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Understanding the survival of honey bees after pesticide exposure is key for environmental risk assessment. Currently, effects on adult honey bees are assessed by Organisation for Economic Co-operation and Development standardized guidelines, such as the acute and chronic oral exposure and acute contact exposure tests. The three different tests are interpreted individually, without consideration that the same compound is investigated in the same species, which should allow for an integrative assessment. In the present study we developed, calibrated, and validated a toxicokinetic-toxicodynamic model with 17 existing data sets on acute and chronic effects for honey bees. The model is based on the generalized unified threshold model for survival (GUTS), which is able to integrate the different exposure regimes, taking into account the physiology of the honey bee: the BeeGUTS model. The model is able to accurately describe the effects over time for all three exposure routes combined within one consistent framework. The model can also be used as a validity check for toxicity values used in honey bee risk assessment and to conduct effect assessments for real-life exposure scenarios. This new integrative approach, moving from single-point estimates of toxicity and exposure to a holistic link between exposure and effect, will allow for a higher confidence of honey bee toxicity assessment in the future. Environ Toxicol Chem 2022;41:2193-2201. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A novel, facultatively anaerobic, Gram-stain-positive, motile, endospore-forming bacterium of the genus Paenibacillus , designated strain 2.1T, was isolated from a colony of Apis mellifera affected by European foulbrood disease in Switzerland. The rod-shaped cells of strain 2.1T were 2.2–6.5 µm long and 0.7–1.1 µm wide. Colonies of strain 2.1T were orange-pigmented under oxic growth conditions on solid basal medium at 35–37 °C. Strain 2.1T showed catalase and cytochrome c oxidase activity. Its polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and phospholipid. The only respiratory quinone was menaquinone 7, and the major cellular fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C15 : 0, iso-C17 : 0 and palmitic acid (C16 : 0), which is consistent with other members of the genus Paenibacillus . The G+C content of the genomic DNA of strain 2.1T was 53.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence similarity showed that strain 2.1T was closely related to Paenibacillus dendritiformis LMG 21716T (99.7 % similarity) and Paenibacillus thiaminolyticus DSM 7262T (98.8 %). The whole-genome average nucleotide identity between strain 2.1T and the type strains of P. dendritiformis and P. thiaminolyticus was 92 and 91 %, respectively, and thus lower than the 95 % threshold value for delineation of genomic prokaryotic species. Based on the results of phylogenetic, genomic, phenotypic and chemotaxonomic analyses we propose the name Paenibacillus melissococcoides sp. nov. for this novel Paenibacillus species. The type strain is 2.1T (=CCOS 2000T=DSM 113619T=LMG 32539T).

Honey bees (Apis) are important pollinators for food crops and wild plants, but are facing great threats from pathogens and parasites, especially an obligate ectoparasitic mite, Varroa destructor. Cell invasion is a key step for V. destructor to reproduce, and the parasite displays remarkable host preference in this process. Varroa destructor made its host-shift from its original host, the Asian honey bee Apis cerana, to the new host, the European honey bee Apis mellifera several decades ago. However, it remains largely unstudied whether V. destructor shows a cell invasion preference between the two host species. Using cell invasion bioassays on a modified four-well arena, we showed that V. destructor significantly preferred to invade the worker and drone larvae of A. mellifera rather than A. cerana, suggesting that the new host is much more attractive to the parasite than the original one. Using gas chromatography-mass spectrometry (GC-MS), we revealed significant differences between the cuticular hydrocarbon (CHC) profiles of worker and drone larvae of the two bee hosts. The amounts of methyl-branched alkanes and alkenes (unsaturated CHCs), but not n-alkanes, were significantly different, and A. mellifera worker and drone larvae were found to express significantly higher amounts of methyl-alkanes, while A. cerana larvae produced higher amounts of alkenes. Cell invasion bioassays with glass dummies showed that the mites preferred the glass dummies coated with the CHCs of A. mellifera worker or drone larvae, which indicates a role of larval CHCs in mediating the preferential cell invasion of Varroa. The findings from this study extend our understanding of the host preference of V. destructor, and can potentially contribute to the development of effective strategies for mite control.

Host shift of parasites may have devastating effects on the novel hosts. One remarkable example is that of the ectoparasitic mite Varroa destructor, which has shifted its host from Eastern honey bees (Apis cerana) to Western honey bees (Apis mellifera) and posed a global threat to apiculture. To identify the genetic factors underlying the reproduction of host-shifted V. destructor on the new host. Genome sequencing was conducted to construct the phylogeny of the host-shifted and non-shifted mites and to screen for genomic signatures that differentiated them. Artificial infestation experiment was conducted to compare the reproductive difference between the mites, and transcriptome sequencing was conducted to find differentially expressed genes (DEGs) during the reproduction process. The host-shifted and non-shifted V. destructor mites constituted two genetically distinct lineages, with 15,362 high-F ST SNPs identified between them. Oogenesis was upregulated in host-shifted mites on the new host A. mellifera relative to non-shifted mites. The transcriptomes of the host-shifted and non-shifted mites differed significantly as early as 1h post-infestation. The DEGs were associated with nine genes carrying nonsynonymous high-F ST SNPs, including mGluR2-like, Lamb2-like and Vitellogenin 6-like, which were also differentially expressed, and eIF4G, CG5800, Dap160 and Sas10, which were located in the center of the networks regulating the DEGs based on protein-protein interaction analysis. The annotated functions of these genes were all associated with oogenesis. These genes appear to be the key genetic determinants of the oogenesis of host-shifted mites on the new host. Further study of these candidate genes will help elucidate the key mechanism underlying the success of host shifts of V. destructor.

