Higher prevalence of sacbrood virus in Apis mellifera (Hymenoptera: Apidae) colonies after pollinating highbush blueberries.

Highbush blueberry pollination depends on managed honey bees (Apis mellifera) L. for adequate fruit sets; however, beekeepers have raised concerns about the poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularly Melisococcus plutonius [(ex. White) Bailey and Collins, Lactobacillales: Enterococcaceae], the causal agent of European foulbrood disease, but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia, Canada, across 2 years (2020 and 2021). Nine viruses, as well as M. plutonius, Vairimorpha ceranae, and V. apis [Tokarev et al., Microsporidia: Nosematidae; formerly Nosema ceranae (Fries et al.) and N. apis (Zander)], were detected by PCR and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to ~6 wk after the beginning of the pollination period). Post hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the near group, not M. plutonius, was the primary driver. Further research is needed to determine if the association of SBV with highbush blueberry pollination is contributing to the health decline that beekeepers observe after pollinating this crop.

Honey bee stressor networks are complex and dependent on crop and region.

Honey bees play a major role in crop pollination but have experienced declining health throughout most of the globe. Despite decades of research on key honey bee stressors (e.g., parasitic Varroa destructor mites and viruses), researchers cannot fully explain or predict colony mortality, potentially because it is caused by exposure to multiple interacting stressors in the field. Understanding which honey bee stressors co-occur and have the potential to interact is therefore of profound importance. Here, we used the emerging field of systems theory to characterize the stressor networks found in honey bee colonies after they were placed in fields containing economically valuable crops across Canada. Honey bee stressor networks were often highly complex, with hundreds of potential interactions between stressors. Their placement in crops for the pollination season generally exposed colonies to more complex stressor networks, with an average of 23 stressors and 307 interactions. We discovered that the most influential stressors in a network-those that substantively impacted network architecture-are not currently addressed by beekeepers. Finally, the stressor networks showed substantial divergence among crop systems from different regions, which is consistent with the knowledge that some crops (e.g., highbush blueberry) are traditionally riskier to honey bees than others. Our approach sheds light on the stressor networks that honey bees encounter in the field and underscores the importance of considering interactions among stressors. Clearly, addressing and managing these issues will require solutions that are tailored to specific crops and regions and their associated stressor networks.

Plant virus diversity in bee and pollen samples from apple (Malus domestica) and sweet cherry (Prunus avium) agroecosystems.

Introduction: Honey bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling. Methods: Plant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis. Results: A wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved. Conclusion: The pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

The effects of protein supplementation, fumagillin treatment, and colony management on the productivity and long-term survival of honey bee (Apis mellifera) colonies.

In this study, we intensively measured the longitudinal productivity and survival of 362 commercially managed honey bee colonies in Canada, over a two-year period. A full factorial experimental design was used, whereby two treatments were repeated across apiaries situated in three distinct geographic regions: Northern Alberta, Southern Alberta and Prince Edward Island, each having unique bee management strategies. In the protein supplemented treatment, colonies were continuously provided a commercial protein supplement containing 25% w/w pollen, in addition to any feed normally provided by beekeepers in that region. In the fumagillin treatment, colonies were treated with the label dose of Fumagilin-B® each year during the fall. Neither treatment provided consistent benefits across all sites and dates. Fumagillin was associated with a large increase in honey production only at the Northern Alberta site, while protein supplementation produced an early season increase in brood production only at the Southern Alberta site. The protein supplement provided no long-lasting benefit at any site and was also associated with an increased risk of death and decreased colony size later in the study. Differences in colony survival and productivity among regions, and among colonies within beekeeping operations, were far larger than the effects of either treatment, suggesting that returns from extra feed supplements and fumagillin were highly contextually dependent. We conclude that use of fumagillin is safe and sometimes beneficial, but that beekeepers should only consider excess protein supplementation when natural forage is limiting.

Environmental metagenetics unveil novel plant-pollinator interactions.

