Colony environment and absence of brood enhance tolerance to a neonicotinoid in winter honey bee workers, Apis mellifera.

In eusocial insects, worker longevity is essential to ensure colony survival in brood-free periods. Trade-offs between longevity and other traits may render long-living workers in brood-free periods more susceptible to pesticides compared to short-lived ones. Further, colony environment (e.g., adequate nutrition) may enable workers to better cope with pesticides, yet data comparing long vs. short-living workers and the role of the colony environment for pesticide tolerance are scarce. Here, we show that long-living honey bee workers, Apis mellifera, are less susceptible to the neonicotinoid thiamethoxam than short-lived workers, and that susceptibility was further reduced when workers were acclimatized under colony compared to laboratory conditions. Following an OECD protocol, freshly-emerged workers were exposed to thiamethoxam in summer and winter and either acclimatized within their colony or in the laboratory. Mortality and sucrose consumption were measured daily and revealed that winter workers were significantly less susceptible than summer workers, despite being exposed to higher thiamethoxam dosages due to increased food consumption. Disparencies in fat body activity, which is key for detoxification, may explain why winter bees were less susceptible. Furthermore, colony acclimatization significantly reduced susceptibility towards thiamethoxam in winter workers likely due to enhanced protein nutrition. Brood absence and colony environment seem to govern workers' ability to cope with pesticides, which should be considered in risk assessments. Since honey bee colony losses occur mostly over winter, long-term studies assessing the effects of pesticide exposure on winter bees are required to better understand the underlying mechanisms.

Thiamethoxam soil contaminations reduce fertility of soil-dwelling beetles, Aethina tumida.

There in increasing evidence for recent global insect declines. This is of major concern as insects play a critical role in ecosystem functionality and human food security. Even though environmental pollutants are known to reduce insect fertility, their potential effects on insect fitness remain poorly understood - especially for soil-dwelling species. Here, we show that fertility of soil-dwelling beetles, Aethina tumida, is reduced, on average, by half due to field-realistic neonicotinoid soil contaminations. In the laboratory, pupating beetles were exposed via soil to concentrations of the neonicotinoid thiamethoxam that reflect global pollution of agricultural and natural habitats. Emerged adult phenotypes and reproduction were measured, and even the lowest concentration reported from natural habitats reduced subsequent reproduction by 50%. The data are most likely a conservative estimate as the beetles were only exposed during pupation. Since the tested concentrations reflect ubiquitous soil pollution, the data reveal a plausible mechanism for ongoing insect declines. An immediate reduction in environmental pollutants is urgently required if our aim is to mitigate the prevailing loss of species biodiversity.

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.

Do pesticide and pathogen interactions drive wild bee declines?

There is clear evidence for wild insect declines globally. Habitat loss, climate change, pests, pathogens and environmental pollution have all been shown to cause detrimental effects on insects. However, interactive effects between these stressors may be the key to understanding reported declines. Here, we review the literature on pesticide and pathogen interactions for wild bees, identify knowledge gaps, and suggest avenues for future research fostering mitigation of the observed declines. The limited studies available suggest that effects of pesticides most likely override effects of pathogens. Bees feeding on flowers and building sheltered nests, are likely less adapted to toxins compared to other insects, which potential susceptibility is enhanced by the reduced number of genes encoding detoxifying enzymes compared with other insect species. However, to date all 10 studies using a fully-crossed design have been conducted in the laboratory on social bees using Crithidia spp. or Nosema spp., identifying an urgent need to test solitary bees and other pathogens. Similarly, since laboratory studies do not necessarily reflect field conditions, semi-field and field studies are essential if we are to understand these interactions and their potential effects in the real-world. In conclusion, there is a clear need for empirical (semi-)field studies on a range of pesticides, pathogens, and insect species to better understand the pathways and mechanisms underlying their potential interactions, in particular their relevance for insect fitness and population dynamics. Such data are indispensable to drive forward robust modelling of interactive effects in different environmental settings and foster predictive science. This will enable pesticide and pathogen interactions to be put into the context of other stressors more broadly, evaluating their relative importance in driving the observed declines of wild bees and other insects. Ultimately, this will enable the development of more effective mitigation measures to protect bees and the ecosystem services they supply.

