Stressors like habitat loss, climate change, pollution, and invasive species have resulted in widespread anthropogenic effects on ecosystems (Butchart et al., 2010; Geldmann et al., 2014). Research on how multiple stressors interact to pressure important ecosystem-service providing species have revealed complex effects, ranging from additive, whereby the effects of two stressors equal their combined individual effects, to synergism for which the combined effect of two stressors is greater than the predicted additive effects, to antagonism, when the combined effect of two stressors is less than the predicted additive effects (Côté et al., 2016). Mechanisms responsible for each type of interaction vary, but they may be influenced by exploitative competition for limited resources (Poulin, 2007) or host stress as a result of tissue pathology or immune suppression (Alaux et al., 2010; Pettis et al., 2013). Understanding and predicting the circumstances surrounding each type of interaction is currently difficult, but is expected to significantly improve as laboratory, field, and meta-analysis investigations on model or important ecosystem-service providing species continue (Boyd and Brown, 2015; Piggott et al., 2015; Kaunisto et al., 2016).

In recent years, it is believed that pressures caused by multiple, possibly interacting, stressors are responsible for consistently high losses of managed western honey bee (Apis mellifera) colonies across the northern hemisphere (Kulhanek et al., 2017; Bruckner et al., 2019; Gray et al., 2019, 2020). Both biotic and abiotic stressors, such as poor nutrition, introduced parasites, heavy metals, and pesticides are blamed (Steinhauer et al., 2018). Individually, they can elicit a range of negative consequences on honey bees, ranging from sub-lethal physiological and behavioral effects to lethal ones that result in reduced survival (Havard et al., 2020). When acting in concert, diverse effects are observed, ranging from antagonistic parasitic-pesticide interactions to synergistic pesticide-pesticide ones (Straub et al., 2020; Bird et al., 2021; Siviter et al., 2021).

Arguably one of the most important biotic stressors for honey bees is the ectoparasitic mite Varroa destructor (Rosenkranz et al., 2010; Traynor et al., 2020). Its life cycle is tightly linked to its honey bee host, and consists of two distinct stages—dispersal and reproduction (Traynor et al., 2020). Mature V. destructor foundress mites produce several offspring that subsequently feed on tissues of developing honey bees (hereafter called brood) until they emerge from brood cells alongside their honey bee hosts (Rosenkranz et al., 2010; Ramsey et al., 2019). Mature female V. destructor mites prefer brood of honey bee males, also called drones, because of specific characteristics such as an extended reproduction period as well as the chemical signals elicited by developing drones (Conte et al., 1989; Fuchs, 1992; Rosenkranz et al., 2010). Mite infestation can negatively affect drone body mass and mating efficiency through increased mortality, reduced flight activity, and low sperm quality (Collins and Pettis, 2001; Duay et al., 2002; Bubalo et al., 2005).

Insecticides like systemic neonicotinoids are important abiotic risk factors (Simon-Delso et al., 2015; Wood and Goulson, 2017). Neonicotinoids are among the most widely applied insecticides globally (Simon-Delso et al., 2015), and are predominantly employed as seed coating treatments translocating throughout the entire plant, including pollen and nectar (Bonmatin et al., 2007; Botías et al., 2015; Pang et al., 2020). Neonicotinoids have been detected in honey bee collected pollen from multiple sources, such as maize (Pilling et al., 2013) and squash (Stoner and Eitzer, 2012), herbaceous plants (Wood et al., 2019), and wild flowers (Botías et al., 2015), as well as in beebread, which is processed pollen stored within the colony (Bonmatin et al., 2015; Mogren and Lundgren, 2016; Tong et al., 2018). Furthermore, these compounds are readily released into the soil and water where they can persist and translocate to adjacent vegetation, posing a risk to non-target organisms (Sur et al., 2003; Bonmatin et al., 2015). The risk of exposure extends from individual honey bee foragers to the entire colony when contaminated resources are shared with other adults and developing individuals (Sanchez-Bayo and Goka, 2014). There is clear evidence that field-relevant concentrations of neonicotinoids have sub-lethal effects on honey bees, eliciting behavioral, physiological, and anatomical changes (Singla et al., 2021). For example, drones experienced reduced development stability and produced fewer living sperm under neonicotinoid exposure, which can ultimately affect colony reproductive potential and overall performance (Straub et al., 2016; Friedli et al., 2020).

