Spider phylosymbiosis: divergence of widow spider species and their tissues' microbiomes.

BACKGROUND: Microbiomes can have profound impacts on host biology and evolution, but to date, remain vastly understudied in spiders despite their unique and diverse predatory adaptations. This study evaluates closely related species of spiders and their host-microbe relationships in the context of phylosymbiosis, an eco-evolutionary pattern where the microbial community profile parallels the phylogeny of closely related host species. Using 16S rRNA gene amplicon sequencing, we characterized the microbiomes of five species with known phylogenetic relationships from the family Theridiidae, including multiple closely related widow spiders (L. hesperus, L. mactans, L. geometricus, S. grossa, and P. tepidariorum). RESULTS: We compared whole animal and tissue-specific microbiomes (cephalothorax, fat bodies, venom glands, silk glands, and ovary) in the five species to better understand the relationship between spiders and their microbial symbionts. This showed a strong congruence of the microbiome beta-diversity of the whole spiders, cephalothorax, venom glands, and silk glands when compared to their host phylogeny. Our results support phylosymbiosis in these species and across their specialized tissues. The ovary tissue microbial dendrograms also parallel the widow phylogeny, suggesting vertical transfer of species-specific bacterial symbionts. By cross-validating with RNA sequencing data obtained from the venom glands, silk glands and ovaries of L. hesperus, L. geometricus, S. grossa, and P. tepidariorum we confirmed that several microbial symbionts of interest are viably active in the host. CONCLUSION: Together these results provide evidence that supports the importance of host-microbe interactions and the significant role microbial communities may play in the evolution and adaptation of their hosts.

Correction: Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History.

[This corrects the article DOI: 10.1371/journal.pbio.2000225.].

Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History.

Phylosymbiosis was recently proposed to describe the eco-evolutionary pattern, whereby the ecological relatedness of host-associated microbial communities parallels the phylogeny of related host species. Here, we test the prevalence of phylosymbiosis and its functional significance under highly controlled conditions by characterizing the microbiota of 24 animal species from four different groups (Peromyscus deer mice, Drosophila flies, mosquitoes, and Nasonia wasps), and we reevaluate the phylosymbiotic relationships of seven species of wild hominids. We demonstrate three key findings. First, intraspecific microbiota variation is consistently less than interspecific microbiota variation, and microbiota-based models predict host species origin with high accuracy across the dataset. Interestingly, the age of host clade divergence positively associates with the degree of microbial community distinguishability between species within the host clades, spanning recent host speciation events (~1 million y ago) to more distantly related host genera (~108 million y ago). Second, topological congruence analyses of each group's complete phylogeny and microbiota dendrogram reveal significant degrees of phylosymbiosis, irrespective of host clade age or taxonomy. Third, consistent with selection on host-microbiota interactions driving phylosymbiosis, there are survival and performance reductions when interspecific microbiota transplants are conducted between closely related and divergent host species pairs. Overall, these findings indicate that the composition and functional effects of an animal's microbial community can be closely allied with host evolution, even across wide-ranging timescales and diverse animal systems reared under controlled conditions.

Establishment of F1 hybrid mortality in real time.

BACKGROUND: Measuring the evolutionary rate of reproductive isolation is essential to understanding how new species form. Tempo calculations typically rely on fossil records, geological events, and molecular evolution analyses. The speed at which genetically-based hybrid mortality arises, or the "incompatibility clock", is estimated to be millions of years in various diploid organisms and is poorly understood in general. Owing to these extended timeframes, seldom do biologists observe the evolution of hybrid mortality in real time. RESULTS: Here we report the very recent spread and fixation of complete asymmetric F1 hybrid mortality within eight years of laboratory maintenance in the insect model Nasonia. The asymmetric interspecific hybrid mortality evolved in an isogenic stock line of N. longicornis and occurs in crosses to N. vitripennis males. The resulting diploid hybrids exhibit complete failure in dorsal closure during embryogenesis. CONCLUSION: These results comprise a unique case whereby a strong asymmetrical isolation barrier evolved in real time. The spread of this reproductive isolation barrier notably occurred in a small laboratory stock subject to recurrent bottlenecks.

