The Protein Paradox: Elucidating the Complex Nutritional Ecology of the Invasive Berry Pest, Spotted-Wing Drosophila (Diptera: Drosophila suzukii)

Spotted-wing drosophila (SWD), Drosophila suzukii, has become one of the most widely studied insect species over the last decade, largely due to its recent invasion and rapid expansion across the Americas and Europe. Unlike other drosophilid species, which colonize rotting fruit, SWD females possess a serrated ovipositor that allows them to lay eggs in intact ripening fruit, causing significant economic problems for fruit/berry producers worldwide. Though an impressive amount of research has been conducted on SWD's ecology and physiology, aspects of their nutritional ecology remain ambiguous. This review synthesizes the research to date to provide a more comprehensive view of SWD's nutritional relationship with its fruit hosts and associated microbes. Overall, data suggest that SWD's ability to utilize novel resources is likely due to changes in their ecological, rather than physiological, niche that are largely mediated by microbial associations. Studies show that SWD's nutrient intake is comparable to other drosophilid species, indicating limited adaptation to feeding on lower-protein resources. Instead, data show that fruit protein content is a reliable predictor of host suitability and that fruit-microbe dynamics have a strong impact on protein availability. In particularly, fruit protein increases after infestation with SWD-associated microbes, suggesting that initially-suboptimal intact fruits can become protein-rich on a timeframe that is relevant for larval nutrition. This body of work suggests that microbial associations between flies and their fruit hosts can compensate for the nutritional differences between intact and rotting fruit, and that these relationships are likely responsible for SWD's expanded nutritional niche.

Correlation Between Increased Homing Flight Duration and Altered Gene Expression in the Brain of Honey Bee Foragers After Acute Oral Exposure to Thiacloprid and Thiamethoxam

Neonicotinoids as thiamethoxam and thiacloprid are suspected to be implicated in the decline of honey bee populations. As nicotinic acetylcholine receptor agonists, they disturb acetylcholine receptor signaling in insects, leading to neurotoxicity and are therefore globally used as insecticides. Several behavioral studies have shown links between neonicotinoid exposure of bees and adverse effects on foraging activity, homing flight performance and reproduction, but the molecular aspects underlying these effects are not well-understood. In the last years, several studies through us and others showed the effects of exposure to neonicotinoids on gene expression in the brain of honey bees. Transcripts of acetylcholine receptors, hormonal regulation, stress markers, detoxification enzymes, immune system related genes and transcripts of the energy metabolism were altered after neonicotinoid exposure. To elucidate the link between homing flight performance and shifts in gene expression in the brain of honey bees after neonicotinoid exposure, we combined homing flight activity experiments applying RFID technology and gene expression analysis. We analyzed the expression of endocrine factors, stress genes, detoxification enzymes and genes linked to energy metabolism in forager bees after homing flight experiments. Three different experiments (experiment I: pilot study; experiment II: “worst-case” study and experiment III: laboratory study) were performed. In a pilot study, we wanted to investigate if we could see differences in gene expression between controls and exposed bees (experiment I). This first study was followed by a so-called “worst-case” study (experiment II), where we investigated mainly differences in the expression of transcripts linked to energy metabolism between fast and slow returning foragers. We found a correlation between homing flight duration and the expression of cytochrome c oxidase subunit 5A, one transcript linked to oxidative phosphorylation. In the third experiment (experiment III), foragers were exposed in the laboratory to 1 ng/bee thiamethoxam and 8 ng/bee thiacloprid followed by gene expression analysis without a subsequent flight experiment. We could partially confirm the induction of cytochrome c oxidase subunit 5A, which we detected in experiment II. In addition, we analyzed the effect of the feeding mode (group feeding vs. single bee feeding) on data scattering and demonstrated that single bee feeding is superior to group feeding as it significantly reduces variability in gene expression. Based on the data, we thus hypothesize that the disruption of energy metabolism may be one reason for a prolongation of homing flight duration in neonicotinoid treated bees.

Haplotype Analysis of Varroa destructor and Deformed Wing Virus Using Long Reads

As a phoretic parasite and virus vector, the mite Varroa destructor and the associated Deformed wing virus (DWV) form a lethal combination to the honey bee, Apis mellifera. Routine acaricide treatment has been reported to reduce the diversity of mites and select for tolerance against these treatments. Further, different DWV strains face selective pressures when transmitted via mites. In this study, the haplotypes of Varroa mites and associated DWV variants were quantified using long reads. A single haplotype dominated the mite mitochondrial gene cytochrome oxidase subunit I, reflecting an ancient bottleneck. However, highly polymorphic genes were present across the mite genome, suggesting the diversity of mites could be actively maintained at a regional level. DWV detected in both mites and honey bees show a dominant variant with only a few low-frequency alternate haplotypes. The relative abundances of DWV haplotypes isolated from honey bees and mites were highly consistent, suggesting that some variants are favored by ongoing selection.

