Real-time monitoring of honeybee colony daily activity and bee loss rates can highlight the risk posed by a pesticide.

Information on honeybee foraging performance and especially bee loss rates at the colony level are crucial for evaluating the magnitude of effects due to pesticide exposure, thereby ensuring that protection goals for honeybee colonies are met (i.e. threshold of acceptable effects). However, current methods for monitoring honeybee foraging activity and mortality are very approximate (visual records) or are time-limited and mostly based on single cohort analysis. We therefore assess the potential of bee counters, that enable a colony-level and continuous monitoring of bee flight activity and mortality, in pesticide risk assessment. After assessing the background activity and bee loss rates, we exposed colonies to two concentrations of sulfoxaflor (a neurotoxic insecticide) in sugar syrup: a concentration that was considered to be field realistic (0.59 µg/ml) and a higher concentration (2.36 µg/ml) representing a worst-case exposure scenario. We did not find any effect of the field-realistic concentration on flight activity and bee loss rates. However, a two-fold decrease in daily flight activity and a 10-fold increase in daily bee losses were detected in colonies exposed to the highest sulfoxaflor concentration as compared to before exposure. When compared to the theoretical trigger values associated with the specific protection goal of 7 % colony-size reduction, the observed fold changes in daily bee losses were often found to be at risk for colonies. In conclusion, the real-time and colony-level monitoring of bee loss rates, combined with threshold values indicating at which levels bee loss rates threaten the colony, have great potential for improving regulatory pesticide risk assessments for honeybees under field conditions.

Toxicity of the Pesticides Imidacloprid, Difenoconazole and Glyphosate Alone and in Binary and Ternary Mixtures to Winter Honey Bees: Effects on Survival and Antioxidative Defenses.

To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.

To Treat or Not to Treat Bees? Handy VarLoad: A Predictive Model for Varroa destructor Load.

The parasitic Varroa destructor is considered a major pathogenic threat to honey bees and to beekeeping. Without regular treatment against this mite, honey bee colonies can collapse within a 2-3-year period in temperate climates. Beyond this dramatic scenario, Varroa induces reductions in colony performance, which can have significant economic impacts for beekeepers. Unfortunately, until now, it has not been possible to predict the summer Varroa population size from its initial load in early spring. Here, we present models that use the Varroa load observed in the spring to predict the Varroa load one or three months later by using easily and quickly measurable data: phoretic Varroa load and capped brood cell numbers. Built on 1030 commercial colonies located in three regions in the south of France and sampled over a three-year period, these predictive models are tools designed to help professional beekeepers' decision making regarding treatments against Varroa. Using these models, beekeepers will either be able to evaluate the risks and benefits of treating against Varroa or to anticipate the reduction in colony performance due to the mite during the beekeeping season.

Pathways for Novel Epidemiology: Plant-Pollinator-Pathogen Networks and Global Change.

Multiple global change pressures, and their interplay, cause plant-pollinator extinctions and modify species assemblages and interactions. This may alter the risks of pathogen host shifts, intra- or interspecific pathogen spread, and emergence of novel population or community epidemics. Flowers are hubs for pathogen transmission. Consequently, the structure of plant-pollinator interaction networks may be pivotal in pathogen host shifts and modulating disease dynamics. Traits of plants, pollinators, and pathogens may also govern the interspecific spread of pathogens. Pathogen spillover-spillback between managed and wild pollinators risks driving the evolution of virulence and community epidemics. Understanding this interplay between host-pathogen dynamics and global change will be crucial to predicting impacts on pollinators and pollination underpinning ecosystems and human wellbeing.

Physiological effects of the interaction between Nosema ceranae and sequential and overlapping exposure to glyphosate and difenoconazole in the honey bee Apis mellifera.

