Reconstructing queen genotypes by pool sequencing colonies in eusocial insects: Statistical Methods and their application to honeybee.

Eusocial insects are crucial to many ecosystems, and particularly the honeybee (Apis mellifera). One approach to facilitate their study in molecular genetics, is to consider whole-colony genotyping by combining DNA of multiple individuals in a single pool sequencing experiment. Cheap and fast, this technique comes with the drawback of producing data requiring dedicated methods to be fully exploited. Despite this limitation, pool sequencing data have been shown to be informative and cost-effective when working on random mating populations. Here, we present new statistical methods for exploiting pool sequencing of eusocial colonies in order to reconstruct the genotypes of the queen of such colony. This leverages the possibility to monitor genetic diversity, perform genomic-based studies or implement selective breeding. Using simulations and honeybee real data, we show that the new methods allow for a fast and accurate estimation of the queen's genetic ancestry, with correlations of about 0.9 to that obtained from individual genotyping. Also, it allows for an accurate reconstruction of the queen genotypes, with about 2% genotyping error. We further validate these inferences using experimental data on colonies with both pool sequencing and individual genotyping of drones. In brief, in this study we present statistical models to accurately estimate the genetic ancestry and reconstruct the genotypes of the queen from pool sequencing data from workers of an eusocial colony. Such information allows to exploit pool sequencing for traditional population genetics analyses, association studies and for selective breeding. While validated in Apis mellifera, these methods are applicable to other eusocial hymenopterans.

Pesticide risk assessment in honeybees: Toward the use of behavioral and reproductive performances as assessment endpoints.

The growing gap between new evidence of pesticide toxicity in honeybees and conventional toxicological assays recommended by regulatory test guidelines emphasizes the need to complement current lethal endpoints with sublethal endpoints. In this context, behavioral and reproductive performances have received growing interest since the 2000s, likely due to their ecological relevance and/or the emergence of new technologies. We review the biological interests and methodological measurements of these predominantly studied endpoints and discuss their possible use in the pesticide risk assessment procedure based on their standardization level, simplicity and ecological relevance. It appears that homing flights and reproduction have great potential for pesticide risk assessment, mainly due to their ecological relevance. If exploratory research studies in ecotoxicology have paved the way toward a better understanding of pesticide toxicity in honeybees, the next objective will then be to translate the most relevant behavioral and reproductive endpoints into regulatory test methods. This will require more comparative studies and improving their ecological relevance. This latter goal may be facilitated by the use of population dynamics models for scaling up the consequences of adverse behavioral and reproductive effects from individuals to colonies.

Measuring ontogenetic shifts in central-place foragers: A case study with honeybees.

Measuring time-activity budgets over the complete individual life span is now possible for many animals with the recent advances of life-long individual monitoring devices. Although analyses of changes in the patterns of time-activity budgets have revealed ontogenetic shifts in birds or mammals, no such technique has been applied to date on insects. We tested an automated breakpoint-based procedure to detect, assess and quantify shifts in the temporal pattern of the flight activities in honeybees. We assumed that the learning and foraging stages of honeybees will differ in several respects, to detect the age at onset of foraging (AOF). Using an extensive dataset covering the life-long monitoring of 1,167 individuals, we compared the AOF outputs with the more conventional approaches based on arbitrary thresholds. We further evaluated the robustness of the different methods comparing the foraging time-activity budget allocations between the presumed foragers and confirmed foragers. We revealed a clear-cut learning-foraging ontogenetic shift that differs in duration, frequency and time of occurrence of flights. Although AOF appeared to be highly plastic among bees, the breakpoint-based procedure seems better capable to detect it than arbitrary threshold-based methods that are unable to deal with inter-individual variation. We developed the aof r-package including a broad range of examples with both simulated and empirical datasets to illustrate the simplicity of use of the procedure. This simple procedure is generic enough to be derived from any individual life-long monitoring devices recording the time-activity budgets, and could propose new ecological applications of bio-logging to detect ontogenetic shifts in the behaviour of central-place foragers.

Neonicotinoid-induced mortality risk for bees foraging on oilseed rape nectar persists despite EU moratorium.

