Exposure to Sublethal Doses of Fipronil and Thiacloprid Highly Increases Mortality of Honeybees Previously Infected by Nosema ceranae

International audienceBACKGROUND: 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

Impact de la microsporidie Nosema ceranae et d'insecticides neurotoxiques sur la santé de l'abeille domestique (Apis mellifera)

Apis mellifera is a pollinator insect playing of major economical and ecological importance. For more than two decades, severe honeybee colony losses have been reported worldwide. The origin of this phenomenon is thought to involve numerous stressors that could interact with each other. The objective of this work was to evaluate the impact on honeybee health of the association between a biotic stressor, namely the microsporidian parasite Nosema ceranae, and an abiotic stressor, here low doses of neurotoxic insecticides. Laboratory studies showed that the N. ceranae-insecticide association leads to a higher mortality in honeybees, and more precisely to a synergistic effect. This effect seemed to be independent of the exposure sequence to stressors. Moreover, when applied at the emergence of honeybees, these stressors had a higher impact on individuals’ mortality. The honeybee response to N. ceranae and to insecticides has also been analysed at a transcriptional level. A midgut transcriptome analysis has been performed combining a global approach, using high-throughput sequencing (RNA-Seq), and a quantitative RT-PCR monitoring of the expression of selected genes. The exposure to N. ceranae and to insecticides led to modifications in the expression of genes involved in honeybee defenses (immunity, detoxification) and in trehalose and chitin metabolisms. Following this work, several prospects can be initiated in order to improve our understanding regarding the honeybee response to various stressors, combining laboratory experiments with field studies.Apis mellifera est un insecte pollinisateur jouant un rôle économique et écologique majeur. Depuis plus d’une vingtaine d’années, d’importantes pertes de colonies d’abeilles ont été recensées à l’échelle mondiale. L’origine de ce phénomène impliquerait de nombreux facteurs de stress qui pourraient en outre interagir entre eux. Ce travail de thèse a eu pour but d’évaluer l’impact sur la santé de l’abeille de l’association entre un facteur biotique, le parasite microsporidien Nosema ceranae, et un facteur abiotique, des insecticides neurotoxiques à faibles doses. Des études en laboratoire ont montré que l’association N. ceranae-insecticide entraine une surmortalité significative, et plus précisément un effet synergique sur la mortalité des abeilles. Cet effet synergique semble indépendant de l’ordre d’exposition des abeilles aux deux facteurs de stress. De plus, lorsqu’ils ont été appliqués dès l’émergence des abeilles, ces facteurs ont eu un impact plus fort sur la mortalité. La réponse de l’abeille à N. ceranae et aux insecticides a ensuite été analysée à l’échelle transcriptomique. L’analyse du transcriptome de l’intestin a été réalisée en combinant une approche globale de séquençage à haut débit (RNA-Seq) et un suivi de l’expression d’une sélection de gènes par qRT-PCR. L’exposition à N. ceranae et aux insecticides a entraîné des modifications de l’expression de gènes impliqués dans les défenses de l’abeille (immunité, détoxication) et dans les métabolismes du tréhalose et de la chitine. De nombreuses perspectives à ce travail sont envisageables dans le but de mieux appréhender la réponse de l’abeille à différents facteurs de stress, notamment en combinant des expérimentations en laboratoire avec des études de terrain

Impact of the microsporidian Nosema ceranae and neurotoxic insecticides on the western honeybee (Apis mellifera) health