Pollen Source Richness May Be a Poor Predictor of Bumblebee (Bombus terrestris) Colony Growth

Agricultural intensification has drastically altered foraging landscapes for bees, with large-scale crop monocultures associated with floral diversity loss. Research on bumblebees and honeybees has shown individuals feeding on pollen from a low richness of floral sources can experience negative impacts on health and longevity relative to higher pollen source richness of similar protein concentrations. Florally rich landscapes are thus generally assumed to better support social bees. Yet, little is known about whether the effects of reduced pollen source richness can be mitigated by feeding on pollen with higher crude protein concentration, and importantly how variation in diet affects whole colony growth, rearing decisions and sexual production. Studying queen-right bumblebee (Bombus terrestris) colonies, we monitored colony development under a polyfloral pollen diet or a monofloral pollen diet with 1.5–1.8 times higher crude protein concentration. Over 6 weeks, we found monofloral colonies performed better for all measures, with no apparent long-term effects on colony mass or worker production, and a higher number of pupae in monofloral colonies at the end of the experiment. Unexpectedly, polyfloral colonies showed higher mortality, and little evidence of any strategy to counteract the effects of reduced protein; with fewer and lower mass workers being reared, and males showing a similar trend. Our findings (i) provide well-needed daily growth dynamics of queenright colonies under varied diets, and (ii) support the view that pollen protein content in the foraging landscape rather than floral species richness per se is likely a key driver of colony health and success.

Acute Imidacloprid Exposure Alters Mitochondrial Function in Bumblebee Flight Muscle and Brain

Mitochondria are intracellular organelles responsible for cellular respiration with one of their major roles in the production of energy in the form of ATP. Activities with increased energetic demand are especially dependent on efficient ATP production, hence sufficient mitochondrial function is fundamental. In bees, flight muscle and the brain have particularly high densities of mitochondria to facilitate the substantial ATP production required for flight activity and neuronal signalling. Neonicotinoids are systemic synthetic insecticides that are widely utilised against crop herbivores but have been reported to cause, by unknown mechanisms, mitochondrial dysfunction, decreasing cognitive function and flight activity among pollinating bees. Here we explore, using high-resolution respirometry, how the neonicotinoid imidacloprid may affect oxidative phosphorylation in the brain and flight muscle of the buff-tailed bumblebee, Bombus terrestris. We find that acute exposure increases routine oxygen consumption in the flight muscle of worker bees. This provides a candidate explanation for prior reports of early declines in flight activity following acute exposure. We further find that imidacloprid increases the maximum electron transport capacity in the brain, with a trend towards increased overall oxygen consumption. However, intra-individual variability is high, limiting the extent to which apparent effects of imidacloprid on brain mitochondria are shown conclusively. Overall, our results highlight the necessity to examine tissue-specific effects of imidacloprid on respiration and energy production.

Occurrence and Phylogenetic Analysis of DWV in Stingless Bee (Apidae sp.) in China: A Case Report

Honey bees play a vital role in providing pollination services for agricultural crops and wild flowering plants. However, the spillover risk of their pathogens to other pollinators or wild insects is becoming a cause for concern. There is some evidence that stingless bees can carry honey bee viruses, but little is known about the presence of honey bee viruses in stingless bees in China. Here, we investigate the occurrence of major honey bee pathogens including bacteria, fungi, and viruses in stingless bees (Apidae: sp.). Our results show that the stingless bees (Apidae: sp.) were mainly infected with DWV-A, but no DWV-B and DWV-C. Phylogenetic analysis on fragments of lp, RdRp, and VP3 of DWV-A indicated that genetic variation in VP3 might an important indicator for host-specific viruses, but it requires further study. Our results indicated that DWV-A is not only the major strain of virus currently circulating in managed bee colonies in China and globally, but in stingless bee species as a whole.

