Experimental Ecotoxicology Procedures Interfere with Honey Bee Life History.

Apis mellifera was used as a model species for ecotoxicological testing. In the present study, we tested the effects of acetone (0.1% in feed), a solvent commonly used to dissolve pesticides, on bees exposed at different developmental stages (larval and/or adult). Moreover, we explored the potential effect of in vitro larval rearing, a commonly used technique for accurately monitoring worker exposure at the larval stage, by combining acetone exposure and treatment conditions (in vitro larval rearing vs. in vivo larval rearing). We then analyzed the life-history traits of the experimental bees using radio frequency identification technology over three sessions (May, June, and August) to assess the potential seasonal dependence of the solvent effects. Our results highlight the substantial influence of in vitro larval rearing on the life cycle of bees, with a 47.7% decrease in life span, a decrease of 0.9 days in the age at first exit, an increase of 57.3% in the loss rate at first exit, and a decrease of 40.6% in foraging tenure. We did not observe any effect of exposure to acetone at the larval stage on the capacities of bees reared in vitro. Conversely, acetone exposure at the adult stage reduced the bee life span by 21.8% to 60%, decreased the age at first exit by 1.12 to 4.34 days, and reduced the foraging tenure by 30% to 37.7%. Interestingly, we found a significant effect of season on acetone exposure, suggesting that interference with the life-history traits of honey bees is dependent on season. These findings suggest improved integration of long-term monitoring for assessing sublethal responses in bees following exposure to chemicals during both the larval and adult stages. Environ Toxicol Chem 2024;00:1-12. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

GeoDanceHive: An Operational Hive for Honeybees Dances Recording.

Honeybees are known for their ability to communicate about resources in their environment. They inform the other foragers by performing specific dance sequences according to the spatial characteristics of the resource. The purpose of our study is to provide a new tool for honeybees dances recording, usable in the field, in a practical and fully automated way, without condemning the harvest of honey. We designed and equipped an outdoor prototype of a production hive, later called "GeoDanceHive", allowing the continuous recording of honeybees' behavior such as dances and their analysis. The GeoDanceHive is divided into two sections, one for the colony and the other serving as a recording studio. The time record of dances can be set up from minutes to several months. To validate the encoding and sampling quality, we used an artificial feeder and visual decoding to generate maps with the vector endpoints deduced from the dance information. The use of the GeoDanceHive is designed for a wide range of users, who can meet different objectives, such as researchers or professional beekeepers. Thus, our hive is a powerful tool for honeybees studies in the field and could highly contribute to facilitating new research approaches and a better understanding landscape ecology of key pollinators.

Honeybee queen exposure to a widely used fungicide disrupts reproduction and colony dynamic.

Pollinators have to cope with a wide range of stressful, not necessarily lethal factors limiting their performance and the ecological services they provide. Among these stressors are pesticides, chemicals that are originally designed to target crop-harming organisms but that also disrupt various functions in pollinators, including flight, communication, orientation and memory. Although all these functions are crucial for reproductive individuals when searching for mates or nesting places, it remains poorly understood how pesticides affect reproduction in pollinators. In this study, we investigated how a widely used fungicide, boscalid, affects reproduction in honey bees (Apis mellifera), an eusocial insect in which a single individual, the queen, fulfills the reproductive functions of the whole colony. Boscalid is a succinate dehydrogenase inhibitor (SDHI) fungicide mainly used on rapeseed flowers to target mitochondrial respiration in fungi but it is also suspected to disrupt foraging-linked functions in bees. We found that immature queen exposure to sublethal, field relevant doses of boscalid disrupted reproduction, as indicated by a dramatic increase in queen mortality during and shortly after the nuptial flights period and a decreased number of spermatozoa stored in the spermatheca of surviving queens. However, we did not observe a decreased paternity frequency in exposed queens that successfully established a colony. Queen exposure to boscalid had detrimental consequences on the colonies they later established regarding brood production, Varroa destructor infection and pollen storage but not nectar storage and population size. These perturbations at the colony-level correspond to nutritional stress conditions, and may have resulted from queen reduced energy provisioning to the eggs. Accordingly, we found that exposed queens had decreased gene expression levels of vitellogenin, a protein involved in egg-yolk formation. Overall, our results indicate that boscalid decreases honey bee queen reproductive quality, thus supporting the need to include reproduction in the traits measured during pesticide risk assessment procedures.

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.

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.

Early steps of cryopreservation of day one honeybee (Apis mellifera) embryos treated with low-frequency sonophoresis.