Despite V. destructor and neonicotinoids considered to ubiquitous stressors to honey bees (Little et al., 2015; Wilfert et al., 2016; Colwell et al., 2017; Mitchell et al., 2017), little is known about their potential interactive effects in honey bees. A handful of studies on non-reproductive honey bee females, the workers, have yielded conflicting results, ranging from no interaction to synergism (Straub et al., 2016; Siede et al., 2018; Morfin et al., 2020; Bird et al., 2021); however, no such work has investigated possible effects on honey bee drones, despite their predicted greater susceptibility to environmental stressors as proposed by the haploid susceptibility hypothesis (O’Donnell and Beshers, 2004), which suggests that drones are likely less resilient to environmental stressors than their worker counterparts due to a lack in allelic variation at important immune related genes (Hamilton, 1964; O’Donnell and Beshers, 2004; Retschnig et al., 2014; Friedli et al., 2020). The availability of high quality drones to mate with reproductive honey bee females, the queens, is crucial for the fitness of those queens and their colonies (Koeniger and Koeniger, 2007), since genetic variation confers benefits such as increased resilience to biotic risk factors (Tarpy, 2003; Tarpy and Seeley, 2006; Delaplane et al., 2015; Simone-Finstrom et al., 2016).

Therefore, for the first time we assessed the effects of simultaneous exposure to neonicotinoid insecticides and the V. destructor mite on honey bee drone emergence mass, adult survival, and sperm quality. Based on previous studies that employed worker honey bees, as well as the haploid susceptibility hypothesis, we expected that both stressors individually would have strong negative effects on drones, and that simultaneous exposure would result in a synergistic negative effect (O’Donnell and Beshers, 2004; Blackmon et al., 2015; Straub et al., 2016, 2019; Maher et al., 2019; Morfin et al., 2020).

Twelve western honey bee (Apis mellifera) packages, each headed by a laying sister queen and 1.5 kg workers, were installed in ten-frame Langstroth hives in Auburn AL, United States on 18 March 2020. To promote growth, colonies were provided ad libitum with sucrose solution (50% weight/volume with water) for 2 weeks before being randomly assigned to either a control or neonicotinoid treatment.

Pressure caused by stressor interactions is believed to be responsible for widespread negative effects on biodiversity (Butchart et al., 2010; Barnosky et al., 2011), including on the economically and ecologically important honey bee (Alaux et al., 2010; Straub et al., 2019). For the first time, we investigated the potential effects of simultaneous pressure from neonicotinoid insecticides and V. destructor mites on adult male honey bee (drone) emergence mass and survival. The data revealed that combined exposure to both stressors resulted in a negative synergistic effect on adult drone survival, but an antagonistic effect on emergence mass. Our results suggest that combined exposure to these two ubiquitous stressors can severely affect the availability of drones for mating, and further highlight the complexity of potential interactive effects of simultaneous stressor pressures on honey bees.

Effects of concurrent exposure from multiple stressors on honey bees have garnered considerable attention of late. This is especially the case considering negative synergistic interactions for which the effect of multiple concurrent factors are worse than the sum of individual effects (Maher et al., 2019). As hypothesized, we observed a synergistic effect of neonicotinoids and V. destructor on adult drone survival. Interestingly, this same interaction was not observed for adult worker survival by a similar experiment performed during the same time of year (Straub et al., 2019). Although that study was performed in a different location, comparative results between the two suggest that differences in observed combined stressor effects on adult drone and worker survival could be explained by the haploid susceptibility hypothesis. This hypothesis states that drones experience higher susceptibility to environmental stressors due to their hemizygosity at loci involved in immune response (O’Donnell and Beshers, 2004; Blackmon et al., 2015). Since both neonicotinoids and V. destructor are known to impair the immune response of honey bees (Claudianos et al., 2006; Prisco et al., 2013; Brandt et al., 2016), a reduction in allelic diversity due to hemizygosity may result in more severe consequences when exposed, although future studies should be performed under the same conditions to further explore this as a reason for differences observed between these two types of honey bees.

In contrast to the observed synergistic negative effect on adult drone survival, we found that the two stressors had an antagonistic effect on drone emergence mass. Similar, contradictory observations were observed by a recent meta-analysis of agrochemical pesticide interactions, whereby proxies of honey bee fitness like behavior and physiology revealed additive or antagonistic effects, whereas synergistic effects were common on honey bee mortality (Siviter et al., 2021). A possible explanation for our observation may be because neonicotinoids are known to affect honey bee carbohydrate and lipid metabolism (Derecka et al., 2013; Cook, 2019). For example, Cook (2019) found that honey bees exposed to low concentrations of clothianidin had high lipid contents compared to controls. Thus, feeding by V. destructor during development may possibly offset the gain in body mass resulting from metabolic dysfunction associated with exposure to neonicotinoids, since V. destructor parasitism alone reduced emergence body mass in our experiment. Unfortunately, due to low sample size as a result of synergistic effects on mortality, we could not sufficiently examine possible interactions of neonicotinoids and V. destructor on sperm traits. Although examining all stressor combinations on multiple proxies of honey bee fitness are not realistic (Côté et al., 2016), the synergistic interaction that we observed on drone mortality by the time of sexual maturity could justify further attempts to understand how these two ubiquitous stressors affect the reproductive health of drones at this important time period, as a reduction in queen health, possibly due to poor mating, is frequently reported as a primary reason for honey bee colony mortality (Pettis et al., 2016).