The implication of neonicotinoids in bee declines led in 2013 to an EU moratorium on three neonicotinoids in bee-attractive crops. However, neonicotinoids are frequently detected in wild flowers or untreated crops suggesting that neonicotinoids applied to cereals can spread into the environment and harm bees. Therefore, we quantified neonicotinoid residues in nectar from winter-sown oilseed rape in western France collected within the five years under the EU moratorium. We detected all three restricted neonicotinoids. Imidacloprid was detected in all years with no clear declining trend but a strong inter- and intra-annual variation and maximum concentrations exceeding reported concentrations in treated crops. No relation to non-organic winter-sown cereals was identified even though these were the only crops treated with imidacloprid, but residue levels depended on soil type and increased with rainfall. Simulating acute and chronic mortality suggests a considerable risk for nectar foraging bees. We conclude that persistent imidacloprid soil residues diffuse on a large scale in the environment and substantially contaminate a major mass-flowering crop. Despite the limitations of case-studies and risk simulations, our findings provide additional support to the recent extension of the moratorium to a permanent ban in all outdoor crops.

Toward the protection of bees and pollination under global change: present and future perspectives in a challenging applied science.

Over the past 30 years (1987-2016), bibliometric data have shown a drastic change in the scientific investigation of threats to bee populations. Bee research efforts committed to studying bioagressors of honeybees (mainly Varroa sp.) were predominant, but now appear to be shifting from bioagressors to global change in the published literature. This rise of global change science reveals prevailing topics, for current and future years: climate change, landscape alteration, agricultural intensification and invasive species. We argue that with increased investment in applied research and development, the scientific, beekeeping and agricultural communities will be able to find management strategies for productive agrosystems and enhanced resilience of pollination and beekeeping. This implies the need for restoring and improving food resources and shelters of bees by ecological intensification of diversified farming systems, and also reconciling sustainable beekeeping with wild pollinator conservation.

Hazard of a neonicotinoid insecticide on the homing flight of the honeybee depends on climatic conditions and Varroa infestation.

The paradigm for all toxicological bioassays in the risk assessment of pesticide registration reflects the principle that experimental conditions should be controlled to avoid any other factors that may affect the endpoint measures. As honeybee colonies can be frequently exposed to bio-aggressors in real conditions, often concomitantly with pesticides, co-exposure to pesticide/bio-aggressors is becoming a concern for regulatory authorities. We investigated the effects of the neonicotinoid insecticide thiamethoxam on the homing performances of foragers emerging from colonies differentiated by health status (infestation with Varroa destructor mites, microsporidian parasite Nosema spp. and Deformed Wing Virus). We designed a homing test that has been recently identified to fill a regulatory gap in the field evaluations of sublethal doses of pesticides before their registration. We also assessed the effect of temperature as an environmental factor. Our results showed that the Varroa mite exacerbates homing failure (HF) caused by the insecticide, whereas high temperatures reduce insecticide-induced HF. Through an analytical Effective Dose (ED) approach, predictive modeling results showed that, for instance, ED level of an uninfested colony, can be divided by 3.3 when the colony is infested by 5 Varroa mites per 100 bees and at a temperature of 24 °C. Our results suggest that the health status of honeybee colonies and climatic context should be targeted for a thorough risk assessment.

A 'Landscape physiology' approach for assessing bee health highlights the benefits of floral landscape enrichment and semi-natural habitats.

Understanding how anthropogenic landscape alteration affects populations of ecologically- and economically-important insect pollinators has never been more pressing. In this context, the assessment of landscape quality typically relies on spatial distribution studies, but, whether habitat-restoration techniques actually improve the health of targeted pollinator populations remains obscure. This gap could be filled by a comprehensive understanding of how gradients of landscape quality influence pollinator physiology. We therefore used this approach for honey bees (Apis mellifera) to test whether landscape patterns can shape bee health. We focused on the pre-wintering period since abnormally high winter colony losses have often been observed. By exposing colonies to different landscapes, enriched in melliferous catch crops and surrounded by semi-natural habitats, we found that bee physiology (i.e. fat body mass and level of vitellogenin) was significantly improved by the presence of flowering catch crops. Catch crop presence was associated with a significant increase in pollen diet diversity. The influence of semi-natural habitats on bee health was even stronger. Vitellogenin level was in turn significantly linked to higher overwintering survival. Therefore, our experimental study, combining landscape ecology and bee physiology, offers an exciting proof-of-concept for directly identifying stressful or suitable landscapes and promoting efficient pollinator conservation.

Variations in the Availability of Pollen Resources Affect Honey Bee Health.

