Hygiene-Eliciting Brood Semiochemicals as a Tool for Assaying Honey Bee (Hymenoptera: Apidae) Colony Resistance to Varroa (Mesostigmata: Varroidae)

Despite numerous interventions, the ectoparasitic mite Varroa (Varroa destructor Anderson and Trueman [Mesostigmata: Varroidae]) and the pathogens it vectors remain a primary threat to honey bee (Apis mellifera Linnaeus [Hymenoptera: Apidae]) health. Hygienic behavior, the ability to detect, uncap, and remove unhealthy brood from the colony, has been bred for selectively for over two decades and continues to be a promising avenue for improved Varroa management. Although hygienic behavior is expressed more in Varroa-resistant colonies, hygiene does not always confer resistance to Varroa. Additionally, existing Varroa resistance selection methods trade efficacy for efficiency, because those achieving the highest levels of Varroa resistance can be time-consuming, and thus expensive and impractical for apicultural use. Here, we tested the hypothesis that hygienic response to a mixture of semiochemicals associated with Varroa-infested honey bee brood can serve as an improved tool for predicting colony-level Varroa resistance. In support of our hypothesis, we demonstrated that a mixture of the compounds (Z)-10-tritriacontene, (Z)-8-hentriacontene, (Z)-8-heptadecene, and (Z)-6-pentadecene triggers hygienic behavior in a two-hour assay, and that high-performing colonies (hygienic response to ≥60% of treated cells) have significantly lower Varroa infestations, remove significantly more introduced Varroa, and are significantly more likely to survive the winter compared to low-performing colonies (hygienic response to <60% of treated cells). We discuss the relative efficacy and efficiency of this assay for facilitating apiary management decisions and selection of Varroa-resistant honey bees, as well as the relevance of these findings to honey bee health, pollination services, and social insect communication.     

Pollen Treated with a Combination of Agrochemicals Commonly Applied During Almond Bloom Reduces the Emergence Rate and Longevity of Honey Bee (Hymenoptera: Apidae) Queens

Honey bee (Apis mellifera L.) colonies that pollinate California’s almond orchards are often exposed to mixtures of agrochemicals. Although agrochemicals applied during almond bloom are typically considered bee-safe when applied alone, their combined effects to honey bees are largely untested. In recent years, beekeepers providing pollination services to California’s almond orchards have reported reductions in queen quality during and immediately after bloom, raising concerns that pesticide exposure may be involved. Previous research identified a synergistic effect between the insecticide active ingredient chlorantraniliprole and the fungicide active ingredient propiconazole to lab-reared worker brood, but their effects to developing queens are unknown. To test the individual and combined effects of these pesticides on the survival and emergence of developing queens, we fed worker honey bees in closed queen rearing boxes with pollen artificially contaminated with formulated pesticides containing these active ingredients as well as the spray adjuvant Dyne-Amic, which contains both organosilicone and alkyphenol ethoxylate. The translocation of pesticides from pesticide-treated pollen into the royal jelly secretions of nurse bees was also measured. Despite consistently low levels of all pesticide active ingredients in royal jelly, the survival of queens from pupation to 7 d post-emergence were reduced in queens reared by worker bees fed pollen containing a combination of formulated chlorantraniliprole (Altacor), propiconazole (Tilt), and Dyne-Amic, as well as the toxic standard, diflubenzuron (Dimilin 2L), applied in isolation. These results support recommendations to protect honey bee health by avoiding application of pesticide tank-mixes containing insecticides and adjuvants during almond bloom.     

Honey Bee (Hymenoptera: Apidae) Nursing Responses to Cuticular Cues Emanating from Short-term Changes in Larval Rearing Environment