Pathogens and pollutants, such as pesticides, are potential stressors to all living organisms, including honey bees. Herbicides and fungicides are among the most prevalent pesticides in beehive matrices, and their interaction with Nosema ceranae is not well understood. In this study, the interactions between N. ceranae, the herbicide glyphosate and the fungicide difenoconazole were studied under combined sequential and overlapping exposure to the pesticides at a concentration of 0.1 µg/L in food. In the sequential exposure experiment, newly emerged bees were exposed to the herbicide from day 3 to day 13 after emerging and to the fungicide from day 13 to day 23. In the overlapping exposure experiment, bees were exposed to the herbicide from day 3 to day 13 and to the fungicide from day 7 to day 17. Infection by Nosema in early adult life stages (a few hours post emergence) greatly affected the survival of honey bees and elicited much higher mortality than was induced by pesticides either alone or in combination. Overlapping exposure to both pesticides induced higher mortality than was caused by sequential or individual exposure. Overlapping, but not sequential, exposure to pesticides synergistically increased the adverse effect of N. ceranae on honey bee longevity. The combination of Nosema and pesticides had a strong impact on physiological markers of the nervous system, detoxification, antioxidant defenses and social immunity of honey bees.

Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic physiological disturbances in winter Apis mellifera honey bees.

Multiple pesticides originating from plant protection treatments and the treatment of pests infecting honey bees are frequently detected in beehive matrices. Therefore, winter honey bees, which have a long life span, could be exposed to these pesticides for longer periods than summer honey bees. In this study, winter honey bees were exposed through food to the insecticide imidacloprid, the fungicide difenoconazole and the herbicide glyphosate, alone or in binary and ternary mixtures, at environmental concentrations (0 (controls), 0.1, 1 and 10 µg/L) for 20 days. The survival of the honey bees was significantly reduced after exposure to these 3 pesticides individually and in combination. Overall, the combinations had a higher impact than the pesticides alone with a maximum mortality of 52.9% after 20 days of exposure to the insecticide-fungicide binary mixture at 1 µg/L. The analyses of the surviving bees showed that these different pesticide combinations had a systemic global impact on the physiological state of the honey bees, as revealed by the modulation of head, midgut and abdomen glutathione-S-transferase, head acetylcholinesterase, abdomen glucose-6-phosphate dehydrogenase and midgut alkaline phosphatase, which are involved in the detoxification of xenobiotics, the nervous system, defenses against oxidative stress, metabolism and immunity, respectively. These results demonstrate the importance of studying the effects of chemical cocktails based on low realistic exposure levels and developing long-term tests to reveal possible lethal and adverse sublethal interactions in honey bees and other insect pollinators.

The prediction of swarming in honeybee colonies using vibrational spectra.

In this work, we disclose a non-invasive method for the monitoring and predicting of the swarming process within honeybee colonies, using vibro-acoustic information. Two machine learning algorithms are presented for the prediction of swarming, based on vibration data recorded using accelerometers placed in the heart of honeybee hives. Both algorithms successfully discriminate between colonies intending and not intending to swarm with a high degree of accuracy, over 90% for each method, with successful swarming prediction up to 30 days prior to the event. We show that instantaneous vibrational spectra predict the swarming within the swarming season only, and that this limitation can be lifted provided that the history of the evolution of the spectra is accounted for. We also disclose queen toots and quacks, showing statistics of the occurrence of queen pipes over the entire swarming season. From this we were able to determine that (1) tooting always precedes quacking, (2) under natural conditions there is a 4 to 7 day period without queen tooting following the exit of the primary swarm, and (3) human intervention, such as queen clipping and the opening of a hive, causes strong interferences with important mechanisms for the prevention of simultaneous rival queen emergence.

Exposure to pollen-bound pesticide mixtures induces longer-lived but less efficient honey bees.

Due to the widespread use of pesticides and their persistence in the environment, non-target organisms are chronically exposed to mixtures of toxic residues. Fungicides, herbicides and insecticides are all found at low doses in the diet of pollinators such as honey bees, but due to the lack of data on the toxicological effects of these mixtures, determining their risk is difficult to assess. We therefore developed a study combining the identification of common pollen-bound pesticide mixtures associated with poor colony development and tested their effects on bee behavior and physiology. We exposed bees to the identified pesticide mixtures during the first days of their adult life, a crucial period for physiological development. Using optic bee counters we recorded the behavior of bees throughout their lives and identified two pesticide mixtures that delay the onset of foraging and slow-down foraging activity. Furthermore, one of these mixtures hampers pollen foraging. As bee longevity is strongly influenced by the time spent foraging, bees exposed to these pesticide mixtures outlived control bees. Physiological analysis revealed that perturbations of the energetic metabolism preceded the altered behavior. In conclusion, we found that early-life exposure to low doses of pesticide mixtures can have long-term effects that translate into longer-lived but slower and less efficient bees. These surprising findings contrast with the commonly reported increase in bee mortality upon pesticide exposure, and demonstrate that exposure that may seem harmless (e.g., very low doses, pesticides not intended to kill insects) can have undesirable effects on non-target organisms.

