Horizontal gene transfer-mediated bacterial strain variation affects host fitness in Drosophila.

BACKGROUND: How microbes affect host fitness and environmental adaptation has become a fundamental research question in evolutionary biology. To better understand the role of microbial genomic variation for host fitness, we tested for associations of bacterial genomic variation and Drosophila melanogaster offspring number in a microbial Genome Wide Association Study (GWAS). RESULTS: We performed a microbial GWAS, leveraging strain variation in the genus Gluconobacter, a genus of bacteria that are commonly associated with Drosophila under natural conditions. We pinpoint the thiamine biosynthesis pathway (TBP) as contributing to differences in fitness conferred to the fly host. While an effect of thiamine on fly development has been described, we show that strain variation in TBP between bacterial isolates from wild-caught D. melanogaster contributes to variation in offspring production by the host. By tracing the evolutionary history of TBP genes in Gluconobacter, we find that TBP genes were most likely lost and reacquired by horizontal gene transfer (HGT). CONCLUSION: Our study emphasizes the importance of strain variation and highlights that HGT can add to microbiome flexibility and potentially to host adaptation.

The role of tissue-specific microbiota in initial establishment success of Pacific oysters.

Microbiota can have positive and negative effects on hosts depending on the environmental conditions. Therefore, it is important to decipher host-microbiota-environment interactions, especially under natural conditions exerting (a)biotic stress. Here, we assess the relative importance of microbiota in different tissues of Pacific oyster for its successful establishment in a new environment. We transplanted oysters from the Southern to the Northern Wadden Sea and controlled for the effects of resident microbiota by administering antibiotics to half of the oysters. We then followed survival and composition of haemolymph, mantle, gill and gut microbiota in local and translocated oysters over 5 days. High mortality was recorded only in non-antibiotic-treated translocated oysters, where high titres of active Vibrio sp. in solid tissues indicated systemic infections. Network analyses revealed the highest connectivity and a link to seawater communities in the haemolymph microbiota. Since antibiotics decreased modularity and increased connectivity of the haemolymph-based networks, we propose that community destabilization in non-treated translocated oysters could be attributed to interactions between resident and external microbiota, which in turn facilitated passage of vibrios into solid tissues and invoked disease. These interactions of haemolymph microbiota with the external and internal environment may thus represent an important component of oyster fitness.

Noninvasive analysis of microbiome dynamics in the fruit fly Drosophila melanogaster.

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.

The nucleotide-binding oligomerization domain-like receptor NLRC5 is involved in IFN-dependent antiviral immune responses.

Nucleotide-binding oligomerization domain-like receptors (NLRs) are a group of intracellular proteins that mediate recognition of pathogen-associated molecular patterns or other cytosolic danger signals. Mutations in NLR genes have been linked to a variety of inflammatory diseases, underscoring their pivotal role in host defense and immunity. This report describes the genomic organization and regulation of the human NLR family member NLRC5 and aspects of cellular function of the encoded protein. We have analyzed the tissue-specific expression of NLRC5 and have characterized regulatory elements in the NLRC5 promoter region that are responsive to IFN-gamma. We show that NLRC5 is upregulated in human fibroblasts postinfection with CMV and demonstrate the role of a JAK/STAT-mediated autocrine signaling loop involving IFN-gamma. We demonstrate that overexpression and enforced oligomerization of NLRC5 protein results in activation of the IFN-responsive regulatory promoter elements IFN-gamma activation sequence and IFN-specific response element and upregulation of antiviral target genes (e.g., IFN-alpha, OAS1, and PRKRIR). Finally, we demonstrate the effect of small interfering RNA-mediated knockdown of NLRC5 on a target gene level in the context of viral infection. We conclude that NLRC5 may represent a molecular switch of IFN-gamma activation sequence/IFN-specific response element signaling pathways contributing to antiviral defense mechanisms.

Characterization of the microbiome of the invasive Asian toad in Madagascar across the expansion range and comparison with a native co-occurring species.

Biological invasions are on the rise, with each invader carrying a plethora of associated microbes. These microbes play important, yet poorly understood, ecological roles that can include assisting the hosts in colonization and adaptation processes or as possible pathogens. Understanding how these communities differ in an invasion scenario may help to understand the host's resilience and adaptability. The Asian common toad, Duttaphrynus melanostictus is an invasive amphibian, which has recently established in Madagascar and is expected to pose numerous threats to the native ecosystems. We characterized the skin and gut bacterial communities of D. melanostictus in Toamasina (Eastern Madagascar), and compared them to those of a co-occurring native frog species, Ptychadena mascareniensis, at three sites where the toad arrived in different years. Microbial composition did not vary among sites, showing that D. melanostictus keeps a stable community across its expansion but significant differences were observed between these two amphibians. Moreover, D. melanostictus had richer and more diverse communities and also harboured a high percentage of total unique taxa (skin: 80%; gut: 52%). These differences may reflect the combination of multiple host-associated factors including microhabitat selection, skin features and dietary preferences.