Intensive agricultural systems often expose honey bees (Apis mellifera L.) to large temporal variations in the availability (quantity, quality and diversity) of nutritional resources. Such nutritional irregularity is expected to affect honey bee health. We therefore tested under laboratory conditions the effect of such variation in pollen availability on honey bee health (survival and nursing physiology-hypopharyngeal gland development and vitellogenin expression). We fed honey bees with different diets composed of pollen pellets collected by honey bees in an agricultural landscape of western France. Slight drops (5-10%) in the availability of oilseed rape (Brassica napus L.) pollen resulted in significant reductions of all tested variables. Despite some variations in taxonomic diversity and nutritional quality, the pollen mixes harvested over the season had a similar positive influence on honey bee health, except for the one collected in late July that induced poor survival and nursing physiology. This period coincided with the mass-flowering of maize (Zea mays L.), an anemophilous crop which produces poor-quality pollen. Therefore, changes in bee health were not connected to variations in pollen diversity but rather to variations in pollen depletion and quality, such as can be encountered in an intensive agricultural system of western France. Finally, even though pollen can be available ad libitum during the mass-flowering of some crops (e.g. maize), it can fail to provide bees with diet adequate for their development.

A Locomotor Deficit Induced by Sublethal Doses of Pyrethroid and Neonicotinoid Insecticides in the Honeybee Apis mellifera.

The toxicity of pesticides used in agriculture towards non-targeted organisms and especially pollinators has recently drawn the attention from a broad scientific community. Increased honeybee mortality observed worldwide certainly contributes to this interest. The potential role of several neurotoxic insecticides in triggering or potentiating honeybee mortality was considered, in particular phenylpyrazoles and neonicotinoids, given that they are widely used and highly toxic for insects. Along with their ability to kill insects at lethal doses, they can compromise survival at sublethal doses by producing subtle deleterious effects. In this study, we compared the bee's locomotor ability, which is crucial for many tasks within the hive (e.g. cleaning brood cells, feeding larvae…), before and after an acute sublethal exposure to one insecticide belonging to the two insecticide classes, fipronil and thiamethoxam. Additionally, we examined the locomotor ability after exposure to pyrethroids, an older chemical insecticide class still widely used and known to be highly toxic to bees as well. Our study focused on young bees (day 1 after emergence) since (i) few studies are available on locomotion at this stage and (ii) in recent years, pesticides have been reported to accumulate in different hive matrices, where young bees undergo their early development. At sublethal doses (SLD48h, i.e. causing no mortality at 48 h), three pyrethroids, namely cypermethrin (2.5 ng/bee), tetramethrin (70 ng/bee), tau-fluvalinate (33 ng/bee) and the neonicotinoid thiamethoxam (3.8 ng/bee) caused a locomotor deficit in honeybees. While the SLD48h of fipronil (a phenylpyrazole, 0.5 ng/bee) had no measurable effect on locomotion, we observed high mortality several days after exposure, an effect that was not observed with the other insecticides. Although locomotor deficits observed in the sublethal range of pyrethroids and thiamethoxam would suggest deleterious effects in the field, the case of fipronil demonstrates that toxicity evaluation requires information on multiple endpoints (e.g. long term survival) to fully address pesticides risks for honeybees. Pyrethroid-induced locomotor deficits are discussed in light of recent advances regarding their mode of action on honeybee ion channels and current structure-function studies.

Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees.

European governments have banned the use of three common neonicotinoid pesticides due to insufficiently identified risks to bees. This policy decision is controversial given the absence of clear consistency between toxicity assessments of those substances in the laboratory and in the field. Although laboratory trials report deleterious effects in honeybees at trace levels, field surveys reveal no decrease in the performance of honeybee colonies in the vicinity of treated fields. Here we provide the missing link, showing that individual honeybees near thiamethoxam-treated fields do indeed disappear at a faster rate, but the impact of this is buffered by the colonies' demographic regulation response. Although we could ascertain the exposure pathway of thiamethoxam residues from treated flowers to honeybee dietary nectar, we uncovered an unexpected pervasive co-occurrence of similar concentrations of imidacloprid, another neonicotinoid normally restricted to non-entomophilous crops in the study country. Thus, its origin and transfer pathways through the succession of annual crops need be elucidated to conveniently appraise the risks of combined neonicotinoid exposures. This study reconciles the conflicting laboratory and field toxicity assessments of neonicotinoids on honeybees and further highlights the difficulty in actually detecting non-intentional effects on the field through conventional risk assessment methods.