Host switching allows parasites to expand their niches. However, successful switching may require suites of adaptations and may decrease performance on the old host. As a result, reductions in gene flow accompany many host switches, driving speciation. Because host switches tend to be rapid, it is difficult to study them in real time and their demographic parameters remain poorly understood. As a result, fundamental factors that control subsequent parasite evolution, such as the size of the switching population or the extent of immigration from the original host, remain largely unknown. To shed light on the host switching process, we explored how host switches occur in independent host shifts by two ectoparasitic honey bee mites (Varroa destructor and V. jacobsoni). Both switched to the western honey bee (Apis mellifera) after it was brought into contact with their ancestral host (Apis cerana), ~70 and ~12 years ago, respectively. Varroa destructor subsequently caused worldwide collapses of honey bee populations. Using whole-genome sequencing on 63 mites collected in their native ranges from both the ancestral and novel hosts, we were able to reconstruct the known temporal dynamics of the switch. We further found multiple previously undiscovered mitochondrial lineages on the novel host, along with genetic equivalent of tens of individuals that were involved in the initial host switch. Despite being greatly reduced, some gene flow remains between mites adapted to different hosts. Our findings suggest that while reproductive isolation may facilitate fixation of traits beneficial for exploitation of the new host, ongoing genetic exchange may allow genetic amelioration of inbreeding effects.

The neuropeptide Pigment-Dispersing Factor (PDF) plays a prominent role in the circadian clock of many insects including honey bees. In the honey bee brain, PDF is expressed in about 15 clock neurons per hemisphere that lie between the central brain and the optic lobes. As in other insects, the bee PDF neurons form wide arborizations in the brain; but certain differences are evident. For example, they arborize only sparsely in the accessory medulla (AME), which serves as important communication center of the circadian clock in cockroaches and flies. Furthermore, all bee PDF neurons cluster together, which makes it impossible to distinguish individual projections. Here, we investigated the developing bee PDF network and found that the first three PDF neurons arise in the 3rd larval instar and form a dense network of varicose fibers at the base of the developing medulla that strongly resembles the AME of hemimetabolous insects. In addition, they send faint fibers toward the lateral superior protocerebrum. In last larval instar, PDF cells with larger somata appear and send fibers toward the distal medulla and the medial protocerebrum. In the dorsal part of the medulla serpentine layer, a small PDF knot evolves from which PDF fibers extend ventrally. This knot disappears during metamorphosis and the varicose arborizations in the putative AME become fainter. Instead a new strongly stained PDF fiber hub appears in front of the lobula. Simultaneously, the number of PDF neurons increases and the PDF neuronal network in the brain gets continuously more complex. This article is protected by copyright. All rights reserved.

Extreme weather events, like high temperatures and droughts, are predicted to become common with climate change, and may negatively impact plant growth. How honey bees (Apis mellifera L. [Hymenoptera: Apidae]) will respond to this challenge is unclear, especially when collecting pollen, their primary source of protein, lipids, and micro-nutrients. We explored this response with a data set from multiple research projects that measured pollen collected by honey bees during 2015–2017 in which above-average temperatures and a drought occurred in 2017. We summarized the abundance and diversity of pollen collected from July to September in replicated apiaries kept at commercial soybean and corn farms in Iowa, in the Midwestern USA. The most commonly collected pollen was from clover (Trifolium spp. [Fabales: Fabaceae]), which dramatically declined in absolute and relative abundance in July 2017 during a period of high temperatures and drought. Due to an apparent lack of clover, honey bees switched to the more drought-tolerant native species (e.g., Chamaecrista fasciculata [Michx.] Greene [Fabales: Fabaceae], Dalea purpurea Vent. [Fabales: Fabaceae], Solidago spp. [Asterales: Asteraceae]), and several species of Asteraceae. This was especially noticeable in August 2017 when C. fasciculata dominated (87%) and clover disappeared from bee-collected pollen. We discuss the potential implications of climate-induced forage dearth on honey bee nutritional health. We also compare these results to a growing body of literature on the use of native, perennial flowering plants found in Midwestern prairies for the conservation of beneficial insects. We discuss the potential for drought resistant-native plants to potentially promote resilience to climate change for the non-native, managed honey bee colonies in the United States.

BACKGROUND Almond production in California is an intensively managed agroecosystem dependent on managed pollination by honey bees, Apis mellifera L. A recent laboratory study reported synergism in honey bees between chlorantraniliprole, a common diamide insecticide used in almond orchards, and the fungicide propiconazole. Indeed, there is an emerging body of evidence that honey bee cytochrome P450 monooxygenases of the CYP9Q subfamily are involved in the detoxification of insecticides across a diverse range of chemical classes. The objective of the present study was to unveil the molecular background of the described synergism and to explore the potential role of CYP9Q enzymes in diamide detoxification. RESULTS Our study confirmed the previously reported synergistic potential of propiconazole on chlorantraniliprole in acute contact toxicity bioassays, whereas no synergism was observed for flubendiamide. Fluorescence-based biochemical assays revealed an interaction of chlorantraniliprole, but not flubendiamide, with functionally expressed CYP9Q2 and CYP9Q3. These findings were validated by an increased chlorantraniliprole tolerance of transgenic Drosophila lines expressing CYP9Q2/3, and an analytically confirmed oxidative metabolism of chlorantraniliprole by recombinantly expressed enzymes. Furthermore, we showed that several triazole fungicides used in almond orchards, including propiconazole, were strong nanomolar inhibitors of functionally expressed honey bee CYP9Q2 and CYP9Q3, whereas other fungicides such as iprodione and cyprodinil did not inhibit these enzymes. CONCLUSION Honey bee CYP9Q enzymes are involved in chlorantraniliprole metabolism and inhibited by triazole fungicides possibly leading to synergism in acute contact toxicity bioassays. Our mechanistic approach has the potential to inform tier I honey bee pesticide risk assessment.