Both neonicotinoids and V. destructor have each been shown to negatively affect drone health and performance (Rangel and Fisher, 2019; Friedli et al., 2020; Straub et al., 2021). On its own, V. destructor parasitism reduced drone emergence body mass by 6% compared to controls during our experiment. This has been shown for both workers and drones (Bowen-Walker and Gunn, 2001; Duay et al., 2003; Blanken et al., 2015; Ramsey et al., 2019; Straub et al., 2019), and is likely the result of mites feeding on the honey bees’ fat bodies during development. In contrast, exposure to neonicotinoids alone resulted in increased emergence body mass of drones (+ 4%). Low concentrations of neonicotinoids are known to affect carbohydrate and lipid metabolism, both which could affect body mass (Derecka et al., 2013). Other examples suggest that hormesis, a biphasic dose response in which biological processes can be stimulated by low concentrations of normally harmful chemical (Calabrese, 2005), can result in increased body mass, possibly at the expense of fitness (Cutler, 2012; Cutler and Rix, 2015). Indeed, our results suggest a negative effect of neonicotinoids on drone survival until sexual maturity, 14 days post emergence. A possible explanation for this could be a reduction in detoxification-competence (Claudianos et al., 2006). Additionally, experimental drones might have been nourished by compromised worker nurses during development, as previous investigations demonstrated that neonicotinoid exposure negatively affected nurse food glands. Additionally, experimental drones might have been nourished by compromised worker nurses during development, as previous investigations demonstrated that neonicotinoid exposure negatively affected nurse food glands (Hatjina et al., 2013). In contrast, V. destructor parasitism had no effect on adult drone survival. This is unexpected, since mites prefer drone brood cells and have been shown to negatively affect adult worker survival (Straub et al., 2019). Surprisingly few efforts have investigated the effects of V. destructor on drone survival (Fuchs, 1992; Rinderer et al., 1999; Collins and Pettis, 2001). For example, Collins and Pettis (2001) found that survival of drones parasitized by more than one reproductive female mite was not impacted when only one female V. destructor was present. Thus, low levels of colony-level infestation in our study could explain why we also did not observe a negative effect of V. destructor alone on drone survival. Despite a lack of a significant reduction in drone survival, it is noteworthy that V. destructor parasitism during development still increased the hazard ratio for drones (35% compared to Controls, Supplementary Table 4); thus, parasitized drones still likely experience increased stress. We were not able to assess effects of V. destructor only on sperm quality traits due to low sample size at the end of the cage trial. Relative to the Control and Neonicotinoid only treatment groups, fewer drones were assigned to this treatment groups at the start of the trail, potentially because parasitized developing drones were removed from their cells prior to artificial emergence (Harbo and Harris, 2005). Low adult survival of drones exposed to V. destructor during development further limited sample size. However, we found that there was no effect of neonicotinoids on sperm quality traits when drones were exposed during development. These results align with Ciereszko et al. (2017), but they contrast Straub et al. (2016) who found negative effects on sperm viability and concentration of living sperm, but not total sperm concentration. Given that spermatogenesis begins during the larval stage and terminates at pupation (Yániz et al., 2020), drones were potentially only exposed to a low dose of insecticides for a portion of the total spermatogenesis process, during the larval stage. This highlights that testing arena and experimental design likely contribute to the complexity of comparing observations among multiple studies, and demonstrates the importance of controlled ring tests performed in multiple laboratories when investigating stressor effects (Medrzycki et al., 2013; van der Sluijs et al., 2015).

 Our results highlight that interaction effects between two important stressors, like V. destructor mites and neonicotinoid insecticides for honey bees, can range from synergism to antagonism depending on the variable measured. According to our findings, neonicotinoids and V. destructor interacted synergistically to induce a severe lethal effect on adult honey bee drones, but interacted antagonistically on drone emergence body mass. The complexity of stressor interactions in the honey bee warrants future work elucidating outcomes of simultaneous pressure from multiple stressors, especially for risk assessment schemes that may otherwise not accurately estimate the interactive effects of stressors. Additionally, extrapolating effects observed under laboratory conditions to the field remain a major challenge and additional field-studies are required to confirm our findings.

Data are available via the Dryad Digital Repository at https://doi.org/10.5061/dryad.gmsbcc2p8.

SB, GW, and PN developed the experimental design. SB performed the experiment and collected the data. SB and LS performed statistical analysis. All authors wrote the manuscript and approved its final submission.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.