Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction.

Background: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. Methods: High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. Results: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. Conclusion: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species.

With a little help from my friends: the roles of microbial symbionts in insect populations and communities.

To understand insect abundance, distribution and dynamics, we need to understand the relevant drivers of their populations and communities. While microbial symbionts are known to strongly affect many aspects of insect biology, we lack data on their effects on populations or community processes, or on insects' evolutionary responses at different timescales. How these effects change as the anthropogenic effects on ecosystems intensify is an area of intense research. Recent developments in sequencing and bioinformatics permit cost-effective microbial diversity surveys, tracking symbiont transmission, and identification of functions across insect populations and multi-species communities. In this review, we explore how different functional categories of symbionts can influence insect life-history traits, how these effects could affect insect populations and their interactions with other species, and how they may affect processes and patterns at the level of entire communities. We argue that insect-associated microbes should be considered important drivers of insect response and adaptation to environmental challenges and opportunities. We also outline the emerging approaches for surveying and characterizing insect-associated microbiota at population and community scales. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Limited congruence in phylogeographic patterns observed for riverine predacious beetles sharing distribution along the mountain rivers.

Riverine predacious beetles (RPB) (Carabidae, Staphylinidae) are highly diverse and numerous elements of riverine ecosystems. Their historical and contemporary distribution and diversity are highly dependent on natural flow regimes and topography of watercourses. Despite broad knowledge of their ecology, data on population genetic diversity and connectivity are lacking. This study aimed to fill this gap in order to solve two principal hypotheses assuming (i) congruence of phylogeographic patterns observed for RPB indicating that they share a common history and the ecological adaptations to the dynamic environment, (ii) genetic structuration of populations according to river basins. The Carpathian populations of four ground beetles and three rove beetles were examined using cytochrome oxidase and arginine kinase sequencing. There are substantial differences in RPB demographic history and current genetic diversity. Star-like phylogeny of Bembidion and complex haplotype networks of Paederus/Paederidus, with some haplotypes being drainage-specific and others found in distant populations, indicate a general lack of isolation by distance. Signs of recent demographic expansion were detected for most RPB with the latest population collapse for some rove beetles. To some extent, migration of examined species has to be limited by watersheds. Observed phylogeographic patterns are essential for correctly understanding RPB meta-population functioning.

Till evolution do us part: The diversity of symbiotic associations across populations of Philaenus spittlebugs.

Symbiotic bacteria have played crucial roles in the evolution of sap-feeding insects and can strongly affect host function. However, their diversity and distribution within species are not well understood; we do not know to what extent environmental factors or associations with other species may affect microbial community profiles. We addressed this question in Philaenus spittlebugs by surveying both insect and bacterial marker gene amplicons across multiple host populations. Host mitochondrial sequence data confirmed morphology-based identification of six species and revealed two divergent clades of Philaenus spumarius. All of them hosted the primary symbiont Sulcia that was almost always accompanied by Sodalis. Interestingly, populations and individuals often differed in the presence of Sodalis sequence variants, suggestive of intra-genome 16S rRNA variant polymorphism combined with rapid genome evolution and/or recent additional infections or replacements of the co-primary symbiont. The prevalence of facultative endosymbionts, including Wolbachia, Rickettsia, and Spiroplasma, varied among populations. Notably, cytochrome I oxidase (COI) amplicon data also showed that nearly a quarter of P. spumarius were infected by parasitoid flies (Verralia aucta). One of the Wolbachia operational taxonomic units (OTUs) was exclusively present in Verralia-parasitized specimens, suggestive of parasitoids as their source and highlighting the utility of host gene amplicon sequencing in microbiome studies.

N-Cadherin Is Critical for the Survival of Germ Cells, the Formation of Steroidogenic Cells, and the Architecture of Developing Mouse Gonads.

Normal gonad development assures the fertility of the individual. The properly functioning gonads must contain a sufficient number of the viable germ cells, possess a correct architecture and tissue structure, and assure the proper hormonal regulation. This is achieved by the interplay between the germ cells and different types of somatic cells. N-cadherin coded by the Cdh2 gene plays a critical role in this interplay. To gain an insight into the role of N-cadherin in the development of mouse gonads, we used the Cre-loxP system to knock out N-cadherin separately in two cell lines: the SF1+ somatic cells and the OCT4+ germ cells. We observed that N-cadherin plays a key role in the survival of both female and male germ cells. However, the N-cadherin is not necessary for the differentiation of the Sertoli cells or the initiation of the formation of testis cords or ovigerous cords. In the later stages of gonad development, N-cadherin is important for the maintenance of testis cord structure and is required for the formation of steroidogenic cells. In the ovaries, N-cadherin is necessary for the formation of the ovarian follicles. These results indicate that N-cadherin plays a major role in gonad differentiation, structuralization, and function.

