Analyse de l'immunoprotéome de l'abeille en réponse à différents stress environnementaux

The loss of honeybee colonies could be explained by multiple stress factors (biotic or abiotic) that could act alone or in synergy to perturb the physiology of the domestic bees. The impact of these stressors on the bee health is widely discussed. In the miscrosporidiosis, an infection due to Nosema, the impact of this stressor on the systemic immune response (haemolymph being the mirror of this response) and the epithelial response (gut tissue as target) remains unsatisfactory documented. In the context of an infection by Nosema spp, the spore multiplication that is occurring at the level of the midgut stimulate the gut epithelial immune response. While at the same time, the stimulation of the systemic immune response remains an opened question. The aim of this thesis is to decipher the cross-talk existing between the host (A. mellifera) and the pathogen (Nosema spp.). We investigated the epithelial (gut tissues) and circulating bee immune response (haemolymph) using complementary mass spectrometry approaches (molecular mass fingerprints by MALDI BeeTyping®, untargeted proteomics and MALDI imaging) in a context of a controlled or natural infection by Nosema spores. We have demonstrated new molecular markers of bee health from hemolymph and bees early infected with Nosema from digestive tract. In addition, we have developed the MALDI Biotyping method on our microsporidia model to identify the species at a lower cost. These different elements constitute the development of new diagnostic and even prognostic tools to assist the beekeeper or the health services in their apiary monitoring.Le phénomène mondial d’effondrement des colonies d’abeilles domestiques peut être expliqué par l’exposition de celles-ci à différents stresseurs (biotiques et abiotiques) qui peuvent agir seuls ou en synergie. L’impact de ces stresseurs sur la santé des abeilles, surtout lorsqu’ils sont multiples, est largement débattu. Dans le cadre de l’infection par Nosema, une microsporidie présente dans le tube digestif de l’abeille, de nombreuses études semblent indiquer que la multiplication des spores affecte l’épithélium de la paroi intestinale et donc stimule une réponse immunitaire épithéliale. Cependant, montrer que la réponse immunitaire systémique (visible au sein de l’hémolymphe) est bien stimulée, reste un cap à franchir particulièrement délicat. Le but de la thèse est de mieux comprendre les interactions qui se créent entre l’hôte (Apis mellifera) et le pathogène (Nosema spp.). Nous avons étudié pour cela le système immunitaire tissulaire (tube digestif) et circulant (hémolymphe) de l’hôte par des approches complémentaires de spectrométrie de masse (empreintes moléculaires massiques par MALDI BeeTyping®, approches de protéomique fine sur des segments de tube digestif et imagerie moléculaire par MALDI) dans un contexte d’infection contrôlée ou naturelle par des spores de Nosema spp. Nous avons mis en évidence de nouveaux marqueurs moléculaires de la santé de l’abeille à partir de l’hémolymphe d’abeilles infectées de manière précoce par Nosema et à partir du tube digestif de ces mêmes abeilles. De plus nous avons développé la méthode de MALDI Biotyping sur notre modèle de microsporidies pour identifier l’espèce à moindre coût. Ces différents éléments permettront le développement de nouveaux outils de diagnostic voir de pronostic pour assister l’apiculteur ou les services sanitaires dans le suivi de leur rucher

Immunoproteome analysis of the honeybee in response to environmental stressors.

