Spiroplasma endosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse fly Glossina fuscipes

Tsetse flies (Glossina spp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within the Glossina genus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in the Morsitans subgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacterium Spiroplasma have been documented in several Palpalis subgroup species, including Glossina fuscipes fuscipes (Gff). While Spiroplasma infections in Gff are known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigate Spiroplasma-mediated factors affecting Gff vector competence, we conducted high-throughput RNA sequencing of the midgut tissue along with functional assays. Our findings reveal elevated oxidative stress in the midgut environment in the presence of Spiroplasma, evidenced by increased expression of nitric oxide synthase, which catalyzes the production of trypanocidal nitric oxide. Additionally, we observed impaired lipid biosynthesis leading to a reduction of this important class of nutrients essential for parasite and host physiologies. In contrast, trypanosome infections in Gffs midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyns first discovered in the stable fly Stomoxys calcitrans. We observed that the Stomoxyn-like locus is exclusive to the genomes of Palpalis subgroup tsetse species. GffStomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic GffStomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetses commensal endosymbiont Sodalis in vitro. Reducing GffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal role in vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed in Spiroplasma-infected Gff may be due to the reduced lipid availability necessary for parasite metabolic maintenance. Furthermore, GffStomoxyn could play a crucial role in the initial immune response(s) against mammalian parasites early in the infection process in the midgut and prevent gut colonization. We discuss the molecular characteristics of GffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity against Trypanosome parasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics. Author SummaryThe tsetse fly, Glossina fuscipes fuscipes (Gff) is of high public health relevance. Gff exhibits strong innate resistance to trypanosomes, especially when infected with the endosymbiotic bacterium Spiroplasma. This study investigated how the bacterium Spiroplasma inside Gff enables them to be resistant to trypanosome infection. Our results indicate alterations in host lipid metabolism with reduction in levels of triglycerides, suggesting a potential metabolic barrier that limits the viability to parasite. In addition, we discovered a small peptide, stomoxyn, exclusively in Gff and related Palpalis tsetse species. We have shown that Gff synthetic Stomoxyn has antibacterial and antitrypanosomal properties and lowering Stomoxyn levels in Gff correlates with increased parasite prevalence. We suggest that strategies to increase Spiroplasma prevalence or enhance stomoxyn expression through paratransgenic approaches could be promising avenues for reducing trypanosomiasis transmission.