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.

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.

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.

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.

Eusocial insect declines: Insecticide impairs sperm and feeding glands in bumblebees.

Insecticides are contributing to global insect declines, thereby creating demand to understand the mechanisms underlying reduced fitness. In the eusocial Hymenoptera, inclusive fitness depends on successful mating of male sexuals (drones) and efficient collaborative brood care by female workers. Therefore, sublethal insecticide effects on sperm and glands used in larval feeding (hypopharyngeal glands (HPG)) would provide key mechanisms for population declines in eusocial insects. However, while negative impacts for bumblebee colony fitness have been documented, the effects of insecticide exposure on individual physiology are less well understood. Here, we show that field-realistic concentrations (4.5-40 ng ml-1) of the neonicotinoid insecticide thiamethoxam significantly impair Bombus terrestris sperm and HPGs, thereby providing plausible mechanisms underlying bumblebee population decline. In the laboratory, drones and workers were exposed to five thiamethoxam concentrations (4.5 to 1000 ng ml-1). Then, survival, food consumption, body mass, HPG development, sperm quantity and viability were assessed. At all concentrations, drones were more exposed than workers due to higher food consumption. Increased body mass was observed in drones starting at 20 ng ml-1 and in workers at 100 ng ml-1. Furthermore, environmentally realistic concentrations (4.5-40 ng ml-1) did not significantly affect survival or consumption for either sex. However, thiamethoxam exposure significantly negatively affected both sperm viability and HPG development at all tested concentrations. Therefore, the results indicate a trade-off between survival and fitness components, possibly due to costly detoxification. Since sperm and HPG are corner stones of colony fitness, the data offer plausible mechanisms for bumblebee population declines. To adequately mitigate ongoing biodiversity declines for the eusocial insects, this study suggests it is essential to evaluate the impact of insecticides on fitness parameters of both sexuals and workers.

The weakest link: Haploid honey bees are more susceptible to neonicotinoid insecticides.

Neonicotinoid insecticides are currently of major concern for the health of wild and managed insects that provide key ecosystem services like pollination. Even though sublethal effects of neonicotinoids are well known, there is surprisingly little information on how they possibly impact developmental stability, and to what extent genetics are involved. This holds especially true for haploid individuals because they are hemizygous at detoxification loci and may be more susceptible. Here we take advantage of haplodiploidy in Western honey bees, Apis mellifera, to show for the first time that neonicotinoids affect developmental stability in diploid females (workers), and that haploid males (drones) are even more susceptible. Phenotypic fore wing venation abnormalities and fluctuating wing asymmetry, as measures of developmental instability, were significantly increased under field-realistic neonicotinoid-exposure of colonies. The higher susceptibility of haploid drones suggests that heterozygosity can play a key role in the ability to buffer the sublethal effects of neonicotinoids. Aiming to improve conservation efforts, our findings highlight the urgent need to better understand the role that genetics plays at enabling non-target organisms to cope with insecticide exposure.

Not every sperm counts: Male fertility in solitary bees, Osmia cornuta.

Reproductive strategies can act as strong selective forces on reproductive traits of male insects, resulting in species-specific variation in sperm quantity and viability. For solitary bees, basic measures of sperm quantity and viability are scarce. Here we evaluated for the first time quantity and viability of sperm in male Osmia cornuta solitary bees at different times after emergence, and how they were affected by male body mass and environmental condition (laboratory or semi-field arena). Sperm viability immediately after adult emergence showed no significant difference compared to four day old individuals, suggesting that O. cornuta males are capable of mating immediately post emergence. However, sperm counts were significantly higher in four day old individuals from the semi-field arena when compared to newly emerged males. This might reflect a final phase of sperm maturation. Regardless of individual male age and body mass differences, O. cornuta males produced on average ~175'000 spermatozoa that were ~65% viable, which are both significantly lower compared to eusocial honeybees and bumblebees. Moreover, sperm quantity, but not viability, was positively correlated with male body mass four days after emergence, while no such relationship was detected immediately after emergence. Even though individuals maintained in semi-field conditions exhibited a significantly greater loss of body mass, experimental arena had no significant effect on male survival, sperm quality or total living sperm produced. This suggests that the proposed laboratory design provides a cost-efficient and simple experimental approach to assess sperm traits in solitary bees. In conclusion, our data suggest a reduced investment in both sperm quantity and quality by male O. cornuta, which appears to be adaptive in light of the life history of this solitary bee.

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.