Sodalis glossinidius and Wolbachia infections in wild population of Glossina morsitans submorsitans caught in the area of Lake Iro in the south of Chad.

Investigations on the bacterial fauna and their association with trypanosome infections in tsetse fly have revealed contrasting results. This study aimed to detect Wolbachia and S. glossinidius in wild populations of G. m. submorsistans and subsequently, understand the influence that these bacteria may have on the vectorial competence of this tsetse species. Tsetse flies were captured in the area of Lake Iro in the south of Chad using biconical traps. After DNA extraction from each tsetse fly, Sodalis glossinidius and Wolbachia were detected using specific primers. Sodalis glossinidius and Wolbachia infection rates were compared and association studies involving trypanosome infections and S. glossinidius or Wolbachia were performed. From 345 G. m. submorsitans analyzed, 9.0% and 14.5% were respectively infected with S. glossinidius and Wolbachia. Only 2.31% of all tsetse flies were co-infected by the 2 bacteria. Of all trypanosome-infected flies, 7.1% and 9.8% harbored, respectively, S. glossinidius and Wolbachia. No association was observed between Wolbachia and trypanosome infections while a significant association (r = 4.992; P = 0.025) was found between S. glossinidius and the presence of trypanosomes. A significant association (r = 3.147; P = 0.043) was also observed between S. glossinidius and T. simiae; and none with T. congolense or T. godfreyi. This study revealed S. glossinidius and Wolbachia in G. m. submorsitans of the area of lake Iro. It showed that co-infections between Wolbachia and S. glossinidius are rare in wild populations of G. m. submorsitans and that the tripartite associations vary according to trypanosome species as well as symbiotic mricroorganisms.

Overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors.

This article presents an overview of paratransgenesis as a strategy to control pathogen transmission by insect vectors. It first briefly summarises some of the disease-causing pathogens vectored by insects and emphasises the need for innovative control methods to counter the threat of resistance by both the vector insect to pesticides and the pathogens to therapeutic drugs. Subsequently, the state of art of paratransgenesis is described, which is a particularly ingenious method currently under development in many important vector insects that could provide an additional powerful tool for use in integrated pest control programmes. The requirements and recent advances of the paratransgenesis technique are detailed and an overview is given of the microorganisms selected for genetic modification, the effector molecules to be expressed and the environmental spread of the transgenic bacteria into wild insect populations. The results of experimental models of paratransgenesis developed with triatomines, mosquitoes, sandflies and tsetse flies are analysed. Finally, the regulatory and safety rules to be satisfied for the successful environmental release of the genetically engineered organisms produced in paratransgenesis are considered.

Prevalence of Trypanosoma and Sodalis in wild populations of tsetse flies and their impact on sterile insect technique programmes for tsetse eradication.

The sterile insect technique (SIT) is an environment friendly and sustainable method to manage insect pests of economic importance through successive releases of sterile irradiated males of the targeted species to a defined area. A mating of a sterile male with a virgin wild female will result in no offspring, and ultimately lead to the suppression or eradication of the targeted population. Tsetse flies, vectors of African Trypanosoma, have a highly regulated and defined microbial fauna composed of three bacterial symbionts that may have a role to play in the establishment of Trypanosoma infections in the flies and hence, may influence the vectorial competence of the released sterile males. Sodalis bacteria seem to interact with Trypanosoma infection in tsetse flies. Field-caught tsetse flies of ten different taxa and from 15 countries were screened using PCR to detect the presence of Sodalis and Trypanosoma species and analyse their interaction. The results indicate that the prevalence of Sodalis and Trypanosoma varied with country and tsetse species. Trypanosome prevalence was higher in east, central and southern African countries than in west African countries. Tsetse fly infection rates with Trypanosoma vivax and T. brucei sspp were higher in west African countries, whereas tsetse infection with T. congolense and T. simiae, T. simiae (tsavo) and T. godfreyi were higher in east, central and south African countries. Sodalis prevalence was high in Glossina morsitans morsitans and G. pallidipes but absent in G. tachinoides. Double and triple infections with Trypanosoma taxa and coinfection of Sodalis and Trypanosoma were rarely observed but it occurs in some taxa and locations. A significant Chi square value (< 0.05) seems to suggest that Sodalis and Trypanosoma infection correlate in G. palpalis gambiensis, G. pallidipes and G. medicorum. Trypanosoma infection seemed significantly associated with an increased density of Sodalis in wild G. m. morsitans and G. pallidipes flies, however, there was no significant impact of Sodalis infection on trypanosome density.