Le phénomène mondial d’effondrement des colonies d’abeilles domestiques peut être expliqué par l’exposition de celles-ci à différents stresseurs (biotiques et abiotiques) qui peuvent agir seuls ou en synergie. L’impact de ces stresseurs sur la santé des abeilles, surtout lorsqu’ils sont multiples, est largement débattu. Dans le cadre de l’infection par Nosema, une microsporidie présente dans le tube digestif de l’abeille, de nombreuses études semblent indiquer que la multiplication des spores affecte l’épithélium de la paroi intestinale et donc stimule une réponse immunitaire épithéliale. Cependant, montrer que la réponse immunitaire systémique (visible au sein de l’hémolymphe) est bien stimulée, reste un cap à franchir particulièrement délicat. Le but de la thèse est de mieux comprendre les interactions qui se créent entre l’hôte (Apis mellifera) et le pathogène (Nosema spp.). Nous avons étudié pour cela le système immunitaire tissulaire (tube digestif) et circulant (hémolymphe) de l’hôte par des approches complémentaires de spectrométrie de masse (empreintes moléculaires massiques par MALDI BeeTyping®, approches de protéomique fine sur des segments de tube digestif et imagerie moléculaire par MALDI) dans un contexte d’infection contrôlée ou naturelle par des spores de Nosema spp. Nous avons mis en évidence de nouveaux marqueurs moléculaires de la santé de l’abeille à partir de l’hémolymphe d’abeilles infectées de manière précoce par Nosema et à partir du tube digestif de ces mêmes abeilles. De plus nous avons développé la méthode de MALDI Biotyping sur notre modèle de microsporidies pour identifier l’espèce à moindre coût. Ces différents éléments permettront le développement de nouveaux outils de diagnostic voir de pronostic pour assister l’apiculteur ou les services sanitaires dans le suivi de leur rucher.The loss of honeybee colonies could be explained by multiple stress factors (biotic or abiotic) that could act alone or in synergy to perturb the physiology of the domestic bees. The impact of these stressors on the bee health is widely discussed. In the miscrosporidiosis, an infection due to Nosema, the impact of this stressor on the systemic immune response (haemolymph being the mirror of this response) and the epithelial response (gut tissue as target) remains unsatisfactory documented. In the context of an infection by Nosema spp, the spore multiplication that is occurring at the level of the midgut stimulate the gut epithelial immune response. While at the same time, the stimulation of the systemic immune response remains an opened question. The aim of this thesis is to decipher the cross-talk existing between the host (A. mellifera) and the pathogen (Nosema spp.). We investigated the epithelial (gut tissues) and circulating bee immune response (haemolymph) using complementary mass spectrometry approaches (molecular mass fingerprints by MALDI BeeTyping®, untargeted proteomics and MALDI imaging) in a context of a controlled or natural infection by Nosema spores. We have demonstrated new molecular markers of bee health from hemolymph and bees early infected with Nosema from digestive tract. In addition, we have developed the MALDI Biotyping method on our microsporidia model to identify the species at a lower cost. These different elements constitute the development of new diagnostic and even prognostic tools to assist the beekeeper or the health services in their apiary monitoring

MALDI Biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema

International audienceHoney bees play a critical role in the maintenance of plant biodiversity and sustainability of food webs. In the past few decades, bees have been subjected to biotic and abiotic threats causing various colony disorders. Therefore, monitoring solutions to help beekeepers to improve bee health are necessary. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) profiling has emerged within this decade as a powerful tool to identify in routine microorganisms and is currently used in real-time clinical diagnosis. MALDI BeeTyping is developed to monitor significant hemolymph molecular changes in honey bees upon infection with a series of entomopathogenic Gram-positive and-negative bacteria. A Serratia marcescens strain isolated from one naturally infected honey bee collected from the field is also considered. A series of hemolymph molecular mass fingerprints is individually recorded and to the authors' knowledge, the first computational model harboring a predictive score of 97.92% and made of nine molecular signatures that discriminate and classify the honey bees' systemic response to the bacteria is built. Hence, the model is challenged by classifying a training set of hemolymphs and an overall recognition of 91.93% is obtained. Through this work, a novel, time and cost saving high-throughput strategy that addresses honey bee health on an individual scale is introduced

Molecular histoproteomy by MALDI mass spectrometry imaging to uncover markers of the impact of Nosema on Apis mellifera

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Proteomics of Anatomical Sections of the Gut of Nosema-Infected Western Honeybee (Apis mellifera) Reveals Different Early Responses to Nosema spp. Isolates.

International audienceHoneybees play an important role in pollinating native plants and agricultural crops and produce valuable hive products. Within the last decade, honeybee colonies have been reported to be in decline, due to both biotic and abiotic stress factors including pathogens and pesticides. This study evaluated the impact of different isolates of Nosema spp. [Nosema apis spores (NA), Nosema ceranae from Apis mellifera from France (NF), N. ceranae from Apis cerana from Thailand (NC1), and N. ceranae from A. mellifera from Thailand (NC2)] on the different gut sections of newly emerged adult A. mellifera bees. With an attempt to decipher the early impact of Nosema spp. on the first barrier against Nosema infection, we used off-gel bottom-up proteomics on the different anatomical sections of the gut four days post inoculation. A total of 2185 identified proteins in the esophagus, 2095 in the crop, 1571 in the midgut, 2552 in the ileum, and 3173 in the rectum were obtained. Using label-free quantification, we observed that the response of the host varies according to the Nosema spp. (N. apis versus N. ceranae) and the geographical origin of Nosema. The proteins in the midgut of A. mellifera, orally inoculated with spores of N. ceranae isolated from France, were the most altered, when compared with controls, exhibiting 50 proteins down-regulated and 16 up-regulated. We thereby established the first mass-spectrometry-based proteomics of different anatomical sections of the gut tissue of Nosema-infected A. mellifera four days post inoculation, following infection by different isolates of Nosema spp. that provoked differential host responses. We reported an alteration of proteins involved in the metabolic pathways and specifically eight proteins of the oxidative phosphorylation pathway. More importantly, we propose that the collagen IV NC1 domain-containing protein may represent an early prognostic marker of the impact of Nosema spores on the A. mellifera health status. Data are available via ProteomeXchange with the identifier PXD021848

