Combined treatment with amitraz and thymol to manage Varroa destructor mites (Acari: Varroidae) in Apis mellifera honey bee colonies (Hymenoptera: Apidae).

The parasitic mite Varroa destructor (Anderson and Trueman) is one of the greatest stressors of Apis mellifera (L.) honey bee colonies. When Varroa infestations reach damaging levels during fall, rapid control is necessary to minimize damage to colonies. We performed a field trial in the US Southeast to determine if a combination of registered treatments (Apivar, amitraz-based; and Apiguard, thymol-based) could provide rapid and effective control of Varroa. We compared colonies that received this combination treatment against colonies that received amitraz-based positive control treatments: (i) Apivar alone; or (ii) amitraz emulsifiable concentrate ("amitraz EC"). While not registered, amitraz EC is used by beekeepers in the United States in part because it is thought to control Varroa more rapidly and effectively than registered products. Based on measurements of Varroa infestation rates of colonies after 21 days of treatment, we found that the combination treatment controlled Varroa nearly as rapidly as the amitraz EC treatment: this or other combinations could be useful for Varroa management. At the end of the 42-day trial, colonies in the amitraz EC group had higher bee populations than those in the Apivar group, which suggests that rapid control helps reduce Varroa damage. Colonies in the combination group had lower bee populations than those in the amitraz EC group, which indicates that the combination treatment needs to be optimized to avoid damage to colonies.

Neonicotinoid exposure increases Varroa destructor (Mesostigmata: Varroidae) mite parasitism severity in honey bee colonies and is not mitigated by increased colony genetic diversity.

Agrochemical exposure is a major contributor to ecological declines worldwide, including the loss of crucial pollinator species. In addition to direct toxicity, field-relevant doses of pesticides can increase species' vulnerabilities to other stressors, including parasites. Experimental field demonstrations of potential interactive effects of pesticides and additional stressors are rare, as are tests of mechanisms via which pollinators tolerate pesticides. Here, we controlled honey bee colony exposure to field-relevant concentrations of 2 neonicotinoid insecticides (clothianidin and thiamethoxam) in pollen and simultaneously manipulated intracolony genetic heterogeneity. We showed that exposure increased rates of Varroa destructor (Anderson and Trueman) parasitism and that while increased genetic heterogeneity overall improved survivability, it did not reduce the negative effect size of neonicotinoid exposure. This study is, to our knowledge, the first experimental field demonstration of how neonicotinoid exposure can increase V. destructor populations in honey bees and also demonstrates that colony genetic diversity cannot mitigate the effects of neonicotinoid pesticides.

No evidence to support the use of glycerol-oxalic acid mixtures delivered via paper towel for controlling Varroa destructor (Mesostigmata: Varroidae) mites in the Southeast United States.

A significant amount of researcher and practitioner effort has focused on developing new chemical controls for the parasitic Varroa destructor mite in beekeeping. One outcome of that has been the development and testing of "glycerol-oxalic acid" mixtures to place in colonies for extended periods of time, an off-label use of the otherwise legal miticide oxalic acid. The majority of circulated work on this approach was led by practitioners and published in nonacademic journals, highlighting a lack of effective partnership between practitioners and scientists and a possible failure of the extension mandate in beekeeping in the United States. Here, we summarize the practitioner-led studies we could locate and partner with a commercial beekeeper in the Southeast of the United States to test the "shop towel-oxalic acid-glycerol" delivery system developed by those practitioners. Our study, using 129 commercial colonies between honey flows in 2017 split into 4 treatment groups, showed no effectiveness in reducing Varroa parasitism in colonies exposed to oxalic acid-glycerol shop towels. We highlight the discrepancy between our results and those circulated by practitioners, at least for the Southeast, and the failure of extension to support practitioners engaged in research.

Assessing Repeated Oxalic Acid Vaporization in Honey Bee (Hymenoptera: Apidae) Colonies for Control of the Ectoparasitic Mite Varroa destructor.

