Spiroplasmaendosymbiont reduction of host lipid synthesis and Stomoxyn-like peptide contribute to trypanosome resistance in the tsetse flyGlossina fuscipes
Authors: Awuoche Erick , Smallenberger Gretchen , Bruzzese Daniel , Orfano Alessandra , Weiss Brian L. , Aksoy Serap 
Published: October 2024

Tsetse flies (Glossinaspp.) vector African trypanosomes that cause devastating diseases in humans and domestic animals. Within theGlossinagenus, species in the Palpalis subgroup exhibit greater resistance to trypanosome infections compared to those in theMorsitanssubgroup. Varying microbiota composition and species-specific genetic traits can significantly influence the efficiency of parasite transmission. Notably, infections with the endosymbiotic bacteriumSpiroplasmahave been documented in several Palpalis subgroup species, includingGlossina fuscipes fuscipes(Gff). WhileSpiroplasmainfections inGffare known to hinder trypanosome transmission, the underlying mechanisms remain unknown. To investigateSpiroplasma-mediated factors affectingGffvector 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 ofSpiroplasma, evidenced by increased expression ofnitric 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 inGff’smidgut significantly upregulated various immunity-related genes, including a small peptide,Stomoxyn-like, homologous to Stomoxyns first discovered in the stable flyStomoxys calcitrans. We observed that theStomoxyn-likelocus is exclusive to the genomes ofPalpalissubgroup tsetse species.GffStomoxyn is constitutively expressed in the cardia (proventriculus) and syntheticGffStomoxyn exhibits potent activity againstEscherichia coliand bloodstream form ofTrypanosoma bruceiparasites, while showing no effect against insect stage procyclic forms or tsetse’s commensal endosymbiontSodalis in vitro. ReducingGffStomoxyn levels significantly increased trypanosome infection prevalence, indicating its potential trypanocidal rolein vivo. Collectively, our results suggest that the enhanced resistance to trypanosomes observed inSpiroplasma-infectedGffmay 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 ofGffStomoxyn, its spatial and temporal expression regulation and its microbicidal activity againstTrypanosomeparasites. Our findings reinforce the nutritional influences of microbiota on host physiology and host-pathogen dynamics.

Author Summary

The 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 bacteriumSpiroplasma. This study investigated how the bacteriumSpiroplasmainsideGffenables 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 inGffand relatedPalpalistsetse species. We have shown thatGffsynthetic Stomoxyn has antibacterial and antitrypanosomal properties and lowering Stomoxyn levels inGffcorrelates with increased parasite prevalence. We suggest that strategies to increaseSpiroplasmaprevalence or enhance stomoxyn expression through paratransgenic approaches could be promising avenues for reducing trypanosomiasis transmission.