Infection with endosymbiotic Spiroplasma disrupts tsetse (Glossina fuscipes fuscipes) metabolic and reproductive homeostasis.

Tsetse flies (Glossina spp.) house a population-dependent assortment of microorganisms that can include pathogenic African trypanosomes and maternally transmitted endosymbiotic bacteria, the latter of which mediate numerous aspects of their host's metabolic, reproductive, and immune physiologies. One of these endosymbionts, Spiroplasma, was recently discovered to reside within multiple tissues of field captured and laboratory colonized tsetse flies grouped in the Palpalis subgenera. In various arthropods, Spiroplasma induces reproductive abnormalities and pathogen protective phenotypes. In tsetse, Spiroplasma infections also induce a protective phenotype by enhancing the fly's resistance to infection with trypanosomes. However, the potential impact of Spiroplasma on tsetse's viviparous reproductive physiology remains unknown. Herein we employed high-throughput RNA sequencing and laboratory-based functional assays to better characterize the association between Spiroplasma and the metabolic and reproductive physiologies of G. fuscipes fuscipes (Gff), a prominent vector of human disease. Using field-captured Gff, we discovered that Spiroplasma infection induces changes of sex-biased gene expression in reproductive tissues that may be critical for tsetse's reproductive fitness. Using a Gff lab line composed of individuals heterogeneously infected with Spiroplasma, we observed that the bacterium and tsetse host compete for finite nutrients, which negatively impact female fecundity by increasing the length of intrauterine larval development. Additionally, we found that when males are infected with Spiroplasma, the motility of their sperm is compromised following transfer to the female spermatheca. As such, Spiroplasma infections appear to adversely impact male reproductive fitness by decreasing the competitiveness of their sperm. Finally, we determined that the bacterium is maternally transmitted to intrauterine larva at a high frequency, while paternal transmission was also noted in a small number of matings. Taken together, our findings indicate that Spiroplasma exerts a negative impact on tsetse fecundity, an outcome that could be exploited for reducing tsetse population size and thus disease transmission.

Modulation of trypanosome establishment in Glossina palpalis palpalis by its microbiome in the Campo sleeping sickness focus, Cameroon.

The purpose of this study was to investigate factors involved in vector competence by analyzing whether the diversity and relative abundance of the different bacterial genera inhabiting the fly's gut could be associated with its trypanosome infection status. This was investigated on 160 randomly selected G. p. palpalis flies - 80 trypanosome-infected, 80 uninfected - collected in 5 villages of the Campo trypanosomiasis focus in South Cameroon. Trypanosome species were identified using specific primers, and the V4 region of the 16S rRNA gene of bacteria was targeted for metabarcoding analysis in order to identify the bacteria and determine microbiome composition. 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 micobiome composition, the statistical analyzes of the 160 tsetse flies showed an association with flies' infection status and the sampling sites. Furthermore, tsetse flies harboring 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. Finally, when comparing the bacterial genera identified in tsetse flies collected during 4 epidemiological surveys, 39 genera were found to be common to flies from at least 2 sampling campaigns.

Bacterial Symbionts of Tsetse Flies: Relationships and Functional Interactions Between Tsetse Flies and Their Symbionts.