Temperature-driven changes in viral loads in the honey bee Apis mellifera.

Many of the physiological traits in insects are shaped by environmental temperatures, which can influence their interactions with pathogens. Therefore, quantifying the thermal responses of the host-pathogen system is crucial for better understanding and predicting their dynamics due to environmental changes. This is particularly important in honey bees, which are experiencing severe colony losses around the world, notably due to infection with the Deformed wing virus (DWV). To investigate the influence of temperature on the honey bee/DWV relationship we exposed adult bees to low or high temperatures and determined the effects on viral titers and bee survival. Emerging bees naturally infected with DWV were reared in vitro at different temperatures ranging from 15 °C to 37 °C. In addition, some bees reared at 37 °C were exposed daily to acute heat treatments (40 and 43 °C). High temperatures significantly decreased DWV titers close to the initial viral load at emergence but increased bee mortality. The lowest temperature resulted in higher mortality, but virus load was not significantly impacted. In conclusion, our results indicate that temperature could contribute to seasonal variations in viral loads but do not suggest temperature to be used as a tool to eliminate viruses, even given that high temperatures limit viral multiplication.

Efficiency of an air curtain as an anti-insect barrier: the honey bee as a model insect.

BACKGROUND: Vector-borne diseases are of high concern for human, animal and plant health. In humans, such diseases are often transmitted by flying insects. Flying insects stop their flight when their kinetic energy cannot compensate for the wind speed. Here, the efficiency of an air curtain in preventing insects from entering a building was studied using the honey bee as a model. RESULTS: Bees were trained to visit a food source placed in a building. The air curtain was tested with strongly motivated bees, when the visiting activity was very high. Airflow velocity was modulated by setting an air curtain device at different voltages. At the nominal voltage, the anti-insect efficiency was 99.9 ± 0.2% compared with both the number of bees at a given time in the absence of the air curtain and the number of bees before the activation of the air curtain. The efficiency decreased as the airflow velocity decreased. CONCLUSION: The results show that an air curtain operating at an airflow velocity of 7.5 m s-1 may prevent a strong flyer with high kinetic energy, such as the honey bee, from entering a building. Thus, air curtains offer an alternative approach for combating vector-borne diseases. © 2018 Society of Chemical Industry.

Environmental heterogeneity and variations in the velocity of bluetongue virus spread in six European epidemics.

Several epidemics caused by different bluetongue virus (BTV) serotypes occurred in European ruminants since the early 2000. Studies on the spatial distribution of these vector-borne infections and the main vector species highlighted contrasted eco-climatic regions characterized by different dominant vector species. However, little work was done regarding the factors associated with the velocity of these epidemics. In this study, we aimed to quantify and compare the velocity of BTV epidemic that have affected different European countries under contrasted eco-climatic conditions and to relate these estimates to spatial factors such as temperature and host density. We used the thin plate spline regression interpolation method in combination with trend surface analysis to quantify the local velocity of different epidemics that have affected France (BTV-8 2007-2008, BTV-1 2008-2009), Italy (BTV-1 2014), Andalusia in Spain (BTV-1 2007) and the Balkans (BTV-4 2014). We found significant differences in the local velocity of BTV spread according to the country and epidemics, ranging from 7.9km/week (BTV-1 2014 Italy) to 24.4km/week (BTV-1 2008 France). We quantify and discuss the effect of temperature and local host density on this velocity.

Colony adaptive response to simulated heat waves and consequences at the individual level in honeybees (Apis mellifera).

Since climate change is expected to bring more severe and frequent extreme weather events such as heat waves, assessing the physiological and behavioural sensitivity of organisms to temperature becomes a priority. We therefore investigated the responses of honeybees, an important insect pollinator, to simulated heat waves (SHW). Honeybees are known to maintain strict brood thermoregulation, but the consequences at the colony and individual levels remain poorly understood. For the first time, we quantified and modelled colony real-time activity and found a 70% increase in foraging activity with SHW, which was likely due to the recruitment of previously inactive bees. Pollen and nectar foraging was not impacted, but an increase in water foragers was observed at the expense of empty bees. Contrary to individual energetic resources, vitellogenin levels increased with SHW, probably to protect bees against oxidative stress. Finally, though immune functions were not altered, we observed a significant decrease in deformed wing virus loads with SHW. In conclusion, we demonstrated that honeybees could remarkably adapt to heat waves without a cost at the individual level and on resource flow. However, the recruitment of backup foraging forces might be costly by lowering the colony buffering capacity against additional environmental pressures.