The American beekeeping industry continually experiences colony mortality with annual losses as high as 43%. A leading cause of this is the exotic, ectoparasitic mite, Varroa destructor Anderson & Trueman (Mesostigmata: Varroidae). Integrated Pest Management (IPM) options are used to keep mite populations from reaching lethal levels, however, due to resistance and/or the lack of suitable treatment options, novel controls for reducing mites are warranted. Oxalic acid for controlling V. destructor has become a popular treatment regimen among commercial and backyard beekeepers. Applying vaporized oxalic acid inside a honey bee hive is a legal application method in the U.S., and results in the death of exposed mites. However, if mites are in the reproductive stage and therefore under the protective wax capping, oxalic acid is ineffective. One popular method of applying oxalic is vaporizing multiple times over several weeks to try and circumvent the problem of mites hiding in brood cells. By comparing against control colonies, we tested oxalic acid vaporization in colonies treated with seven applications separated by 5 d (35 d total). We tested in apiaries in Georgia and Alabama during 2019 and 2020, totaling 99 colonies. We found that adult honey bees Linnaeus (Hymenoptera: Apidae), and developing brood experienced no adverse impacts from the oxalic vaporization regime. However, we did not find evidence that frequent periodic application of oxalic during brood-rearing periods is capable of bringing V. destructor populations below treatment thresholds.

Deformed wings of small hive beetle independent of virus infections and mites.

Insect wing deformities can be caused by viruses, mites and other environmental stressors during development. Here we conducted differential diagnostics of deformed wings in small hive beetles, Aethina tumida (SHB). Adult SHB with and without deformed wings from individual and mass reared scenarios were evaluated for the mite Tyrophagus putrescentiae and for deformed wing virus. Viral load and mite number were similar for SHB with deformed wings and unaffected beetles. Because deformed wings were only observed in individually pupating SHB, a humidity challenge most likely explains the observed clinical symptoms. Our observations support the importance of differential diagnostics.

Neonicotinoid insecticides can serve as inadvertent insect contraceptives.

There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.

The ectoparasitic mite Tropilaelaps mercedesae reduces western honey bee, Apismellifera, longevity and emergence weight, and promotes Deformed wing virus infections.

Historically an ectoparasite of the native Giant honey bee Apis dorsata, the mite Tropilaelaps mercedesae has switched hosts to the introduced western honey bee Apis mellifera throughout much of Asia. Few data regarding lethal and sub-lethal effects of T. mercedesae on A. mellifera exist, despite its similarity to the devastating mite Varroa destructor. Here we artificially infested worker brood of A. mellifera with T. mercedesae to investigate lethal (longevity) and sub-lethal (emergence weight, Deformed wing virus (DWV) levels and clinical symptoms of DWV) effects of the mite on its new host. The data show that T. mercedesae infestation significantly reduced host longevity and emergence weight, and promoted both DWV levels and associated clinical symptoms. Our results suggest that T. mercedesae is a potentially important parasite to the economically important A. mellifera honey bee.

Effects, but no interactions, of ubiquitous pesticide and parasite stressors on honey bee (Apis mellifera) lifespan and behaviour in a colony environment.

Interactions between pesticides and parasites are believed to be responsible for increased mortality of honey bee (Apis mellifera) colonies in the northern hemisphere. Previous efforts have employed experimental approaches using small groups under laboratory conditions to investigate influence of these stressors on honey bee physiology and behaviour, although both the colony level and field conditions play a key role for eusocial honey bees. Here, we challenged honey bee workers under in vivo colony conditions with sublethal doses of the neonicotinoid thiacloprid, the miticide tau-fluvalinate and the endoparasite Nosema ceranae, to investigate potential effects on longevity and behaviour using observation hives. In contrast to previous laboratory studies, our results do not suggest interactions among stressors, but rather lone effects of pesticides and the parasite on mortality and behaviour, respectively. These effects appear to be weak due to different outcomes at the two study sites, thereby suggesting that the role of thiacloprid, tau-fluvalinate and N. ceranae and interactions among them may have been overemphasized. In the future, investigations into the effects of honey bee stressors should prioritize the use of colonies maintained under a variety of environmental conditions in order to obtain more biologically relevant data.

Thiacloprid-Nosema ceranae interactions in honey bees: host survivorship but not parasite reproduction is dependent on pesticide dose.

Interactions between stressors contribute to the recently reported increase in losses of honey bee colonies. Here we demonstrated that a synergistic effect on mortality by the low toxic, commonly used neonicotinoid thiacloprid and the nearly ubiquitous gut parasite Nosemaceranae is dependent on the pesticide dose. Furthermore, thiacloprid had a negative influence on N.ceranae reproduction. Our results highlight that interactions among honey bee health stressors can be dynamic and should be studied across a broader range of combinations.