Honey bees are efficient pollinators of flowering plants, aiding in the plant reproductive cycle and acting as vehicles for evolutionary processes. Their role as agents of selection and drivers of gene flow is instrumental to the structure of plant populations, but historically, our understanding of their influence has been limited to predominantly insect-dispersed flowering species. Recent metagenetic work has provided evidence that honey bees also forage on pollen from anemophilous species, suggesting that their role as vectors for transmission of plant genetic material is not confined to groups designated as entomophilous, and leading us to ask: could honey bees act as dispersal agents for non-flowering plant taxa? Using an extensive pollen metabarcoding dataset from Canada, we discovered that honey bees may serve as dispersal agents for an array of sporophytes (Anchistea, Claytosmunda, Dryopteris, Osmunda, Osmundastrum, Equisetum) and bryophytes (Funaria, Orthotrichum, Sphagnum, Ulota). Our findings also suggest that honey bees may occasionally act as vectors for the dispersal of aquatic phototrophs, specifically Coccomyxa and Protosiphon, species of green algae. Our work has shed light on the broad resource-access patterns that guide plant-pollinator interactions and suggests that bees could act as vectors of gene flow, and potentially even agents of selection, across Plantae.

Queen quality, performance, and winter survival of imported and domestic honey bee queen stocks.

Canadian beekeepers have faced high colony mortality each winter over the last decade. Frequently citing "poor queen quality" as a top contributing factor to colony loss, Canadian beekeepers report needing to replace half their queens each year. Domestic queen production exists throughout Canada but is limited due to the short season and can be further limited when colony mortality is high. Consequently, Canadian beekeepers import over 260,000 queens annually, primarily from locations with warmer climates. In this study, newly mated imported queens from Hawaii (USA) and New Zealand were compared to domestic Canadian queens produced in British Columbia; these stocks were evaluated on their morphological and sperm storage characteristics. Stock quality was also evaluated in the field at two locations in Alberta, Canada over two production seasons. Our results show initial variation in queen morphology and fertility among imported and domestic queen stocks. Most striking, the New Zealand queens weighed 10-13% less than the Hawaii and British Columbia queens, respectively upon arrival. Colony performance over a two-year field study suggests: (1) brood pattern solidness has a positive nonlinear correlation with honey production regardless of queen stock and environment; (2) environment (i.e., apiary location) and queen stock variably predict colony health and productivity depending on year; specifically, apiary site appears to be a stronger predictor of colony health and productivity than queen stock in year one, but in year two, queen stock appears to be a stronger predictor than apiary site; (3) high clinical symptoms of chalkbrood may explain the prevalence of poor brood patterns in colonies headed by queens from New Zealand; (4) domestic queens are 25% more likely to survive winter in Alberta than imported queens. Therefore, it is important to consider possible mismatches in disease immunity and climate conditioning of imported queen stocks heading colonies in temperate regions that face drastically different seasonal climates and disease ecology dynamics.

Area Wide Monitoring of Plant and Honey Bee (Apis mellifera) Viruses in Blueberry (Vaccinium corymbosum) Agroecosystems Facilitated by Honey Bee Pollination.

Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee (Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.

Investigation of Melissococcus plutonius isolates from 3 outbreaks of European foulbrood disease in commercial beekeeping operations in western Canada.

European foulbrood (EFB) disease is an economically important bacterial disease of honey bee larvae caused by enteric infection with Melissococcus plutonius. In this study, we investigated 3 clinical outbreaks of EFB disease in commercial beekeeping operations in western Canada in the summer of 2020 and characterized the Melissococcus plutonius isolates cultured from these outbreaks according to genetic multi-locus sequence type and i n vitro larval pathogenicity. We isolated M. plutonius sequence type 19 from EFB outbreaks in British Columbia and Alberta, and a novel M. plutonius sequence type 36 from an EFB outbreak in Saskatchewan. In vitro larval infection with each M. plutonius isolate was associated with decreased larval survival in vitro by 58.3 to 70.8% (P < 0.001) compared to non-infected controls. Further elucidation of mechanisms of virulence of M. plutonius, paired with epidemiologic investigation, is imperative to improve EFB management strategies and mitigate risks of EFB outbreaks in western Canada.

