Vincent Doublet

The Assay for Transposase‐Accessible Chromatin with sequencing (ATAC‐seq) is a versatile and widely utilized method for identifying potential regulatory regions, such as promoters and enhancers, within a genome. ATAC‐seq has been successfully applied to a wide range of established and emerging model organisms. However, implementing this method in emerging model systems, such as arthropods, can be challenging due to several factors that influence data quality. These factors include the availability of a sufficient amount and quality of tissue or cells, the need for species‐ and tissue‐specific protocol optimization, the completeness and accuracy of the reference genome, and the quality of the genome annotation. In this article, we emphasize the key steps in the ATAC‐seq protocol that, based on our experience, have the greatest impact on data quality when adapting this method for emerging model organisms. Specifically, we discuss the importance of nuclei isolation, the incubation conditions of the Tn5 transposase, and PCR amplification of the library. Furthermore, we outline essential quality checkpoints during the bioinformatic analysis of ATAC‐seq data to assist in assessing data integrity and consistency. Given that many emerging model organisms may not be readily available in laboratory cultures, we also emphasize the importance of evaluating how different preservation methods affect ATAC‐seq data quality. Based on examples in one spider and one ant species, we demonstrate that replication and thorough quality controls at all steps of the protocol and data analysis are essential to assess the usability of ATAC‐seq data. Our data highlights the importance of isolating the right number of intact nuclei, as well as ensuring optimal amplification conditions during library preparation to obtain good‐quality sequence data for downstream analyses. We recommend using fresh tissue samples if possible because we show that direct cryopreservation of the tissue may affect chromatin integrity. This effect could be avoided or reduced by preserving the homogenate in cell culture medium. Overall, we explain the ATAC‐seq protocol and downstream analyses in detail and give step‐by‐step advice to researchers who are new to the field and want to implement this method. With careful planning and validation, ATAC‐seq can reveal the regulatory landscape of a genome and aid in identifying elements that govern gene expression.

The composition of viral communities (i.e. viromes) can be dynamic and complex. Co-evolution may lead to virome host-specificity. However, eco-evolutionary factors may influence virome dynamics in wild host communities, potentially leading to disease emergence. Social bees are relevant models to address the drivers of virome composition: these important pollinators form multi-species assemblages, with high niche overlap and strong seasonality in their biotic interactions. We applied a microbial community approach to disentangle the role of host phylogeny and host ecology in shaping bee viromes, combining plant-pollinator networks with meta-transcriptomics, and small interfering RNAs as proxies for viral replication in pollinators and pollen. We identified over a hundred insect and plant viral sequences from ca. 4500 insect pollinator samples across three time points in one year. While host genetic distance drives the distribution of bee viruses, we find that plant-pollinator interactions and phenology drive plant virus communities collected by bees. This reveals the opportunities for virus spread in the bee assemblage. However, we show that transmission to multiple hosts is only realized for a fraction of insect viruses, with even fewer found to be actively replicating in multiple species, including the particularly virulent multi-host acute bee paralysis virus.

The growing urbanization process is accompanied by the emergence of new habitats for wildlife, and cities are sometimes seen as refuges for pollinators such as wild bees compared to intensively cultivated rural habitats. However, the contrasting living conditions that combine high fragmentation, exposure to pollutants, and heat island effects, with low pesticide use and potentially high availability of resources, make it difficult to predict the overall effect of urban living on the health of wild bees. Moreover, if the responses of wild bee populations in terms of species richness and diversity have been the focus of many recent studies, individual responses to urbanization have been more rarely investigated. More specifically, data on the impacts on individual bee physiology and health are lacking. To help fill this gap, we collected red‐tailed bumblebee (Bombus lapidarius) workers along a gradient of urbanization defined by the level of soil imperviousness, and estimated environmental (air quality) and ecological (pathogens' prevalence and loads; local competition) pressures they locally experienced. In parallel, we quantified the expression of selected immune marker genes. We measured how the immune system of bumblebees responds to urbanization gradient and which local parameters best explain the observed changes in immune gene expression. We evidenced three immune markers, tightly linked with cellular metabolism, whose expressions increase with the level of urbanization, independently of individual infection and pollution exposure. We suggest that induction of their expression reveals a shift in wild bee immunometabolism, supposedly in response to the stressful conditions experienced in areas with high built‐up cover. The induction of these genes is likely at the root of any immune activation; they could thus be used as markers to estimate the levels of urban stress locally experienced by pollinators.

Ecological restoration programs are established to reverse land degradation, mitigate biodiversity loss, and reinstate ecosystem services. Following recent agricultural intensification that led to a decrease in flower diversity and density in rural areas and subsequently to the decline of many insects, conservation measures targeted at pollinators have been established, including sown wildflower strips (WFS) along field margins. Historically successful in establishing a high density of generalist bees and increasing pollinator diversity, the impact of enhanced flower provision on wider ecological interactions and the structure of pollinator networks has been rarely investigated. Here, we tested the effects of increasing flower species richness and flower density in agricultural landscapes on bee‐plant interaction networks. We measured plant species richness and flower density and surveyed honeybee and bumblebee visits on flowers across a range of field margins on 10 UK farms that applied different pollinator conservation measures. We found that both flower species richness and flower density significantly increased bee abundance, in early and late summer, respectively. At the network level, we found that higher flower species richness did not significantly alter bee species' generality indices, but significantly reduced network connectance and marginally reduced niche overlap across honeybees and bumblebee species, a proxy for insect competition. While higher connectance and niche overlap is believed to strengthen network robustness and often is the aim for the restoration of pollinator networks, we argue that carefully designed WFS may benefit bees by partitioning their foraging niche, limiting competition for resources and the potential for disease transmission via shared floral use. We also discuss the need to extend WFS and their positive effects into spring when wild bee populations are established.