Apis mellifera est un insecte pollinisateur jouant un rôle économique et écologique majeur. Depuis plus d’une vingtaine d’années, d’importantes pertes de colonies d’abeilles ont été recensées à l’échelle mondiale. L’origine de ce phénomène impliquerait de nombreux facteurs de stress qui pourraient en outre interagir entre eux. Ce travail de thèse a eu pour but d’évaluer l’impact sur la santé de l’abeille de l’association entre un facteur biotique, le parasite microsporidien Nosema ceranae, et un facteur abiotique, des insecticides neurotoxiques à faibles doses. Des études en laboratoire ont montré que l’association N. ceranae-insecticide entraine une surmortalité significative, et plus précisément un effet synergique sur la mortalité des abeilles. Cet effet synergique semble indépendant de l’ordre d’exposition des abeilles aux deux facteurs de stress. De plus, lorsqu’ils ont été appliqués dès l’émergence des abeilles, ces facteurs ont eu un impact plus fort sur la mortalité. La réponse de l’abeille à N. ceranae et aux insecticides a ensuite été analysée à l’échelle transcriptomique. L’analyse du transcriptome de l’intestin a été réalisée en combinant une approche globale de séquençage à haut débit (RNA-Seq) et un suivi de l’expression d’une sélection de gènes par qRT-PCR. L’exposition à N. ceranae et aux insecticides a entraîné des modifications de l’expression de gènes impliqués dans les défenses de l’abeille (immunité, détoxication) et dans les métabolismes du tréhalose et de la chitine. De nombreuses perspectives à ce travail sont envisageables dans le but de mieux appréhender la réponse de l’abeille à différents facteurs de stress, notamment en combinant des expérimentations en laboratoire avec des études de terrain.Apis mellifera is a pollinator insect playing of major economical and ecological importance. For more than two decades, severe honeybee colony losses have been reported worldwide. The origin of this phenomenon is thought to involve numerous stressors that could interact with each other. The objective of this work was to evaluate the impact on honeybee health of the association between a biotic stressor, namely the microsporidian parasite Nosema ceranae, and an abiotic stressor, here low doses of neurotoxic insecticides. Laboratory studies showed that the N. ceranae-insecticide association leads to a higher mortality in honeybees, and more precisely to a synergistic effect. This effect seemed to be independent of the exposure sequence to stressors. Moreover, when applied at the emergence of honeybees, these stressors had a higher impact on individuals’ mortality. The honeybee response to N. ceranae and to insecticides has also been analysed at a transcriptional level. A midgut transcriptome analysis has been performed combining a global approach, using high-throughput sequencing (RNA-Seq), and a quantitative RT-PCR monitoring of the expression of selected genes. The exposure to N. ceranae and to insecticides led to modifications in the expression of genes involved in honeybee defenses (immunity, detoxification) and in trehalose and chitin metabolisms. Following this work, several prospects can be initiated in order to improve our understanding regarding the honeybee response to various stressors, combining laboratory experiments with field studies

Impact de la microsporidie Nosema ceranae et d'insecticides neurotoxiques sur la santé de l'abeille domestique (Apis mellifera)

Apis mellifera est un insecte pollinisateur jouant un rôle économique et écologique majeur. Depuis plus d une vingtaine d années, d importantes pertes de colonies d abeilles ont été recensées à l échelle mondiale. L origine de ce phénomène impliquerait de nombreux facteurs de stress qui pourraient en outre interagir entre eux. Ce travail de thèse a eu pour but d évaluer l impact sur la santé de l abeille de l association entre un facteur biotique, le parasite microsporidien Nosema ceranae, et un facteur abiotique, des insecticides neurotoxiques à faibles doses. Des études en laboratoire ont montré que l association N. ceranae-insecticide entraine une surmortalité significative, et plus précisément un effet synergique sur la mortalité des abeilles. Cet effet synergique semble indépendant de l ordre d exposition des abeilles aux deux facteurs de stress. De plus, lorsqu ils ont été appliqués dès l émergence des abeilles, ces facteurs ont eu un impact plus fort sur la mortalité. La réponse de l abeille à N. ceranae et aux insecticides a ensuite été analysée à l échelle transcriptomique. L analyse du transcriptome de l intestin a été réalisée en combinant une approche globale de séquençage à haut débit (RNA-Seq) et un suivi de l expression d une sélection de gènes par qRT-PCR. L exposition à N. ceranae et aux insecticides a entraîné des modifications de l expression de gènes impliqués dans les défenses de l abeille (immunité, détoxication) et dans les métabolismes du tréhalose et de la chitine. De nombreuses perspectives à ce travail sont envisageables dans le but de mieux appréhender la réponse de l abeille à différents facteurs de stress, notamment en combinant des expérimentations en laboratoire avec des études de terrain.Apis mellifera is a pollinator insect playing of major economical and ecological importance. For more than two decades, severe honeybee colony losses have been reported worldwide. The origin of this phenomenon is thought to involve numerous stressors that could interact with each other. The objective of this work was to evaluate the impact on honeybee health of the association between a biotic stressor, namely the microsporidian parasite Nosema ceranae, and an abiotic stressor, here low doses of neurotoxic insecticides. Laboratory studies showed that the N. ceranae-insecticide association leads to a higher mortality in honeybees, and more precisely to a synergistic effect. This effect seemed to be independent of the exposure sequence to stressors. Moreover, when applied at the emergence of honeybees, these stressors had a higher impact on individuals mortality. The honeybee response to N. ceranae and to insecticides has also been analysed at a transcriptional level. A midgut transcriptome analysis has been performed combining a global approach, using high-throughput sequencing (RNA-Seq), and a quantitative RT-PCR monitoring of the expression of selected genes. The exposure to N. ceranae and to insecticides led to modifications in the expression of genes involved in honeybee defenses (immunity, detoxification) and in trehalose and chitin metabolisms. Following this work, several prospects can be initiated in order to improve our understanding regarding the honeybee response to various stressors, combining laboratory experiments with field studies.CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

New insights on the genetic diversity of the honeybee parasite Nosema ceranae based on multilocus sequence analysis.

