Metabolisation of thiamethoxam (a neonicotinoid pesticide) and interaction with the Chronic bee paralysis virus in honeybees.

Pathogens and pesticides are likely to co-occur in honeybee hives, but much remains to be investigated regarding their potential interactions. Here, we first investigated the metabolisation kinetics of thiamethoxam in chronically fed honeybees. We show that thiamethoxam, at a dose of 0.25ng/bee/day, is quickly and effectively metabolised into clothianidin, throughout a 20day exposure period. Using a similar chronic exposure to pesticide, we then studied, in a separate experiment, the impact of thiamethoxam and Chronic bee paralysis virus (CBPV) co-exposure in honeybees. The honeybees were exposed to the virus by contact, mimicking the natural transmission route in the hive. We demonstrate that a high dose of thiamethoxam (5.0ng/bee/day) can cause a synergistic increase in mortality in co-exposed honeybees after 8 to 10days of exposure, with no increase in viral loads. At a lower dose (2.5ng/bee/day), there was no synergistic increase of mortality, but viral loads were significantly higher in naturally dead honeybees, compared with sacrificed honeybees exposed to the same conditions. These results show that the interactions between pathogens and pesticides in honeybees can be complex: increasing pesticide doses may not necessarily be linked to a rise in viral loads, suggesting that honeybee tolerance to the viral infection might change with pesticide exposure.

Evidence for positive selection and recombination hotspots in Deformed wing virus (DWV).

Deformed wing virus (DWV) is considered one of the most damaging pests in honey bees since the spread of its vector, Varroa destructor. In this study, we sequenced the whole genomes of two virus isolates and studied the evolutionary forces that act on DWV genomes. The isolate from a Varroa-tolerant bee colony was characterized by three recombination breakpoints between DWV and the closely related Varroa destructor virus-1 (VDV-1), whereas the variant from the colony using conventional Varroa management was similar to the originally described DWV. From the complete sequence dataset, nine independent DWV-VDV-1 recombination breakpoints were detected, and recombination hotspots were found in the 5' untranslated region (5' UTR) and the conserved region encoding the helicase. Partial sequencing of the 5' UTR and helicase-encoding region in 41 virus isolates suggested that most of the French isolates were recombinants. By applying different methods based on the ratio between non-synonymous (dN) and synonymous (dS) substitution rates, we identified four positions that showed evidence of positive selection. Three of these positions were in the putative leader protein (Lp), and one was in the polymerase. These findings raise the question of the putative role of the Lp in viral evolution.

A New Stratified Sampling Procedure which Decreases Error Estimation of Varroa Mite Number on Sticky Boards.

A new procedure of stratified sampling is proposed in order to establish an accurate estimation of Varroa destructor populations on sticky bottom boards of the hive. It is based on the spatial sampling theory that recommends using regular grid stratification in the case of spatially structured process. The distribution of varroa mites on sticky board being observed as spatially structured, we designed a sampling scheme based on a regular grid with circles centered on each grid element. This new procedure is then compared with a former method using partially random sampling. Relative error improvements are exposed on the basis of a large sample of simulated sticky boards (n=20,000) which provides a complete range of spatial structures, from a random structure to a highly frame driven structure. The improvement of varroa mite number estimation is then measured by the percentage of counts with an error greater than a given level.

Varroa destructor changes its cuticular hydrocarbons to mimic new hosts.

Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host's cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.

Propolis chemical composition and honeybee resistance against Varroa destructor.

Propolis is known as honeybee chemical defence against infections and parasites. Its chemical composition is variable and depends on the specificity of the local flora. However, there are no data concerning the relationship between propolis chemical composition and honeybee colony health. We tried to answer this question, studying the chemical composition of propolis of bee colonies from an apiary near Avignon, which are tolerant to Varroa destructor, comparing it with colonies from the same apiary which are non-tolerant to the mites. The results indicated that non-tolerant colonies collected more resin than the tolerant ones. The percentage of four biologically active compounds - caffeic acid and pentenyl caffeates - was higher in propolis from tolerant colonies. The results of this study pave the way to understanding the effect of propolis in individual and social immunity of the honeybees. Further studies are needed to clarify the relationship between propolis chemical composition and honeybee colony health.

A selective sweep in a microsporidian parasite Nosema-tolerant honeybee population, Apis mellifera.