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.

Honey bees and climate explain viral prevalence in wild bee communities on a continental scale.

Viruses are omnipresent, yet the knowledge on drivers of viral prevalence in wild host populations is often limited. Biotic factors, such as sympatric managed host species, as well as abiotic factors, such as climatic variables, are likely to impact viral prevalence. Managed and wild bees, which harbor several multi-host viruses with a mostly fecal-oral between-species transmission route, provide an excellent system with which to test for the impact of biotic and abiotic factors on viral prevalence in wild host populations. Here we show on a continental scale that the prevalence of three broad host viruses: the AKI-complex (Acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus), Deformed wing virus, and Slow bee paralysis virus in wild bee populations (bumble bees and solitary bees) is positively related to viral prevalence of sympatric honey bees as well as being impacted by climatic variables. The former highlights the need for good beekeeping practices, including Varroa destructor management to reduce honey bee viral infection and hive placement. Furthermore, we found that viral prevalence in wild bees is at its lowest at the extreme ends of both temperature and precipitation ranges. Under predicted climate change, the frequency of extremes in precipitation and temperature will continue to increase and may hence impact viral prevalence in wild bee communities.

Thiamethoxam as an inadvertent anti-aphrodisiac in male bees.

Sexual reproduction is common to almost all multi-cellular organisms and can be compromised by environmental pollution, thereby affecting entire populations. Even though there is consensus that neonicotinoid insecticides can impact non-target animal fertility, their possible impact on male mating success is currently unknown in bees. Here, we show that sublethal exposure to a neonicotinoid significantly reduces both mating success and sperm traits of male bumblebees. Sexually mature male Bombus terrestris exposed to a field-realistic concentration of thiamethoxam (20 ng g-1) or not (controls) were mated with virgin gynes in the laboratory. The results confirm sublethal negative effects of thiamethoxam on sperm quantity and viability. While the latency to mate was reduced, mating success was significantly impaired in thiamethoxam-exposed males by 32% probably due to female choice. Gynes mated by exposed males revealed impaired sperm traits compared to their respective controls, which may lead to severe constraints for colony fitness. Our laboratory findings demonstrate for the first time that neonicotinoid insecticides can negatively affect male mating success in bees. Given that holds true for the field, this provides a plausible mechanism contributing to declines of wild bee populations globally. The widespread prophylactic use of neonicotinoids may therefore have previously overlooked inadvertent anti-aphrodisiac effects on non-target animals, thereby limiting conservation efforts.

Varying impact of neonicotinoid insecticide and acute bee paralysis virus across castes and colonies of black garden ants, Lasius niger (Hymenoptera: Formicidae).

Pesticides and pathogens are known drivers of declines in global entomofauna. However, interactions between pesticides and viruses, which could range from antagonistic, over additive to synergistic, are poorly understood in ants. Here, we show that in ants the impact of single and combined pesticide and virus stressors can vary across castes and at the colony level. A fully-crossed laboratory assay was used to evaluate interactions between a sublethal dose of the neonicotinoid thiamethoxam and Acute bee paralysis virus (ABPV) in black garden ants, Lasius niger. After monitoring colonies over 64 weeks, body mass, neonicotinoid residues and virus titres of workers and queens, as well as worker behavioural activity were measured. ABPV, but not thiamethoxam, reduced activity of workers. Neonicotinoid exposure resulted in reduced body mass of workers, but not of queens. Further, thiamethoxam facilitated ABPV infections in queens, but not in workers. Overall, virus exposure did not compromise detoxification and body mass, but one colony showed high virus titres and worker mortality. Although the data suggest additive effects at the level of individuals and castes, co-exposure with both stressors elicited antagonistic effects on colony size. Our results create demand for long-term holistic risk assessment of individual stressors and their interactions to protect biodiversity.

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 neonicotinoid thiamethoxam impairs male fertility in solitary bees, Osmia cornuta.