Tissue-specific knockout of E-cadherin (Cdh1) in developing mouse gonads causes germ cells loss.

The normal course of gonad development is critical for the sexual development and reproductive capacity of the individual. During development, an incipient bipotential gonad which consists of unorganized aggregate of cells, must differentiate into highly structured testis or ovary. Cell adhesion molecules (CAMs) are a group of proteins crucial for segregation and aggregation of different cell types to form different tissues. E-cadherin (Cdh1) is one of the CAMs expressed in the developing gonads. We used tissue-specific knockout of Cdh1 gene in OCT4+ germ cells and, separately, in SF1+ somatic cells of developing gonads. The knockout of E-cadherin in somatic cells caused decrease in the number of germ cells, while the knockout in the germ cells caused their almost complete loss. Thus, the presence of E-cadherin in both the germ and somatic cells is necessary for the survival of germ cells. Although the lack of E-cadherin did not impair cell proliferation, it enhanced apoptosis, which was a possible cause of germ cell loss. However, the somatic cells of the gonad differentiated normally into Sertoli cells in the testis cords, and into follicular cells in the ovaries. The testis and ovigerous cords maintained their integrity; they were covered by continuous basement membranes. The testicular interstitium with steroidogenic fetal Leydig cells did not show any noticeable changes. However, in the female gonads, because of the lack of germ cells, the ovarian follicles were absent. The sex determination and sexual differentiation of the gonad were not impaired. These results underscore an important role of E-cadherin in germ cell survival and gonad development.

How Hosts Taxonomy, Trophy, and Endosymbionts Shape Microbiome Diversity in Beetles.

Bacterial communities play a crucial role in the biology, ecology, and evolution of multicellular organisms. In this research, the microbiome of 24 selected beetle species representing five families (Carabidae, Staphylinidae, Curculionidae, Chrysomelidae, Scarabaeidae) and three trophic guilds (carnivorous, herbivorous, detrivorous) was examined using 16S rDNA sequencing on the Illumina platform. The aim of the study was to compare diversity within and among species on various levels of organization, including evaluation of the impact of endosymbiotic bacteria. Collected data showed that beetles possess various bacterial communities and that microbiota of individuals of particular species hosts are intermixed. The most diverse microbiota were found in Carabidae and Scarabaeidae; the least diverse, in Staphylinidae. On higher organization levels, the diversity of bacteria was more dissimilar between families, while the most distinct with respect to their microbiomes were trophic guilds. Moreover, eight taxa of endosymbiotic bacteria were detected including common genera such as Wolbachia, Rickettsia, and Spiroplasma, as well as the rarely detected Cardinium, Arsenophonus, Buchnera, Sulcia, Regiella, and Serratia. There were no correlations among the abundance of the most common Wolbachia and Rickettsia; a finding that does not support the hypothesis that these bacteria occur interchangeably. The abundance of endosymbionts only weakly and negatively correlates with diversity of the whole microbiome in beetles. Overall, microbiome diversity was found to be more dependent on host phylogeny than on the abundance of endosymbionts. This is the first study in which bacteria diversity is compared between numerous species of beetles in a standardized manner.

Using host species traits to understand the Wolbachia infection distribution across terrestrial beetles.

Knowledge of Wolbachia prevalence with respect to its hosts is restricted mainly to taxonomic/phylogenetic context. In contrast, relations between infection and most host's ecological and biological traits are poorly understood. This study aimed to elaborate on relations between bacteria and its beetle hosts in taxonomic and the ecological contexts. In particular, the goal is to verify which ecological and biological traits of beetles could cause them to be prone to be infected. Verification of Wolbachia infection status across 297 beetle taxa showed that approximately 27% of taxa are infected by supergroups A and B. Only minor support for coevolution between bacteria and its beetle hosts was observed in some genera of beetles, but in general coevolution between beetles and Wolbachia was rejected. Some traits of beetles were found to be unrelated to Wolbachia prevalence (type of range and thermal preferences); some traits were related with ambiguous effects (habitats, distribution, mobility and body size); some were substantially related (reproduction mode and trophy). The aforementioned summary does not show obvious patterns of Wolbachia prevalence and diversity in relation to host taxonomy, biology, and ecology. As both Wolbachia and Coleoptera are diverse groups, this lack of clear patterns is probably a reflection of nature, which is characterised by highly diversified and probably unstable relations.