Honey bee larvae are dependent on the social structure of colony for their provisioning and survival. With thousands of larvae being managed collectively by groups of foragers (collecting food resources) and nurse bees (processing food and provisioning larvae), coordination of colony efforts in rearing brood depends on multiple dynamic cues of larval presence and needs. Much of these cues appear to be chemical, with larvae producing multiple pheromones, major being brood ester pheromone (BEP; nonvolatile blend of fatty acid esters) that elicits both short-term releaser effects and long-term primer effects. While BEP can affect colony food collection and processing with the signaling of larval presence, it is unclear if BEP signals individual larval needs. To understand this aspect, in a series of experiments we manipulated larval feeding environment by depriving larvae from adult bee contact for 4-h period and examined (1) nurse bee interactions with contact-deprived and nondeprived larvae and larval extracts; (2) forager bee responses to contact-deprived and nondeprived larval extracts. We also characterized BEP of contact-deprived and nondeprived larvae. We found that nurse honey bees tend to aggregate more over contact-deprived larvae when compared with nondeprived larvae, but that these effects were not found in response to whole hexane extracts. Our analytical results suggest that BEP components changed in both quantity and quality over short period of contact deprivation. These changes affected foraging behavior, but did not appear to directly affect nursing behavior, suggesting that different chemical cues are involved in regulating nursing effort to individual larvae.     

North American Prairie Is a Source of Pollen for Managed Honey Bees (Hymenoptera: Apidae)

Prairie was a dominant habitat within large portions of North America before European settlement. Conversion of prairies to farmland resulted in the loss of a large proportion of native floral resources, contributing to the decline of native pollinator populations. Efforts to reconstruct prairie could provide honey bees (Apis mellifera) a source of much-needed forage, especially in regions dominated by crop production. To what extent honey bees, which were introduced to North America by European settlers, use plants native to prairies is unclear. We placed colonies with pollen traps within reconstructed prairies in central Iowa to determine which and how much pollen is collected from prairie plants. Honey bee colonies collected more pollen from nonnative than native plants during June and July. During August and September, honey bee colonies collected more pollen from plants native to prairies. Our results suggest that honey bees’ use of native prairie plants may depend upon the seasonality of both native and nonnative plants present in the landscape. This finding may be useful for addressing the nutritional health of honey bees, as colonies in this region frequently suffer from a dearth of forage contributing to colony declines during August and September when crops and weedy plants cease blooming. These results suggest that prairie can be a significant source of forage for honey bees in the later part of the growing season in the Midwestern United States; we discuss this insight in the context of honey bee health and biodiversity conservation.     

Surfing the Sweet Wave: Migrating Giant Honey Bees (Hymenoptera: Apidae: Apis dorsata) Display Spatial and Temporal Fidelity to Annual Stopover Site in Thailand

Apis dorsata F. (Hymenoptera: Apidae), the giant honey bee of southern Asia, is an important pollinator of crops and non-cultivated angiosperms, and a producer of honey and beeswax. Its populations are in decline in many areas. Colonies migrate seasonally between highland and lowland nesting sites, taking advantage of available food sources. In 2009, a stopover site was discovered in Thailand where numerous migrating colonies bivouacked near one another. Bivouacs used the site again in 2010. I went to the site in 2016 to test the hypothesis that bees use the site regularly as part of an annual migration. I witnessed many bivouacs, spanning almost precisely the same time period and occupying the same area as in 2010. Here I describe their migratory dances in preparation for departure and their subsequent flights as well as periodic mass flight and defensive behavior. Analysis of photographs indicated that the bivouacking bees aged slowly and may thus live long enough to be capable of intergenerational transmission of migratory route knowledge. I describe attributes of the stopover site, e.g., abundant food and water availability, its location along a major river, and other possible navigational cues. Although the site is the only one of its kind so far known to researchers, such stopover sites probably exist wherever giant honey bees undertake long seasonal migrations. I recommend searching for bivouacking sites, particularly along rivers, wherever giant honey bees migrate. Stopover sites are undoubtedly essential to the life history and health of migratory bee populations, and thus warrant conservation policies.     

Honey Bees (Apis mellifera Hymenoptera: Apidae) Preferentially Avoid Sugar Solutions Supplemented with Field-Relevant Concentrations of Hydrogen Peroxide Despite High Tolerance Limits