Stress response in honeybees is associated with changes in task-related physiology and energetic metabolism.

In a rapidly changing environment, honeybee colonies are increasingly exposed to diverse sources of stress (e.g., new parasites, pesticides, climate warming), which represent a challenge to individual and social homeostasis. However, bee physiological responses to stress remain poorly understood. We therefore exposed bees specialised in different tasks (nurses, guards and foragers) to ancient (immune and heat stress) or historically more recent sources of stress (pesticides), and we determined changes in the expression of genes linked to behavioural maturation (vitellogenin - vg and juvenile hormone esterase - jhe) as well as in energetic metabolism (glycogen level, expression level of the receptor to the adipokinetic hormone - akhr, and endothermic performance). While acute exposure to sublethal doses of two pesticides did not affect vg and jhe expression, immune and heat challenges caused a decrease and increase in both genes, respectively, suggesting that bees had responded to ecologically relevant stressors. Since vg and jhe are expressed to a higher level in nurses than in foragers, it is reasonable to assume that an immune challenge stimulated behavioural maturation to decrease potential contamination risk and that a heat challenge promoted a nurse profile for brood thermoregulation. All behavioural castes responded in the same way. Though endothermic performances did not change upon stress exposure, the akhr level dropped in immune and heat-challenged individuals. Similarly, the abdomen glycogen level tended to decline in immune-challenged bees. Altogether, these results suggest that bee responses are stress specific and adaptive but that they tend to entail a reduction of energetic metabolism that needs to be studied on a longer timescale.

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.

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle.

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.

Parasitic and immune modulation of flight activity in honey bees tracked with optical counters.

Host-parasite interactions are often characterized by changes in the host behaviour, which are beneficial to either the parasite or the host, or are a non-adaptive byproduct of parasitism. These interactions are further complicated in animal society because individual fitness is associated with group performance. However, a better understanding of host-parasite interaction in animal society first requires the identification of individual host behavioural modification. Therefore, we challenged honey bee (Apis mellifera) workers with the parasite Nosema ceranae or an immune stimulation and tracked their flight activity over their lifetime with an optic counter. We found that bees responded differently to each stress: both Nosema-infected and immune-challenged bees performed a lower number of daily flights compared with control bees, but the duration of their flights increased and decreased over time, respectively. Overall, parasitized bees spent more time in the field each day than control bees, and the inverse was true for immune-challenged bees. Despite the stress of immune challenge, bees had a survival similar to that of control bees likely because of their restricted activity. We discuss how those different behavioural modifications could be adaptive phenotypes. This study provides new insights into how biological stress can affect the behaviour of individuals living in society and how host responses have evolved.

Did vaccination slow the spread of bluetongue in France?

Vaccination is one of the most efficient ways to control the spread of infectious diseases. Simulations are now widely used to assess how vaccination can limit disease spread as well as mitigate morbidity or mortality in susceptible populations. However, field studies investigating how much vaccines decrease the velocity of epizootic wave-fronts during outbreaks are rare. This study aimed at investigating the effect of vaccination on the propagation of bluetongue, a vector-borne disease of ruminants. We used data from the 2008 bluetongue virus serotype 1 (BTV-1) epizootic of southwest France. As the virus was newly introduced in this area, natural immunity of livestock was absent. This allowed determination of the role of vaccination in changing the velocity of bluetongue spread while accounting for environmental factors that possibly influenced it. The average estimated velocity across the country despite restriction on animal movements was 5.4 km/day, which is very similar to the velocity of spread of the bluetongue virus serotype 8 epizootic in France also estimated in a context of restrictions on animal movements. Vaccination significantly reduced the propagation velocity of BTV-1. In comparison to municipalities with no vaccine coverage, the velocity of BTV-1 spread decreased by 1.7 km/day in municipalities with immunized animals. For the first time, the effect of vaccination has been quantified using data from a real epizootic whilst accounting for environmental factors known to modify the velocity of bluetongue spread. Our findings emphasize the importance of vaccination in limiting disease spread across natural landscape. Finally, environmental factors, specifically those related to vector abundance and activity, were found to be good predictors of the velocity of BTV-1 spread, indicating that these variables need to be adequately accounted for when evaluating the role of vaccination on bluetongue spread.