Brain transcriptional regulatory network for behavior demonstrates that brain gene expression in the honey bee can be accurately predicted from the expression transcription factors (TFs), but roles for specific TFs are less understood. Mushroom bodies (MBs) are important for learning, memory and sensory integration in the honey bee brain. A transcription factor, Mblk-1, expressed preferentially in the large-type Kenyon cells of the honeybee MBs is predicted to be involved in brain function by regulating transcription of its target genes in honey bee. However its function and the mechanism of regulation in behavior of honey bee is still obscure. Here we show that Mblk-1 had significantly higher expression in the brains of forager bees relative to nurse bees. Mblk-1 was significantly inhibited in bees fed siRNA. In addition, inhibition of Mblk-1 decreased sucrose responsiveness in foragers. Finally, we determined that Mblk-1 regulated the mRNA of AmGR1. These findings suggest that Mblk-1 may target AmGR1 to regulate the sucrose responsiveness of foragers. This article is protected by copyright. All rights reserved.

Recapping of Varroa destructor-infested brood cells is a trait that has recently attracted interest in honey bee breeding to select mite-resistant Apis mellifera colonies. To investigate the genetic architecture of this trait, we evaluated a sample of A. mellifera mellifera colonies (N = 155) from Switzerland and France and performed a genome-wide association study, using a pool of 500 workers per colony for next-generation sequencing. The results revealed that two QTL were significantly (P < 0.05) associated with recapping of V. destructor-infested brood cells. The best-associated QTL is located on chromosome 5 in a region previously found to be associated with grooming behaviour, a resistance trait against V. destructor, in A. mellifera and Apis cerana. The second best-associated QTL is located on chromosome 4 in an intron of the Dscam gene, which is involved in neuronal wiring. Previous research demonstrated that genes involved in neuronal wiring are associated with recapping and varroa sensitive hygiene. Therefore, our study confirms the role of a gene region on chromosome 5 in social immunity and simultaneously provides novel insights into genetic interactions between common mite resistance traits in honey bees.

Honey bee (Apis mellifera) health and hive transport are regulated by local apiary programs composed of apiary inspectors. Inspectors monitor and ensure the health of honey bees through field visits to apiaries where they inspect, identify, diagnose, and provide recommendations for the treatment of honey bee health issues. Laws and regulations pertaining to beekeeping and honey bee health are present in most states, territories, and provinces. Veterinarians are encouraged to establish a relationship with their local apiary inspector to further support beekeepers and the management of healthy honey bee colonies.

Honey bee colony health has received considerable attention in recent years, with many studies highlighting multifactorial issues contributing to colony losses. Disease and weather are consistently highlighted as primary drivers of colony loss, yet little is understood about how they interact. Here, we combined disease records from government honey bee health inspections with meteorological data from the CEDA to identify how weather impacts EFB, AFB, CBP, varroosis, chalkbrood and sacbrood. Using R-INLA, we determined how different meteorological variables influenced disease prevalence and disease risk. Temperature caused an increase in the risk of both varroosis and sacbrood, but overall, the weather had a varying effect on the six honey bee diseases. The risk of disease was also spatially varied and was impacted by the meteorological variables. These results are an important step in identifying the impacts of climate change on honey bees and honey bee diseases.

The honey bee Apis mellifera is a model organism for sociogenomics and one of the most important managed pollinators. High mortalities experienced by honey bee colonies over the past several decades are expected to have a substantive effect on crop pollination and global food security. These threats and the availability of a growing number of genomic resources for the honey bee have motivated research on how genetics and genomics can be practically applied to manage bee health. The authors review 3 such applications: (1) Certification of bee lineages using single-polymorphism markers; (2) breeding bees using marker-assisted selection; (3) diagnosing honey bee stressors using biomarkers.

Honey bees are faced with many diseases, some more serious than others. Observing irregularities during routine hive inspection may indicate potential problems. Not all disorders are equally important; some are more detrimental and need immediate attention, whereas others may only need time to clear up. It is important to be observant to be able to recognize these diseases and differentiate between them so the correct treatment may be done in a timely manner when needed to maintain the health of the colony. Colonies need to be healthy to survive and prosper.

Varroa destructor is the main pest of the honey bee Apis mellifera, causing colony losses. We investigated the effect of temperature on the autumn abundance of V. destructor in bee colonies over 1991–2020 in Central Europe. We tested the hypothesis that temperature can affect autumn mite populations with different time-lags modulating the bee abundance and brood availability. We showed that raised spring (March–May) and autumn (October) temperatures reinforce autumn V. destructor infestation in the bee colonies. The critical temperature signals embrace periods of bee activity, i.e., just after the first cleansing flights and just before the last observed bee flights, but no direct effects of phenological changes on V. destructor abundance were found. These effects were potentially associated with increased bee reproduction in the specific periods of the year and not with the extended period of activity or accelerated spring onset. We found significant effects of autumn bee abundance, autumn capped brood abundance, and the number of colonies merged on autumn mite infestation. We also observed differences in V. destructor abundance between bees derived from different subspecies. We indicated that climatic effects, through influence on the bee abundance and brood availability, are one of the main drivers regulating V. destructor abundance.

Optimal nutrition is crucial for honey bee colony growth and robust immune systems. Honey bee nutrition is complex and depends on the floral composition of the landscape. Foraging behavior of honey bees depends on both colony environment and external environment. There are significant gaps in knowledge regarding honey bee nutrition, and hence no optimal diet is available for honey bees, as there is for other livestock. In this review, we discuss (1) foraging behavior of honey bees, (2) nutritional needs, (3) nutritional supplements used by beekeepers, (4) probiotics, and (5) supplemental forage and efforts integrating floral diversity into cropping systems.

Honey bee colonies can be afflicted by serious conditions beyond infectious etiologies. Noninfectious conditions, such as starvation, laying worker colonies, and environmental dysregulation, can be as devastating as any disease. Improper hive monitoring and care often are the underlying causes of noninfectious conditions and each condition may be prevented by instituting best management practices.