Disease defence through generations: leaf-cutter ants and their symbiotic bacteria.

Microbial ecology of animals is taking on significance in the modern dialogue for the biology of species. Similar to a nuclear genome, the entire bacterial assemblage maintains an ancestral signal of the host's evolution leading to cophylogeny between the host and the microbes they harbour (Brucker & Bordenstein 2012b). The stability of such associations is of great interest as they provide a means for species to acquire new traits and genetic diversity that their own genomes lack (McFall-Ngai et al. 2013). The role of gut microbiota, for example, in host health and nutrition is widely recognized and a shared characteristic among animals. The role of bacteria colonizing the outside surfaces of animals is less well understood, but rather than random colonization, these microbes on skin, cuticles, scales and feathers in many cases provide benefits to the host. The symbiosis of leaf-cutter ants, their fungus gardens and their microbiota is a fascinating and complex system. Whether culture-independent bacterial diversity on the cuticle of leaf-cutter ants is high or highly constrained by subcuticular gland secretions is one prominent question. In this issue of Molecular Ecology, Andersen et al. (2013) show that leaf-cutting ants, Acromyrmex echinatior, maintain a dominant and colony-specific bacterium called Pseudonocardia on their cuticles (the laterocervical plates in particular). This bacterium is involved in protecting the ants and their fungal gardens from disease. Other fungus-gardening attine species as well as soil and vegetation can harbour Pseudonocardia. However, it was previously unknown how stable the bacterial strain-ant colony association was through the lifetime of the colony.

An optimized method for Nasonia germ-free rearing.

A germ-free rearing system is a crucial method for host-microbiota interactions using Nasonia as a model system. The previous rearing media in 2012 introduced toxic factors like bleach and antibiotics, required significant effort and volume of media preparation, and the rearing protocols in 2012 and 2016 often resulted in embryos, larvae, and enclosing pupae drowning, underfed, or desiccating. In this work, we optimize the germ-free rearing media that excludes the toxic factors and provide a substrate for the developing animals to have constant access to media without the risk of drowning or desiccation. The new process resulted in an increase in full maturation of larvae to adults from 33 to 65%, with no effect on the rate of growth or final adult size. This significantly improves the applicability of germ-free rearing of Nasonia and potentially other parasitoids.

Disruption of the termite gut microbiota and its prolonged consequences for fitness.

The disruption of host-symbiont interactions through the use of antibiotics can help elucidate microbial functions that go beyond short-term nutritional value. Termite gut symbionts have been studied extensively, but little is known about their impact on the termite's reproductive output. Here we describe the effect that the antibiotic rifampin has not only on the gut microbial diversity but also on the longevity, fecundity, and weight of two termite species, Zootermopsis angusticollis and Reticulitermes flavipes. We report three key findings: (i) the antibiotic rifampin, when fed to primary reproductives during the incipient stages of colony foundation, causes a permanent reduction in the diversity of gut bacteria and a transitory effect on the density of the protozoan community; (ii) rifampin treatment reduces oviposition rates of queens, translating into delayed colony growth and ultimately reduced colony fitness; and (iii) the initial dosages of rifampin had severe long-term fitness effects on Z. angusticollis. Taken together, our findings demonstrate that the antibiotic-induced perturbation of the microbial community is associated with prolonged reductions in longevity and fecundity. A causal relationship between these changes in the gut microbial population structures and fitness is suggested by the acquisition of opportunistic pathogens and incompetence of the termites to restore a pretreatment, native microbiota. Our results indicate that antibiotic treatment significantly alters the termite's microbiota, reproduction, colony establishment, and ultimately colony growth and development. We discuss the implications for antimicrobials as a new application to the control of termite pest species.

When your host shuts down: larval diapause impacts host-microbiome interactions in Nasonia vitripennis.