Enquête sur des isolats de Melissococcus plutonius provenant de trois éclosions de loque e uropéenne dans des exploitations apicoles commerciales de l'Ouest canadien. La loque européenne (EFB) est une maladie bactérienne économiquement importante des larves d'abeilles mellifères causée par une infection entérique par Melissococcus plutonius. Dans cette étude, nous avons enquêté sur trois éclosions cliniques de la maladie EFB dans des exploitations apicoles commerciales dans l'ouest du Canada à l'été 2020 et caractérisé les isolats de Melissococcus plutonius cultivés à partir de ces éclosions selon le typage génomique multilocus et la pathogénicité larvaire in vitro. Nous avons isolé le type de séquence 19 de M. plutonius des éclosions d'EFB en Colombie-Britannique et en Alberta, et une nouvelle séquence de type 36 de M. plutonius d'une éclosion d'EFB en Saskatchewan. L'infection larvaire in vitro avec chaque isolat de M. plutonius était associée à une diminution de la survie larvaire in vitro de 58,3 à 70,8 % (P < 0,001) par rapport aux témoins non infectés. Une élucidation plus poussée des mécanismes de virulence de M. plutonius, associée à une enquête épidémiologique, est impérative pour améliorer les stratégies de gestion de l'EFB et atténuer les risques d'épidémies d'EFB dans l'Ouest canadien.(Traduit par Dr Serge Messier).

Evaluating approved and alternative treatments against an oxytetracycline-resistant bacterium responsible for European foulbrood disease in honey bees.

European foulbrood (EFB) is a disease of honey bee larvae caused by Melissococcus plutonius. In North America, oxytetracycline (OTC) is approved to combat EFB disease though tylosin (TYL) and lincomycin (LMC) are also registered for use against American foulbrood disease. Herein, we report and characterize an OTC-resistant M. plutonius isolate from British Columbia, Canada, providing an antimicrobial sensitivity to the three approved antibiotics and studying their abilities to alter larval survival in an in vitro infection model. Specifically, we investigated OTC, TYL, and LMC as potential treatment options for EFB disease using laboratory-reared larvae infected with M. plutonius. The utility of the three antibiotics were compared through an experimental design that either mimicked metaphylaxis or antimicrobial intervention. At varying concentrations, all three antibiotics prevented clinical signs of EFB disease following infection with M. plutonius 2019BC1 in vitro. This included treatment with 100 µg/mL of OTC, a concentration that was ~ 3× the minimum inhibitory concentration measured to inhibit the strain in nutrient broth. Additionally, we noted high larval mortality in groups treated with doses of OTC corresponding to ~ 30× the dose required to eliminate bacterial growth in vitro. In contrast, TYL and LMC were not toxic to larvae at concentrations that exceed field use. As we continue to investigate antimicrobial resistance (AMR) profiles of M. plutonius from known EFB outbreaks, we expect a range of AMR phenotypes, reiterating the importance of expanding current therapeutic options along with alternative management practices to suppress this disease.

Phenomic analysis of the honey bee pathogen-web and its dynamics on colony productivity, health and social immunity behaviors.

