Bumble bee microcolonies have been used to investigate a range of research questions including pesticide effects. As a toxicity test, the microcolony can be a tool to observe concentration-based endpoints and has the flexibility for delivering a test substance via sucrose solution, pollen provision, or both. However, there is no regulatory guideline available nor an academic standard experimental design for bumble bee microcolonies and test performance can be influenced by pollen source, pollen amount, and worker selection. Our objectives were to evaluate the impact of worker size and age, as well as pollen quantity and quality, on microcolony test performance of common eastern bumble bee (Bombus impatiens Cresson). A cumulative 5 g of pollen provision during the first 10 d of colony establishment resulted in a higher number of emerged males at the end of the test compared to 2 g. The number of emerged males was different depending on the source and nutritional composition of the pollen. Mixed sizes and ages of workers yielded good test performance and are recommended based upon our findings. These collective results improve test performance (ie maximize the number of produced drones, maintain worker survival, and enable consumption of sucrose solution and pollen provisions, the latter as potential vehicles of test items in a toxicity test) when conducting a bumble bee microcolony study. These findings enhance the reliability of bumble bee microcolony studies and provide essential insights for developing standardized testing protocols that can better assess the impacts of environmental stressors on pollinator health.

Ecological risk assessment is a key component of the regulatory process required for registration of crop protection products around the world. The western honey bee (Apis mellifera) is the model organism for pesticide risk assessments for bees, but there are uncertainties over whether it is predictive of risks to other bees. Consequently, efforts are underway to develop test methodologies for other non-Apis bees. We conducted a semi-field colony-feeding study with Bombus impatiens colonies to develop a colony-level methodology for bumble bees. We exposed commercially available bumble bee colonies to diets consisting of 4 concentration treatments of dimethoate insecticide (0.05, 0.19, 0.75, and 3.0 mg a.i./L) via supplemental sugar solution for 6 wk and compared exposed colonies to untreated controls. Each treatment group had 10 replicate colonies, with 1 replicate per treatment group represented at each of the 10 study rural locations. We collected data on various colony-level endpoints including production of female reproductive (gyne) offspring, colony weight, foraging activity, and consumption of provisioned sugar solution. Our results indicated that the test design could be used to derive concentration-response relationships for several endpoints including the most sensitive, colony mass (No Observed Adverse Effect Concentration = 0.05 mg a.i./L). Overall, our study provides the foundation for a semi-field, colony-feeding study test design for bumble bees, thus adding to the growing body of studies that may be used to assess the protectiveness of the honey bee risk assessment framework for non-Apis bees exposed to pesticides.

Orchard bees of the genus Osmia Panzer are important pollinators of fruit trees in various regions of the world, with some species commercially available in the United States and Europe. In addition to their pollination services, Osmia lignaria, Osmia cornifrons, Osmia bicornis, and Osmia cornuta have been identified as potential model species for solitary bees in pesticide risk assessment and have been used for the development of new methods to test acute lethal effects via contact and oral routes of exposure. Our goal was to expand the available methodology to characterize the toxicity of pesticides for these solitary bees through a chronic oral test for adult bees. Chronic oral toxicity of pesticides to orchard bees has been reported, but methods differ among research groups. In our study, O. lignaria, O. cornifrons, O. bicornis, and O. cornuta female bees had access to sucrose solution ad libitum in separate, species-specific 10-day tests. Mean body mass, mean daily consumption, and survival differed among the studied bee species. The dose-response test design was validated with dimethoate, a reference toxic compound, and chronic toxicity endpoints were estimated for the 4 Osmia species. The median lethal daily doses normalized by weight for O. lignaria, O. bicornis, O. cornuta, and O. cornifrons were within the same order of magnitude at 0.23, 0.26, 0.49, and 0.61 mu g dimethoate/g bee/day, respectively. The methodology described here was aligned as much as possible with the available honey bee and bumble bee standard methods to facilitate the comparison of chronic toxicity profiles among bee species.