Why did bluetongue spread the way it did? Environmental factors influencing the velocity of bluetongue virus serotype 8 epizootic wave in France.

Understanding where and how fast an infectious disease will spread during an epidemic is critical for its control. However, the task is a challenging one as numerous factors may interact and drive the spread of a disease, specifically when vector-borne diseases are involved. We advocate the use of simultaneous autoregressive models to identify environmental features that significantly impact the velocity of disease spread. We illustrate this approach by exploring several environmental factors influencing the velocity of bluetongue (BT) spread in France during the 2007-2008 epizootic wave to determine which ones were the most important drivers. We used velocities of BT spread estimated in 4,495 municipalities and tested sixteen covariates defining five thematic groups of related variables: elevation, meteorological-related variables, landscape-related variables, host availability, and vaccination. We found that ecological factors associated with vector abundance and activity (elevation and meteorological-related variables), as well as with host availability, were important drivers of the spread of the disease. Specifically, the disease spread more slowly in areas with high elevation and when heavy rainfall associated with extreme temperature events occurred one or two months prior to the first clinical case. Moreover, the density of dairy cattle was correlated negatively with the velocity of BT spread. These findings add substantially to our understanding of BT spread in a temperate climate. Finally, the approach presented in this paper can be used with other infectious diseases, and provides a powerful tool to identify environmental features driving the velocity of disease spread.

Estimating front-wave velocity of infectious diseases: a simple, efficient method applied to bluetongue.

Understanding the spatial dynamics of an infectious disease is critical when attempting to predict where and how fast the disease will spread. We illustrate an approach using a trend-surface analysis (TSA) model combined with a spatial error simultaneous autoregressive model (SAR(err) model) to estimate the speed of diffusion of bluetongue (BT), an infectious disease of ruminants caused by bluetongue virus (BTV) and transmitted by Culicoides. In a first step to gain further insight into the spatial transmission characteristics of BTV serotype 8, we used 2007-2008 clinical case reports in France and TSA modelling to identify the major directions and speed of disease diffusion. We accounted for spatial autocorrelation by combining TSA with a SAR(err) model, which led to a trend SAR(err) model. Overall, BT spread from north-eastern to south-western France. The average trend SAR(err)-estimated velocity across the country was 5.6 km/day. However, velocities differed between areas and time periods, varying between 2.1 and 9.3 km/day. For more than 83% of the contaminated municipalities, the trend SAR(err)-estimated velocity was less than 7 km/day. Our study was a first step in describing the diffusion process for BT in France. To our knowledge, it is the first to show that BT spread in France was primarily local and consistent with the active flight of Culicoides and local movements of farm animals. Models such as the trend SAR(err) models are powerful tools to provide information on direction and speed of disease diffusion when the only data available are date and location of cases.

Diseases and reproductive success in a wild mammal: example in the alpine chamois.

Density-dependent and climatic factors affect reproduction and dynamics of wild ungulates. Parasites can also decrease reproductive success through either a direct abortive effect or a negative impact on host growth and body condition. However, few studies have investigated the effect of parasitism on fecundity of ungulates in natural conditions. We studied three bacterial infections caused by Salmonella enterica serovar Abortusovis, Chlamydophila abortus and Coxiella burnetii. These bacteria are leading causes of reproductive failure in sheep, goat and cattle, which raises the question of their influence on population dynamics of wild ungulates. A long-term study of demography and epidemiology of an alpine chamois (Rupicapra rupicapra, L.) population (Les Bauges Reserve, France) and a generalized linear modeling approach were used to analyze the reproductive success of chamois according to population density, weather conditions and the prevalence of antibodies against the three bacteria in females. This approach enabled us to identify the confounding effect of weather and parasitism on fecundity in a natural population. After accounting for density, the prevalence of antibodies against the three bacteria explained 36% of the annual variation in reproductive success, and weather conditions explained an additional 31%. This study was, to our knowledge, the first to compare the decrease in fecundity due to bacterial infections and weather conditions in a population of wild mountain ungulates.