The ongoing loss of global biodiversity is endangering ecosystem functioning and human food security. While environmental pollutants are well known to reduce fertility, the potential effects of common neonicotinoid insecticides on insect fertility remain poorly understood. Here, we show that field-realistic neonicotinoid exposure can drastically impact male insect fertility. In the laboratory, male and female solitary bees Osmia cornuta were exposed to four concentrations of the neonicotinoid thiamethoxam to measure survival, food consumption, and sperm traits. Despite males being exposed to higher dosages of thiamethoxam, females revealed an overall increased hazard rate for survival; suggesting sex-specific differences in toxicological sensitivity. All tested sublethal concentrations (i.e., 1.5, 4.5 and 10 ng g-1) reduced sperm quantity by 57% and viability by 42% on average, with the lowest tested concentration leading to a reduction in total living sperm by 90%. As the tested sublethal concentrations match estimates of global neonicotinoid pollution, this reveals a plausible mechanism for population declines, thereby reflecting a realistic concern. An immediate reduction in environmental pollutants is required to decelerate the ongoing loss of biodiversity.

Positive Correlation between Pesticide Consumption and Longevity in Solitary Bees: Are We Overlooking Fitness Trade-Offs?

The ubiquitous use of pesticides is one major driver for the current loss of biodiversity, and the common practice of simultaneously applying multiple agrochemicals may further contribute. Insect toxicology currently has a strong focus on survival to determine the potential hazards of a chemical routinely used in risk evaluations. However, studies revealing no effect on survival or even indicating enhanced survival are likely to be misleading, if potential trade-offs between survival and other physiological factors are overlooked. Here, we used standard laboratory experiments to investigate the sublethal (i.e., food consumption) and lethal (i.e., survival) effects of two common agricultural pesticides (Roundup® and clothianidin) on adult female solitary bees, Osmia bicornis. The data showed no significant effect of the treatment on cumulative survival; however, a significant positive correlation between herbicide and insecticide exposure and age was revealed, i.e., bees exposed to higher dosages lived longer. As no significant differences in daily food consumption were observed across treatment groups, increased food intake can be excluded as a factor leading to the prolonged survival. While this study does not provide data on fitness effects, two previous studies using solitary bees observed significant negative effects of neonicotinoid insecticides on fitness, yet not on survival. Thus, we conjecture that the observed non-significant effects on longevity may result from a trade-off between survival and reproduction. The data suggest that a focus on survival can lead to false-negative results and it appears inevitable to include fitness or at least tokens of fitness at the earliest stage in future risk assessments.

Gene Expression and Functional Analyses of Odorant Receptors in Small Hive Beetles (Aethina tumida).

Olfaction is key to many insects. Odorant receptors (ORs) stand among the key chemosensory receptors mediating the detection of pheromones and kairomones. Small hive beetles (SHBs), Aethina tumida, are parasites of social bee colonies and olfactory cues are especially important for host finding. However, how interactions with their hosts may have shaped the evolution of ORs in the SHB remains poorly understood. Here, for the first time, we analyzed the evolution of SHB ORs through phylogenetic and positive selection analyses. We then tested the expression of selected OR genes in antennae, heads, and abdomens in four groups of adult SHBs: colony odor-experienced/-naive males and females. The results show that SHBs experienced both OR gene losses and duplications, thereby providing a first understanding of the evolution of SHB ORs. Additionally, three candidate ORs potentially involved in host finding and/or chemical communication were identified. Significantly different downregulations of ORs between the abdomens of male and female SHBs exposed to colony odors may reflect that these expression patterns might also reflect other internal events, e.g., oviposition. Altogether, these results provide novel insights into the evolution of SHB ORs and provide a valuable resource for analyzing the function of key genes, e.g., for developing biological control. These results will also help in understanding the chemosensory system in SHBs and other beetles.

Long-term effects of neonicotinoid insecticides on ants.

The widespread prophylactic usage of neonicotinoid insecticides has a clear impact on non-target organisms. However, the possible effects of long-term exposure on soil-dwelling organisms are still poorly understood especially for social insects with long-living queens. Here, we show that effects of chronic exposure to the neonicotinoid thiamethoxam on black garden ant colonies, Lasius niger, become visible before the second overwintering. Queens and workers differed in the residue-ratio of thiamethoxam to its metabolite clothianidin, suggesting that queens may have a superior detoxification system. Even though thiamethoxam did not affect queen mortality, neonicotinoid-exposed colonies showed a reduced number of workers and larvae indicating a trade-off between detoxification and fertility. Since colony size is a key for fitness, our data suggest long-term impacts of neonicotinoids on these organisms. This should be accounted for in future environmental and ecological risk assessments of neonicotinoid applications to prevent irreparable damages to ecosystems.