Evaluation of cage designs and feeding regimes for honey bee (Hymenoptera: Apidae) laboratory experiments.

The aim of this study was to improve cage systems for maintaining adult honey bee (Apis mellifera L.) workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Prevention of honey bee COlony LOSSes), on the physiology and survival of honey bees. We identified three cages that promoted good survival of honey bees. The bees from cages that exhibited greater survival had relatively lower titers of deformed wing virus, suggesting that deformed wing virus is a significant marker reflecting stress level and health status of the host. We also determined that a leak- and drip-proof feeder was an integral part of a cage system and a feeder modified from a 20-ml plastic syringe displayed the best result in providing steady food supply to bees. Finally, we also demonstrated that the addition of protein to the bees' diet could significantly increase the level ofvitellogenin gene expression and improve bees' survival. This international collaborative study represents a critical step toward improvement of cage designs and feeding regimes for honey bee laboratory experiments.

Managing the parasitic honey bee mite Tropilaelaps mercedesae through combined cultural and chemical control methods.

The western honey bee (Apis mellifera) is severely impacted by the parasitic Tropilaelaps mercedesae mite, which has the capacity to outcompete Varroa destructor mites (the current leading cause of colony losses) and more rapidly overwhelm colonies. While T. mercedesae is native to Asia, it has recently expanded its geographic range and has the potential to devastate beekeeping worldwide if introduced to new regions. Our research exploited the dependence of T. mercedesae on developing honey bees (brood) by combining a cultural technique (brood break) with U.S. registered chemical products (oxalic acid or formic acid) to manage T. mercedesae infestation. To evaluate this approach, we compared four treatment groups: (1) Brood Break; (2) Brood Break + Formic Acid (FormicPro®); (3) Brood Break + Oxalic Acid dribble (Api-Bioxal®); and (4) untreated Control. We found that the mite infestation rate of worker brood in Control colonies rose from 0.4 to 15.25% over 60 days, whereas all other treatment groups had infestation rates under 0.11% on Day 60. Mite fall assessments showed similar results, whereby Control colonies had 15.48 mites fall per 24 h on day 60 compared to less than 0.2 mites for any other treatment group. Evaluation of colony strength revealed that Brood Break + Formic Acid colonies had slightly reduced adult honey bee populations. No treatment eliminated all mites, so additional measures may be needed to eradicate T. mercedesae if detected in countries that do not currently have T. mercedesae.

Shift in virus composition in honeybees (Apis mellifera) following worldwide invasion by the parasitic mite and virus vector Varroa destructor.

Invasive vectors can induce dramatic changes in disease epidemiology. While viral emergence following geographical range expansion of a vector is well known, the influence a vector can have at the level of the host's pathobiome is less well understood. Taking advantage of the formerly heterogeneous spatial distribution of the ectoparasitic mite Varroa destructor that acts as potent virus vector among honeybees Apis mellifera, we investigated the impact of its recent global spread on the viral community of honeybees in a retrospective study of historical samples. We hypothesized that the vector has had an effect on the epidemiology of several bee viruses, potentially altering their transmissibility and/or virulence, and consequently their prevalence, abundance, or both. To test this, we quantified the prevalence and loads of 14 viruses from honeybee samples collected in mite-free and mite-infested populations in four independent geographical regions. The presence of the mite dramatically increased the prevalence and load of deformed wing virus, a cause of unsustainably high colony losses. In addition, several other viruses became more prevalent or were found at higher load in mite-infested areas, including viruses not known to be actively varroa-transmitted, but which may increase opportunistically in varroa-parasitized bees.

Negative but antagonistic effects of neonicotinoid insecticides and ectoparasitic mites Varroa destructor on Apis mellifera honey bee food glands.

Collaborative brood care by workers is essential for the functionality of eusocial Apis mellifera honey bee colonies. The hypopharyngeal food glands of workers play a crucial role in this context. Even though there is consensus that ubiquitous ectoparasitic mites Varroa destructor and widespread insecticides, such as neonicotinoids, are major stressors for honey bee health, their impact alone and in combination on the feeding glands of workers is poorly understood. Here, we show that combined exposure to V. destructor and neonicotinoids antagonistically interacted on hypopharyngeal gland size, yet they did not interact on emergence body mass or survival. While the observed effects of the antagonistic interaction were less negative than expected based on the sum of the individual effects, hypopharyngeal gland size was still significantly reduced. Alone, V. destructor parasitism negatively affected emergence body mass, survival, and hypopharyngeal gland size, whereas neonicotinoid exposure reduced hypopharyngeal gland size only. Since size is associated with hypopharyngeal gland functionality, a reduction could result in inadequate brood care. As cooperative brood care is a cornerstone of eusociality, smaller glands could have adverse down-stream effects on inclusive fitness of honey bee colonies. Therefore, our findings highlight the need to further study how ubiquitous stressors like V. destructor and neonicotinoids interact to affect honey bees.