Honey bees (Apis mellifera L. Hymeoptera: Apidae) use hydrogen peroxide (synthesized by excreted glucose oxidase) as an important component of social immunity. However, both tolerance of hydrogen peroxide and the production of glucose oxidase in honey is costly. Hydrogen peroxide may also be encountered by honey bees at high concentrations in nectar while foraging, however despite its presence both in their foraged and stored foods, it is unclear if and how bees monitor concentrations of, and their behavioral responses to, hydrogen peroxide. The costs of glucose oxidase production and the presence of hydrogen peroxide in both nectar and honey suggest hypotheses that honey bees preferentially forage on hydrogen peroxide supplemented feed syrups at certain concentrations, and avoid feed syrups supplemented with hydrogen peroxide at concentrations above some tolerance threshold. We test these hypotheses and find that, counter to expectation, honey bees avoid glucose solutions supplemented with field-relevant hydrogen peroxide concentrations and either avoid or don’t differentiate supplemented sucrose solutions when given choice assays. This is despite honey bees showing high tolerance for hydrogen peroxide in feed solutions, with no elevated mortality until concentrations of hydrogen peroxide exceed 1% (v/v) in solution, with survival apparent even at concentrations up to 10%. The behavioral interaction of honey bees with hydrogen peroxide during both within-colony synthesis in honey and when foraging on nectar therefore likely relies on interactions with other indicator molecules, and maybe constrained evolutionarily in its plasticity, representing a constitutive immune mechanism.     

Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions

This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4°C) or heat (37°C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H₂O₂) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H₂O₂. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress.     

A Visual Tracking System for Honey Bee (Hymenoptera: Apidae) 3D Flight Trajectory Reconstruction and Analysis

We describe the development, field testing, and results from an automated 3D insect flight detection and tracking system for honey bees (Apis mellifera L.) (Hymenoptera: Apidae) that is capable of providing remarkable insights into airborne behavior. It comprises two orthogonally mounted video cameras with an observing volume of over 200 m³ and an offline analysis software system that outputs 3D space trajectories and inflight statistics of the target honey bees. The imaging devices require no human intervention once set up and are waterproof, providing high resolution and framerate videos. The software module uses several forms of modern image processing techniques with GPU-enabled acceleration to remove both stationary and moving artifact while preserving flight track information. The analysis system has thus far provided information not only on flight statistics (such as speeds and accelerations), but also on subtleties associated with flight behavior by generating heat maps of density and classifying flight patterns according to patrol and foraging behavior. Although the results presented here focus on behavior in the locale of a beehive, the system could be adapted to study a wide range of airborne insect activity.     

Parallel Mechanism Composed of Abdominal Cuticles and Muscles Simulates the Complex and Diverse Movements of Honey Bee (Apis mellifera L.) Abdomen

The abdominal intersegmental structures allow insects, such as honey bees, dragonflies, butterflies, and drosophilae, to complete diverse behavioral movements. In order to reveal how the complex abdominal movements of these insects are produced, we use the honey bee (Apis mellifera L.) as a typical insect to study the relationship between intersegmental structures and abdominal motions. Microstructure observational experiments are performed by using the stereoscope and the scanning electron microscope. We find that a parallel mechanism, composed of abdominal cuticle and muscles between the adjacent segments, produces the complex and diverse movements of the honey bee abdomen. These properties regulate multiple behavioral activities such as waggle dance and flight attitude adjustment. The experimental results demonstrate that it is the joint efforts of the muscles and membranes that connected the adjacent cuticles together. The honey bee abdomen can be waggled, expanded, contracted, and flexed with the actions of the muscles. From the view point of mechanics, a parallel mechanism is evolved from the intersegmental connection structures of the honey bee abdomen. Here, we conduct a kinematic analysis of the parallel mechanism to simulate the intersegmental abdominal motions.     

Integrated Pest Management Control of Varroa destructor (Acari: Varroidae), the Most Damaging Pest of (Apis mellifera L. (Hymenoptera: Apidae)) Colonies