The honey bee ectoparasite Varroa destructor is considered the major threat to apiculture, as untreated colonies of Apis mellifera usually collapse within a few years. In order to control this mite, many beekeepers rely on a limited number of approved synthetic acaricides, including the pyrethroids tau-fluvalinate and flumethrin. Due to the intensive use of these products, resistance is now commonplace in many beekeeping regions across the world. In the present study, the occurrence of amino acid substitutions at residue L925 of the voltage-gate sodium channel-the pyrethroid target site-was studied in Varroa populations collected throughout Flanders, Belgium. Dose-response bioassays supported the involvement of the frequently observed L925V substitution in flumethrin resistance, resulting in a 12.64-fold increase of the LC50 in a Varroa population mostly consisting of homozygous 925 V/V mites. With the presence of L925 substitutions in about four out of 10 screened apiaries, the use of pyrethroid-based varroacides in Flanders, including the recently released PolyVar® Yellow, should be carefully considered.

Pollinators are in decline thanks to the combined stresses of disease, pesticides, habitat loss, and climate. Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Understanding host-pathogen interactions and virulence of DWV in honey bees is slowed by the lack of cost-effective high-throughput screening methods for viral infection. Currently, analysis of virus infection in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we describe virus clones tagged with green fluorescent protein (GFP) or nanoluciferase (nLuc) that provide high-throughput detection and quantification of virus infections. GFP fluorescence is measured noninvasively in living bees via commonly available long-wave UV light sources and a smartphone camera, or a standard ultraviolet transilluminator gel imaging system. Nonlethal monitoring with GFP allows continuous screening of virus growth and serves as a direct breeding tool for identifying honey bee parents with increased antiviral resistance. Expression using the nLuc reporter strongly correlates with virus infection levels and is especially sensitive. Using multiple reporters, it is also possible to visualize competition, differential virulence, and host tissue targeting by co-occuring pathogens. Finally, it is possible to directly assess the risk of cross-species "spillover" from honey bees to other pollinators and vice versa.

Research Highlight: Norton, A. M., Remnant, E. J., Tom, J., Buchmann, G., Blacquiere, T., & Beekman, M. (2021). Adaptation to vector-based transmission in a honeybee virus. Journal of Animal Ecology, 90, https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2656.13493. In their paper on the adaptation to vector-based transmission via the mite Varroa destructor in a honeybee virus, Norton et al. study how high versus low levels of a viral vector affect viral load and potential competition between two strains of Deformed Wing Virus, an important highly virulent bee virus with the potential to spill-over into other pollinators and bee-associated insect species. This paper addresses two very timely issues, on the one hand on viral evolutionary ecology in response to vector-borne transmission, and on the other hand providing much needed information on an important honey bee pathogen. Using a complex natural system, this study shows that vector-borne transmission, and the control of the vector, can select for complex host-pathogen-vector interactions and that adaptations to changing transmission landscapes in fast evolving pathogens can create conditions for emerging pathogens to transition to endemic diseases.

18 pages, 6 figures.-- This article is licensed under a Creative Commons Attribution 4.0 International License, Honeybee health and the species’ gut microbiota are interconnected. Also noteworthy are the multiple niches present within hives, each with distinct microbiotas and all coexisting, which we termed “apibiome”. External stressors (e.g. anthropization) can compromise microbial balance and bee resilience. We hypothesised that (1) the bacterial communities of hives located in areas with different degrees of anthropization differ in composition, and (2) due to interactions between the multiple microbiomes within the apibiome, changes in the community of a niche would impact the bacteria present in other hive sections. We characterised the bacterial consortia of different niches (bee gut, bee bread, hive entrance and internal hive air) of 43 hives from 3 different environments (agricultural, semi-natural and natural) through 16S rRNA amplicon sequencing. Agricultural samples presented lower community evenness, depletion of beneficial bacteria, and increased recruitment of stress related pathways (predicted via PICRUSt2). The taxonomic and functional composition of gut and hive entrance followed an environmental gradient. Arsenophonus emerged as a possible indicator of anthropization, gradually decreasing in abundance from agriculture to the natural environment in multiple niches. Importantly, after 16 days of exposure to a semi-natural landscape hives showed intermediate profiles, suggesting alleviation of microbial dysbiosis through reduction of anthropization., This work was funded by the Dept. of Economic Development and Competitiveness of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza), R&D&I grants for the agricultural, food and fishing sectors of the Basque Autonomous Community (37-2017-00044), and the Research Group IT1233-19 of the Basque University System. JG was supported by the Department of Agriculture, Fisheries and Food of the Basque Government (Gobierno Vasco/Eusko Jaurlaritza) through a subsidy programme of training aid and support