Many pathogens and parasites have evolved to overwhelm and suppress their host's immune system. Nevertheless, the interactive effects of these agents on colony productivity and wintering success have been relatively unexplored, particularly in large-scale phenomic studies. As a defense mechanism, honey bees have evolved remarkable social behaviors to defend against pathogen and parasite challenges, which reduce the impact of disease and improve colony health. To investigate the complex role of pathogens, parasites and social immunity behaviors in relation to colony productivity and outcomes, we extensively studied colonies at several locations across Canada for two years. In 2016 and 2017, colonies founded with 1-year-old queens of diverse genetic origin were evaluated, which represented a generalized subset of the Canadian bee population. During each experimental year (May through April), we collected phenotypic data and sampled colonies for pathogen analysis in a standardized manner. Measures included: colony size and productivity (colony weight, cluster size, honey production, and sealed brood population), social immunity traits (hygienic behavior, instantaneous mite population growth rate, and grooming behavior), as well as quantification of gut parasites (Nosema spp., and Lotmaria passim), viruses (DWV-A, DWV-B, BQCV and SBV) and external parasites (Varroa destructor). Our goal was to examine: 1) correlations between pathogens and colony phenotypes; 2) the dynamics of pathogens and parasites on colony phenotypes and productivity traits; and 3) the effects of social immunity behaviors on colony pathogen load. Our results show that colonies expressing high levels of some social immunity behaviors were associated with low levels of pathogens/parasites, including viruses, Nosema spp., and V. destructor. In addition, we determined that elevated viral and Nosema spp. levels were associated with low levels of colony productivity, and that five out of six pathogenic factors measured were negatively associated with colony size and weight in both fall and spring periods. Finally, this study also provides information about the incidence and abundance of pathogens, colony phenotypes, and further disentangles their inter-correlation, so as to better understand drivers of honey bee colony health and productivity.

Impacts of COVID-19 on Canadian Beekeeping: Survey Results and a Profitability Analysis.

To gauge the impact of COVID-19 on the Canadian beekeeping sector, we conducted a survey of over 200 beekeepers in the fall of 2020. Our survey results show Canadian beekeepers faced two major challenges: 1) disrupted importation of honey bees (Hymenoptera: Apidae) (queen and bulk bees) that maintain populations; and 2) disrupted arrival of temporary foreign workers (TFWs). Disruptions in the arrival of bees and labor resulted in fewer colonies and less colony management, culminating in higher costs and lower productivity. Using the survey data, we develop a profitability analysis to estimate the impact of these disruptions on colony profit. Our results suggest that a disruption in either foreign worker or bee arrival allows beekeepers to compensate and while colony profits are lower, they remain positive. When both honey bee and foreign workers arrivals are disrupted for a beekeeper, even when the beekeeper experiences less significant colony health and cost impacts, a colony with a single pollination contract is no longer profitable, and a colony with two pollination contracts has significantly reduced profitability. As COVID-19 disruptions from 2020 and into 2021 become more significant to long-term colony health and more costly to a beekeeping operation, economic losses could threaten the industry's viability as well as the sustainability of pollination-dependent crop sectors across the country. The economic and agricultural impacts from the COVID-19 pandemic have exposed a vulnerability within Canada's beekeeping industry stemming from its dependency on imported labor and bees. Travel disruptions and border closures pose an ongoing threat to Canadian agriculture and apiculture in 2021 and highlight the need for Canada's beekeeping industry to strengthen domestic supply chains to minimize future risks.

Honey Bee Queen Production: Canadian Costing Case Study and Profitability Analysis.

The decline in managed honey bee (Hymenoptera: Apidae) colony health worldwide has had a significant impact on the beekeeping industry. To mitigate colony losses, beekeepers in Canada and around the world introduce queens into replacement colonies; however, Canada's short queen rearing season has historically limited the production of early season queens. As a result, Canadian beekeepers rely on the importation of foreign bees, particularly queens from warmer climates. Importing a large proportion of (often mal-adapted) queens each year creates a dependency on foreign bee sources, putting beekeeping, and pollination sectors at risk in the event of border closures, transportation issues, and other restrictions as is currently happening due to the 2020 Covid-19 pandemic. Although traditional Canadian queen production is unable to fully meet early season demand, increasing domestic queen production to meet mid- and later season demand would reduce Canada's dependency. As well, on-going studies exploring the potential for overwintering queens in Canada may offer a strategy to have early season domestic queens available. Increasing the local supply of queens could provide Canadian beekeepers, farmers, and consumers with a greater level of agricultural stability and food security. Our study is the first rigorous analysis of the economic feasibility of queen production. We present the costs of queen production for three Canadian operations over two years. Our results show that it can be profitable for a beekeeping operation in Canada to produce queen cells and mated queens and could be one viable strategy to increase the sustainability of the beekeeping industry.