Artificial pollination of kiwifruit (Actinidia chinensis Planch. var. chinensis) (Ericales: Actinidiaceae) results in greater fruit set compared to flowers pollinated by managed bees (Apis mellifera L. (Hymenoptera: Apidae) and Bombus impatiens Cresson (Hymenoptera: Apidae)).

Due to a lack of knowledge on the pollination requirements of kiwifruit cultivars grown within the United States, farmers simultaneously implement multiple pollination methods, like the rental of managed bee species or artificial pollination to achieve high fruit yields. However, implementing multiple pollination methods is costly and possibly an inefficient use of resources. We assessed the contribution of two managed bees (Apis mellifera and Bombus impatiens) to the pollination of kiwifruit by i) determining the relative abundance of kiwifruit pollen collected by foragers of each bee species, and ii) comparing fruit set and fruit quality among insect and artificially pollinated flowers through an insect exclusion experiment. A significant difference was observed between the mean relative abundance of kiwifruit pollen carried in the corbicula of A. mellifera and B. impatiens, with B. impatiens carrying on average 46% more kiwifruit pollen than A. mellifera. Artificially pollinated kiwifruit flowers set significantly greater numbers of fruit per flower at four weeks post-bloom and at harvest compared to insect pollination, wind pollination, and pollen exclusion treatment. Artificial pollination produced fruits of greater weight, size, and seed number compared to insect-pollinated flowers, and few fruits were produced in the pollen exclusion and wind pollination treatments. Kiwifruit producers experiencing similar conditions to ours should focus on artificially pollinating their crops rather than relying on managed or wild insects for kiwifruit pollination. Future research should evaluate other methods of artificial pollination to determine their effectiveness, efficiency, and economics in the pollination of kiwifruit grown within the United States.

Multiple mating by both sexes in an invasive insect species, Aethina tumida (Coleoptera: Nitidulidae).

Multiple mating by both sexes is common among sexually reproducing animals. Small hive beetles (SHB), Aethina tumida, are parasites of bee nests endemic to sub-Saharan Africa and have become a widespread invasive species. Despite the considerable economic damages they can cause, their basic biology remains poorly understood. Here we show that male and female small hive beetles can mate multiple times, suggesting that costs for mating are low in this species. In an invasive A. tumida population in the United States, a combination of laboratory experiments for males and paternity analysis with eight polymorphic DNA microsatellite markers for field-caught females were used to estimate the number of mating by both sexes. The data show that females and males can mate multiple times-females mated with up to eight males, whereas males mated with at least seven females. The results also showed that A. tumida displayed a skewed paternity, although this was not consistent among the tested females. Thus, first or last male advantage seem to be unlikely in A. tumida. Our observations that individuals of both sexes of A. tumida can mate multiple times opens new research avenues for examining drivers of multiple mating and determining the role it may play in promoting biological invasions.

Honey bee colony loss linked to parasites, pesticides and extreme weather across the United States.

Honey bee (Apis mellifera) colony loss is a widespread phenomenon with important economic and biological implications, whose drivers are still an open matter of investigation. We contribute to this line of research through a large-scale, multi-variable study combining multiple publicly accessible data sources. Specifically, we analyzed quarterly data covering the contiguous United States for the years 2015-2021, and combined open data on honey bee colony status and stressors, weather data, and land use. The different spatio-temporal resolutions of these data are addressed through an up-scaling approach that generates additional statistical features which capture more complex distributional characteristics and significantly improve modeling performance. Treating this expanded feature set with state-of-the-art feature selection methods, we obtained findings that, nation-wide, are in line with the current knowledge on the aggravating roles of Varroa destructor and pesticides in colony loss. Moreover, we found that extreme temperature and precipitation events, even when controlling for other factors, significantly impact colony loss. Overall, our results reveal the complexity of biotic and abiotic factors affecting managed honey bee colonies across the United States.

Buffered fitness components: Antagonism between malnutrition and an insecticide in bumble bees.