Genetic variation for both resistance and disease tolerance has been described in a range of species infected with bacterial, viral and fungal pathogens. In Drosophila melanogaster , genetic variation in mortality following systemic Drosophila C Virus (DCV) infection has been shown to be driven by large effect polymorphisms in the viral restriction factor pastrel (pst) . However, it is unclear if pst impacts variation in DCV titres (i.e. resistance), or if it also contributes to disease tolerance. We investigated systemic infection across a range of DCV challenge doses spanning nine orders of magnitude, in males and females of ten Drosophila Genetic Reference Panel (DGRP) lines carrying either a susceptible (S) or resistant (R) pst allele. Our results uncover among-line variation in fly survival, viral titers, and disease tolerance measured both as the ability to maintain survival (mortality tolerance) and reproduction (fecundity tolerance). We confirm the role of pst in resistance, as fly lines with the resistant (R) pst allele experienced lower viral titers, and we uncover novel effects of pst on host vigor, as flies carrying the R allele exhibited higher survival and fecundity even in the absence of infection. Finally, we found significant variation in the expression of the JAK-STAT ligand upd3 and the epigenetic regulator of JAK-STAT G9a . While G9a has been previously shown to mediate tolerance of DCV infection, we found no correlation between the expression of either upd3 or G9a on fly tolerance or resistance. Our work highlights the importance of both resistance and tolerance in viral defence.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission byVarroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

The ratio of amino acids to carbohydrates (AA:C) that bumble bees consume has been reported to affect their survival. However, it is unknown how dietary AA:C ratio affects other bumble bee fitness traits (e.g., fecundity, condition) and possible trade-offs between them. Moreover, while individual AAs affect phenotype in many species, the effects of AA blend on bumble bee fitness and food intake are unclear. We test how the AA:C ratio that bumble bees (Bombus terrestris) consume affects their condition (abdomen lipid and dry mass), survival following food removal, and ovarian activation. We then compare ovarian activation and food intake in bees fed identical AA:C ratios, but where the blend of AAs in diets differ, i.e., diets contained the same 10 AAs in an equimolar ratio or in the same ratio as in bee collected pollen. We found that AA:C ratio did not significantly affect survival following food removal or ovarian activation; however, high AA intake increased body mass, which is positively correlated with multiple fitness traits in bumble bees. AA blend (i.e., equimolar versus pollen) did not significantly affect overall ovarian activation or consumption of each experimental diet. However, there was an interaction between AA mix and dietary AA:C ratio affecting survival during the feeding experiment, and signs that there may have been weak, interactive effects of AA mix and AA:C ratio on food consumption. These results suggest that the effect of total AA intake on bumble bee phenotype may depend on the blend of individual AAs in experimental diets. We suggest that research exploring how AA blend affects bumble bee performance and dietary intake is warranted, and highlight that comparing research on bee nutrition is complicated by even subtle variation in experimental diet composition.

In the past centuries, viruses have benefited from globalization to spread across the globe, infecting new host species and populations. A growing number of viruses have been documented in the western honey bee, Apis mellifera. Several of these contribute significantly to honey bee colony losses. This review synthetizes the knowledge of the diversity and distribution of honey-bee-infecting viruses, including recent data from high-throughput sequencing (HTS). After presenting the diversity of viruses and their corresponding symptoms, we surveyed the scientific literature for the prevalence of these pathogens across the globe. The geographical distribution shows that the most prevalent viruses (deformed wing virus, sacbrood virus, black queen cell virus and acute paralysis complex) are also the most widely distributed. We discuss the ecological drivers that influence the distribution of these pathogens in worldwide honey bee populations. Besides the natural transmission routes and the resulting temporal dynamics, global trade contributes to their dissemination. As recent evidence shows that these viruses are often multihost pathogens, their spread is a risk for both the beekeeping industry and the pollination services provided by managed and wild pollinators.

Deformed wing virus (DWV) is an emerging infectious disease of the honey bee (Apis mellifera) that is considered a major cause of elevated losses of honey bee colonies. DWV comprises two widespread genotypes: the originally described genotype A, and genotype B. In adult honey bees, DWV-B has been shown to be more virulent than DWV-A. However, their comparative effects on earlier host developmental stages are unknown. Here, we experimentally inoculated honey bee pupae and tested for the relative impact of DWV-A versus DWV-B on mortality and wing deformities in eclosing adults. DWV-A and DWV-B caused similar, and only slightly elevated, pupal mortality (mean 18% greater mortality than control). Both genotypes caused similarly high wing deformities in eclosing adults (mean 60% greater wing deformities than control). Viral titer was high in all of the experimentally inoculated eclosing adults, and was independent of wing deformities, suggesting that the phenotype ‘deformed wings’ is not directly related to viral titer or viral genotype. These viral traits favor the emergence of both genotypes of DWV by not limiting the reproduction of its vector, the ectoparasitic Varroa destructor mite, in infected pupae, and thereby facilitating the spread of DWV in honey bees infested by the mite.