International audienceThe microsporidian parasite Nosema ceranae is a common pathogen of the Western honeybee (Apis mellifera) whose variable virulence could be related to its genetic polymorphism and/or its polyphenism responding to environmental cues. Since the genotyping of N. ceranae based on unique marker sequences had been unsuccessful, we tested whether a multilocus approach, assessing the diversity of ten genetic markers - encoding nine proteins and the small ribosomal RNA subunit - allowed the discrimination between N. ceranae variants isolated from single A. mellifera individuals in four distant locations. High nucleotide diversity and allele content were observed for all genes. Most importantly, the diversity was mainly present within parasite populations isolated from single honeybee individuals. In contrast the absence of isolate differentiation precluded any taxa discrimination, even through a multilocus approach, but suggested that similar populations of parasites seem to infect honeybees in distant locations. As statistical evolutionary analyses showed that the allele frequency is under selective pressure, we discuss the origin and consequences of N. ceranae heterozygosity in a single host and lack of population divergence in the context of the parasite natural and evolutionary history

Transcriptome analyses of the honeybee response to Nosema ceranae and insecticides.

International audienceHoneybees (Apis mellifera) are constantly exposed to a wide variety of environmental stressors such as parasites and pesticides. Among them, Nosema ceranae and neurotoxic insecticides might act in combination and lead to a higher honeybee mortality. We investigated the molecular response of honeybees exposed to N. ceranae, to insecticides (fipronil or imidacloprid), and to a combination of both stressors. Midgut transcriptional changes induced by these stressors were measured in two independent experiments combining a global RNA-Seq transcriptomic approach with the screening of the expression of selected genes by quantitative RT-PCR. Although N. ceranae-insecticide combinations induced a significant increase in honeybee mortality, we observed that they did not lead to a synergistic effect. According to gene expression profiles, chronic exposure to insecticides had no significant impact on detoxifying genes but repressed the expression of immunity-related genes. Honeybees treated with N. ceranae, alone or in combination with an insecticide, showed a strong alteration of midgut immunity together with modifications affecting cuticle coatings and trehalose metabolism. An increasing impact of treatments on gene expression profiles with time was identified suggesting an absence of stress recovery which could be linked to the higher mortality rates observed

Honeybee genes whose expression is significantly modified in response to at least one treatment (<i>N. ceranae</i> infection, chronic exposure to fipronil or a combination of both stressors) compared to the untreated control, 1 or 7 days after the experiment initiation.

<p>The log₂ ratio of the normalized transcript content relative to the control at the same day is given together with the adjusted p-value in parentheses. Significant expression changes (adjusted p-value <0.1) are shown in bold.</p

Expression levels of 24 honeybee genes in response to various stressors, alone or in combination, 7 or 11 days after experiment initiation (d.a.i).

<p>Log fold change is given together with the p-value in parentheses. Significant expression changes (p-value ≤0.05) are shown in bold.</p

Effect of <i>N. ceranae</i> and fipronil, alone or in combination, on honeybee survival.

<p>Data give the cumulative proportion of surviving honeybees exposed to no treatment (blue), <i>N. ceranae</i> (red), fipronil (green), or a <i>N. ceranae</i>-fipronil combination (pink). <i>N. ceranae</i>-treated honeybees were individually infected at their emergence (day 0) and fipronil-treated ones were chronically and orally exposed to fipronil (1.3 μg/L) from day 0 to day 7. Data from 165 honeybees per experimental condition were analysed using the Kaplan-Meier method.</p

Impact of <i>N. ceranae</i> and fipronil on gene expression or enzyme activity in the honeybee.

<p>The figure presents a selection of genes or enzymes whose expression or activity has been shown to significantly increase (<b>↑</b>) or decrease (<b>↓</b>) under exposure to <i>N. ceranae</i> (red), to fipronil (green), or to a <i>N. ceranae</i>-fipronil combination (purple) in the present work or in previous studies: ¹Antunez <i>et al.</i>, 2009; ²Chaimanee <i>et al.</i>, 2012; ³Dussaubat <i>et al.</i>, 2012; ⁴Vidau <i>et al.</i>, 2011.</p