Essai d’accroissement de la résistance des glossines comme méthode de lutte antivectorielle contre les trypanosomoses africaines (THA et Nagana).

Abstract

Glossins (tsetse flies) are bloodsucking insects host and vectors of species/ subspecies of African trypanosome responsible for either Human African Trypanosomiasis (HAT) / sleeping sickness or African Animal Trypanosomiasis (AAT) / Nagana, sura, dourine... Of medical/veterinary and agricultural importance, these Diptera are found in 36 countries in subSaharan Africa and have serious repercussions both on human (HAT) and animal health (AAT) and severely affect the economy of the populations of these regions. In the absence of a vaccine, the control of the disease is based on diagnosis, chemotherapy and mainly on the control of insect vectors. Being a part of the control of vector populations, new biological control strategies aim to use as a biological agent Sodalis glossinidius (secondary glossin’s symbiont). This symbiont is transmitted to the progeny and has the particularity to be cultivated and genetically modified in order to deliver in situ anti-trypanosome molecules in the gut of glossins. Although the refractory nature of glossins is already high, it could be "supplemented" by para-transgenesis techniques based on an in-depth analysis of the molecular dialogue of glossin, symbiont and parasite interactions in order to block the chain transmission. This study was divided into two axes. The first axis aimed to investigate factors involved in vector competence by analysing whether the diversity and relative abundance of the different bacterial genera inhabiting the fly’s gut could be associated with its trypanosome infection status. Metabarcoding analysing of fly’s gut identified a total of 261 bacterial genera were identified of which only 114 crossed two barriers: A threshold of 0.01% relative abundance and the presence at least in 5 flies. The secondary symbiont Sodalis glossinidius was identified in 50% of the flies but it was not considered since its relative abundance was much lower than the 0.01% relative abundance threshold. The primary symbiont Wigglesworthia displayed 87% relative abundance, the remaining 13% were prominently constituted by the genera Spiroplasma, Tediphilus, Acinetobacter and Pseudomonas. Despite a large diversity in bacterial genera and in their abundance observed in microbiome composition, the statistical analyses of the 160 tsetse flies showed an association with flies’ infection status and the sampling sites. Furthermore, tsetse flies harbouring Trypanosoma congolense Savanah type displayed a different composition of bacterial flora compared to uninfected flies. In addition, our study revealed that 36 bacterial genera were present only in uninfected flies, which could therefore suggest a possible involvement in flies’ refractoriness; with the exception of Cupriavidus, they were however of low relative abundance. Some genera, including Acinetobacter, Cutibacterium, Pseudomonas and Tepidiphilus, although present both in infected and uninfected flies, were found to be associated with uninfected status of tsetse flies. Hence their effective role deserves to be further evaluated in order to determine whether some of them could become targets for tsetse control of fly vector competence and consequently for the control of the disease. The second axis consisted of in vivo functional analysis by para-transgenesis of cecropin and rTbgTCTP on the establishment of Trypanosoma brucei gambiense. In this study, we successfully cloned genes encoding our proteins of interest into Sodalis strains. These recombinant Sodalis (recSodalis) have been successfully micro-injected into the L3 larval stages of glossins. It should also be noted that the cloned genes were stable even after a 20th generation. In the case of glossins hosting recSodalis expressing cecropin, we observed a lower rate than the various controls (control without micro-injection and control micro-injected with physiological water) although this is not statistically significant. As for the glossins hosting the recSodalis expressing the rTbgTCTP, it turned out that this protein had a lethal action on the development of larvae; no protective effects were found in those that emerged. Our study is one of the first on protein assays that can modulate the installation of trypanosomes on Glossina palpalis. The results obtained are quite encouraging and open the way to other tests that may lead to the blocking of the transmission of trypanosomes by vectors.