Understanding the frequency of non-additive effects of pesticides (synergism and antagonism) is important in the context of risk assessment. The goal of this study was to investigate the prevalence of non-additive effects of pesticides to honey bees (Apis mellifera). We investigated a large set of mixtures including insecticides and fungicides of different chemical modes of action and classes. The mixtures included represent a relevant sample of pesticides that are currently used globally. We investigated whether the experimental toxicity of the mixtures could be predicted based on the Concentration Addition (CA) model for acute contact and oral adult bee toxicity tests. We measured the degree of deviation from the additivity predictions of the experimental toxicity based on the well-known Mixture Deviation Ratio (MDR). Further, we investigated the appropriate MDR thresholds that should be used for the identification of non-additive effects based on acceptable rates for false positive (alpha) and true positive (beta) findings. We found that a deviation factor of MDR = 5 is a sound reference for labeling potential non-additive effects in acute adult bee experimental designs when assuming a typical Coefficient of Variation (CV%) = 100 in the determination of the LD50 of a pesticide (a factor of 2× deviation in the LD 50 resulting from inter-experimental variability). We found that only 2.4 % and 9 % of the mixtures evaluated had an MDR > 5 and MDR < 0.2, respectively. The frequency and magnitude of deviation from additivity found for bees in this study are consistent with those of other terrestrial and aquatic taxa. Our findings suggest that additivity is a good baseline for predicting the toxicity of pesticide mixtures to bees, and that the rare cases of synergy of pesticide mixtures to bees are not random but have a mechanistic basis.

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991–1003. © 2022 SETAC

Risk assessment for chemicals in the United States relies upon the honey bee (Apis meliffera L. [Hymenoptera: Apidae]) as a surrogate for other bee species. There is uncertainty in extrapolating honey bee toxicity data to bumble bees due to differences in life history strategies, food consumption, and nest structure. Here we evaluated the design of a queenless bumble bee microcolony test that could be considered for generating larval toxicity data. Three microcolony studies were conducted with Bombus impatiens to evaluate the effects of exposure to 1) diflubenzuron in pollen, 2) dimethoate in pollen, and 3) dimethoate in sucrose. Immature drone bee emergence, worker survival, pollen, and sucrose utilization were measured throughout the study duration. For dimethoate, a 10-d chronic adult bumble bee study was also conducted to compare microcolony endpoints to toxicity endpoints on individual adults. Microcolonies exposed to 10 mg diflubenzuron/kg pollen produced fewer adult drones despite no effects on worker survival. Microcolonies treated with dimethoate at ≥3 mg a.i./kg pollen and ≥0.1 mg a.i./kg sucrose solution produced fewer drones. Exposure to dimethoate in the 10-d chronic adult study resulted in direct mortality to the adult workers at ≥0.1 mg a.i./kg diet. Results from the 10-d study suggest direct effects of dimethoate on workers in the microcolony will alter provisioning of diet to the brood, resulting in lower drone production in the microcolony. Our data suggest that the microcolony study is only appropriate to assess brood effects to bumble bees for substances with low toxicity to adults, as demonstrated with diflubenzuron.

s: Oral Presentation 46 Julius-Kühn-Archiv, 465, 2020 2.2 Progress on the Osmia acute oral test findings of the ICPPR Non-Apis subgroup solitary bee laboratory testing Ivo Roessink1, Nicole Hanewald2, Christof Schneider2, Anja Quambusch3, Nina Exeler3, Ana R. Cabrera4, AnnaMaria Molitor5, Verena Tänzler6, Bettina Hodapp7, Matthias Albrecht8, Annely Brandt9, Steven Vinall10, Anne-Kathrin Rathke11, Hervé Giffard12, Eugenia Soler13, Alexander Schnurr14, Michael Patnaude15, Elodie Couture16, David Lehman17 1Wageningen Environmental Research -The Netherlands, 2BASF-Germany, 3BAYER-Germany,4BAYER-United States, 5Eurofins Agroscience Services Ecotox GmbH-Germany, 6IBACON GmbH-Germany, 7Innovative Environmental Services (IES) Ltd-Switzerland, 8Agroscope-Switzerland, 9Landesbetrieb Landwirtschaft HessenGermany, 10MamboTox-United Kingdom, 11Noack laboratorien-Germany, 12TestApi-France, 13Trialcamp SLUSpain, 14Biochem Agrar GmbH-Germany, 15Smithers-United States, 16SynTech-France, 17US Environmental Protection Agency-United States DOI 10.5073/jka.2020.465.017