Nosema is a microsporidian parasite of the honeybee, which infects the epithelial cells of the gut. In Denmark, honeybee colonies have been selectively bred for the absence of Nosema over decades, resulting in a breeding line that is tolerant toward Nosema infections. As the tolerance toward the Nosema infection is a result of artificial selection, we screened chromosome 14 for a selective sweep with microsatellite markers, where a major quantitative trait locus (QTL) had been identified to be involved in the reduction in Nosema spores in the honeybees. By comparing the genetic variability of 10 colonies of the selected honeybee strain with a population sample from 22 unselected colonies, a selective sweep was revealed within the previously identified QTL region. The genetic variability of the swept loci was not only reduced in relation to the flanking markers on chromosome 14 within the selected strain but also significantly reduced compared with the same region in the unselected honeybees. This confirmed the results of the previous QTL mapping for reduced Nosema infections. The success of the selective breeding may have driven the selective sweep found in our study.

Social immunity in honeybees (Apis mellifera): transcriptome analysis of varroa-hygienic behaviour.

Honeybees have evolved a social immunity consisting of the cooperation of individuals to decrease disease in the hive. We identified a set of genes involved in this social immunity by analysing the brain transcriptome of highly varroa-hygienic bees, who efficiently detect and remove brood infected with the Varroa destructor mite. The function of these candidate genes does not seem to support a higher olfactory sensitivity in hygienic bees, as previously hypothesized. However, comparing their genomic profile with those from other behaviours suggests a link with brood care and the highly varroa-hygienic Africanized honeybees. These results represent a first step toward the identification of genes involved in social immunity and thus provide first insights into the evolution of social immunity.

Pheromones in a superorganism: from gene to social regulation.

Analogous to the importance of hormones in controlling organism homoeostasis, pheromones play a major role in the regulation of group homoeostasis at the social level. In social insects, pheromones coordinate the association of "unitary" organisms into a coherent social unit or so called "superorganism." For many years, honey bees have been a convincing model for studying pheromone regulation of social life. In addition, with the recent sequencing of its genome, a global view of pheromone communication is starting to emerge, and it is now possible to decipher this complex chemical language from the molecular to the social level. We review here the different pheromones regulating the main biological functions of the superorganism and detail their respective action on the genome, physiology and behavior of nestmates. Finally, we suggest some future research that may improve our understanding of the remarkably rich syntax of pheromone communication at the social level.

Regulation of brain gene expression in honey bees by brood pheromone.

Pheromones are very important in animal communication. To learn more about the molecular basis of pheromone action, we studied the effects of a potent honey bee pheromone on brain gene expression. Brood pheromone (BP) caused changes in the expression of hundreds of genes in the bee brain in a manner consistent with its known effects on behavioral maturation. Brood pheromone exposure in young bees causes a delay in the transition from working in the hive to foraging, and we found that BP treatment tended to upregulate genes in the brain that are upregulated in bees specialized on brood care but downregulate genes that are upregulated in foragers. However, the effects of BP were age dependent; this pattern was reversed when older bees were tested, consistent with the stimulation of foraging by BP in older bees already competent to forage. These results support the idea that one way that pheromones influence behavior is by orchestrating large-scale changes in brain gene expression. We also found evidence for a relationship between cis and BP regulation of brain gene expression, with several cis-regulatory motifs statistically overrepresented in the promoter regions of genes regulated by BP. Transcription factors that target a few of these motifs have already been implicated in the regulation of bee behavior. Together these results demonstrate strong connections between pheromone effects, behavior, and regulation of brain gene expression.

Climate change: impact on honey bee populations and diseases.

The European honey bee, Apis mellifera, is the most economically valuable pollinator of agricultural crops worldwide. Bees are also crucial in maintaining biodiversity by pollinating numerous plant species whose fertilisation requires an obligatory pollinator. Apis mellifera is a species that has shown great adaptive potential, as it is found almost everywhere in the world and in highly diverse climates. In a context of climate change, the variability of the honey bee's life-history traits as regards temperature and the environment shows that the species possesses such plasticity and genetic variability that this could give rise to the selection of development cycles suited to new environmental conditions. Although we do not know the precise impact of potential environmental changes on honey bees as a result of climate change, there is a large body of data at our disposal indicating that environmental changes have a direct influence on honey bee development. In this article, the authors examine the potential impact of climate change on honey bee behaviour, physiology and distribution, as well as on the evolution of the honey bee's interaction with diseases. Conservation measures will be needed to prevent the loss of this rich genetic diversity of honey bees and to preserve ecotypes that are so valuable for world biodiversity.

Differential gene expression of the honey bee Apis mellifera associated with Varroa destructor infection.