From antagonism to synergism: Extreme differences in stressor interactions in one species.

Interactions between stressors are involved in the decline of wild species and losses of managed ones. Those interactions are often assumed to be synergistic, and per se of the same nature, even though susceptibility can vary within a single species. However, empirical measures of interaction effects across levels of susceptibility remain scarce. Here, we show clear evidence for extreme differences in stressor interactions ranging from antagonism to synergism within honeybees, Apis mellifera. While female honeybee workers exposed to both malnutrition and the pathogen Nosema ceranae showed synergistic interactions and increased stress, male drones showed antagonistic interactions and decreased stress. Most likely sex and division of labour in the social insects underlie these findings. It appears inevitable to empirically test the actual nature of stressor interactions across a range of susceptibility factors within a single species, before drawing general conclusions.

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.

Bees and flowers: How to feed an invasive beetle species.

Invasive species may exploit a wide range of food sources, thereby fostering their success and hampering mitigation, but the actual degree of opportunism is often unknown. The small hive beetle (SHB), Aethina tumida, is a parasite of honeybee colonies endemic to sub-Saharan Africa. SHBs have now spread on all habitable continents and can also infest colonies of other social bees. To date, the possible role of solitary bee nests as alternative hosts is unknown. Similarly, flowers as possible alternative food sources are not well understood. Here, we show that SHBs can complete an entire life cycle in association with nests of solitary bees Megachile rotundata. The data also show that flowers can serve as alternative food sources. These results support the opportunistic nature of this invasive species, thereby generating further obstacles for mitigation efforts in the field. It also suggests that SHB invasions may result in more serious consequences for endemic bee fauna than previously thought. This provides further motivation to slow down the global spread of this pest, and to improve its management in areas, where it is established.

Trypanosomatid parasites infecting managed honeybees and wild solitary bees.

The parasite Crithidia mellificae (Kinetoplastea: Trypanosomatidae) infects honeybees, Apis mellifera. No pathogenic effects have been found in individual hosts, despite positive correlations between infections and colony mortalities. The solitary bee Osmia cornuta might constitute a host, but controlled infections are lacking to date. Here, we challenged male and female O. cornuta and honeybee workers in laboratory cages with C. mellificae. No parasite cells were found in any control. Parasite numbers increased 6.6 fold in honeybees between days 6 and 19 p.i. and significantly reduced survival. In O. cornuta, C. mellificae numbers increased 2-3.6 fold within cages and significantly reduced survival of males, but not females. The proportion of infected hosts increased in O. cornuta cages with faeces, but not in honeybee cages without faeces, suggesting faecal - oral transmission. The data show that O. cornuta is a host of C. mellificae and suggest that males are more susceptible. The higher mortality of infected honeybees proposes a mechanism for correlations between C. mellificae infections and colony mortalities.

Neonicotinoids and ectoparasitic mites synergistically impact honeybees.

The Western honeybee, Apis mellifera, is the most important managed pollinator globally and has recently experienced unsustainably high colony losses. Synergistic interactions among stressors are believed to be primarily responsible. However, despite clear evidence of strong effect on honeybee longevity of widely-employed neonicotinoid insecticides and of the ubiquitous ectoparasitic mite Varroa destructor, no data exist to show synergistic effects between these two stressors. Even though neonicotinoids had no significant impact by themselves, we here show for the first time a synergistic time-lag interaction between mites and neonicotinoids that resulted in significantly reduced survival of long-lived winter honeybees. Even though these mites are potent vectors of viruses, the virus-insecticide interaction had no significant impact. The data suggest a previously overlooked mechanism possibly explaining recent unsustainably high losses of managed A. mellifera honeybee colonies in many regions of the world. Future mitigation efforts should concentrate on developing sustainable agro-ecosystem management schemes that incorporate reduced use of neonicotinoids and sustainable solutions for V. destructor mites.

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.