Serap Aksoy

Tsetse flies ( Glossina spp.) transmit African trypanosomes, the causative agents of human African and African animal trypanosomiases (HAT and AAT, respectively). These neglected tropical diseases impose significant public health and economic burdens across sub-Saharan Africa. Trypanosome transmission by tsetse flies is influenced by multiple factors, including host genetic background, ecological factors, and interactions with heritable microbial endosymbionts. Spiroplasma glossinidia has recently emerged as an important modulator of tsetse reproductive fitness and vector competence, making it a potential target for symbiont-based vector control strategies. In this review, we summarize the current knowledge of the tsetse- Spiroplasma symbiosis. We detail Spiroplasma’s spatial and temporal infection dynamics in laboratory-reared and natural populations. Additionally, we highlight key aspects of the bacterium’s genomics, phylogenetics, and physiological interactions with its tsetse host, including influences on host gene expression reproductive physiology, and vector competence. Finally, we discuss how the tsetse– Spiroplasma symbiosis could be harnessed to develop innovative, biological-based vector control and trypanosome transmission-blocking strategies, and we identify critical gaps that must be addressed to translate these findings into effective disease control interventions.

Habitat fragmentation, climate change, poaching, human-wildlife conflicts, and infectious diseases are the main threats to biodiversity conservation. They alter host-pathogen dynamics, reduce viable conservation areas, and promote genetic isolation, resulting in physiological stress among animal populations. Moreover, increased proximity between domestic and wild animals further facilitates disease spillovers exposing naïve host species and ecosystems to new pathogens. Of the more than 200 known zoonotic diseases, approximately 60% originate from animals, contributing significantly to the global infectious disease burden. Here, we describe the development of an innovative non-invasive approach for biological sampling that has been validated in mice and shelter cats. Our device consists of a disposable plastic cassette that through odor attractants lures animals to lick a filter paper. This saliva collection approach allowed for the detection of RNA viruses by RTqPCR and third-generation sequencing. RTqPCR oral swab and licked paper results showed that both methods significantly predicted serological status. Our sequencing results revealed the richness of the gene space, demonstrating the potential of this device for discovering rare or unknown species circulating in the saliva donor, enabling this player to be recognized as an environmental sentinel. This study demonstrates the feasibility of deploying this device in sheltered/captive animal settings as well as under laboratory simulations of different environments, providing necessary foundations for future field applications. Our methodology holds great potential for monitoring zoonotic pathogens in both captive and free-ranging animals, to even possibly allow proactive mitigation measures prior to spillover, without interfering with the natural animal behaviour and social structures.

Peptidoglycan Recognition Proteins (PGRPs) are conserved proteins regulating immune responses to the peptidoglycan microbe-associated molecular patterns (MAMPs). Compared to other dipterans, the tsetse fly ( Glossina morsitans morsitans ) genome encodes only five PGRPs- PGRP-LA, -LB, -LC, -SA, and -SB – far fewer than most dipterans, likely reflecting its sterile blood diet and streamlined microbiota. Here, we identify PGRP-LA as a critical regulator of peritrophic matrix (PM) integrity in the cardia (proventriculus), the tissue responsible for PM production. The PM is a chitinous sleeve-like barrier that separates the midgut epithelium from the ingested bloodmeal, supporting digestive homeostasis and infection resistance. We show that pgrp-la is prominently expressed in the cardia, transiently induced after a bloodmeal in newly eclosed flies, and reinduced following subsequent feedings, likely in response to blood- constituents or mechanical stretch. This induction is sustained during microbial exposure and prolonged in trypanosome-infected flies. RNAi-mediated reduction of pgrp-la significantly increased the prevalence of midgut trypanosome infections, indicating a protective role during early infection. PGRP-LA did not mediate infection resistance via canonical IMD pathway signaling, as its silencing did not affect antimicrobial peptide expression. Instead, PGRP-LA modulated the expression of PM-associated genes and gut barrier integrity. Silencing pgrp-la reduced PM structure, increased midgut weights and enhanced fly survival following oral challenge with entomopathogen Serratia marcescens , likely due to earlier epithelial immune responses through a compromised PM. Similar phenotypes were observed when flies were fed anti-PGRP-LA antibodies, supporting a structural role for PGRP-LA. In addition, soluble variant surface glycoproteins (sVSGs) from trypanosomes and knockdown of microRNA-275 (miR-275 ) , also decreased pgrp-la expression, suggesting that PGRP-LA is part of a broader regulatory network, including the miR-275/Wingless signaling. Collectively, our results identify PGRP-LA as novel regulator of PM biogenesis and vector competence in tsetse, expanding the functional repertoire of PGRPs in insect gut barrier maintenance beyond canonical immune signaling pathways.

Glossina fuscipes fuscipes is a riverine tsetse fly species, the primary vector of human and animal trypanosomiasis in Sub-Saharan Africa. Controlling tsetse fly populations is crucial for mitigating the socio-economic impact of this disease, as effective treatments remain challenging. Indeed, the development of control strategies is hindered by the species’ unique reproductive biology: adenotrophic viviparity, in which the female retains and nourishes the developing larva in her uterus throughout the pregnancy. The discovery of Spiroplasma in some G. f. fuscipes populations has drawn attention as a potential tool to enhance tsetse fly control strategies. Although Spiroplasma does not exhibit in G. f. fuscipes the male-killing phenotype observed in Drosophila melanogaster , evidence suggests that it may confer refractoriness to Trypanosoma infection. This has led to further investigations into its broader effects on G. f. fuscipes biology, particularly its potential impact on Glossina reproductive fitness. In this study, we considered Spiroplasma effect on the male reproduction. For this, we performed a differential gene expression analysis on testes and male accessory glands (MAGs) between Spiroplasma -infected and uninfected males. A significant downregulation of genes was observed in testes while a minor effect has been detected on MAGs. Downregulation of testes genes associated with functions related to sperm motility, energy metabolism, and mitochondrial function has been observed. Additionally, differentially expressed genes involved in antimicrobial activity and circadian rhythm regulation were observed. These findings provide valuable insights into the potential fitness costs of Spiroplasma infection for the fly and its implications for the bacterium use as biological control strategies targeting G. f. fuscipes .