BACKGROUND: The life cycles of many insect species include an obligatory or facultative diapause stage with arrested development and low metabolic activity as an overwintering strategy. Diapause is characterised by profound physiological changes in endocrine activity, cell proliferation and nutrient metabolism. However, little is known regarding host-microbiome interactions during diapause, despite the importance of bacterial symbionts for host nutrition and development. In this work, we investigated (i) the role of the microbiome for host nutrient allocation during diapause and (ii) the impact of larval diapause on microbiome dynamics in the parasitoid wasp Nasonia vitripennis, a model organism for host-microbiome interactions. RESULTS: Our results demonstrate that the microbiome is essential for host nutrient allocation during diapause in N. vitripennis, as axenic diapausing larvae had consistently lower glucose and glycerol levels than conventional diapausing larvae, especially when exposed to cold temperature. In turn, microbiome composition was altered in diapausing larvae, potentially due to changes in the surrounding temperature, host nutrient levels and a downregulation of host immune genes. Importantly, prolonged larval diapause had a transstadial effect on the adult microbiome, with unknown consequences for host fitness. Notably, the most dominant microbiome member, Providencia sp., was drastically reduced in adults after more than 4 months of larval diapause, while potential bacterial pathogens increased in abundance. CONCLUSION: This work investigates host-microbiome interactions during a crucial developmental stage, which challenges both the insect host and its microbial associates. The impact of diapause on the microbiome is likely due to several factors, including altered host regulatory mechanisms and changes in the host environment. Video Abstract.

Coadaptation between host genome and microbiome under long-term xenobiotic-induced selection.

One of the most difficult experimental challenges today is testing the evolutionary dynamics shaping complex host-microbiome interactions. We investigated host-microbiome codiversification in response to xenobiotic-induced selection using an experimental evolution approach. To this end, we exposed the parasitoid wasp Nasonia vitripennis to sublethal concentrations of the widely used herbicide atrazine for 85 generations. Our results reveal that atrazine exposure not only mediated adaptive changes within the microbiome, which conferred host resistance to atrazine toxicity, but also exerted selective pressure on the host genome and altered host gene expression and immune response. Furthermore, microbiome transplant experiments reveal a decreased survival of adults from the control population after exposure to the evolved microbiome of the atrazine-exposed population, while no such decrease occurred in the reciprocal transplant. These results indicate that xenobiotic-induced selection mediated host-microbiome coadaptation, ultimately leading to a new host genome-microbiome equilibrium.

Changes in Microbiome Confer Multigenerational Host Resistance after Sub-toxic Pesticide Exposure.

The gut is a first point of contact with ingested xenobiotics, where chemicals are metabolized directly by the host or microbiota. Atrazine is a widely used pesticide, but the role of the microbiome metabolism of this xenobiotic and the impact on host responses is unclear. We exposed successive generations of the wasp Nasonia vitripennis to subtoxic levels of atrazine and observed changes in the structure and function of the gut microbiome that conveyed atrazine resistance. This microbiome-mediated resistance was maternally inherited and increased over successive generations, while also heightening the rate of host genome selection. The rare gut bacteria Serratia marcescens and Pseudomonas protegens contributed to atrazine metabolism. Both of these bacteria contain genes that are linked to atrazine degradation and were sufficient to confer resistance in experimental wasp populations. Thus, pesticide exposure causes functional, inherited changes in the microbiome that should be considered when assessing xenobiotic exposure and as potential countermeasures to toxicity.

The bacterially produced metabolite violacein is associated with survival of amphibians infected with a lethal fungus.

The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 microM on a salamander's skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.

Genome Sequence of Providencia rettgeri NVIT03, Isolated from Nasonia vitripennis.

Providencia rettgeri is a common insect-associated Gram-negative bacterium. Here, we present the draft genome sequence of P. rettgeri NVIT03, the most common bacterial symbiont of the insect hymenopteran model Nasonia vitripennis. This symbiont is also part of the Sarcophaga bullata pupal microbiome that Nasonia spp. parasitize and that critically influences the development of the wasp.

Genome Sequence of Enterococcus faecalis NVIT04, Isolated from Nasonia vitripennis.