Tsetse flies (Glossina spp.) act as the sole vectors of the African trypanosome species that cause Human African Trypanosomiasis (HAT or African Sleeping Sickness) and Nagana in animals. These flies have undergone a variety of specializations during their evolution including an exclusive diet consisting solely of vertebrate blood for both sexes as well as an obligate viviparous reproductive biology. Alongside these adaptations, Glossina species have developed intricate relationships with specific microbes ranging from mutualistic to parasitic. These relationships provide fundamental support required to sustain the specializations associated with tsetse's biology. This chapter provides an overview on the knowledge to date regarding the biology behind these relationships and focuses primarily on four bacterial species that are consistently associated with Glossina species. Here their interactions with the host are reviewed at the morphological, biochemical and genetic levels. This includes: the obligate symbiont Wigglesworthia, which is found in all tsetse species and is essential for nutritional supplementation to the blood-specific diet, immune system maturation and facilitation of viviparous reproduction; the commensal symbiont Sodalis, which is a frequently associated symbiont optimized for survival within the fly via nutritional adaptation, vertical transmission through mating and may alter vectorial capacity of Glossina for trypanosomes; the parasitic symbiont Wolbachia, which can manipulate Glossina via cytoplasmic incompatibility and shows unique interactions at the genetic level via horizontal transmission of its genetic material into the genome in two Glossina species; finally, knowledge on recently observed relations between Spiroplasma and Glossina is explored and potential interactions are discussed based on knowledge of interactions between this bacterial Genera and other insect species. These flies have a simple microbiome relative to that of other insects. However, these relationships are deep, well-studied and provide a window into the complexity and function of host/symbiont interactions in an important disease vector.

Conjugal DNA Transfer in Sodalis glossinidius, a Maternally Inherited Symbiont of Tsetse Flies.

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis-a zoonotic disease that incurs a high socioeconomic cost in regions of endemicity. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here, we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies.IMPORTANCE Tsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness-a zoonotic disease that inflicts a substantial economic cost on a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies, allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here, we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.

Quorum sensing sets the stage for the establishment and vertical transmission of Sodalis praecaptivus in tsetse flies.

Bacterial virulence factors facilitate host colonization and set the stage for the evolution of parasitic and mutualistic interactions. The Sodalis-allied clade of bacteria exhibit striking diversity in the range of both plant and animal feeding insects they inhabit, suggesting the appropriation of universal molecular mechanisms that facilitate establishment. Here, we report on the infection of the tsetse fly by free-living Sodalis praecaptivus, a close relative of many Sodalis-allied symbionts. Key genes involved in quorum sensing, including the homoserine lactone synthase (ypeI) and response regulators (yenR and ypeR) are integral for the benign colonization of S. praecaptivus. Mutants lacking ypeI, yenR and ypeR compromised tsetse survival as a consequence of their inability to repress virulence. Genes under quorum sensing, including homologs of the binary insecticidal toxin PirAB and a putative symbiosis-promoting factor CpmAJ, demonstrated negative and positive impacts, respectively, on tsetse survival. Taken together with results obtained from experiments involving weevils, this work shows that quorum sensing virulence suppression plays an integral role in facilitating the establishment of Sodalis-allied symbionts in diverse insect hosts. This knowledge contributes to the understanding of the early evolutionary steps involved in the formation of insect-bacterial symbiosis. Further, despite having no established history of interaction with tsetse, S. praecaptivus can infect reproductive tissues, enabling vertical transmission through adenotrophic viviparity within a single host generation. This creates an option for the use of S. praecaptivus in the biocontrol of insect disease vectors via paratransgenesis.

Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the genus Sodalis.

Insect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living species of the genus Sodalis, Sodalis praecaptivus, provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus and empirically verify its predictions. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken from this starting point after becoming internalized. The order in which key genes are lost is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides a detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

Trypanosoma brucei colonizes the tsetse gut via an immature peritrophic matrix in the proventriculus.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2-4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies.

Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.

Large-scale and significant expression from pseudogenes in Sodalis glossinidius - a facultative bacterial endosymbiont.

