Differential expression pattern of Vago in bumblebee (Bombus terrestris), induced by virulent and avirulent virus infections

Viruses are one of the main drivers of the decline of domesticated and wild bees but the mechanisms of antiviral immunity in pollinators are poorly understood. Recent work has suggested that next to the small interfering RNA (siRNA) pathway other immune-related pathways play a role in the defense of the bee hosts against viral infection. In addition, Vago plays a role in the cross-talk between the innate immune pathways in Culex mosquito cells. Here we describe the Vago orthologue in bumblebees of Bombus terrestris, and investigated its role upon the infection of two different bee viruses, the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that BtVago was downregulated upon the infection of IAPV that killed all bumblebees, but not with SBPV where the workers survived the virus infection. Thus, for the first time, Vago/Vago-like expression appears to be associated with the virulence of virus and may act as a modulator of antiviral immunity

Induction of RNAi core machinery's gene expression by exogenous dsRNA and the effects of pre-exposure to dsRNA on the gene silencing efficiency in the pea aphid (Acyrthosiphon pisum)

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and biological model organism, and RNA interference (RNAi) is an important tool for functional genomics and for insect pest management. However, the efficiency of RNAi in pea aphids is variable, limiting its application in aphids. In this study, we present optimized conditions for inducing and increasing the gene silencing efficiency of RNAi in pea aphids. The optimal gene silencing of the target Aphunchback gene was achieved by injecting 600 ng double-stranded (ds) RNA, and the highest mRNA depletion rate (74%) was detected at 36 h after injection. Moreover, the same gene silencing conditions were used to achieve transcript silencing for nine different genes in the pea aphid, although the silencing efficiencies for the different genes varied. Furthermore, the pre-exposure of aphids to dsRNA (600 ng dsGFP) led to significant hunchback silencing following a secondary exposure to 60 ng of dshunchback, a dose which did not lead to gene silencing when independently injected. The information presented here can be exploited to develop more efficient RNAi bioassays for pea aphids, both as gene functional study tools and an insect pest control strategy

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Infections of virulent and avirulent viruses differentially influenced the expression of dicer-1, ago-1, and microRNAs in Bombus terrestris

The microRNA (miRNA) pathway is well established to be involved in host-pathogen interactions. As key insect pollinators, bees are suffering from widely spreading viruses, especially honeybees and bumblebees. In order to better understand bee-virus interaction, we comparatively analyzed the involvement of the bumblebee miRNA pathway upon infection by two different viruses. In our setup, an avirulent infection is induced by slow bee paralysis virus (SBPV) and a virulent infection is induced by Israeli acute paralysis virus (IAPV). Our results showed the increased expressions of dicer-1 and ago-1 upon SBPV infection. There were 17 and 12 bumblebee miRNAs differentially expressed upon SBPV and IAPV infections, respectively. These results may indicate the involvement of the host miRNA pathway in bumblebee-virus interaction. However, silencing of dicer-1 did not influence the genome copy number of SBPV. Target prediction for these differentially expressed miRNAs showed their possible involvement in targeting viral genomic RNA and in the regulation of networks in bumblebee. Our study opens a new insight into bee-virus interaction meditated by host miRNAs

A depauperate immune repertoire precedes evolution of sociality in bees

Background Sociality has many rewards, but can also be dangerous, as high population density and low genetic diversity, common in social insects, is ideal for parasite transmission. Despite this risk, honeybees and other sequenced social insects have far fewer canonical immune genes relative to solitary insects. Social protection from infection, including behavioral responses, may explain this depauperate immune repertoire. Here, based on full genome sequences, we describe the immune repertoire of two ecologically and commercially important bumblebee species that diverged approximately 18 million years ago, the North American Bombus impatiens and European Bombus terrestris. Results We find that the immune systems of these bumblebees, two species of honeybee, and a solitary leafcutting bee, are strikingly similar. Transcriptional assays confirm the expression of many of these genes in an immunological context and more strongly in young queens than males, affirming Bateman’s principle of greater investment in female immunity. We find evidence of positive selection in genes encoding antiviral responses, components of the Toll and JAK/STAT pathways, and serine protease inhibitors in both social and solitary bees. Finally, we detect many genes across pathways that differ in selection between bumblebees and honeybees, or between the social and solitary clades. Conclusions The similarity in immune complement across a gradient of sociality suggests that a reduced immune repertoire predates the evolution of sociality in bees. The differences in selection on immune genes likely reflect divergent pressures exerted by parasites across social contexts

Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris

To date, there are no validated internal reference genes for the normalization of RT-qPCR data from virus infection experiments with pollinating insects. In this study we evaluated the stability of five candidate internal reference genes: elongation factor-1 -alpha (ELF1 alpha), peptidylprolyl isomerase A (PPIA), 60S ribosomal protein L23 (RPL23), TATA-binding protein (TBP) and polyubiquitin (UBI), in relation to Israeli acute paralysis virus (IAPV) infection of Bombus terrestris. We investigated the stability of these genes: in whole bodies and individual body parts, as well as in whole bodies collected at different time intervals after infection with IAPV. Our data identified PPIA as the single, most-optimal internal reference gene and the combination of PPAI-RPL23-UBI as a fully-sufficient multiple internal reference genes set for IAPV infection experiments in B. terrestris

RNAi in Insects: A Revolution in Fundamental Research and Pest Control Applications

In this editorial for the Special Issue on ‘RNAi in insect pest control’, three important applications of RNA interference (RNAi) in insects are briefly discussed and linked to the different studies published in this Special Issue. The discovery of the RNAi mechanism revolutionized entomological research, as it presented researchers with a tool to knock down genes, which is easily applicable in a wide range of insect species. Furthermore, RNAi also provides crop protection with a novel and promising pest control mode-of-action. The sequence-dependent nature allows RNAi-based control strategies to be highly species selective and the active molecule, a natural biological molecule known as double-stranded RNA (dsRNA), has a short environmental persistence. However, more research is needed to investigate different cellular and physiological barriers, such as cellular uptake and dsRNA degradation in the digestive system in insects, in order to provide efficient control methods against a wide range of insect pest species. Finally, the RNAi pathway is an important part of the innate antiviral immune defence of insects, and could even lead to applications targeting viruses in beneficial insects such as honeybees in the future.</jats:p

RNAi in Insects: A Revolution in Fundamental Research and Pest Control Applications

In this editorial for the Special Issue on &lsquo;RNAi in insect pest control&rsquo;, three important applications of RNA interference (RNAi) in insects are briefly discussed and linked to the different studies published in this Special Issue. The discovery of the RNAi mechanism revolutionized entomological research, as it presented researchers with a tool to knock down genes, which is easily applicable in a wide range of insect species. Furthermore, RNAi also provides crop protection with a novel and promising pest control mode-of-action. The sequence-dependent nature allows RNAi-based control strategies to be highly species selective and the active molecule, a natural biological molecule known as double-stranded RNA (dsRNA), has a short environmental persistence. However, more research is needed to investigate different cellular and physiological barriers, such as cellular uptake and dsRNA degradation in the digestive system in insects, in order to provide efficient control methods against a wide range of insect pest species. Finally, the RNAi pathway is an important part of the innate antiviral immune defence of insects, and could even lead to applications targeting viruses in beneficial insects such as honeybees in the future