Enterococcus faecalis is a Gram-positive, lactic acid-producing coccus which can be found as a member of the gut microbiome in many animal species and is a potential pathogen in humans. Here, we describe the genome sequence of an E. faecalis strain isolated from the gut microbiome of the hymenopteran model Nasonia vitripennis.

Distinct mucosal microbial communities in infants with surgical necrotizing enterocolitis correlate with age and antibiotic exposure.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most common surgical emergency in preterm infants, and pathogenesis associates with changes in the fecal microbiome. As fecal samples incompletely represent microbial communities in intestinal mucosa, we sought to determine the NEC tissue-specific microbiome and assess its contribution to pathogenesis. DESIGN: We amplified and sequenced the V1-V3 hypervariable region of the bacterial 16S rRNA gene extracted from intestinal tissue and corresponding fecal samples from 12 surgical patients with NEC and 14 surgical patients without NEC. Low quality and non-bacterial sequences were removed, and taxonomic assignment was made with the Ribosomal Database Project. Operational taxonomic units were clustered at 97%. We tested for differences between NEC and non-NEC samples in microbiome alpha- and beta-diversity and differential abundance of specific taxa between NEC and non-NEC samples. Additional analyses were performed to assess the contribution of other demographic and environmental confounding factors on the infant tissue and fecal microbiome. RESULTS: The fecal and tissue microbial communities were different. NEC was associated with a distinct microbiome, which was characterized by low diversity, higher abundances of Staphylococcus and Clostridium_sensu_stricto, and lower abundances of Actinomyces and Corynebacterium. Infant age and vancomycin exposure correlated with shifts in the tissue microbiome. CONCLUSION: The observed low diversity in NEC tissues suggests that NEC is associated with a bacterial bloom and a distinct mucosal bacterial community. The exact bacterial species that constitute the bloom varied by infant and were strongly influenced by age and exposure to vancomycin.

Bacterial DNA is present in the fetal intestine and overlaps with that in the placenta in mice.

Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.

Mosquito Microbiome Dynamics, a Background for Prevalence and Seasonality of West Nile Virus.

Symbiotic microbial communities augment host phenotype, including defense against pathogen carriage and infection. We sampled the microbial communities in 11 adult mosquito host species from six regions in southern Ontario, Canada over 3 years. Of the factors examined, we found that mosquito species was the largest driver of the microbiota, with remarkable phylosymbiosis between host and microbiota. Seasonal shifts of the microbiome were consistently repeated over the 3-year period, while region had little impact. Both host species and seasonal shifts in microbiota were associated with patterns of West Nile virus (WNV) in these mosquitoes. The highest prevalence of WNV, with a seasonal spike each year in August, was in the Culex pipiens/restuans complex, and high WNV prevalence followed a decrease in relative abundance of Wolbachia in this species. Indeed, mean temperature, but not precipitation, was significantly correlated with Wolbachia abundance. This suggests that at higher temperatures Wolbachia abundance is reduced leading to greater susceptibility to WNV in the subsequent generation of C. pipiens/restuans hosts. Different mosquito genera harbored significantly different bacterial communities, and presence or abundance of Wolbachia was primarily associated with these differences. We identified several operational taxonomic units (OTUs) of Wolbachia that drive overall microbial community differentiation among mosquito taxa, locations and timepoints. Distinct Wolbachia OTUs were consistently found to dominate microbiomes of Cx. pipiens/restuans, and of Coquilletidia perturbans. Seasonal fluctuations of several other microbial taxa included Bacillus cereus, Enterococcus, Methylobacterium, Asaia, Pantoea, Acinetobacter johnsonii, Pseudomonas, and Mycoplasma. This suggests that microbiota may explain some of the variation in vector competence previously attributed to local environmental processes, especially because Wolbachia is known to affect carriage of viral pathogens.

Disentangling a Holobiont - Recent Advances and Perspectives in Nasonia Wasps.