Honey bee subspecies originate from specific geographical areas in Africa, Europe and the Middle East, and beekeepers interested in specific phenotypes have imported genetic material to regions outside of the bees' original range for use either in pure lines or controlled crosses. Moreover, imported drones are present in the environ-ment and mate naturally with queens from the local subspecies. The resulting ad-mixture complicates population genetics analyses, and population stratification can be a major problem for association studies. To better understand Western European honey bee populations, we produced a whole genome sequence and single nucleotide polymorphism (SNP) genotype data set from 870 haploid drones and demonstrate its utility for the identification of nine genetic backgrounds and various degrees of admixture in a subset of 629 samples. Five backgrounds identified correspond to sub-species, two to isolated populations on islands and two to managed populations. We also highlight several large haplotype blocks, some of which coincide with the position of centromeres. The largest is 3.6 Mb long and represents 21% of chromosome 11, with two major haplotypes corresponding to the two dominant genetic backgrounds identified. This large naturally phased data set is available as a single vcf file that can now serve as a reference for subsequent populations genomics studies in the honey bee, such as (i) selecting individuals of verified homogeneous genetic backgrounds as references, (ii) imputing genotypes from a lower-density data set generated by an SNP- chip or by low- pass sequencing, or (iii) selecting SNPs compatible with the re-quirements of genotyping chips. This work was performed in collaboration with the GeT platform, Toulouse (France), a partner of the National Infrastructure France Génomique, thanks to support by the Commissariat aux Grands Invetissements (ANR-10- INBS-0009). Bioinformatics analyses were performed on the GenoToul Bioinfo computer cluster. This work was funded by a grant from the INRA Département de Génétique Animale (INRA Animal Genetics division) and by the SeqApiPop pro-gramme, funded by the FranceAgriMer grant 14-21- AT. We thank John Kefuss for helpful discussions. We thank Andrew Abrahams for providing honey bee samples from Colonsay (Scotland), the Association Conservatoire de l'Abeille Noire Bretonne (ACANB) for samples from Ouessant (France), CETA de Savoie for sample from Savoie, ADAPI for samples from Porquerolles and all beekeepers and bee breeders who kindly participated in this study by providing sam-ples from their colonies. info:eu-repo/semantics/publishedVersion

Flower morphologies shape the accessibility to nectar and pollen, two major traits that determine plant–pollinator interactions and reproductive success. Melon is an economically important crop whose reproduction is completely pollinator-dependent and, as such, is a valuable model for studying crop-ecological functions. High-resolution imaging techniques, such as micro-computed tomography (micro-CT), have recently become popular for phenotyping in plant science. Here, we implemented micro-CT to study floral morphology and honey bees in the context of nectar-related traits without a sample preparation to improve the phenotyping precision and quality. We generated high-quality 3D models of melon male and female flowers and compared the geometric measures. Micro-CT allowed for a relatively easy and rapid generation of 3D volumetric data on nectar, nectary, flower, and honey bee body sizes. A comparative analysis of male and female flowers showed a strong positive correlation between the nectar gland volume and the volume of the secreted nectar. We modeled the nectar level inside the flower and reconstructed a 3D model of the accessibility by honey bees. By combining data on flower morphology, the honey bee size and nectar volume, this protocol can be used to assess the flower accessibility to pollinators in a high resolution, and can readily carry out genotypes comparative analysis to identify nectar-pollination-related traits.

Intragenic DNA methylation, also called gene body methylation, is an evolutionarily-conserved epigenetic mechanism in animals and plants. In social insects, gene body methylation is thought to contribute to behavioral plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in a subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

Host-parasite co-evolution is a process of reciprocal, adaptive genetic change. In natural conditions, parasites can shift to other host species, given both host and parasite genotypes allow this. Even though host-parasite co-evolution has been extensively studied both theoretically and empirically, few studies have focused on parasite gene flow between native and novel hosts. Nosema ceranae is a native parasite of the Asian honey bee Apis cerana, which infects epithelial cells of mid-guts. This parasite successfully switched to the European honey bee Apis mellifera, where high virulence has been reported. In this study, we used the parasite N. ceranae and both honey bee species as model organisms to study the impacts of two-host habitat sharing on parasite diversity and virulence. SNVs (Single Nucleotide Variants) were identified from parasites isolated from native and novel hosts from sympatric populations, as well as novel hosts from a parapatric population. Parasites isolated from native hosts showed the highest levels of polymorphism. By comparing the parasites isolated from novel hosts between sympatric and parapatric populations, habitat sharing with the native host significantly enhanced parasite diversity, suggesting there is continuing gene flow of parasites between the two host species in sympatric populations.

Pollinator refuges such as wildflower strips are planted on farms with the goals of mitigating wild pollinator declines and promoting crop pollination services. It is unclear, however, whether or how these goals are impacted by managed honey bee (Apis mellifera L.) hives on farms. We examined how wildflower strips and honey bee hives and/or their interaction influence wild bee communities and the fruit count of two pollinator-dependent crops across 21 farms in the Mid-Atlantic U.S. Although wild bee species richness increased with bloom density within wildflower strips, populations did not differ significantly between farms with and without them whereas fruit counts in both crops increased on farms with wildflower strips during one of 2 years. By contrast, wild bee abundance decreased by 48%, species richness by 20%, and strawberry fruit count by 18% across all farm with honey bee hives regardless of wildflower strip presence, and winter squash fruit count was consistently lower on farms with wildflower strips with hives as well. This work demonstrates that honey bee hives could detrimentally affect fruit count and wild bee populations on farms, and that benefits conferred by wildflower strips might not offset these negative impacts. Keeping honey bee hives on farms with wildflower strips could reduce conservation and pollination services.

Background MicroRNA (miRNA) is a class of small noncoding RNAs, which targets on thousands of mRNA and thus plays important roles in many biological processes. It has been reported that miRNA has cross-species regulation functions between parasitoid-host, or plant-animal, etc. For example, several plant miRNAs enter into the honey bees and regulate gene expression. However, whether cross-species regulation function of miRNAs is a universal mechanism remains a debate question. Results We have evaluated transmission of miRNAs from sunflower and sedr plants into the midgut of honey bee using RNA-Seq analyses complemented with confirmation by RT-qPCR. The results showed that at least 11 plant miRNAs were found in the midgut of honey bee feeding by sunflower and sedr pollen. Among which, nine miRNAs, including miR-30d, miR-143, miR-148a, miR-21, let-7 g, miR-26a, miR-126, miR-27a, and miR-203, were shared between the sunflower- and sedr-fed honey bees, suggesting they might have essential roles in plant-insect interactions. Moreover, existence of these co-shared miRNAs presents a strong evidence to support the successful transmission of miRNAs into the midgut of the insect. In total, 121 honeybee mRNAs were predicted to be the target of these 11 plant-derived miRNAs. Interestingly, a sedr-derived miRNA, miR-206, targets on 53 honeybee genes. Kyoto Encyclopedia of Genes and Genome (KEGG) analyses showed that these target genes are significantly involved in hippo signaling pathway-fly, Wnt signaling pathway, and N-Glycan biosynthesis. Conclusions In summary, these results provide evidence of cross-species regulation function of miRNA between honeybee and flowering host plants, extending our understanding of the molecular interactions between plants and animals.