Global insect biodiversity declines due to reduced fitness are linked to interactions between environmental stressors. In social insects, inclusive fitness depends on successful mating of reproductives, i.e. males and queens, and efficient collaborative brood care by workers. Therefore, interactive effects between malnutrition and environmental pollution on sperm and feeding glands (hypopharyngeal glands (HPGs)) would provide mechanisms for population declines, unless buffered against due to their fitness relevance. However, while negative effects for bumble bee colony fitness are known, the effects of malnutrition and insecticide exposure singly and in combination on individuals are poorly understood. Here we show, in a fully-crossed laboratory experiment, that malnutrition and insecticide exposure result in neutral or antagonistic interactions for spermatozoa and HPGs of bumble bees, Bombus terrestris, suggesting strong selection to buffer key colony fitness components. No significant effects were observed for mortality and consumption, but significant negative effects were revealed for spermatozoa traits and HPGs. The combined effects on these parameters were not higher than the individual stressor effects, which indicates an antagonistic interaction between both. Despite the clear potential for additive effects, due to the individual stressors impairing muscle quality and neurological control, simultaneous malnutrition and insecticide exposure surprisingly did not reveal an increased impact compared to individual stressors, probably due to key fitness traits being resilient. Our data support that stressor interactions require empirical tests on a case-by-case basis and need to be regarded in context to understand underlying mechanisms and so adequately mitigate the ongoing decline of the entomofauna.

The Pollination and Fruit Quality of Two Kiwifruit Cultivars (Actinidia chinensis var. chinensis 'AU Golden Sunshine' and 'AU Gulf Coast Gold') (Ericales: Actinidiaceae) Grown in the Southeastern United States.

Kiwifruit is a new emerging crop for the southeastern United States that requires cross-pollination to set fruit. However, the pollination requirements for varieties grown in the southeastern United States are unknown. Through insect surveys and a bagging experiment, we assessed the pollination requirements of two female kiwifruit cultivars (Actinidia chinensis var. chinensis 'AU Golden Sunshine' and A. chinensis var. chinensis 'AU Gulf Coast Gold'). For each, fruit quantity (fruit set) and fruit quality (weight, size, seed count, firmness, soluble solid content, and dry matter) were compared among three pollination treatments (wind, insect, and artificial pollination). Low abundances of insects were observed visiting female flowers of both kiwifruit cultivars, and therefore likely minimally influenced kiwifruit pollination. Artificial pollination resulted in the greatest percentages of fruit set and marketable fruits, followed by insect and wind pollination. Artificial pollination resulted in fruits that were greater in weight, size, and contained more seeds, than insect- and wind-pollinated fruits. Firmness and soluble solid content did not vary greatly between pollination treatments, yet were greater in 'AU Golden Sunshine'. Dry matter content did not vary greatly between pollination treatments or between each cultivar. To maximize yields and optimize fruit quality, these results suggest that kiwifruit producers should place more effort into artificial pollination compared to wind and insect pollination. Future research should explore the use of managed bees (e.g., honey bees and bumble bees) within kiwifruit orchards to determine ways to utilize them as a secondary source for pollination needs.

Cuticular Hydrocarbon Profile of Parasitic Beetles, Aethina tumida (Coleoptera: Nitidulidae).

Cuticular hydrocarbons (CHCs) cover insects' bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs), Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.

Long-term field-realistic exposure to a next-generation pesticide, flupyradifurone, impairs honey bee behaviour and survival.

The assessment of pesticide risks to insect pollinators have typically focused on short-term, lethal impacts. The environmental ramifications of many of the world's most commonly employed pesticides, such as those exhibiting systemic properties that can result in long-lasting exposure to insects, may thus be severely underestimated. Here, seven laboratories from Europe and North America performed a standardised experiment (a ring-test) to study the long-term lethal and sublethal impacts of the relatively recently approved 'bee safe' butenolide pesticide flupyradifurone (FPF, active ingredient in Sivanto®) on honey bees. The emerging contaminant, FPF, impaired bee survival and behaviour at field-realistic doses (down to 11 ng/bee/day, corresponding to 400 µg/kg) that were up to 101-fold lower than those reported by risk assessments (1110 ng/bee/day), despite an absence of time-reinforced toxicity. Our findings raise concerns about the chronic impact of pesticides on pollinators at a global scale and support a novel methodology for a refined risk assessment.