s: Poster 132 Julius-Kühn-Archiv, 465, 2020 4.10.P Development and validation of a bumble bee adult chronic oral test Nina Exeler1, Anja Quambusch1, Nicole Hanewald2, Arnaud Zicot3, Eugenia Soler4, Annette Kling5, Steven Vinall6, K. Dressler7, Verena Tänzler8, Stefan Kimmel9, David M. Lehmann10, Michael Patnaude11, Ana R. Cabrera12 1Bayer AG, Monheim am Rhein, Germany, 2BASF SE, Limburgerhof, Germany, 3SynTech, Nimes, France, 4Eurofins Trialcamp, Alcàsser-Valencia, Spain, 5Eurofins Agroscience Services, Niefern-Öschelbronn, Germany, 6Mambo-Tox Ltd., Southampton, UK, 7BioChem agrar GmbH, Machern OT Gerichshain, Germany 8Ibacon, Rossdorf, Germany, 9IES Ltd, Witterswil, Switzerland, 10USEPA, Research Triangle Park, NC, USA 11Smithers Viscient US, Wareham, MA, USA, 12Bayer CropScience LP, Chesterfield, MO, USA DOI 10.5073/jka.2020.465.058

The honey bee, Apis mellifera L. (Hymenoptera: Apidae), is a model organism for pollinators in risk assessment frameworks globally. The acute toxicity tests with adult honey bees for contact and oral exposure are part of the requirements for pesticide registration and are typically conducted with the active ingredient. A question often asked is if the typical end-use product (TEP) is more toxic than the technical grade active ingredient (TGAI) to honey bees. We explored this question by mining publicly available databases from regulatory agencies worldwide, where testing with the TEP is required. The objective of this study was to determine whether TEPs are comparable in toxicity to the TGAI. The dataset was analyzed via a 3 × 3 contingency table with toxicity categories, as the data cannot be computed for regression analysis. Of the 151 active ingredients with reported endpoints for contact exposure, 28 were classified as either moderately or highly toxic, 123 were classified as practically nontoxic, and 3 were inconclusive. Only two (1.3%) were reclassified from nontoxic to moderately toxic as the TEP. Of the 141 active ingredients with reported endpoints for oral exposure, 23 were classified as moderately or highly toxic, 113 were classified as practically nontoxic, and 5 were inconclusive. Only five (3.6%) were reclassified from nontoxic to moderately toxic as the TEP. Fewer than 5% of the total TEPs evaluated (contact and oral) were shown to be more toxic than the TGAI, suggesting that the risk assessments of TGAIs would be sufficiently protective to pollinators at the screening laboratory level.

To date, regulatory pesticide risk assessments have relied on the honey bee (Apis mellifera L.) (Hymenoptera: Apidae) as a surrogate test species for estimating the risk of pesticide exposure to all bee species. However, honey bees and non-Apis bees may differ in their susceptibility and exposure to pesticides. In 2017, a workshop (‘Pesticide Exposure Assessment Paradigm for Non-Apis Bees’) was held to assess if honey bee risk assessment frameworks are reflective of non-Apis bee pesticide exposure. In this article, we summarize the workshop discussions on bumble bees (Bombus spp.). We review the life history and foraging behavior of bumble bees and honey bees and discuss how these traits may influence routes and levels of exposure for both taxa. Overall, the major pesticide exposure routes for bumble bees and honey bees are similar; however, bumble bees face additional exposure routes (direct exposure of foraging queens and exposure of larvae and adults to soil residues). Furthermore, bumble bees may receive comparatively higher pesticide doses via contact or oral exposure. We conclude that honey bee pesticide risk assessments may not always be protective of bumble bees, especially queens, in terms of exposure. Data needed to reliably quantify pesticide exposure for bumble bees (e.g., food consumption rates, soil residue levels) are lacking. Addressing these knowledge gaps will be crucial before bumble bee exposure can be incorporated into the pesticide risk assessment process. Because bumble bees exhibit appreciable interspecific variation in colony and behavioral characteristics, data relevant to pesticide exposure should be generated for multiple species.