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50 % pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple 'omic' strategies, combining Illumina and Pacific Biosciences Single-Molecule Real-Time DNA sequencing and annotation, stranded RNA sequencing and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53 and 74 % of the Sodalis transcriptome remains active in cell-free culture. The mean sense transcription from coding domain sequences (CDSs) is four times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40 % of the 2729 genes in the core genome, suggesting that they are stable and/or that Sodalis is a recent introduction across the genus Glossina as a facultative symbiont. These data shed further light on the importance of transcriptional and translational control in deciphering host-microbe interactions. The combination of genomics, transcriptomics and proteomics gives a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

Thermal stress responses of Sodalis glossinidius, an indigenous bacterial symbiont of hematophagous tsetse flies.

Tsetse flies (Diptera: Glossinidae) house a taxonomically diverse microbiota that includes environmentally acquired bacteria, maternally transmitted symbiotic bacteria, and pathogenic African trypanosomes. Sodalis glossinidius, which is a facultative symbiont that resides intra and extracellularly within multiple tsetse tissues, has been implicated as a mediator of trypanosome infection establishment in the fly's gut. Tsetse's gut-associated population of Sodalis are subjected to marked temperature fluctuations each time their ectothermic fly host imbibes vertebrate blood. The molecular mechanisms that Sodalis employs to deal with this heat stress are unknown. In this study, we examined the thermal tolerance and heat shock response of Sodalis. When grown on BHI agar plates, the bacterium exhibited the most prolific growth at 25oC, and did not grow at temperatures above 30oC. Growth on BHI agar plates at 31°C was dependent on either the addition of blood to the agar or reduction in oxygen levels. Sodalis was viable in liquid cultures for 24 hours at 30oC, but began to die upon further exposure. The rate of death increased with increased temperature. Similarly, Sodalis was able to survive for 48 hours within tsetse flies housed at 30oC, while a higher temperature (37oC) was lethal. Sodalis' genome contains homologues of the heat shock chaperone protein-encoding genes dnaK, dnaJ, and grpE, and their expression was up-regulated in thermally stressed Sodalis, both in vitro and in vivo within tsetse fly midguts. Arrested growth of E. coli dnaK, dnaJ, or grpE mutants under thermal stress was reversed when the cells were transformed with a low copy plasmid that encoded the Sodalis homologues of these genes. The information contained in this study provides insight into how arthropod vector enteric commensals, many of which mediate their host's ability to transmit pathogens, mitigate heat shock associated with the ingestion of a blood meal.

Spatio-temporal distribution of Spiroplasma infections in the tsetse fly (Glossina fuscipes fuscipes) in northern Uganda.

Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.

Mutualist-Provisioned Resources Impact Vector Competency.

Many symbionts supplement their host's diet with essential nutrients. However, whether these nutrients also enhance parasitism is unknown. In this study, we investigated whether folate (vitamin B9) production by the tsetse fly (Glossina spp.) essential mutualist, Wigglesworthia, aids auxotrophic African trypanosomes in completing their life cycle within this obligate vector. We show that the expression of Wigglesworthia folate biosynthesis genes changes with the progression of trypanosome infection within tsetse. The disruption of Wigglesworthia folate production caused a reduction in the percentage of flies that housed midgut (MG) trypanosome infections. However, decreased folate did not prevent MG trypanosomes from migrating to and establishing an infection in the fly's salivary glands, thus suggesting that nutrient requirements vary throughout the trypanosome life cycle. We further substantiated that trypanosomes rely on symbiont-generated folate by feeding this vitamin to Glossina brevipalpis, which exhibits low trypanosome vector competency and houses Wigglesworthia incapable of producing folate. Folate-supplemented G. brevipalpis flies were significantly more susceptible to trypanosome infection, further demonstrating that this vitamin facilitates parasite infection establishment. Our cumulative results provide evidence that Wigglesworthia provides a key metabolite (folate) that is "hijacked" by trypanosomes to enhance their infectivity, thus indirectly impacting tsetse species vector competency. Parasite dependence on symbiont-derived micronutrients, which likely also occurs in other arthropod vectors, represents a relationship that may be exploited to reduce disease transmission.IMPORTANCE Parasites elicit several physiological changes in their host to enhance transmission. Little is known about the functional association between parasitism and microbiota-provisioned resources typically dedicated to animal hosts and how these goods may be rerouted to optimize parasite development. This study is the first to identify a specific symbiont-generated metabolite that impacts insect vector competence by facilitating parasite establishment and, thus, eventual transmission. Specifically, we demonstrate that the tsetse fly obligate mutualist Wigglesworthia provisions folate (vitamin B9) that pathogenic African trypanosomes exploit in an effort to successfully establish an infection in the vector's MG. This process is essential for the parasite to complete its life cycle and be transmitted to a new vertebrate host. Disrupting metabolic contributions provided by the microbiota of arthropod disease vectors may fuel future innovative control strategies while also offering minimal nontarget effects.