Honey bee diet in intensive farmland habitats reveals an unexpectedly high flower richness and a major role of weeds.

In intensive farmland habitats, pollination of wild flowers and crops may be threatened by the widespread decline of pollinators. The honey bee decline, in particular, appears to result from the combination of multiple stresses, including diseases, pathogens, and pesticides. The reduction of semi-natural habitats is also suspected to entail floral resource scarcity for bees. Yet, the seasonal dynamics and composition of the honey bee diet remains poorly documented to date. In this study, we studied the seasonal contribution of mass-flowering crops (rapeseed and sunflower) vs. other floral resources, as well as the influence of nutritional quality and landscape composition on pollen diet composition over five consecutive years. From April to October, the mass of pollen and nectar collected by honey bees followed a bimodal seasonal trend, marked by a two-month period of low food supply between the two oilseed crop mass-flowerings (ending in May for rapeseed and July for sunflower). Bees collected nectar mainly from crops while pollen came from a wide diversity of herbaceous and woody plant species in semi-natural habitats or from weeds in crops. Weed species constituted the bulk of the honey bee diet between the mass flowering crop periods (up to 40%) and are therefore suspected to play a critical role at this time period. The pollen diet composition was related to the nutritional value of the collected pollen and by the local landscape composition. Our study highlights (1) a food supply depletion period of both pollen and nectar resources during late spring, contemporaneously with the demographic peak of honey bee populations, (2) a high botanical richness of pollen diet, mostly proceeding from trees and weeds, and (3) a pollen diet composition influenced by the local landscape composition. Our results therefore support the Agri-Environmental Schemes intended to promote honey bees and beekeeping sustainability through the enhancement of flower availability in agricultural landscapes.

Larval Exposure to the Juvenile Hormone Analog Pyriproxyfen Disrupts Acceptance of and Social Behavior Performance in Adult Honeybees.

BACKGROUND: Juvenile hormone (JH) plays an important role in honeybee development and the regulation of age-related division of labor. However, honeybees can be exposed to insect growth regulators (IGRs), such as JH analogs developed for insect pest and vector control. Although their side effects as endocrine disruptors on honeybee larval or adult stages have been studied, little is known about the subsequent effects on adults of a sublethal larval exposure. We therefore studied the impact of the JH analog pyriproxyfen on larvae and resulting adults within a colony under semi-field conditions by combining recent laboratory larval tests with chemical analysis and behavioral observations. Oral and chronic larval exposure at cumulative doses of 23 or 57 ng per larva were tested. RESULTS: Pyriproxyfen-treated bees emerged earlier than control bees and the highest dose led to a significant rate of malformed adults (atrophied wings). Young pyriproxyfen-treated bees were more frequently rejected by nestmates from the colony, inducing a shorter life span. This could be linked to differences in cuticular hydrocarbon (CHC) profiles between control and pyriproxyfen-treated bees. Finally, pyriproxyfen-treated bees exhibited fewer social behaviors (ventilation, brood care, contacts with nestmates or food stocks) than control bees. CONCLUSION: Larval exposure to sublethal doses of pyriproxyfen affected several life history traits of the honeybees. Our results especially showed changes in social integration (acceptance by nestmates and social behaviors performance) that could potentially affect population growth and balance of the colony.

Delayed and time-cumulative toxicity of imidacloprid in bees, ants and termites.

Imidacloprid, one of the most commonly used insecticides, is highly toxic to bees and other beneficial insects. The regulatory challenge to determine safe levels of residual pesticides can benefit from information about the time-dependent toxicity of this chemical. Using published toxicity data for imidacloprid for several insect species, we construct time-to-lethal-effect toxicity plots and fit temporal power-law scaling curves to the data. The level of toxic exposure that results in 50% mortality after time t is found to scale as t(1.7) for ants, from t(1.6) to t(5) for honeybees, and from t(1.46) to t(2.9) for termites. We present a simple toxicological model that can explain t(2) scaling. Extrapolating the toxicity scaling for honeybees to the lifespan of winter bees suggests that imidacloprid in honey at 0.25 µg/kg would be lethal to a large proportion of bees nearing the end of their life.

Pesticide risk assessment in free-ranging bees is weather and landscape dependent.