For honey bees (Apis mellifera), colony maintenance and growth are highly dependent on worker foragers obtaining sufficient resources from flowering plants year round. Despite the importance of floral diversity for proper bee nutrition, urban development has drastically altered resource availability and diversity for these important pollinators. Therefore, understanding the floral resources foraged by bees in urbanized areas is key to identifying and promoting plants that enhance colony health in those environments. In this study, we identified the pollen foraged by bees in four developed areas of the U.S., and explored whether there were spatial or temporal differences in the types of floral sources of pollen used by honey bees in these landscapes. To do this, pollen was collected every month for up to one year from colonies located in developed (urban and suburban) sites in California, Texas, Florida, and Michigan, except during months of pollen dearth or winter. Homogenized pollen samples were acetolyzed and identified microscopically to the lowest taxonomic level possible. Once identified, each pollen type was classified into a frequency category based on its overall relative abundance. Species richness and diversity indices were also calculated and compared across states and seasons. We identified up to 64 pollen types belonging to 39 plant families in one season (California). Species richness was highest in CA and lowest in TX, and was highest during spring in every state. In particular, "predominant" and "secondary" pollen types belonged to the families Arecaceae, Sapindaceae, Anacardiaceae, Apiaceae, Asteraceae, Brassicaceae, Fabaceae, Fagaceae, Lythraceae, Myrtaceae, Rhamnaceae, Rosaceae, Rutaceae, Saliaceae, and Ulmaceae. This study will help broaden our understanding of honey bee foraging ecology and nutrition in urban environments, and will help promote the use of plants that serve the dual purpose of providing aesthetic value and nutritious forage for honey bee colonies placed in developed landscapes.

Bumble bee adult chronic toxicity studies and bioassays to assess larval development in the laboratory are currently undergoing method validation and standardization through ring-testing. These test designs will contribute valuable data required for Tier 1 risk assessments for this significant and commercially valuable pollinator. While laboratory assays allow for a conservative, highly controlled, and standardized evaluation of the relationship between test item dose and organism response, they do not reflect field-realistic exposure scenarios and cannot adequately address potential impacts on whole colony development. Semi-field, landscape-level field, or feeding studies are more suitable to describe whole-colony health and development and potential impacts from pesticide exposure in an agricultural setting. However, evaluation end-points need to be clearly characterized and the associated assessment methodology should minimize variation across studies. This is especially true for field studies, where genetic and environmental variability will cause significant impacts on study results. Here, we seek to provide a comprehensive review of available bumble bee colony end-points, assess their relevance and suitability for higher tier studies examining field-realistic exposure scenarios, and identify data, method, and knowledge gaps that may guide future research activity.

A novel feeding protocol for delivery of bio-active agents to Varroa mites was developed by providing mites with honey bee larva hemolymph supplemented with cultured insect cells and selected materials delivered on a fibrous cotton substrate. Mites were starved, fed on treated hemolymph to deliver selected agents and then returned to bee larvae. Transcript levels of two reference genes, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as well as for nine selected genes involved in reproductive processes showed that the starvation and feeding protocol periods did not pose a high level of stress to the mites as transcript levels remained comparable between phoretic mites and those completing the protocol. The feeding protocol was used to deliver molecules such as hormone analogs or plasmids. Mites fed with Tebufenozide, an ecdysone analog, had higher transcript levels of shade than untreated or solvent treated mites. In order to extend this feeding protocol, cultured insect cells were incorporated to a final ratio of 1 part cells and 2 parts hemolymph. Although supplementation with Bombyx mori Bm5 cells increased the amount of hemolymph consumed per mite, there was a significant decrease in the percentage of mites that fed and survived. On the other hand, Drosophila melanogaster S2 cells reduced significantly the percentage of mites that fed and survived as well as the amount of hemolymph consumed. The feeding protocol provides a dynamic platform with which to challenge the Varroa mite to establish efficacy of control agents for this devastating honey bee pest.