Host Genotype and Tissue Type Determine DWV Infection Intensity

Varroa mite-vectored viruses such as Deformed wing virus (DWV) are of great concern for honey bee health as they can cause disease in individuals and increase colony mortality. Two genotypes of DWV (A and B) are prevalent in the United States and may have differential virulence and pathogenicity. Honey bee genetic stocks bred to resist Varroa mites also exhibit differential infection responses to the Varroa mite-vectored viruses. The goal of this project was to determine if interactions between host genotype could influence the overall infection levels and dissemination of DWV within honey bees. To do this, we injected DWV isolated from symptomatic adult bees into mite-free, newly emerged adult bees from five genetic stocks with varying levels of resistance to Varroa mites. We measured DWV-A and DWV-B dissemination among tissues chosen based on relevance to general health outcomes for 10 days. Injury from sham injections did not increase DWV-A levels but did increase DWV-B infections. DWV injection increased both DWV-A and DWV-B levels over time with significant host stock interactions. While we did not observe any differences in viral dissemination among host stocks, we found differences in virus genotype dissemination to different body parts. DWV-A exhibited the highest initial levels in heads and legs while the highest initial levels of DWV-B were found in heads and abdomens. These interactions underscore the need to evaluate viral genotype and tissue specificity in conjunction with host genotype, particularly when the host has been selected for traits relative to virus-vector and virus resistance.

Individual vs. Combined Short-Term Effects of Soil Pollutants on Colony Founding in a Common Ant Species

Insects are integral to terrestrial life and provide essential ecosystem functions such as pollination and nutrient cycling. Due to massive declines in insect biomass, abundance, or species richness in recent years, the focus has turned to find their causes. Anthropogenic pollution is among the main drivers of insect declines. Research addressing the effects of pollutants concentrates on aquatic insects and pollinators, despite the apparent risk of contaminated soils. Pollutants accumulating in the soil might pose a significant threat because concentrations tend to be high and different pollutants are present simultaneously. Here, we exposed queens of the black garden ant Lasius niger at the colony founding stage to different concentrations and combinations of pollutants (brake dust, soot, microplastic particles and fibers, manure) to determine dose-dependent effects and interactions between stressors. As proxies for colony founding success, we measured queen survival, the development time of the different life stages, the brood weight, and the number of offspring. Over the course of the experiment queen mortality was very low and similar across treatments. Only high manure concentrations affected the colony founding success. Eggs from queens exposed to high manure concentrations took longer to hatch, which resulted in a delayed emergence of workers. Also, fewer pupae and workers were raised by those queens. Brake dust, soot and plastic particles did not visibly affect colony founding success, neither as single nor as multiple stressors. The application of manure, however, affected colony founding in L. niger negatively underlining the issue of excessive manure application to our environment. Even though anthropogenic soil pollutants seem to have little short-term effects on ant colony founding, studies will have to elucidate potential long-term effects as a colony grows.

The Honey Bee Gene Bee Antiviral Protein-1 Is a Taxonomically Restricted Antiviral Immune Gene

Insects have evolved a wide range of strategies to combat invading pathogens, including viruses. Genes that encode proteins involved in immune responses often evolve under positive selection due to their co-evolution with pathogens. Insect antiviral defense includes the RNA interference (RNAi) mechanism, which is triggered by recognition of non-self, virally produced, double-stranded RNAs. Indeed, insect RNAi genes (e.g., dicer and argonaute-2) are under high selective pressure. Honey bees (Apis mellifera) are eusocial insects that respond to viral infections via both sequence specific RNAi and a non-sequence specific dsRNA triggered pathway, which is less well-characterized. A transcriptome-level study of virus-infected and/or dsRNA-treated honey bees revealed increased expression of a novel antiviral gene, GenBank: MF116383, and in vivo experiments confirmed its antiviral function. Due to in silico annotation and sequence similarity, MF116383 was originally annotated as a probable cyclin-dependent serine/threonine-protein kinase. In this study, we confirmed that MF116383 limits virus infection, and carried out further bioinformatic and phylogenetic analyses to better characterize this important gene—which we renamed bee antiviral protein-1 (bap1). Phylogenetic analysis revealed that bap1 is taxonomically restricted to Hymenoptera and Blatella germanica (the German cockroach) and that the majority of bap1 amino acids are evolving under neutral selection. This is in-line with the results from structural prediction tools that indicate Bap1 is a highly disordered protein, which likely has relaxed structural constraints. Assessment of honey bee gene expression using a weighted gene correlation network analysis revealed that bap1 expression was highly correlated with several immune genes—most notably argonaute-2. The coexpression of bap1 and argonaute-2 was confirmed in an independent dataset that accounted for the effect of virus abundance. Together, these data demonstrate that bap1 is a taxonomically restricted, rapidly evolving antiviral immune gene. Future work will determine the role of bap1 in limiting replication of other viruses and examine the signal cascade responsible for regulating the expression of bap1 and other honey bee antiviral defense genes, including coexpressed ago-2, and determine whether the virus limiting function of bap1 acts in parallel or in tandem with RNAi.