Varroa destructor is among the greatest biological threats to western honey bee (Apis mellifera L.) health worldwide. Beekeepers routinely use chemical treatments to control this parasite, though overuse and mismanagement of these treatments have led to widespread resistance in Varroa populations. Integrated Pest Management (IPM) is an ecologically based, sustainable approach to pest management that relies on a combination of control tactics that minimize environmental impacts. Herein, we provide an in-depth review of the components of IPM in a Varroa control context. These include determining economic thresholds for the mite, identification of and monitoring for Varroa, prevention strategies, and risk conscious treatments. Furthermore, we provide a detailed review of cultural, mechanical, biological, and chemical control strategies, both longstanding and emerging, used against Varroa globally. For each control type, we describe all available treatments, their efficacies against Varroa as described in the primary scientific literature, and the obstacles to their adoption. Unfortunately, reliable IPM protocols do not exist for Varroa due to the complex biology of the mite and strong reliance on chemical control by beekeepers. To encourage beekeeper adoption, a successful IPM approach to Varroa control in managed colonies must be an improvement over conventional control methods and include cost-effective treatments that can be employed readily by beekeepers. It is our intention to provide the most thorough review of Varroa control options available, ultimately framing our discussion within the context of IPM. We hope this article is a call-to-arms against the most damaging pest managed honey bee colonies face worldwide.     

Reproductive and Morphological Quality of Commercial Honey Bee (Hymenoptera: Apidae) Drones in the United States

Exploration into reproductive quality in honey bees (Apis mellifera Linneaus (Hymenoptera: Apidae) largely focuses on factors that affect queens, with drones primarily being considered insofar as they pass on effects of environmental stressors to the queen and subsequent offspring. In those studies that consider drone quality explicitly, a primary focus has been on the dimorphic nature of drones laid in worker cells (either through rare queen error or worker reproduction) as compared to drones laid by the queen in the slightly larger drone cells. The implication from these studies is that that there exists a bimodality of drone morphological quality that is related to reproductive quality and competitive ability during mating. Our study quantifies the presence of such small drones in commercial populations, finding that rates of ‘low-quality’ drones are far higher than theoretically predicted under optimum conditions. Observations from commercial colonies also show significant inter-colony variation among the size and fecundity of drones produced, prompting speculation as to the mechanisms inducing such variation and the potential use of drone-quality variation for the colony- or apiary-level exposure to nutrition, agrichemical, or parasitic stressors.     

Effects of Dinotefuran on Brain miRNA Expression Profiles in Young Adult Honey Bees (Hymenopptera: Apidae)

Honey bees are important pollinators of wild plants and crops. MicroRNAs (miRNAs) are endogenous regulators of gene expression. In this study, we initially determined that the lethal concentration 50 (LC₅₀) of dinotefuran was 0.773 mg/l. Then, the expression profiles and differentially expressed miRNAs (DE miRNAs) in honey bee brains after 1, 5, and 10 d of treatment with the lethal concentration 10 (LC₁₀) of dinotefuran were explored via deep small-RNA sequencing and bioinformatics. In total, 2, 23, and 27 DE miRNAs were identified after persistent exposure to the LC₁₀ of dinotefuran for 1, 5, and 10 d, respectively. Some abundant miRNAs, such as ame-miR-375-3p, ame-miR-281-5p, ame-miR-3786-3p, ame-miR-10-5p, and ame-miR-6037-3p, were extremely significantly differentially expressed. Enrichment analysis suggested that the candidate target genes of the DE miRNAs are involved in the regulation of biological processes, cellular processes, and behaviors. These results expand our understanding of the regulatory roles of miRNAs in honey bee Apis mellifera (Hymenopptera: Apidae) responses to neonicotinoid insecticides and facilitate further studies on the functions of miRNAs in honey bees.     

Physicochemical Parameters and Antioxidant Activity of Bee Honey Enriched With Herbs

Three groups of products enriched with herbs were studied: (1) commercial herb honeys (n?=?5) produced by bees fed a syrup with an herbal extract, (2) natural herbal honey (n?=?3) produced by bees from the nectar of herbs, and (3) creamed multifloral honey with added dried herbs (n?=?5). As a control, multifloral honey (n?=?5) was used. The physicochemical parameters (i.e., sugar extract, water content, specific rotation, conductivity, hydroxymethylfurfural content, pH and acidity), sugar profiles (HPLC analysis), antioxidant activity and total phenolic compounds content of the studied samples were compared. Although great diversity in the basic properties of the studied products was observed, they were comparable to multifloral honey and complied with honey regulations. Significant differences in sugar composition were observed, and adversely positive rotation (excluding nettle herb honey) was detected in group 1, likely resulting from the change in bee feeding. The best antioxidant activity for creamed honeys with dried herbs (group 2) was investigated, whereas herb honeys (group 1) exhibited similar antioxidant properties as multifloral honey. The use of controlled feeding of bees appears to be an effective method of enriching honey with desirable plant bioactive components to create innovative bee products.     