Honeybees, major providers of pollination, are endangered in many areas. Embryo cryopreservation may be a very useful tool to maintain their genetic diversity. However, it is complex in insects, because embryos are chill sensitive and are surrounded by two protectant membranes, the chorion and vitelline. These membranes prevent penetration of cryoprotectant in the embryos. This study aimed to test different conditions of embryo preparation before cryopreservation, including low-frequency sonophoresis, a physical method of permeabilization, and passages through cryoprotectant solutions. Apis mellifera ligustica embryos were collected in artificial cell plugs 7.5 h after queens had been caged, in two different seasons (winter, spring) and were then incubated in vitro overnight (16.5 h). Embryos were individually sonicated and then incubated in three cryoprotectant baths (B1 = 10%, B2 = 20% and B3 = 40% of cryoprotectant) and quenched in liquid nitrogen. Artificial cell plugs and in vitro incubation device were efficient in producing future embryos hatching. Embryos stained ruby red with rhodamine B after sonophoresis treatment indicated that low-frequency ultrasound had permeabilized embryos. According to the treatment, different significant hatching rates were obtained after sonophoresis (up to 25%). After three cryoprotectant incubations, best hatching rates were obtained after 10 min in B1 and B2, and 40 s in B3. These results show that sonophoresis is an efficient tool to permeabilize the chorion and vitelline membrane of the day one honeybee embryo allowing a hatching rate of more than 20%. They also show that the season is an important variability factor.

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.

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.

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.

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.

Honeybee tracking with microchips: a new methodology to measure the effects of pesticides.

Losses of foraging bees are sometimes attributed to altered flight pattern between a meliferous plant treated with an insecticide and the hive. Only a limited number of studies has investigated the impact of pesticides on homing flight due to the difficulty of measuring the flight time between the food source and the hive. Monitoring the flights of the foraging bees needs their individual identification. The number of bees monitored simultaneously and the time span during which observations can be made limit most of the monitoring techniques. However, techniques of automatic tracking and identification of individuals have the potential to revolutionize the study of the ecotoxicological effects of xenobiotics on the bee behaviors. Radio Frequency Identification (RFID) offer numerous advantages such as an unlimited number of codes, a large number of simultaneous recording, and a quick reading, especially through materials (e.g., wood). The aim of this study was to show how the RFID device can be used to study the effects of pesticides on both the behavioral traits and the lifespan of bees. In this context, we have developed a method under tunnel to automatically record the displacements of foragers individualized with RFID tags and to detect the alteration of the flight pattern between an artificial feeder and the hive. Fipronil was selected as test substance due to the lack of information on the effects of this insecticide on the foraging behavior of free-flying bees. We showed that oral treatment of 0.3 ng of fipronil per bee (LD50/20) reduced the number of foraging trips. The strengths of our approach were briefly discussed.

Validation of a method using queenless Bombus terrestris micro-colonies for testing the nutritive value of commercial pollen mixes by comparison with queenright colonies.

The nutritive value of pollen blends purchased by bumble bee producers from beekeepers is a key factor of successful mass rearing. We have already devised a method for quality ranking of pollen diets. It was a 1-mo bioassay using queenless micro-colonies of three callow workers (Bombus terrestris L.; Apidae: Bombinae). In the current study, we tested three pollen diets through the micro-colony method and compared the results to the development of queenright colonies supplied with the same diets. The three diets were "A," a spring blend with a protein rate of 15.7% and a dominance of Corylus avellana L.(46%) and Buxus sempervivens L. (35%); "B," a summer assemblage with alower crude protein content (14.4%), with a dominance of Helianthus annuus L. (37%), Zea mays L. (35%), and Poaceae (21%); and "C," a 50/50 mixture by weight of A and B, with a crude protein content of 15.8%. In micro-colonies, the more sensitive parameter for diet ranking was the mean weight of a larva, whereas in queenright colonies, the discrimination between treatments was enabled by considering the body size of new queens (weight and length of the radial cell) and the slope of the sigmoidal curve of the pollen consumption at the inflection point reflecting brood growth. Both testing methods assigned the lower rank to diet B compared with diet A. Despite the closeness of the three diets in terms of nitrogen content, we concluded that micro-colonies were a good estimate of colony development when nutritive value of pollen was tested.

Toxicity of dimethoate and fenoxycarb to honey bee brood (Apis mellifera), using a new in vitro standardized feeding method.

A new in vitro method was devised to assess the effects of pesticides on honey bee brood. The method allowed the quantification of doses ingested by larvae and the assessment of larval and pupal mortality. Larval mortality in control samples was lower than 10%. Two active substances were tested: dimethoate and fenoxycarb. The LD(50) of dimethoate was 1.9 microg larva(-1) 48 h after oral exposure of larvae at day 4. Additional dose-related effects on pupal mortality were noted. After a chronic intoxication, the NOAEC (No Observed Adverse Effect Concentration) for larval mortality at day 7 was 2.5 mg kg(-1), whereas a NOAEC of 5 mg kg(-1) was found at day 22 for delayed effects on the reduction of adult emergence. Fenoxycarb applied at day 4 showed no effect on larvae, whereas emergence of adults was affected at doses higher than 6 ng larva(-1).