Jean-Luc Brunet

Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera)

Global pollinators, like honeybees, are declining in abundance and diversity, which can adversely affect natural ecosystems and agriculture. Therefore, we tested the current hypotheses describing honeybee losses as a multifactorial syndrome, by investigating integrative effects of an infectious organism and an insecticide on honeybee health. We demonstrated that the interaction between the microsporidia Nosema and a neonicotinoid (imidacloprid) significantly weakened honeybees. In the short term, the combination of both agents caused the highest individual mortality rates and energetic stress. By quantifying the strength of immunity at both the individual and social levels, we showed that neither the haemocyte number nor the phenoloxidase activity of individuals was affected by the different treatments. However, the activity of glucose oxidase, enabling bees to sterilize colony and brood food, was significantly decreased only by the combination of both factors compared with control, Nosema or imidacloprid groups, suggesting a synergistic interaction and in the long term a higher susceptibility of the colony to pathogens. This provides the first evidences that interaction between an infectious organism and a chemical can also threaten pollinators, interactions that are widely used to eliminate insect pests in integrative pest management.

Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae.

Background The honeybee, Apis mellifera, is undergoing a worldwide decline whose origin is still in debate. Studies performed for twenty years suggest that this decline may involve both infectious diseases and exposure to pesticides. Joint action of pathogens and chemicals are known to threaten several organisms but the combined effects of these stressors were poorly investigated in honeybees. Our study was designed to explore the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. Methodology/Finding Five days after their emergence, honeybees were divided in 6 experimental groups: (i) uninfected controls, (ii) infected with N. ceranae, (iii) uninfected and exposed to fipronil, (iv) uninfected and exposed to thiacloprid, (v) infected with N. ceranae and exposed 10 days post-infection (p.i.) to fipronil, and (vi) infected with N. ceranae and exposed 10 days p.i. to thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. Surprisingly, exposures to fipronil and thiacloprid had opposite effects on microsporidian spore production. Analysis of the honeybee detoxification system 10 days p.i. showed that N. ceranae infection induced an increase in glutathione-S-transferase activity in midgut and fat body but not in 7-ethoxycoumarin-O-deethylase activity. Conclusions/Significance After exposure to sublethal doses of fipronil or thiacloprid a higher mortality was observed in N. ceranae-infected honeybees than in uninfected ones. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

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 Nosema ceranae 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.

Neural effects of insecticides in the honey bee

During their foraging activity, honey bees are often exposed to direct and residual contacts with pesticides, especially insecticides, all substances specifically designed to kill, repel, attract or perturb the vital functions of insects. Insecticides may elicit lethal and sublethal effects of different natures that may affect various biological systems of the honey bee. The first step in the induction of toxicity by a chemical is the interaction between the toxic compound and its molecular target. The action on the molecular target can lead to the induction of observable or non-visible effects. The toxic substance may impair important processes involved in cognitive functions, behaviour or integrity of physiological functions. This review is focused on the neural effects of insecticides that have repercussions on (a) cognitive functions, including learning and memory, habituation, olfaction and gustation, navigation and orientation; (b) behaviour, including foraging and (c) physiological functions, including thermoregulation and muscle activity.

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).

Standard methods for toxicology research in Apis mellifera

Summary Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.

This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at -20°C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD(50), 51.16 ng bee(-1)) and two sublethal doses, LD(50)/10 (5.12 ng bee(-1)) and LD(50)/20 (2.56 ng bee(-1)). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee(-1). We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides.

Fipronil is a powerful uncoupler of oxidative phosphorylation that triggers apoptosis in human neuronal cell line SHSY5Y

Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.

Nosema spp. Infection Alters Pheromone Production in Honey Bees (Apis mellifera)

Pheromones in social insects play a key role in the regulation of group homoeostasis. It is well-established that parasites can modify hormone signaling of their host, but less is known about the effect of parasites on pheromone signaling in insect societies. We, thus, tested in honey bees (Apis mellifera) the effect of the widespread parasite Nosema spp. on the production of ethyl oleate (EO), the only identified primer pheromone in honey bee workers. Since environmental stressors like pesticides also can weaken honey bees, we also analyzed the effect of imidacloprid, a neonicotinoid widely used in agriculture, on EO production. We show that, contrary to imidacloprid, Nosema spp. significantly altered EO production. In addition, the level of Nosema infection was correlated positively with the level of EO production. Since EO is involved in the regulation of division of labor among workers, our result suggests that the changes in EO signaling induced by parasitism have the potential to disturb the colony homoeostasis.