Gammarus elvirae and Escherichia coli from water samples, respectively. The metallo-ß-lactamase CphA was found in Aeromonas veronii and Aeromonas hydrophila strains isolated from the predator Dina lineata. The presence of a large plasmid was ascertained only in E. coli strains isolated from water. In all strains studied, an integrase I typical of class I integrin was found. In contaminated freshwater habitats, ARB and antibiotic resistance genes could be disseminated through trophic links with important ecological implications. Transmission through the food chain may contribute to spreading and transferring antibiotic resistance not only in freshwater ecosystems but also outside the aquatic habitat.

Compositional variation between high and low prokaryotic diversity coral reef biotopes translates to different predicted metagenomic gene content.

In a previous study, we identified host species that housed high and low diversity prokaryotic communities. In the present study, we expand on this and assessed the prokaryotic communities associated with seawater, sediment and 11 host species from 7 different phyla in a Taiwanese coral reef setting. The host taxa sampled included hard, octo- and black corals, molluscs, bryozoans, flatworms, fish and sea urchins. There were highly significant differences in composition among host species and all host species housed distinct communities from those found in seawater and sediment. In a hierarchical clustering analysis, samples from all host species, with the exception of the coral Galaxea astreata, formed significantly supported clusters. In addition to this, the coral G. astreata and the bryozoan Triphyllozoon inornatum on the one hand and the coral Tubastraea coccinea, the hermit crab Calcinus laevimanus and the flatworm Thysanozoon nigropapillosum on the other formed significantly supported clusters. In addition to composition, there were highly pronounced differences in richness and evenness among host species from the most diverse species, the bryozoan T. inornatum at 2518 ± 240 OTUs per 10,000 sequences to the least diverse species, the octocoral Cladiella sp. at 142 ± 14 OTUs per 10,000 sequences. In line with the differences in composition, there were significant differences in predicted metagenomic gene counts among host species. Furthermore, there were pronounced compositional and predicted functional differences between high diversity hosts (Liolophura japonica, G. astreata, T. coccinea, C. laevimanus, T. inornatum) and low diversity hosts (Antipathes sp., Pomacentrus coelestis, Modiolus auriculatus, T. nigropapillosum, Cladiella sp. and Diadema savigny). In particular, we found that all tested low diversity hosts were predicted to be enriched for the phosphotransferase system compared to high diversity hosts.

Unraveling the Detoxification Mechanism of 2,4-Dichlorophenol by Marine-Derived Mesophotic Symbiotic Fungi Isolated from Marine Invertebrates.

Chlorophenols (CPs) are environmental pollutants that are produced through various anthropogenic activities and introduced in the environment. Living organisms, including humans, are exposed to these toxic xenobiotics and suffer from adverse health effects. More specifically, 2,4-dichlorophenol (2,4-DCP) is released in high amounts in the environment and has been listed as a priority pollutant by the US Environmental Protection Agency. Bioremediation has been proposed as a sustainable alternative to conventional remediation methods for the detoxification of phenolic compounds. In this work, we studied the potential of fungal strains isolated as symbionts of marine invertebrates from the underexplored mesophotic coral ecosystems. Hence, the unspecific metabolic pathways of these fungal strains are being explored in the present study, using the powerful analytical capabilities of a UHPLC-HRMS/MS. The newly identified 2,4-DCP metabolites add significantly to the knowledge of the transformation of such pollutants by fungi, since such reports are scarce.

Microbes and macro-invertebrates show parallel ß-diversity but contrasting α-diversity patterns in a marine natural experiment.

Documenting ecological patterns across spatially, temporally and taxonomically diverse ecological communities is necessary for a general understanding of the processes shaping biodiversity. A major gap in our understanding remains the comparison of diversity patterns across a broad spectrum of evolutionarily and functionally diverse organisms, particularly in the marine realm. Here, we aim to narrow this gap by comparing the diversity patterns of free-living microbes and macro-invertebrates across a natural experiment provided by the marine lakes of Palau: geographically discrete and environmentally heterogeneous bodies of seawater with comparable geological and climatic history, and a similar regional species pool. We find contrasting patterns of α-diversity but remarkably similar patterns of ß-diversity between microbial and macro-invertebrate communities among lakes. Pairwise dissimilarities in community composition among lakes are positively correlated between microbes and macro-invertebrates, and influenced to a similar degree by marked gradients in oxygen concentration and salinity. Our findings indicate that a shared spatio-temporal and environmental context may result in parallel patterns of ß-diversity in microbes and macro-invertebrates, in spite of key trait differences between these organisms. This raises the possibility that parallel processes also influence transitions among regional biota across the tree of life, at least in the marine realm.

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.