Honey bee colony losses worldwide call for a more in-depth understanding of the pathogenic and mutualistic components of the honey bee microbiota and their relation with the environment. In this descriptive study, we characterized the yeast and bacterial communities that arise from six substrates associated with honey bees: corbicular pollen, beebread, hive debris, intestinal contents, body surface of nurses and forager bees, comparing two different landscapes, Minas Gerais, Brazil and Maryland, United States. The sampling of five hives in Brazil and four in the USA yielded 217 yeast and 284 bacterial isolates. Whereas the yeast community, accounted for 47 species from 29 genera, was dominated in Brazil by Aureobasidium sp. and Candida orthopsilosis, the major yeast recovered from the USA was Debaryomyces hansenii. The bacterial community was more diverse, encompassing 65 species distributed across 31 genera. Overall, most isolates belonged to Firmicutes, genus Bacillus. Among LAB, species from Lactobacillus were the most prevalent. Cluster analysis evidenced high structuration of the microbial communities, with two distinguished microbial groups between Brazil and the United States. In general, the higher difference among sites and substrates were dependents on the turnover effect (~ 93% of the beta diversity), with a more pronounced effect of nestedness (~ 28%) observed from Brazil microbiota change. The relative abundance of yeasts and bacteria also showed the dissimilarity of the microbial communities between both environments. These results provide a comprehensive view of microorganisms associated with A. mellifera, highlighting the importance of the environment in the establishment of the microbiota associated with honey bees. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00539-7.

In recent years, many pollinators have experienced large population declines, which threaten food security and the stability of natural ecosystems. Bumble bees are particularly important because their ability to “buzz” pollinate and tolerate cooler conditions make them critical pollinators for certain plants and regions. Here, we apply a conservation genomics approach to study the vulnerable Bombus terricola. We sequenced RNA from 30 worker abdomens, 18 of which were collected from agricultural sites and 12 of which were collected from nonagricultural sites. We found transcriptional signatures associated with exposure to insecticides, with gene expression patterns suggesting that bumble bees were exposed to neonicotinoids and/or fipronil—two compounds known to negatively impact bees. We also found transcriptional signatures associated with pathogen infections. In addition to the transcriptomic analysis, we carried out a metatranscriptomic analysis and detected five pathogens in the abdomens of workers, three of which are common in managed honey bee and bumble bee colonies. Our conservation genomics study provides functional support for the role of pesticides and pathogen spillover in the decline of B. terricola. We demonstrate that conservation genomics is an invaluable tool which allows researchers to quantify the effects of multiple stressors that impact pollinator populations in the wild.

Background Dinotefuran (CAS No. 165252–70-0), a neonicotinoid insecticide, has been used to protect various crops against invertebrate pests and has been associated with numerous negative sublethal effects on honey bees. Long noncoding RNAs (lncRNAs) play important roles in mediating various biological and pathological processes, involving transcriptional and gene regulation. The effects of dinotefuran on lncRNA expression and lncRNA function in the honey bee brain are still obscure. Results Through RNA sequencing, a comprehensive analysis of lncRNAs and mRNAs was performed following exposure to 0.01 mg/L dinotefuran for 1, 5, and 10 d. In total, 312 lncRNAs and 1341 mRNAs, 347 lncRNAs and 1458 mRNAs, and 345 lncRNAs and 1155 mRNAs were found to be differentially expressed (DE) on days 1, 5 and 10, respectively. Gene set enrichment analysis (GSEA) indicated that the dinotefuran-treated group showed enrichment in carbohydrate and protein metabolism and immune-inflammatory responses such as glycine, serine and threonine metabolism, pentose and glucuronate interconversion, and Hippo and transforming growth factor-β (TGF-β) signaling pathways. Moreover, the DE lncRNA TCONS_00086519 was shown by fluorescence in situ hybridization (FISH) to be distributed mainly in the cytoplasm, suggesting that it may serve as a competing endogenous RNA and a regulatory factor in the immune response to dinotefuran. Conclusion This study characterized the expression profile of lncRNAs upon exposure to neonicotinoid insecticides in young adult honey bees and provided a framework for further study of the role of lncRNAs in honey bee growth and the immune response.

Honey bee queen health is crucial for colony health and productivity, and pesticides have been previously associated with queen loss and premature supersedure. Prior research has investigated the effects of indirect pesticide exposure on queens via workers, as well as direct effects on queens during development. However, as adults, queens are in constant contact with wax as they walk on comb and lay eggs; therefore, direct pesticide contact with adult queens is a relevant but seldom investigated exposure route. Here, we conducted laboratory and field experiments to investigate the impacts of topical pesticide exposure on adult queens. We tested six pesticides commonly found in wax: coumaphos, tau-fluvalinate, atrazine, 2,4-DMPF, chlorpyriphos, chlorothalonil, and a cocktail of all six, each administered at 1, 4, 8, 16, and 32 times the concentrations typically found in wax. We found no effect of any treatment on queen mass, sperm viability, or fat body protein expression. In a field trial testing queen topical exposure of a pesticide cocktail, we found no impact on egg-laying pattern, queen mass, emergence mass of daughter workers, and no proteins in the spermathecal fluid were differentially expressed. These experiments consistently show that pesticides commonly found in wax have no direct impact on queen performance, reproduction, or quality metrics at the doses tested. We suggest that previously reported associations between high levels of pesticide residues in wax and queen failure are most likely driven by indirect effects of worker exposure (either through wax or other hive products) on queen care or queen perception.