Innate immunity in the tsetse fly (Glossina), vector of African trypanosomes.

Tsetse flies (Glossina sp.) are medically and veterinary important vectors of African trypanosomes, protozoan parasites that cause devastating diseases in humans and livestock in sub-Saharan Africa. These flies feed exclusively on vertebrate blood and harbor a limited diversity of obligate and facultative bacterial commensals. They have a well-developed innate immune system that plays a key role in protecting the fly against invading pathogens and in modulating the fly's ability to transmit African trypanosomes. In this review, we briefly summarize our current knowledge on the tsetse fly innate immune system and its interaction with the bacterial commensals and the trypanosome parasite.

The composition and abundance of bacterial communities residing in the gut of Glossina palpalis palpalis captured in two sites of southern Cameroon.

BACKGROUND: A number of reports have demonstrated the role of insect bacterial flora on their host's physiology and metabolism. The tsetse host and vector of trypanosomes responsible for human sleeping sickness (human African trypanosomiasis, HAT) and nagana in animals (African animal trypanosomiasis, AAT) carry bacteria that influence its diet and immune processes. However, the mechanisms involved in these processes remain poorly documented. This underscores the need for increased research into the bacterial flora composition and structure of tsetse flies. The aim of this study was to identify the diversity and relative abundance of bacterial genera in Glossina palpalis palpalis flies collected in two trypanosomiasis foci in Cameroon. METHODS: Samples of G. p. palpalis which were either negative or naturally trypanosome-positive were collected in two foci located in southern Cameroon (Campo and Bipindi). Using the V3V4 and V4 variable regions of the small subunit of the 16S ribosomal RNA gene, we analyzed the respective bacteriome of the flies' midguts. RESULTS: We identified ten bacterial genera. In addition, we observed that the relative abundance of the obligate endosymbiont Wigglesworthia was highly prominent (around 99%), regardless of the analyzed region. The remaining genera represented approximately 1% of the bacterial flora, and were composed of Salmonella, Spiroplasma, Sphingomonas, Methylobacterium, Acidibacter, Tsukamurella, Serratia, Kluyvera and an unidentified bacterium. The genus Sodalis was present but with a very low abundance. Globally, no statistically significant difference was found between the bacterial compositions of flies from the two foci, and between positive and trypanosome-negative flies. However, Salmonella and Serratia were only described in trypanosome-negative flies, suggesting a potential role for these two bacteria in fly refractoriness to trypanosome infection. In addition, our study showed the V4 region of the small subunit of the 16S ribosomal RNA gene was more efficient than the V3V4 region at describing the totality of the bacterial diversity. CONCLUSIONS: A very large diversity of bacteria was identified with the discovering of species reported to secrete anti-parasitic compounds or to modulate vector competence in other insects. For future studies, the analyses should be enlarged with larger sampling including foci from several countries.

Colonization of the tsetse fly midgut with commensal Kosakonia cowanii Zambiae inhibits trypanosome infection establishment.