Common structuring principles of the Drosophila melanogaster microbiome on a continental scale and between host and substrate.

The relative importance of host control, environmental effects and stochasticity in the assemblage of host-associated microbiomes is being debated. We analysed the microbiome among fly populations that were sampled across Europe by the European Drosophila Population Genomics Consortium (DrosEU). In order to better understand the structuring principles of the natural D. melanogaster microbiome, we combined environmental data on climate and food-substrate with dense genomic data on host populations and microbiome profiling. Food-substrate, temperature, and host population structure correlated with microbiome structure. Microbes, whose abundance was co-structured with host populations, also differed in abundance between flies and their substrate in an independent survey. This finding suggests common, host-related structuring principles of the microbiome on different spatial scales.

Microbial embryonal colonization during pipefish male pregnancy.

While originally acquired from the environment, a fraction of the microbiota is transferred from parents to offspring. The immune system shapes the microbial colonization, while commensal microbes may boost host immune defences. Parental transfer of microbes in viviparous animals remains ambiguous, as the two transfer routes (transovarial vs. pregnancy) are intermingled within the maternal body. Pipefishes and seahorses (syngnathids) are ideally suited to disentangle transovarial microbial transfer from a contribution during pregnancy due to their maternal egg production and their unique male pregnancy. We assessed the persistency and the changes in the microbial communities of the maternal and paternal reproductive tracts over proceeding male pregnancy by sequencing microbial 16S rRNA genes of swabs from maternal gonads and brood pouches of non-pregnant and pregnant fathers. Applying parental immunological activation with heat-killed bacteria, we evaluated the impact of parental immunological status on microbial development. Our data indicate that maternal gonads and paternal brood pouches harbor distinct microbial communities, which could affect embryonal development in a sex-specific manner. Upon activation of the immune system, a shift of the microbial community was observed. The activation of the immune system induced the expansion of microbiota richness during late pregnancy, which corresponds to the time point of larval mouth opening, when initial microbial colonization must take place.

Microbiota-based analysis reveals specific bacterial traits and a novel strategy for the diagnosis of infectious infertility.

Tubal factor infertility (TFI) accounts for more than 30% of the cases of female infertility and mostly resides from an inflammatory process triggered by an infection. Clinical appearances largely differ, and very often infections are not recognized or remain completely asymptomatic over time. Here, we characterized the microbial pattern in females diagnosed with infectious infertility (ININF) in comparison to females with non-infectious infertility (nININF), female sex workers (FSW) and healthy controls (fertile). Females diagnosed with infectious infertility differed significantly in the seroprevalence of IgG antibodies against the C. trachomatis proteins MOMP, OMP2, CPAF and HSP60 when compared to fertile females. Microbiota analysis using 16S amplicon sequencing of cervical swabs revealed significant differences between ININF and fertile controls in the relative read count of Gardnerella (10.08% vs. 5.43%). Alpha diversity varies among groups, which are characterized by community state types including Lactobacillus-dominated communities in fertile females, an increase in diversity in all the other groups and Gardnerella-dominated communities occurring more often in ININF. While all single parameters did not allow predicting infections as the cause of infertility, including C. trachomatis IgG/IgA status together with 16S rRNA gene analysis of the ten most frequent taxa a total of 93.8% of the females were correctly classified. Further studies are needed to unravel the impact of the cervical microbiota in the pathogenesis of infectious infertility and its potential for identifying females at risk earlier in life.

Spatial and Temporal Dynamics of Pacific Oyster Hemolymph Microbiota across Multiple Scales.

Unveiling the factors and processes that shape the dynamics of host associated microbial communities (microbiota) under natural conditions is an important part of understanding and predicting an organism's response to a changing environment. The microbiota is shaped by host (i.e., genetic) factors as well as by the biotic and abiotic environment. Studying natural variation of microbial community composition in multiple host genetic backgrounds across spatial as well as temporal scales represents a means to untangle this complex interplay. Here, we combined a spatially-stratified with a longitudinal sampling scheme within differentiated host genetic backgrounds by reciprocally transplanting Pacific oysters between two sites in the Wadden Sea (Sylt and Texel). To further differentiate contingent site from host genetic effects, we repeatedly sampled the same individuals over a summer season to examine structure, diversity and dynamics of individual hemolymph microbiota following experimental removal of resident microbiota by antibiotic treatment. While a large proportion of microbiome variation could be attributed to immediate environmental conditions, we observed persistent effects of antibiotic treatment and translocation suggesting that hemolymph microbial community dynamics is subject to within-microbiome interactions and host population specific factors. In addition, the analysis of spatial variation revealed that the within-site microenvironmental heterogeneity resulted in high small-scale variability, as opposed to large-scale (between-site) stability. Similarly, considerable within-individual temporal variability was in contrast with the overall temporal stability at the site level. Overall, our longitudinal, spatially-stratified sampling design revealed that variation in hemolymph microbiota is strongly influenced by site and immediate environmental conditions, whereas internal microbiome dynamics and oyster-related factors add to their long-term stability. The combination of small and large scale resolution of spatial and temporal observations therefore represents a crucial but underused tool to study host-associated microbiome dynamics.