Funding: Biotechnology and Biological Sciences Research Council (BBSRC), BB/M00337X/2. Deformed wing virus (DWV) is the most important globally distributed pathogen of honey bees and, when vectored by the ectoparasite Varroa destructor, is associated with high levels of colony losses. Divergent DWV types may differ in their pathogenicity and are reported to exhibit superinfection exclusion upon sequential infections, an inevitability in a Varroa-infested colony. We used a reverse genetic approach to investigate competition and interactions between genetically distinct or related virus strains, analysing viral load over time, tissue distribution with reporter gene-expressing viruses and recombination between virus variants. Transient competition occurred irrespective of the order of virus acquisition, indicating no directionality or dominance. Over longer periods, the ability to compete with a pre-existing infection correlated with the genetic divergence of the inoculae. Genetic recombination was observed throughout the DWV genome with recombinants accounting for ~2% of the population as determined by deep sequencing. We propose that superinfection exclusion, if it occurs at all, is a consequence of a cross-reactive RNAi response to the viruses involved, explaining the lack of dominance of one virus type over another. A better understanding of the consequences of dual- and superinfection will inform development of cross-protective honey bee vaccines and landscape-scale DWV transmission and evolution. Publisher PDF

In June of 2013 an application of dinotefuran on an ornamental planting of European linden trees (Tilia cordata Mill. [Malvales: Malvalceae]) in a shopping mall parking lot in Wilsonville, Oregon provoked the largest documented pesticide kill of bumble bees in North America. Based on geographic information systems and population genetic analysis, we estimate that between 45,830 and 107,470 bumble bees originating from between 289 and 596 colonies were killed during this event. Dinotefuran is a neonicotinoid that is highly effective in exterminating and/or harming target pest insects and non-target beneficial insects. Analysis to detect the concentration of pesticides in flowers that received foliar application revealed that the minimum reported dinotefuran concentration of a sampled T. cordata flower was 7.4 ppm, or in excess of 737% above the LC50 of the beneficial pollinator, the honey bee (Apis mellifera Linnaeus, 1758 [Hymenoptera: Apidae]). Furthermore, sampled Vosnesensky bumble bees (Bombus vosnesenskii Radoskowski, 1862 [Hymenoptera: Apidae]) were found to have an average dinotefuran concentration of 0.92 ppm at the time of death, which exceeds the maximum LC50 of A. mellifera (0.884 ppm). Our study underscores the lethal impact of the neonicotinoid pesticide dinotefuran on pollinating insect populations in a suburban environment. To our knowledge, the documentation and impact of pesticide kills on wild populations of beneficial insects has not been widely reported in the scientific literature. It is likely that the vast majority of mass pesticide kills of beneficial insects across other environments go unnoticed and unreported.

Pollinator-dependent crops rely on the activity of managed and wild pollinators. While farm management and surrounding landscape can influence wild pollinator contributions, managed pollinator contributions may be primarily driven by their stocking densities, though this is not well studied across crops. We selected 20 southern highbush blueberry farms along two independent gradients of honey bee Apis mellifera L. (Hymenoptera: Apidae) stocking density (~1–11 hives/acre) and bumble bee Bombus impatiens Cresson (Hymenoptera: Apidae) stocking density (0 - 3 colonies/acre) ensuring that stocking densities were not correlated with farm or landscape attributes. Across farms, we observed managed and wild bee visitation rates, and measured yield estimates. Farms with greater bumble bee stocking densities had higher bumble bee visitation rates and yield estimates, but farms with higher honey bee stocking densities only received higher honey bee visitation rates at the end of bloom and did not have higher yield estimates. The main wild pollinator, the southeastern blueberry bee Habropoda laboriosa (Fabricius) (Hymenoptera: Apidae), showed higher visitation rates on organic farms and in late bloom. In general, higher visitation rates by honey bees, bumble bees, and H. laboriosa were correlated with higher yields. Our results suggest that yields are limited by bee visitation rates, and that within the stocking density ranges studied, increasing managed bumble bees, but not honey bees, increases their visitation rates. While H. laboriosa had the greatest effect on yield estimates, its activity appears to be limited by both a phenological mismatch with crop bloom and farm management.

Bees provide pollination services to managed and wild ecosystems but are threatened globally due to multiple stressors, including exposure to contaminants. Perfluorooctane sulfonate (PFOS) is a widely detected and persistent contaminant that accumulates and biomagnifies in food chains. In this exposure effect study, small whole colonies of Apis mellifera (1000 bees) were exposed to PFOS using a purpose-built cage system over a 4-week period. The PFOS exposure concentrations were provided to bees in sugar syrup at concentrations detected in the environment, ranging from 0 to 1.6 mg L-1 . A range of biological and behavioral responses were monitored. Bee tissue, honey, and fecal matter were analyzed using isotope dilution combined with liquid chromatography-tandem mass spectrometry adapted for bee and honey matrix analysis. Bee mortality increased significantly with PFOS exposure at 0.8 mg L-1 or greater, and brood development ceased entirely at 0.02 mg L-1 or greater. Colony activity, temperament, hive maintenance, and defense were adversely affected in all PFOS exposure treatments compared with the control, even at the lowest PFOS exposure of 0.02 mg L-1 . Perfluorooctane sulfonate was detected in bee tissue with a mean bioaccumulation factor of 0.3, and it was also identified in honey and in feces collected from the hive cages. These findings provide the first evidence that PFOS exposure adversely affects honey bee colonies and may transfer to honey. With PFOS contaminating thousands of sites worldwide, our study has implications for exposed bee populations under natural conditions, pollination services, the honey industry, and human health. Integr Environ Assess Manag 2021;17:673-683. © 2021 SETAC.