Tsetse flies (Glossina spp.) vector pathogenic trypanosomes (Trypanosoma spp.) in sub-Saharan Africa. These parasites cause human and animal African trypanosomiases, which are debilitating diseases that inflict an enormous socio-economic burden on inhabitants of endemic regions. Current disease control strategies rely primarily on treating infected animals and reducing tsetse population densities. However, relevant programs are costly, labor intensive and difficult to sustain. As such, novel strategies aimed at reducing tsetse vector competence require development. Herein we investigated whether Kosakonia cowanii Zambiae (Kco_Z), which confers Anopheles gambiae with resistance to Plasmodium, is able to colonize tsetse and induce a trypanosome refractory phenotype in the fly. Kco_Z established stable infections in tsetse's gut and exhibited no adverse effect on the fly's survival. Flies with established Kco_Z infections in their gut were significantly more refractory to infection with two distinct trypanosome species (T. congolense, 6% infection; T. brucei, 32% infection) than were age-matched flies that did not house the exogenous bacterium (T. congolense, 36% infected; T. brucei, 70% infected). Additionally, 52% of Kco_Z colonized tsetse survived infection with entomopathogenic Serratia marcescens, compared with only 9% of their wild-type counterparts. These parasite and pathogen refractory phenotypes result from the fact that Kco_Z acidifies tsetse's midgut environment, which inhibits trypanosome and Serratia growth and thus infection establishment. Finally, we determined that Kco_Z infection does not impact the fecundity of male or female tsetse, nor the ability of male flies to compete with their wild-type counterparts for mates. We propose that Kco_Z could be used as one component of an integrated strategy aimed at reducing the ability of tsetse to transmit pathogenic trypanosomes.

Molecular identification of Wolbachia and Sodalis glossinidius in the midgut of Glossina fuscipes quanzensis from the Democratic Republic of Congo.

During the last 30 years, investigations on the microbiome of different tsetse species have generated substantial data on the bacterial flora of these cyclical vectors of African trypanosomes, with the overarching goal of improving the control of trypanosomiases. It is in this context that the presence of Wolbachia and Sodalis glossinidius was studied in wild populations of Glossina fuscipes quanzensis from the Democratic Republic of Congo. Tsetse flies were captured with pyramidal traps. Of the 700 Glossina f. quanzensis captured, 360 were dissected and their midguts collected and analyzed. Sodalis glossinidius and Wolbachia were identified by PCR. The Wolbachia-positive samples were genetically characterized with five molecular markers. PCR revealed 84.78% and 15.55% midguts infected by Wolbachia and S. glossinidius, respectively. The infection rates varied according to capture sites. Of the five molecular markers used to characterize Wolbachia, only the fructose bis-phosphate aldolase gene was amplified for about 60% of midguts previously found with Wolbachia infections. The sequencing results confirmed the presence of Wolbachia and revealed the presence of S. glossinidius in the midgut of Glossina f. quanzensis. A low level of midguts were naturally co-infected by both bacteria. The data generated in this study open a framework for investigations aimed at understanding the contribution of these symbiotic microorganisms to the vectorial competence of Glossina fuscipes quanzensis.

Endosymbionts interaction with trypanosomes in Palpalis group of Glossina captured in southwest Nigeria.

Glossina species epidemiological studies were conducted in "fly-belt" endemic zone of southwest Nigeria. Two major study areas were identified and four Nzi traps were set in each site for tsetse collection. This study was conducted to determine the prevalence of endosymbionts (Wigglesworthia glossinidia, Sodalis glossinidius and Wolbachia) in natural field-trapped populations of G. p. palpalis and G. tachinoides and investigate the corresponding interactions with African trypanosomes. A total of 64 tsetse flies were collected, these included G. p. palpalis (n = 28) and G. tachinoides (n = 36). Trypanosome infection and endosymbionts of these flies were determined using polymerase chain reaction (PCR) amplification. The infection rates of W. glossinidia was 100.0% in both species, no flies were positive for Wolbachia. Sodalis glossinidius prevalence was similar between the two-tsetse species, with G. p. palpalis and G. tachinoides showing prevalence of 35.7% (95%CI: 20.7-54.2) and 27.8% (95%CI: 15.9-44.0) respectively. No relationship was found between the endosymbionts and trypanosomes in trapped tsetse flies. More studies are needed to enhance the potential control interventions mediated by endosymbionts to reduce parasitic infections.