High-Throughput, Amplicon-Based Sequencing of the CREBBP Gene as a Tool to Develop a Universal Platform-Independent Assay.

High-throughput sequencing technologies are widely used to analyse genomic variants or rare mutational events in different fields of genomic research, with a fast development of new or adapted platforms and technologies, enabling amplicon-based analysis of single target genes or even whole genome sequencing within a short period of time. Each sequencing platform is characterized by well-defined types of errors, resulting from different steps in the sequencing workflow. Here we describe a universal method to prepare amplicon libraries that can be used for sequencing on different high-throughput sequencing platforms. We have sequenced distinct exons of the CREB binding protein (CREBBP) gene and analysed the output resulting from three major deep-sequencing platforms. platform-specific errors were adjusted according to the result of sequence analysis from the remaining platforms. Additionally, bioinformatic methods are described to determine platform dependent errors. Summarizing the results we present a platform-independent cost-efficient and timesaving method that can be used as an alternative to commercially available sample-preparation kits.

Draft Genome Sequences of 11 Staphylococcus epidermidis Strains Isolated from Wild Mouse Species.

We report here the draft genome sequences of 11 strains of Staphylococcus epidermidis, a common bacterium inhabiting the skin of humans and other animals. These isolates, obtained from five mouse species, provide valuable information on the native Staphylococcus spp. of this important model organism and form a basis for studying host-bacterial interactions in their natural environment.

Female mate choice can drive the evolution of high frequency echolocation in bats: a case study with Rhinolophus mehelyi.

Animals employ an array of signals (i.e. visual, acoustic, olfactory) for communication. Natural selection favours signals, receptors, and signalling behaviour that optimise the received signal relative to background noise. When the signal is used for more than one function, antagonisms amongst the different signalling functions may constrain the optimisation of the signal for any one function. Sexual selection through mate choice can strongly modify the effects of natural selection on signalling systems ultimately causing maladaptive signals to evolve. Echolocating bats represent a fascinating group in which to study the evolution of signalling systems as unlike bird songs or frog calls, echolocation has a dual role in foraging and communication. The function of bat echolocation is to generate echoes that the calling bat uses for orientation and food detection with call characteristics being directly related to the exploitation of particular ecological niches. Therefore, it is commonly assumed that echolocation has been shaped by ecology via natural selection. Here we demonstrate for the first time using a novel combined behavioural, ecological and genetic approach that in a bat species, Rhinolophus mehelyi: (1) echolocation peak frequency is an honest signal of body size; (2) females preferentially select males with high frequency calls during the mating season; (3) high frequency males sire more off-spring, providing evidence that echolocation calls may play a role in female mate choice. Our data refute the sole role of ecology in the evolution of echolocation and highlight the antagonistic interplay between natural and sexual selection in shaping acoustic signals.

Environmentally determined differences in the murine lung microbiota and their relation to alveolar architecture.

Commensal bacteria control the micro-ecology of metazoan epithelial surfaces with pivotal effect on tissue homeostasis and host defense. In contrast to the upper respiratory tract, the lower respiratory tract of healthy individuals has largely been considered free of microorganisms. To understand airway micro-ecology we studied microbiota of sterilely excised lungs from mice of different origin including outbred wild mice caught in the natural environment or kept under non-specific-pathogen-free (SPF) conditions as well as inbred mice maintained in non-SPF, SPF or germ-free (GF) facilities. High-throughput pyrosequencing of reverse transcribed 16S rRNA revealed metabolically active murine lung microbiota in all but GF mice. The overall composition across samples was similar at the phylum and family level. However, species richness was significantly different between lung microbiota from SPF and non-SPF mice. Non-cultivatable Betaproteobacteria such as Ralstonia spp. made up the major constituents and were also confirmed by 16S rRNA gene cloning analysis. Additionally, Pasteurellaceae, Enterobacteria and Firmicutes were isolated from lungs of non-SPF mice. Bacterial communities were detectable by fluorescent in situ hybridization (FISH) at alveolar epithelia in the absence of inflammation. Notably, higher bacterial abundance in non-SPF mice correlated with more and smaller size alveolae, which was corroborated by transplanting Lactobacillus spp. lung isolates into GF mice. Our data indicate a common microbial composition of murine lungs, which is diversified through different environmental conditions and affects lung architecture. Identification of the microbiota of murine lungs will pave the path to study their influence on pulmonary immunity to infection and allergens using mouse models.