In Vitro Effects of Pesticides on European Foulbrood in Honeybee Larvae.

Neonicotinoid and fungicide exposure has been linked to immunosuppression and increased susceptibility to disease in honeybees (Apis mellifera). European foulbrood, caused by the bacterium Melissococcus plutonius, is a disease of honeybee larvae which causes economic hardship for commercial beekeepers, in particular those whose colonies pollinate blueberries. We report for the first time in Canada, an atypical variant of M. plutonius isolated from a blueberry-pollinating colony. With this isolate, we used an in vitro larval infection system to study the effects of pesticide exposure on the development of European foulbrood disease. Pesticide doses tested were excessive (thiamethoxam and pyrimethanil) or maximal field-relevant (propiconazole and boscalid). We found that chronic exposure to the combination of thiamethoxam and propiconazole significantly decreased the survival of larvae infected with M. plutonius, while larvae chronically exposed to thiamethoxam and/or boscalid or pyrimethanil did not experience significant increases in mortality from M. plutonius infection in vitro. Based on these results, individual, calculated field-realistic residues of thiamethoxam and/or boscalid or pyrimethanil are unlikely to increase mortality from European foulbrood disease in honeybee worker brood, while the effects of field-relevant exposure to thiamethoxam and propiconazole on larval mortality from European foulbrood warrant further study.

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

A Bio-Economic Case Study of Canadian Honey Bee (Hymenoptera: Apidae) Colonies: Marker-Assisted Selection (MAS) in Queen Breeding Affects Beekeeper Profits.

Over the past decade in North America and Europe, winter losses of honey bee (Hymenoptera: Apidae) colonies have increased dramatically. Scientific consensus attributes these losses to multifactorial causes including altered parasite and pathogen profiles, lack of proper nutrition due to agricultural monocultures, exposure to pesticides, management, and weather. One method to reduce colony loss and increase productivity is through selective breeding of queens to produce disease-, pathogen-, and mite-resistant stock. Historically, the only method for identifying desirable traits in honey bees to improve breeding was through observation of bee behavior. A team of Canadian scientists have recently identified markers in bee antennae that correspond to behavioral traits in bees and can be tested for in a laboratory. These scientists have demonstrated that this marker-assisted selection (MAS) can be used to produce hygienic, pathogen-resistant honey bee colonies. Based on this research, we present a beekeeping case study where a beekeeper's profit function is used to evaluate the economic impact of adopting colonies selected for hygienic behavior using MAS into an apiary. Our results show a net profit gain from an MAS colony of between 2% and 5% when Varroa mites are effectively treated. In the case of ineffective treatment, MAS generates a net profit benefit of between 9% and 96% depending on the Varroa load. When a Varroa mite population has developed some treatment resistance, we show that MAS colonies generate a net profit gain of between 8% and 112% depending on the Varroa load and degree of treatment resistance.

Correlation of proteome-wide changes with social immunity behaviors provides insight into resistance to the parasitic mite, Varroa destructor, in the honey bee (Apis mellifera).