Gamma irradiation-induced offspring masculinization is associated with epigenetic changes in female zebrafish

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Proteomics and Immune Response Differences in Apis mellifera and Apis cerana Inoculated with Three Nosema ceranae Isolates

International audienceNosema ceranae infects midgut epithelial cells of Apis species and jumped from its original host A. cerana to A. mellifera worldwide asking questions on how the new host is responding. We compared A. mellifera and A. cerana responses to N. ceranae isolates from A. cerana, A. mellifera from Thailand and A. mellifera from France. Proteomics and transcriptomics results were merged to deeper understand the impact on immunity of the two species. This represents the first combination of omics analysis to evaluate the impact of N. ceranae spores from different origin and provides new insights into the differential immune responses in honeybees inoculated by N. ceranae from original A. cerana. No difference in the antimicrobial peptides (AMP) was observed in A. mellifera, while these peptides were altered in A. cerana when compared to controls. Inoculation of A. mellifera or A. cerana with N. ceranae caused up-regulation of the AMP genes and cellular mediated immune genes but did not greatly alter apoptosis-related gene expression. A. cerana showed a stronger immune response than A. mellifera after different N. ceranae isolate inoculation. N. ceranae from A. cerana caused a highly negative impact on A. mellifera and A. cerana health compared to other Nosema isolates

Altered ovarian transcriptome is linked to early mortality and abnormalities in zebrafish embryos after maternal exposure to gamma irradiation

Recent laboratory studies focusing on multigenerational approach demonstrated drastic phenotypic effects after chronic fish irradiation exposure. No irradiation effect at phenotypic scale was observed for F0 (reproductive performances) while early mortality and malformations were observed in F1 offspring whether they were irradiated or not. The objective was to study molecular mechanisms likely to be involved in these phenotypic effects induced by parental irradiation. Thus, F0 adult zebrafish were irradiated for ten days until reproduction and maternal involvement in offspring development was assessed. Levels of maternal provided cortisol and vitellogenin, needed for embryo development, were not impacted by irradiation. However, maternal transcriptome highlighted irradiation effect on processes involved in oocyte development, as well as on essential maternal factors needed for offspring development. Therefore, this study highlighted the importance of parental exposure on offspring fate and of the importance of multigenerational exposure in risk assessment

IGFBP-rP1, a strongly conserved member of the androgenic hormone signalling pathway in Isopoda

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Circulating MicroRNAs Indicative of Sex and Stress in the European Seabass (Dicentrarchus labrax): Toward the Identification of New Biomarkers

MicroRNAs (miRNAs) constitute a new category of biomarkers. Studies on miRNAs in non-mammalian species have drastically increased in the last few years. Here, we explored the use of miRNAs as potential, poorly invasive markers, to identify sex and characterize acute stress in fish. The European seabass (Dicentrarchus labrax) was chosen as a model because of its rapid response to stress and its specific sex determination system, devoid of sexual chromosomes. We performed a small RNA-sequencing analysis in the blood plasma of male and female European seabass (mature and immature) as well as in the blood plasma of juveniles submitted to an acute stress and sampled throughout the recovery period (at 0 h, 0.5 h, 1.5 h and 6 h). In immature individuals, both miR-1388-3p and miR-7132a-5p were up-regulated in females, while miR-499a-5p was more abundant in males. However, no miRNAs were found to be differentially expressed between sexes in the blood plasma of mature individuals. For the acute stress analysis, five miRNAs (miR-155-5p, miR-200a-3p, miR-205-1-5p, miR-143-3p, and miR-223-3p) followed cortisol production over time. All miRNAs identified were tested and validated by RT-qPCR on sequenced samples. A complementary analysis on the 3′UTR sequences of the European seabass allowed to predict potential mRNA targets, some of them being particularly relevant regarding stress regulation, e.g., the glucocorticoid receptor 1 and the mineralocorticoid receptor. The present study provides new avenues and recommendations on the use of miRNAs as biomarkers of sex or stress of the European seabass, with potential application on other fish species