Flupyradifurone (Sivanto) is a novel systemic insecticide from the butenolide class developed by Bayer. Based on available data (USEPA 2014), this insecticide appears to have a favorable safety profile for honey bee colonies. As a result, the label permits the product to be applied during prebloom and bloom in various crops, including citrus, except when mixed with azole fungicides during the blooming period. We placed 24 honey bee (Apis mellifera L.) colonies adjacent to eight flowering buckwheat (Fagopyrum esculentum Moench) fields that either had been sprayed with the maximum label rate of flupyradifurone or with water only (control fields), with three colonies placed adjacent to each field. We conducted colony strength assessments during which the number of adult bees, eggs, uncapped brood cells, capped brood cells, food storage cells, and weights of honey supers and brood chambers were determined prior to, during, and after the flowering period. We also analyzed bee-collected pollen and nectar for flupyradifurone residues. Overall, there were no differences in any colony strength parameter for colonies placed at control and flupyradifurone-treated buckwheat fields. Residue analyses showed that pollen (x =  565.8 ppb) and nectar (x  =  259.4 ppb) gathered by bees on fields treated with flupyradifurone contained significantly higher flupyradifurone residues than did bee bread and unprocessed nectar collected by bees from control fields (75% of samples <LOD). Within the conditions set forth by our experimental design, our collective data suggest no adverse effects of flupyradifurone on honey bee colonies when following label directions.

Global declines of bumble bees and other pollinator populations are of concern because of their critical role for crop production and maintenance of wild plant biodiversity. Although the consensus among scientists is that the interaction of many factors, including habitat loss, forage scarcity, diseases, parasites, and pesticides, potentially plays a role in causing these declines, pesticides have received considerable attention and scrutiny. In response, regulatory agencies have introduced more stringent pollinator testing requirements for registration and reregistration of pesticides, to ensure that the risks to pollinators are minimized. In this context, guidelines for testing bumble bees (Bombus spp.) in regulatory studies are not yet available, and a pressing need exists to develop suitable protocols for routine higher-tier studies with these non-Apis sp., social bees. To meet this need, Bayer CropScience LP, Syngenta Crop Protection LLC US, and Valent USA. Corporation organized a workshop bringing together a group of global experts on bumble bee behavior, ecology, and ecotoxicology to discuss and develop draft protocols for both semi-field (Tier II) and field (Tier III) studies. The workshop was held May 8-9, 2014, at the Bayer Bee Care Center, North Carolina, USA. The participants represented academic, consulting, and industry scientists from Europe, Canada, the United States, and Brazil. The workshop identified a clear protection goal and generated proposals for basic experimental designs, relevant measurements, and endpoints for both semifield (tunnel) and field tests. These initial recommendations are intended to form the basis of discussions to help advance the development of appropriate protocol guidelines.

Declines of bumble bees and other pollinator populations in Europe and North America are of concern because of their critical role for crop production and biodiversity maintenance. Although the consensus in the scientific community is that the interaction of many factors including habitat loss, forage scarcity, diseases, parasites and pesticides probably play a role in causing these declines, pesticides have received considerable public attention and scrutiny. In response regulatory agencies have introduced more stringent pollinator testing requirements for registration and re-registration of plant protection products, to ensure the risks to pollinators are minimised. Guidelines for testing bumble bees in regulatory studies are not yet available and there is a pressing need to develop suitable protocols for routine studies with these non-Apis, social bees. As a first step, Bayer CropScience, Syngenta Crop Protection and Valent U.S.A. Corporation organized a workshop bringing together a global team of bumble bee ecotoxicology experts to discuss and develop draft protocols for both semi-field (Tier II) and field (Tier III) studies. The workshop was held at the Bayer Bee Care Center, in Research Triangle Park, North Carolina during May 8-9, 2014. The participants represented academia, consulting and industry from Europe, Canada, United States and Brazil. The workshop identified a clear protection goal, and generated proposals for basic experimental layouts, relevant measurements and endpoints for both semifield (tunnel) and field tests. The workshop participants intend to disseminate this information as widely as possible to interested researchers and regulatory officers, who can advance the development of protocol guidelines based on these initial recommendations.