The parasitoid wasp genus Nasonia (Hymenoptera: Chalcidoidea) is a well-established model organism for insect development, evolutionary genetics, speciation, and symbiosis. The host-microbiota assemblage which constitutes the Nasonia holobiont (a host together with all of its associated microbes) consists of viruses, two heritable bacterial symbionts and a bacterial community dominated in abundance by a few taxa in the gut. In the wild, all four Nasonia species are systematically infected with the obligate intracellular bacterium Wolbachia and can additionally be co-infected with Arsenophonus nasoniae. These two reproductive parasites have different transmission modes and host manipulations (cytoplasmic incompatibility vs. male-killing, respectively). Pioneering studies on Wolbachia in Nasonia demonstrated that closely related Nasonia species harbor multiple and mutually incompatible Wolbachia strains, resulting in strong symbiont-mediated reproductive barriers that evolved early in the speciation process. Moreover, research on host-symbiont interactions and speciation has recently broadened from its historical focus on heritable symbionts to the entire microbial community. In this context, each Nasonia species hosts a distinguishable community of gut bacteria that experiences a temporal succession during host development and members of this bacterial community cause strong hybrid lethality during larval development. In this review, we present the Nasonia species complex as a model system to experimentally investigate questions regarding: (i) the impact of different microbes, including (but not limited to) heritable endosymbionts, on the extended phenotype of the holobiont, (ii) the establishment and regulation of a species-specific microbiota, (iii) the role of the microbiota in speciation, and (iv) the resilience and adaptability of the microbiota in wild populations subjected to different environmental pressures. We discuss the potential for easy microbiota manipulations in Nasonia as a promising experimental approach to address these fundamental aspects.

Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.

Getting the Hologenome Concept Right: an Eco-Evolutionary Framework for Hosts and Their Microbiomes.

Given the complexity of host-microbiota symbioses, scientists and philosophers are asking questions at new biological levels of hierarchical organization-what is a holobiont and hologenome? When should this vocabulary be applied? Are these concepts a null hypothesis for host-microbe systems or limited to a certain spectrum of symbiotic interactions such as host-microbial coevolution? Critical discourse is necessary in this nascent area, but productive discourse requires that skeptics and proponents use the same lexicon. For instance, critiquing the hologenome concept is not synonymous with critiquing coevolution, and arguing that an entity is not a primary unit of selection dismisses the fact that the hologenome concept has always embraced multilevel selection. Holobionts and hologenomes are incontrovertible, multipartite entities that result from ecological, evolutionary, and genetic processes at various levels. They are not restricted to one special process but constitute a wider vocabulary and framework for host biology in light of the microbiome.

Using "Omics" and Integrated Multi-Omics Approaches to Guide Probiotic Selection to Mitigate Chytridiomycosis and Other Emerging Infectious Diseases.

Emerging infectious diseases in wildlife are responsible for massive population declines. In amphibians, chytridiomycosis caused by Batrachochytrium dendrobatidis, Bd, has severely affected many amphibian populations and species around the world. One promising management strategy is probiotic bioaugmentation of antifungal bacteria on amphibian skin. In vivo experimental trials using bioaugmentation strategies have had mixed results, and therefore a more informed strategy is needed to select successful probiotic candidates. Metagenomic, transcriptomic, and metabolomic methods, colloquially called "omics," are approaches that can better inform probiotic selection and optimize selection protocols. The integration of multiple omic data using bioinformatic and statistical tools and in silico models that link bacterial community structure with bacterial defensive function can allow the identification of species involved in pathogen inhibition. We recommend using 16S rRNA gene amplicon sequencing and methods such as indicator species analysis, the Kolmogorov-Smirnov Measure, and co-occurrence networks to identify bacteria that are associated with pathogen resistance in field surveys and experimental trials. In addition to 16S amplicon sequencing, we recommend approaches that give insight into symbiont function such as shotgun metagenomics, metatranscriptomics, or metabolomics to maximize the probability of finding effective probiotic candidates, which can then be isolated in culture and tested in persistence and clinical trials. An effective mitigation strategy to ameliorate chytridiomycosis and other emerging infectious diseases is necessary; the advancement of omic methods and the integration of multiple omic data provide a promising avenue toward conservation of imperiled species.