| Titre : | Implication of an odorant-binding protein in precopulatory behaviour and interaction with the bacterial microbiota |
| Titre original: | Implication d'une protéine de liaison aux odorants dans le comportement précopulatoire et l'interaction avec le microbiote bactérien |
| Auteurs : | E. Aruçi, - Auteur ; INRAE Centre Bourgogne Franche-Comté (FRA) Centre des Sciences du Goût et de l'Alimentation (Dijon, FRA), - Auteur ; Université de Bourgogne Franche Comté, Ecole doctorale Environnements, Santé (Dijon, FRA), Organisme de soutenance |
| Type de document : | thèse/mémoire |
| Année de publication : | 2023 |
| Format : | 217 p. |
| Langues: | = Anglais |
| Mots-clés: | DROSOPHILA ; CHEMORECEPTION ; OBP ; GUSTATORY SYSTEM ; GUT MICROBIOTA ; COURTSHIP BEHAVIOUR |
| Résumé : |
Chemoreception allows animals to detect nutritive food and avoid toxic compounds. Volatile and non-volatile chemical compounds, which are detected by olfactory and gustatory sensory organs, can trigger feeding and reproductive behaviours in animals such as Drosophila melanogaster. Inside chemosensory organs, perireceptor proteins like odorant-binding proteins (OBPs) serve to transport odorant and tastant molecule to dedicated receptors. OBPs are not only involved in chemoreception but also in several other functions. A recent study revealed an interaction between insect microbiota and OBP expression while another one showed that microbes can promote nutrient harvesting on food.
In our project, we used Drosophila melanogaster as a model organism, to find a link between the presence of bacteria, the expression of OBPs and behavioural performance of flies. More particularly, we investigated the expression of OBP56d and OBP56g, both present in the adult chemosensory system and in the gut, and we measured their influence on reproductive-related behaviours. We used both in vitro and in vivo approaches to characterize several aspects pertaining to OBP expression in adult gustatory appendages, gut and reproductive organs. The expression of OBP56g and OBP56d was measured and compared in the thorax, head, and gut of virgin females and males and of mated females subjected to fasting of different durations (0h, 2h, 16h, and 30h). Beside the weak effect of fasting duration, our RT-qPCR analysis showed that the two OBPs are expressed in the male gut with OBP56d showing a higher expression than OBP56g which, in turn, was more expressed in the head of females than males. A focus on OBP56d expression revealed a particularly strong expression in the male hindgut and in the proboscis of both sexes. To test the effect of bacterial microbiota on OBP56d and OBP56g expression, our first experiment consisted to raise a control D.melanogaster strain, during two generations on an antibiotic-rich diet. This manipulation eliminated most of the bacteria (including the endosymbiotic Wolbachia bacteria) and induced decreased OBP56d expression in the male gut, and decreased OBP56g expression in the female head. Then, in the axenic food, we added separately two bacteria species normally present in flies, or grinded guts from control females or control males. The quantification of OBP expression resulting of these four manipulations showed few significant variations which were more difficult to interpret than those shown by flies only raised on axenic diet. With the help of an external collaboration, we also sequenced the microbiome to compare control, axenic and OBP56d- mutant female and male flies. Some differences were found, specially between these three treatments. However, the presence of Wolbachia introduced a strong bias in the interpretation of these differences. Both OBPs were cloned, produced with a bacterial heterologous expression system and purified. Their binding properties were studied with a competitive fluorescence binding assay. Among many compounds tested OBP56d, and to a lesser extend OBP56g, showed a strong affinity for long chain fatty acids with no clear preference according to the presence and number of unsaturation(s). The use of in situ fluorescent probes confirmed the expression of OBP56d in the male hindgut. Moreover, we discovered OBP56d expression in the testis, and this led us to test mating behaviour. More particularly, we tested copulation in pairs of flies combining control and OBP56d- mutant flies. The OBP56d- mutation strongly decreased female copulation in experiments lasting 2 hours but not in those lasting 12 hours indicating that the OBP56d- mutation was likely slowing down female capacity to detect or to interpret male stimulating pheromone(s) whose identity remains still unknown. However, the OBP56d- mutation did not affect fly fertility and fecundity. Overall, our PhD project provides novel insights on the roles of OBPs in precopulatory behaviour and some hints on the impact of microbiota on their expression. |
| Diplôme : | Thèse (Dr. d'Université), option Biochimie, Biologie Moléculaire |
| En ligne : | https://tel.archives-ouvertes.fr/tel-03989045 |
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