Tomato (Solanum lycopersicum L.) is among the most valuable agricultural products, but Meloidogyne spp. (root-knot nematode) infestations result in serious crop losses. In tomato, resistance to root-knot nematodes is controlled by the gene Mi-1, but heat stress interferes with Mi-1-associated resistance. Inconsistent results in published field and greenhouse experiments led us to test the effect of short-term midday heat stress on tomato susceptibility to Meloidogyne incognita race 1. Under controlled day/night temperatures of 25°C/21°C, 'Amelia', which was verified as possessing the Mi-1 gene, was deemed resistant (4.1 ± 0.4 galls/plant) and Rutgers, which does not possess the Mi-1 gene, was susceptible (132 ± 9.9 galls/plant) to M. incognita infection. Exposure to a single 3 hr heat spike of 35°C was sufficient to increase the susceptibility of 'Amelia' but did not affect Rutgers. Despite this change in resistance, Mi-1 gene expression was not affected by heat treatment, or nematode infection. The heat-induced breakdown of Mi-1 resistance in 'Amelia' did recover with time regardless of additional heat exposures and M. incognita infection. These findings would aid in the development of management strategies to protect the tomato crop at times of heightened M. incognita susceptibility.

The complete genomic region and corresponding transcript of the most abundant protein in phoretic varroa mites, Varroa destructor (Anderson & Trueman), were sequenced and have homology with acarine hemelipoglycoproteins and the large lipid transfer protein (LLTP) super family. The genomic sequence of VdLLTP included 14 introns and the mature transcript coded for a predicted polypeptide of 1575 amino acid residues. VdLLTP shared a minimum of 25% sequence identity with acarine LLTPs. Phylogenetic assessment showed VdLLTP was most closely related to Metaseiulus occidentalis vitellogenin and LLTP proteins of ticks; however, no heme binding by VdLLTP was detected. Analysis of lipids associated with VdLLTP showed that it was a carrier for free and esterified C12-C22 fatty acids from triglycerides, diacylglycerides and monoacylglycerides. Additionally, cholesterol and β-sitosterol were found as cholesterol esters linked to common fatty acids. Transcript levels of VdLLTP were 42 and 310 times higher in phoretic female mites when compared with males and quiescent deutonymphs, respectively. Coincident with initiation of the reproductive phase, VdLLTP transcript levels declined to a third of those in phoretic female mites. VdLLTP functions as an important lipid transporter and should provide a significant RNA interference target for assessing the control of varroa mites.

When a female varroa mite, Varroa destructor (Anderson & Trueman), invades a honey bee brood cell, the physiology rapidly changes from feeding phoretic to reproductive. Changes in foraging and malvolio transcript levels in the brain have been associated with modulated intra-specific food searching behaviors in insects and other invertebrates. Transcription profiles for both genes were examined during and immediately following brood cell invasion to assess their role as potential control elements. Vdfor and Vdmvl transcripts were found in all organs of varroa mites with the highest Vdfor transcript levels in ovary-lyrate organs and the highest Vdmvl in Malpighian tubules. Changes in transcript levels of Vdfor and Vdmvl in synganglia were not associated with the cell invasion process, remaining comparable between early reproductive mites (collected from the pre-capping brood cells) and phoretic mites. However, Vdfor and Vdmvl transcript levels were lowered by 37 and 53%, respectively, in synganglia from reproductive mites compared to early reproductive mites, but not significantly different to levels in synganglia from phoretic mites. On the other hand, in whole body preparations the Vdfor and Vdmvl had significantly higher levels of transcript in reproductive mites compared to phoretic and early reproductive, mainly due to the presence of both transcripts accumulating in the eggs carried by the ovipositing mite. Varroa mites are a critical component for honey bee population decline and finding varroa mite genes associated with brood cell invasion, reproduction, ion balance and other physiological processes will facilitate development of novel control avenues for this honey bee parasite.