International audience; Understanding the neural principles governing taste perception in species that bear economic importance or serve as research models for other sensory modalities constitutes a strategic goal. Such is the case of the honey bee (Apis mellifera), which is environmentally and socioeconomically important, given its crucial role as pollinator agent in agricultural landscapes and which has served as a traditional model for visual and olfactory neurosciences and for research on communication, navigation, and learning and memory. Here we review the current knowledge on honey bee gustatory receptors to provide an integrative view of peripheral taste detection in this insect, highlighting specificities and commonalities with other insect species. We describe behavioral and electrophysiological responses to several tastant categories and relate these responses, whenever possible, to known molecular receptor mechanisms. Overall, we adopted an evolutionary and comparative perspective to understand the neural principles of honey bee taste and define key questions that should be answered in future gustatory research centered on this insect.

Honey bees (Apis mellifera) provide invaluable benefits for food production and maintenance of biodiversity of natural environments through pollination. They are widely spread across the world, being adapted to different climatic conditions. To survive the winter in cold temperate regions, honey bees developed different strategies including storage of honey and pollen, confinement of individuals during the winter, and an annual cycle of colony growth and reproduction. Under these conditions, winter honey bees experience physiological changes, including changes in immunity and the composition of honey bee gut microbiota. However, under tropical or subtropical climates, the life cycle can experience alterations, i.e., queens lay eggs during almost all the year and new honey bees emerge constantly. In the present study, we characterized nurses' honey bee gut microbiota in colonies under subtropical region through a year, combining qPCR, PCR-DGGE, and 16S rDNA high-throughput sequencing. We also identified environmental variables involved in those changes. Our results showed that under the mentioned conditions, the number of bacteria is stable throughout the year. Diversity of gut microbiota is higher in spring and lower in summer and winter. Gradual changes in compositions occur between seasons: Lactobacillus spp. predominate in spring while Gilliamella apicola and Snodgrasella alvi predominate in summer and winter. Environmental variables (mainly precipitations) affected the composition of the honey bee gut microbiota. Our findings provide new insights into the dynamics of honey bee gut microbiota and may be useful to understand the adaptation of bees to different environmental conditions.

Since lithium salts were demonstrated to be very effective for the potential control of Varroa destructor, a highly detrimental parasite of honey bee (Apis mellifera), no studies have been reported on their comparison with any commonly used varroicides in commercial bee colonies. In this study we compared the effectiveness of lithium chloride to that of oxalic acid, a widely used miticide. The results of the present study confirm that lithium has superior efficacy to oxalic acid sublimation both as a main or a supplementary pre-wintering treatment at moderate infestation levels, restricted to certain pre-wintering conditions. Considering its easy implementation in apicultural practice and its twofold mode of action, trickling would be the preferred way of administration after the use of lithium salts as varroicides is authorised.

Background Sacbrood is an infectious disease of the honey bee caused by Scbrood virus (SBV) which belongs to the family Iflaviridae and is especially lethal for Asian honeybee Apis cerana. Chinese Sacbrood virus (CSBV) is a geographic strain of SBV. Currently, there is a lack of an effective antiviral agent for controlling CSBV infection in honey bees. Methods Here, we explored the antiviral effect of a Chinese medicinal herb Radix isatidis on CSBV infection in A. cerana by inoculating the 3rd instar larvae with purified CSBV and treating the infected bee larvae with R. isatidis extract at the same time. The growth, development, and survival of larvae between the control and treatment groups were compared. The CSBV copy number at the 4th instar, 5th instar, and 6th instar larvae was measured by the absolute quantification PCR method. Results Bioassays revealed that R. isatidis extract significantly inhibited the replication of CSBV, mitigated the impacts of CSBV on larval growth and development, reduced the mortality of CSBV-infected A. cerana larvae, and modulated the expression of immune transcripts in infected bees. Conclusion Although the mechanism underlying the inhibition of CSBV replication by the medicine plant will require further investigation, this study demonstrated the antiviral activity of R. isatidis extract and provides a potential strategy for controlling SBV infection in honey bees.

Background The varroa mite is one of the main causes of honey bee mortality. An important mechanism by which honey bees increase their resistance against this mite is the expression of suppressed mite reproduction. This trait describes the physiological inability of mites to produce viable offspring and was found associated with eight genomic variants in previous research. Results This paper presents the development and validation of high-throughput qPCR assays with dual-labeled probes for discriminating these eight single-nucleotide variants. Amplicon sequences used for assay validation revealed additional variants in the primer/probe binding sites in four out of the eight assays. As for two of these the additional variants interfered with the genotyping outcome supplementary primers and/or probes were developed. Inclusion of these primers and probes in the assay mixes allowed for the correct genotyping of all eight variants of interest within our bee population. Conclusion These outcomes underline the importance of checking for interfering variants in designing qPCR assays. Ultimately, the availability of this assay allows genotyping for the suppressed mite reproduction trait and paves the way for marker assisted selection in breeding programs.

While there is widespread concern regarding the impact of pesticides on honey bees, well-replicated field experiments, to date, have failed to provide clear insights on pesticide effects. Here, we adopt a systems biology approach to gain insights into the web of interactions amongst the factors influencing honey bee health. We put the focus on the properties of the system that depend upon its architecture and not on the strength, often unknown, of each single interaction. Then we test in vivo, on caged honey bees, the predictions derived from this modelling analysis. We show that the impact of toxic compounds on honey bee health can be shaped by the concurrent stressors affecting bees. We demonstrate that the immune-suppressive capacity of the widespread pathogen of bees, deformed wing virus, can introduce a critical positive feed-back loop in the system causing bistability, i.e., two stable equilibria. Therefore, honey bees under similar initial conditions can experience different consequences when exposed to the same stressor, including prolonged survival or premature death. The latter can generate an increased vulnerability of the hive to dwindling and collapse. Our conclusions reconcile contrasting field-testing outcomes and have important implications for the application of field studies to complex systems.