Peptidoglycan Recognition Proteins (PGRPs) are conserved pattern-recognition receptors that detect microbe-associated molecular patterns (MAMPs) and activate host immune responses. Compared to other dipterans, the tsetse fly ( Glossina morsitans morsitans ) genome encodes only five PGRPs-PGRP-LA, -LB, -LC, -SA, and -SB – far fewer than most dipterans, likely reflecting its sterile blood diet and streamlined microbiota. Here, we identify PGRP-LA as a critical regulator of peritrophic matrix (PM) integrity in the cardia (proventriculus), the tissue responsible for PM production. The PM is a chitinous sleeve-like barrier that separates the midgut epithelium from the ingested bloodmeal, supporting digestive homeostasis and infection resistance. We show that pgrp-la is prominently expressed in the cardia, transiently induced after a bloodmeal in newly eclosed flies, and reinduced following subsequent feedings, likely in response to blood-constituents or mechanical stretch. This induction is sustained during microbial exposure and prolonged in trypanosome-infected flies. RNAi-mediated reduction of pgrp-la significantly increased the prevalence of midgut trypanosome infections, indicating a protective role during early infection. PGRP-LA did not mediate infection resistance via canonical IMD pathway signaling, as its silencing did not affect antimicrobial peptide expression. Instead, PGRP-LA modulated the expression of PM-associated genes and gut barrier integrity. Silencing pgrp-la reduced PM structure, increased midgut weights and enhanced fly survival following oral challenge with entomopathogen Serratia marcescens , likely due to earlier epithelial immune responses through a compromised PM. Similar phenotypes were observed when flies were fed anti-PGRP-LA antibodies, supporting a structural role for PGRP-LA. In addition, soluble variant surface glycoproteins (sVSGs) from trypanosomes and knockdown of microRNA-275 (miR-275 ) , also decreased pgrp-la expression, suggesting that PGRP-LA is part of a broader regulatory network, including the miR-275/Wingless signaling. Collectively, our results identify PGRP-LA as novel regulator of PM biogenesis and vector competence in tsetse, expanding the functional repertoire of PGRPs in insect gut barrier maintenance beyond canonical immune signaling pathways. Author Summary: Insect vectors such as tsetse flies can be infected with pathogens that cause devastating disease in mammals. To protect themselves insect vectors rely on pattern recognition receptors (PRRs) that detect pathogens and activate the production of antimicrobial peptides (AMPs). Physical barriers in the gut also play an important role in limiting infections. One such barrier is the peritrophic matrix (PM), a sleeve-like structure that lines the insect gut and separates the blood meal and its contents from the underlying cells. For trypanosome parasites, which cause sleeping sickness in humans, the PM is the first barrier they must traverse to colonize the tsetse’s gut. In this study, we identified a PRR, PGRP-LA, that, unlike related proteins in other insects that activate AMPs, regulates the integrity of the tsetse’s protective PM barrier. When PGRP-LA was disrupted, the gut barrier weakened, and flies became more susceptible to trypanosome infection. Our work highlights a previously unrecognized role for PGRP-LA in maintaining gut barrier integrity and suggest that targeting this pathway could be a strategy to help reduce parasite transmission.

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 gut tissue along with functional assays. Our findings reveal elevated oxidative stress in the gut 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 Gff’s midgut significantly upregulated various immunity-related genes, including a small peptide, Stomoxyn-like, homologous to Stomoxyn 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 tsetse’s 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 gut 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.

The protozoan parasite Trypanosoma brucei evades clearance by the host immune system through antigenic variation of its dense variant surface glycoprotein (VSG) coat, periodically ‘switching’ expression of the VSG using a large genomic repertoire of VSG-encoding genes^1–6. Recent studies of antigenic variation in vivo have focused near exclusively on parasites in the bloodstream^6–8, but research has shown that many, if not most, parasites reside in the interstitial spaces of tissues^9–13. We sought to explore the dynamics of antigenic variation in extravascular parasite populations using VSG-seq⁷, a high-throughput sequencing approach for profiling VSGs expressed in populations of T. brucei. Here we show that tissues, not the blood, are the primary reservoir of antigenic diversity during both needle- and tsetse bite-initiated T. brucei infections, with more than 75% of VSGs found exclusively within extravascular spaces. We found that this increased diversity is correlated with slower parasite clearance in tissue spaces. Together, these data support a model in which the slower immune response in extravascular spaces provides more time to generate the antigenic diversity needed to maintain a chronic infection. Our findings reveal the important role that extravascular spaces can have in pathogen diversification.

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 Gff’s 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. Gff Stomoxyn is constitutively expressed in the cardia (proventriculus) and synthetic Gff Stomoxyn exhibits potent activity against Escherichia coli and bloodstream form of Trypanosoma brucei parasites, while showing no effect against insect stage procyclic forms or tsetse’s commensal endosymbiont Sodalis in vitro . Reducing Gff Stomoxyn 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, Gff Stomoxyn 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 Gff Stomoxyn, 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 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 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.