BACKGROUND: The parasitic mite, Varroa destructor, is the most serious pest of the western honey bee, Apis mellifera, and has caused the death of millions of colonies worldwide. This mite reproduces in brood cells and parasitizes immature and adult bees. We investigated whether Varroa infestation induces changes in Apis mellifera gene expression, and whether there are genotypic differences that affect gene expression relevant to the bee's tolerance, as first steps toward unravelling mechanisms of host response and differences in susceptibility to Varroa parasitism. RESULTS: We explored the transcriptional response to mite parasitism in two genetic stocks of A. mellifera which differ in susceptibility to Varroa, comparing parasitized and non-parasitized full-sister pupae from both stocks. Bee expression profiles were analyzed using microarrays derived from honey bee ESTs whose annotation has recently been enhanced by results from the honey bee genome sequence. We measured differences in gene expression in two colonies of Varroa-susceptible and two colonies of Varroa-tolerant bees. We identified a set of 148 genes with significantly different patterns of expression: 32 varied with the presence of Varroa, 116 varied with bee genotype, and 2 with both. Varroa parasitism caused changes in the expression of genes related to embryonic development, cell metabolism and immunity. Bees tolerant to Varroa were mainly characterized by differences in the expression of genes regulating neuronal development, neuronal sensitivity and olfaction. Differences in olfaction and sensitivity to stimuli are two parameters that could, at least in part, account for bee tolerance to Varroa; differences in olfaction may be related to increased grooming and hygienic behavior, important behaviors known to be involved in Varroa tolerance. CONCLUSION: These results suggest that differences in behavior, rather than in the immune system, underlie Varroa tolerance in honey bees, and give an indication of the specific physiological changes found in parasitized bees. They provide a first step toward better understanding molecular pathways involved in this important host-parasite relationship.

Modifications of the cuticular hydrocarbon profile of Apis mellifera worker bees in the presence of the ectoparasitic mite Varroa jacobsoni in brood cells.

Varroa jacobsoni is an ectoparasite of Apis mellifera which invades brood cells, on 8-day-old larvae several hours before cell capping. Reproduction of the parasite takes place in the capped brood cells during the nymphose of the bee. Cuticular hydrocarbons of unparasitized bees and of bees parasitized by Varroa jacobsoni were extracted and analysed by gas chromatography (GC) coupled with mass spectrometry (GC-MS). Three developmental stages of worker honey bees were studied: larvae, pupae and emergent adults. The comparison between unparasitized and parasitized hosts was performed with Principal Components Analysis coupled with a multivariate variance analysis. The cuticular hydrocarbon profiles of honey bees were qualitatively similar, for the 3 developmental stages and regardless of the presence of Varroa in the cells. Nevertheless, comparison of the relative proportions of hydrocarbons showed that the cuticular profiles of pupae and emergent adults parasitized by 1 mite and of larvae parasitized by 2 mites were significantly different from the corresponding unparasitized individuals. Such modifications could be regarded (i) as a cause of the multi-infestation in larvae during invasion of brood and (ii) as a consequence of stress and/or removal of proteins contained in the haemolymph of the host during its development.

Variations in chemical mimicry by the ectoparasitic mite Varroa jacobsoni according to the developmental stage of the host honey-bee Apis mellifera.

The ectoparasitic mite Varroa jacobsoni poses a major threat to the survival of European honey-bee populations. Development of effective control methods is therefore much needed. Study of interspecific chemical communication between the parasite and host is a particularly promising avenue of research. Previous study has shown that the cuticular hydrocarbons of the parasite mite Varroa jacobsoni are qualitatively identical to those of its honey-bee host Apis mellifera (Nation J.L., Sanford M.T., Milne K., 1992. Cuticular hydrocarbons from Varroa jacobsoni. Experimental and Applied Acarology 16, 331-344). The purpose of the present study was to compare the cuticular hydrocarbon patterns of the two species at different stages of bee development. Cuticular components were identified by gas chromatography/mass spectrometry. The proportion of each component was calculated at three stages of bee development (larvae, pupa, emerging bee). The degree of chemical mimicry between the parasite and host was evaluated by multivariate analyses using the resulting proportions for each category of individuals. There were four main findings. The first was that the proportions of some components are different at the larval, pupal and imago stage of bee development. Second, Varroa profiles vary depending on the developmental stage of the host. Third, the cuticular profile of adult mites is more similar to that of the stage of the host than that of later and/or earlier stages except for parasites collected from emerging adult bees. Fourth, the degree of mimicry by Varroa is greater during larval and pupal stages than during the emerging adult bee stages. The role of chemical mimicry - although it is not perfect - in enabling parasites to infest bee colonies by the parasite is discussed.