The Complete Mitochondrial Genome of Lepidotrigona flavibasis (Hymenoptera: Meliponini) and High Gene Rearrangement in Lepidotrigona Mitogenomes

We reported the sequence and characteristics of the complete mitochondrial genome of an ecologically important stingless bee, Lepidotrigona flavibasis (Hymenoptera: Meliponini), that has suffered serious population declines in recent years. A phylogenetic analysis based on complete mitogenomes indicated that L. flavibasis was first clustered with another Lepidotrigona species (L. terminata) and then joined with the other two Melipona (Hymenoptera: Meliponini) stingless bees (M. scutellaris and M. bicolor), forming a single clade of stingless bees. The stingless bee clade has a closer relationship with bumblebees (Bombus) (Hymenoptera: Apidae) than with honeybees (Apis) (Hymenoptera: Apidae). Extremely high gene rearrangements involving tRNAs, rRNAs, D-loop regions, and protein-coding genes were observed in the Lepidotrigona mitogenomes, suggesting an overactive evolutionary status in Lepidotrigona species. These mitogenomic organization variations could provide a good system with which to understand the evolutionary history of Meliponini.     

Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee,Bombus terrestris (Hymenoptera: Apidae)

Hexamerins are members of the hemocyanin superfamily and play essential roles in providing amino acids and energy for the nonfeeding stages of insects. In this study, we cloned and analyzed the expression patterns of four hexamerin genes (hex 70a, hex 70b, hex 70c, and hex 110) at different worker development stages and queen diapause statuses in the bumble bee, Bombus terrestris. The results of this study showed that hex 110 has the longest open reading frame (ORF; 3,297 bp) compared to the ORFs of hex 70a (2,034 bp), hex 70b (2,067 bp), and hex 70c (2,055 bp). The putative translation product of Hex 70a, Hex 70b, Hex70c, and Hex 110 has 677, 688, 684, and 1,098aa with predicted molecular mass of 81.13, 79.69, 81.58, and 119 kDa. In the development stages of workers, the expression levels of hex 70a, hex 70b, and hex 70c increased gradually from the larval stage and exhibited high expression levels at the pink eyed and brown eyed pupae stage, whereas hex 110 exhibited the highest expression level at the larval period. Four hexamerin genes were highly expressed at the prediapause status of queen (P < 0.05), and compared to the eclosion queen, the lowest upregulation was 3.7-fold, and the highest upregulation was 1,742-fold. The expression levels of hex 70b, hex 70c, and hex 110 at diapause were significantly higher than those at postdiapause (P < 0.05). In conclusion, hexamerins may play important roles in queen diapause and metamorphosis of larval and pupal stages.     

Genetic and Usurpation Data Support High Incidence of Bumble Bee Nest Invasion by Socially Parasitic Bumble Bee,Bombus insularis

Cuckoo bumble bees (Psithyrus) (Lepeletier, 1832) (Hymenoptera: Apidae) are a unique lineage of bees that depend exclusively on a host bumble bee species to provide nesting material, nutritional resources, and labor to rear offspring. In this study, we document usurpation incidence and population genetic data of Bombus insularis (Smith, 1861) (Hymenoptera: Apidae), a bumble bee species in the Psithyrus subgenus, on field-deployed B. huntii colonies in northern Utah, United States. Within 12 d of deploying B. huntii Greene, 1860 (Hymenoptera: Apidae) colonies at two field sites, 13 of the 16 colonies contained at least one established B. insularis female. Although our results demonstrate that field-deployed bumble bee colonies are highly susceptible to B. insularis usurpation, applying a fabricated excluder to prevent the inquiline from invading a colony was 100% effective. Sibship analysis using microsatellite genotype data of 59 B. insularis females estimates that they originated from at least 49 unique colonies. Furthermore, sibship analysis found siblings distributed between the field sites that were 7.04 km apart. Our result suggests that B. insularis females have the capacity to disperse across the landscape in search of host colonies at distances of at least 3.52 km and up to 7.04 km. Our study underscores the detrimental impact B. insularis usurpation has on the host bumble bee colony. As B. insularis significantly impacts the success of bumble bee colonies, we briefly discuss how the utilization of excluders may be useful for commercial bumble bee colonies that are used to pollinate open field crops.     