The risk assessment of plant protection products on pollinators is currently based on the evaluation of lethal doses through repeatable lethal toxicity laboratory trials. Recent advances in honeybee toxicology have, however, raised interest on assessing sublethal effects in free-ranging individuals. Here, we show that the sublethal effects of a neonicotinoid pesticide are modified in magnitude by environmental interactions specific to the landscape and time of exposure events. Field sublethal assessment is therefore context dependent and should be addressed in a temporally and spatially explicit way, especially regarding weather and landscape physiognomy. We further develop an analytical Effective Dose (ED) framework to help disentangle context-induced from treatment-induced effects and thus to alleviate uncertainty in field studies. Although the ED framework involves trials at concentrations above the expected field exposure levels, it allows to explicitly delineating the climatic and landscape contexts that should be targeted for in-depth higher tier risk assessment.

Crop-emptying rate and the design of pesticide risk assessment schemes in the honey bee and wild bees (Hymenoptera: Apidae).

Recent scientific literature and reports from official sanitary agencies have pointed out the deficiency of current pesticide risk assessment processes regarding sublethal effects on pollinators. Sublethal effects include troubles in learning performance, orientation skills, or mobility, with possible contribution to substantial dysfunction at population scale. However, the study of sublethal effects is currently limited by considerable knowledge gaps, particularly for the numerous pollinators other than the honey bee Apis mellifera L.--the traditional model for pesticide risk assessment in pollinators. Here, we propose to use the crop-emptying time as a rule of thumb to guide the design of oral exposure experiments in the honey bee and wild bees. The administration of contaminated sucrose solutions is typically followed by a fasting time lapse to allow complete assimilation before the behavioral tests. The fasting duration should at least encompass the crop-emptying time, because no absorption takes place in the crop. We assessed crop-emptying rate in fasted bees and how it relates 1) with sucrose solution concentration in the honey bee and 2) with body mass in wild bees. Fasting duration required for complete crop emptying in honey bees fed 20 microl of a 50% sucrose solution was nearly 2 h. Actual fasting durations are usually shorter in toxicological studies, suggesting incomplete crop emptying, and therefore partial assimilation of experimental solutions that could imply underestimation of sublethal effects. We also found faster crop-emptying rates in large wild bees compared with smaller wild bees, and suggest operative rules to adapt sublethal assessment schemes accordingly.

Influence of pollen nutrition on honey bee health: do pollen quality and diversity matter?

Honey bee colonies are highly dependent upon the availability of floral resources from which they get the nutrients (notably pollen) necessary to their development and survival. However, foraging areas are currently affected by the intensification of agriculture and landscape alteration. Bees are therefore confronted to disparities in time and space of floral resource abundance, type and diversity, which might provide inadequate nutrition and endanger colonies. The beneficial influence of pollen availability on bee health is well-established but whether quality and diversity of pollen diets can modify bee health remains largely unknown. We therefore tested the influence of pollen diet quality (different monofloral pollens) and diversity (polyfloral pollen diet) on the physiology of young nurse bees, which have a distinct nutritional physiology (e.g. hypopharyngeal gland development and vitellogenin level), and on the tolerance to the microsporidian parasite Nosemaceranae by measuring bee survival and the activity of different enzymes potentially involved in bee health and defense response (glutathione-S-transferase (detoxification), phenoloxidase (immunity) and alkaline phosphatase (metabolism)). We found that both nurse bee physiology and the tolerance to the parasite were affected by pollen quality. Pollen diet diversity had no effect on the nurse bee physiology and the survival of healthy bees. However, when parasitized, bees fed with the polyfloral blend lived longer than bees fed with monofloral pollens, excepted for the protein-richest monofloral pollen. Furthermore, the survival was positively correlated to alkaline phosphatase activity in healthy bees and to phenoloxydase activities in infected bees. Our results support the idea that both the quality and diversity (in a specific context) of pollen can shape bee physiology and might help to better understand the influence of agriculture and land-use intensification on bee nutrition and health.

A common pesticide decreases foraging success and survival in honey bees.

Nonlethal exposure of honey bees to thiamethoxam (neonicotinoid systemic pesticide) causes high mortality due to homing failure at levels that could put a colony at risk of collapse. Simulated exposure events on free-ranging foragers labeled with a radio-frequency identification tag suggest that homing is impaired by thiamethoxam intoxication. These experiments offer new insights into the consequences of common neonicotinoid pesticides used worldwide.