Transcriptional profiling validates involvement of extracellular matrix and proteinases genes in mouse gonad development.

Extracellular matrix (ECM) plays an important scaffolding role in the establishment of organs structure during development. A great number of ECM components and enzymes (proteinases) regulating formation/degradation of ECM during organ remodeling have been identified. In order to study the role of ECM in the mouse gonad development, especially during sexual differentiation of the gonads when the structure of the testis and ovary becomes established, we performed a global analysis of transcriptome in three main cell types of developing gonad (supporting, interstitial/stromal and germ cells) using transgenic mice, cell sorting and microarray. The genes coding for ECM components were mostly expressed in two gonadal cell lines: supporting and interstitial/stromal cells. These two cell lines differed in the expression pattern of ECM components, which suggests that ECM components might be crucial for differentiation of gonad compartments (for example testis cords vs. interstitium in XY gonads). Collagens and proteoglycans coding genes were mainly expressed in the interstitium/stromal cells, while non-collagen glycoproteins and matricellular coding genes were expressed in both cell lines. We also analyzed the expression of genes encoding ECM enzymes that are secreted to the ECM where they remodel the scaffolding of developing organs. We found that the ECM enzyme genes were also mostly expressed in supporting and interstitial/stromal cells. In contrast to the somatic cells, the germ cells expressed only limited number of ECM components and enzymes. This suggests that the germ line cells do not participate, or play only a minor role, in the sculpting of the gonad structure via ECM synthesis and remodeling. Importantly, the supporting cells showed the sex-specific pattern of expression of ECM components. However, the pattern of expression of most ECM enzymes in the somatic and germ cells is independent on the sex of the gonad. Further studies are required to elucidate the exact roles of identified genes in sexual differentiation of the gonads.

Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary.

Reconstructed historical distribution and phylogeography unravels non-steppic origin of Caucasotachea vindobonensis (Gastropoda: Helicidae).

Existing data on the phylogeography of European taxa of steppic provenance suggests that species were widely distributed during glacial periods but underwent range contraction and fragmentation during interglacials into "warm-stage refugia." Among the steppe-related invertebrates that have been examined, the majority has been insects, but data on the phylogeography of snails is wholly missing. To begin to fill this gap, phylogeographic and niche modeling studies on the presumed steppic snail Caucasotachea vindobonensis were conducted. Surprisingly, reconstruction of ancestral areas suggests that extant C. vindobonensis probably originated in the Balkans and survived there during the Late Pleistocene glaciations, with a more recent colonization of the Carpatho-Pannonian and the Ponto-Caspian regions. In the Holocene, C. vindobonensis colonized between the Sudetes and the Carpathians to the north, where its recent and current distribution may have been facilitated by anthropogenic translocations. Together, these data suggest a possible non-steppic origin of C. vindobonensis. Further investigation may reveal the extent to which the steppic snail assemblages consist partly of Holocene newcomers.

No evidence for reproductive isolation through sexual conflict in the bulb mite Rhizoglyphus robini.

Sexual conflict leading to sexual antagonistic coevolution has been hypothesized to drive reproductive isolation in allopatric populations and hence lead to speciation. However, the generality of this speciation mechanism is under debate. We used experimental evolution in the bulb mite Rhizoglyphusrobini to investigate whether sexual conflict promotes reproductive isolation measured comprehensively to include all possible pre- and post-zygotic mechanisms. We established replicate populations in which we either enforced monogamy, and hence removed sexual conflict by making male and female evolutionary interests congruent, or allowed promiscuity. After 35 and 45 generations of experimental evolution, we found no evidence of reproductive isolation between the populations in any of the mating systems. Our results indicate that sexual conflict does not necessarily drive fast reproductive isolation and it may not be a ubiquitous mechanism leading to speciation.