Evidence for Bombus occidentalis (Hymenoptera: Apidae) Populations in the Olympic Peninsula,the Palouse Prairie,and Forests of Northern Idaho

Since the mid-1990s, Bombus occidentalis (Green) has declined from being one of the most common to one of the rarest bumble bee species in the Pacific Northwest of the United States. Although its conservation status is unresolved, a petition to list this species as endangered or threatened was recently submitted to the U.S. Fish and Wildlife Service. To shed light on the conservation situation and inform the U.S. Fish and Wildlife Service decision, we report on the detection and abundance of B. occidentalis following bumble bee collection between 2012 and 2014 across the Pacific Northwest. Collection occurred from the San Juan Islands and Olympic peninsula east to northern Idaho and northeastern Oregon, excluding the arid region in central Washington. B. occidentalis was observed at 23 collection sites out of a total of 234. With the exception of three sites on the Olympic peninsula, all of these were in the southeastern portion of the collection range.     

Woody habitats promote pollinators and complexity of plant–pollinator interactions in homegardens located in rice terraces of the Philippine Cordilleras

Bees are important pollinators of wild plants and crops, but little is known about bee habitat requirements and pollinator management in tropical mountainous agricultural regions. Here, smallholder farmers produce fruits and vegetables in homegardens that depend upon or benefit from bee pollination. We hypothesized that abundance and richness of wild and domesticated bees and the complexity of plant–pollinator interactions are higher in homegardens surrounded by woody habitats than in homegardens found farther from woodlands. Bees were sampled in 20 homegardens in the rice terraces of the Philippine Cordilleras. We used linear mixed effect models to analyse effects of woody habitats around homegardens on bee richness and abundance. Based on pooled observations for each garden category, we built pollinator–plant interactions networks to illustrate shifts in interaction frequencies. We recorded 354 bee individuals of 13 wild and one domesticated bee species (Apis cerana). Wild bee richness was significantly higher in homegardens surrounded by woody habitats. Bee abundance increased significantly with increasing flower cover. Wild bees visited cultivated plants significantly more often than domesticated bees. Six vegetable species and 76% of all flower visits on cultivated plants in total were performed by wild bees and three plant species and 24% by domesticated bees. Pollinator–plant networks were more complex in homegardens surrounded by woody habitats. We conclude that woody habitats increase abundance and richness of wild and domesticated bees. Increasing availability of floral resources also promotes bee abundance. In order to promote pollination services in the landscape mosaic of smallholder rice farms, woody habitats and forest fragments together with numerous floral resources should be protected and restored.     

Risk Assessment for the Establishment of Vespa mandarinia (Hymenoptera: Vespidae) in the Pacific Northwest,United States

The recent introduction of the Asian giant hornet, Vespa mandarinia Smith, in the United States in late 2019 has raised concerns about its establishment in the Pacific Northwest and its potential deleterious effects on honey bees, Apis spp., and their pollination services in the region. Therefore, we conducted a risk assessment of the establishment of V. mandarinia in Washington, Oregon, Montana, and Idaho on a county-by-county basis. Our highly conservative tier-1 qualitative and semiquantitative risk assessment relied on the biological requirements and ecological relationships of V. mandarinia in the environments of the Pacific Northwest. Our risk characterization was based on climate and habitat suitability estimates for V. mandarinia queens to overwinter and colonize nests, density and distribution of apiaries, and locations of major human-mediated introduction pathways that may increase establishment of the hornet in the counties. Our results suggest that 32 counties in the region could be at low risk, 120 at medium risk, and 23 at high risk of establishment. Many of the western counties in the region were estimated to be at the highest risk of establishment mainly because of their suitable climate for queens to overwinter, dense forest biomass for nest colonization, and proximity to major port and freight hubs in the region. By design, our tier-1 risk assessment most likely overestimates the risk of establishment, but considering its negative effects, these counties should be prioritized in ongoing monitoring and eradication efforts of V. mandarinia.