BACKGROUND: Disease is a major factor driving the evolution of many organisms. In honey bees, selection for social behavioral responses is the primary adaptive process facilitating disease resistance. One such process, hygienic behavior, enables bees to resist multiple diseases, including the damaging parasitic mite Varroa destructor. The genetic elements and biochemical factors that drive the expression of these adaptations are currently unknown. Proteomics provides a tool to identify proteins that control behavioral processes, and these proteins can be used as biomarkers to aid identification of disease tolerant colonies. RESULTS: We sampled a large cohort of commercial queen lineages, recording overall mite infestation, hygiene, and the specific hygienic response to V. destructor. We performed proteome-wide correlation analyses in larval integument and adult antennae, identifying several proteins highly predictive of behavior and reduced hive infestation. In the larva, response to wounding was identified as a key adaptive process leading to reduced infestation, and chitin biosynthesis and immune responses appear to represent important disease resistant adaptations. The speed of hygienic behavior may be underpinned by changes in the antenna proteome, and chemosensory and neurological processes could also provide specificity for detection of V. destructor in antennae. CONCLUSIONS: Our results provide, for the first time, some insight into how complex behavioural adaptations manifest in the proteome of honey bees. The most important biochemical correlations provide clues as to the underlying molecular mechanisms of social and innate immunity of honey bees. Such changes are indicative of potential divergence in processes controlling the hive-worker maturation.

Alternative reading frame selection mediated by a tRNA-like domain of an internal ribosome entry site.

The dicistrovirus intergenic region internal ribosome entry site (IRES) utilizes a unique mechanism, involving P-site tRNA mimicry, to directly assemble 80S ribosomes and initiate translation at a specific non-AUG codon in the ribosomal A site. A subgroup of dicistrovirus genomes contains an additional stem-loop 5'-adjacent to the IRES and a short open reading frame (ORFx) that overlaps the viral structural polyprotein ORF (ORF2) in the +1 reading frame. Using mass spectrometry and extensive mutagenesis, we show that, besides directing ORF2 translation, the Israeli acute paralysis dicistrovirus IRES also directs ORFx translation. The latter is mediated by a UG base pair adjacent to the P-site tRNA-mimicking domain. An ORFx peptide was detected in virus-infected honey bees by multiple reaction monitoring mass spectrometry. Finally, the 5' stem-loop increases IRES activity and may couple translation of the two major ORFs of the virus. This study reveals a novel viral strategy in which a tRNA-like IRES directs precise, initiator Met-tRNA-independent translation of two overlapping ORFs.

Ecological adaptation of diverse honey bee (Apis mellifera) populations.

BACKGROUND: Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology. Proteomics provides tools unique among the major 'omics disciplines for identifying the mechanisms employed by an organism in adapting to environmental challenges. RESULTS: Through proteome profiling of adult honey bee midgut from geographically dispersed, domesticated populations combined with multiple parallel statistical treatments, the data presented here suggest some of the major cellular processes involved in adapting to different climates. These findings provide insight into the molecular underpinnings that may confer an advantage to honey bee populations. Significantly, the major energy-producing pathways of the mitochondria, the organelle most closely involved in heat production, were consistently higher in bees that had adapted to colder climates. In opposition, up-regulation of protein metabolism capacity, from biosynthesis to degradation, had been selected for in bees from warmer climates. CONCLUSIONS: Overall, our results present a proteomic interpretation of expression polymorphisms between honey bee ecotypes and provide insight into molecular aspects of local adaptation or selection with consequences for honey bee management and breeding. The implications of our findings extend beyond apiculture as they underscore the need to consider the interdependence of animal populations and their agro-ecological context.

Proteome profile and lentiviral transduction of cultured honey bee (Apis mellifera L.) cells.

Honey bees (Apis mellifera L.) play a vital role in agriculture as pollinators, and serve as model organisms of social behaviour and immunity. The lack of both immortalized cell lines and methods to introduce recombinant DNA reliably into primary cells hinders cellular and molecular studies in this organism. We hereby demonstrate the expression of a GFP gene delivered by lentivirus transduction to cultured embryonic cells. The success of this approach indicates that viral transduction could be used to deliver constitutively active oncogenes in order to immortalize honey bee cells. We were able to revive cells successfully after several months of cryogenic storage and we show how the proteome varies between freshly collected and cultured embryonic cells.

Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.