Primer effects of a brood pheromone on honeybee behavioural development.

Primer pheromones are thought to act in a variety of vertebrates and invertebrates but only a few have been chemically identified. We report that a blend of ten fatty-acid esters found on the cuticles of honeybee larvae, already known as a kairomone, releaser pheromone and primer pheromone, also act as a primer pheromone in the regulation of division of labour among adult workers. Bees in colonies receiving brood pheromone initiated foraging at significantly older ages than did bees in control colonies in five out of five trials. Laboratory and additional field tests also showed that exposure to brood pheromone significantly depressed blood titres of juvenile hormone. Brood pheromone exerted more consistent effects on age at first foraging than on juvenile hormone, suggesting that the primer effects of this pheromone may occur via other, unknown, mechanisms besides juvenile hormone. These results bring the number of social factors known to influence honeybee division of labour to three: worker-worker interactions, queen mandibular pheromone and brood pheromone.

Does the spatial distribution of the parasitic mite varroa jacobsoni oud. (Mesostigmata: varroidae) in worker brood of honey bee apis mellifera L. (Hymenoptera: apidae) rely on an aggregative process?

Varroa jacobsoni is an ectoparasite of honey bees which reproduces in capped brood cells. Multi-infestation is frequently observed in worker brood and can be interpreted as an aggregative phenomenon. The aim of this study was to determine whether the distribution of V. jacobsoni in worker brood cells relies on a random or an aggregative process. We studied the distribution of Varroa females in capped worker brood at similar age by comparing, by a Monte Carlo test, the observed frequency distribution of mites per cell to simulated distributions based on a random process. A complementary approach, using the "nearest neighbor distances" (NND) with Monte Carlo tests, was investigated to study the spatial distribution (a) between mites in different cells and (b) between infested cells in brood. The observed distributions did not differ significantly from that expected by a random process, and we conclude that there is no aggregation during invasion of V. jacobsoni in worker brood.

Microsatellite analysis of sperm admixture in honeybee.

Sperm usage was investigated in an instrumentally inseminated honeybee queen. Her progeny were examined in the first 3 months of the egg-laying period using a microsatellite marker. Frequencies of different subfamilies differed significantly from one month to another. However, there was no evidence for sperm displacement or sperm precedence of a specific male in the worker progeny. The variance of subfamily proportions decreased over time suggesting that sperm admixture in the spermatheca was incomplete at the beginning of the egg-laying period of the queen and improved progressively during the first months after mating.

Effect of a brood pheromone on honeybee hypopharyngeal glands.

In a honeybee colony, brood stimulates development of hypopharyngeal glands of nurse bees. A chemical signal, a blend of 10 fatty acid esters, has been identified on larval cuticle. We demonstrate that the blend of 10 esters, ethyl oleate, and methyl palmitate stimulates the protein synthesis of hypopharyngeal glands of nurses. Thus, in Apis mellifera the chemical signal from the brood acts as a primer pheromone in addition to its previously shown role as a releaser pheromone.

Semiochemical basis of infestation of honey bee brood byVarroa jacobsoni.

Capping of workerApis mellifera cells is elicited by four fatty acid methyl esters (Methyl palmitate, methyl oleate, methyl linoleate, and methyl linolenate) that are present on the surface of the worker and drone larvae only a few hours before the cell is closed. The amount of the pheromone reaches its maximum value when the cell has just been capped, at 8.5 and at 10.25 days of age, respectively, for worker and drone larvae. Thereafter, the amount of the pheromone decreases to its initial level. These data suggest that the esters also have a role in the capping of the drone cells, the temporal signal allowing the worker bees to recognize the age of the larvae and then to do the appropriate behavior. Two pheromonal components, methyl palmitate and methyl linolenate, and the inactive ethyl palmitate are kairomones attractive toVarroa females. Their secretion by the larvae follows the same pattern of development as the pheromonal signal. The longer and greater kairomonal signal in drone larvae, compared to worker secretion, could explain the preference ofVarroa towards drone brood.

Attraction of the parasitic mite varroa to the drone larvae of honey bees by simple aliphatic esters.

An important parasitic threat to honey bees, the mite Varroa jacobsoni, is attracted to its major prey, drone larvae, by methyl and ethyl esters of straight-chain fatty acids, in particular methyl palmitate. These esters were extracted from drone larvae with n-hexane and were identified by gas chromatography-mass spectrometry. Their behavioral effect was evaluated with the use of a four-arm airflow olfactometer.