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The 0.719 Mb genome of the tsetse endosymbiont, Wigglesworthia glossinidia, from Glossina palpalis gambiensis is presented. This Wigglesworthia genome retains 611 protein-coding sequences and a 25.3% GC content. A cryptic plasmid is conserved, between Wigglesworthia isolates, suggesting functional significance. This genome adds a further dimension to characterize Wigglesworthia lineage-based differences.

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BACKGROUND: Tsetse control is considered an effective and sustainable tactic for the control of cyclically transmitted trypanosomosis in the absence of effective vaccines and inexpensive, effective drugs. The sterile insect technique (SIT) is currently used to eliminate tsetse fly populations in an area-wide integrated pest management (AW-IPM) context in Senegal. For SIT, tsetse mass rearing is a major milestone that associated microbes can influence. Tsetse flies can be infected with microorganisms, including the primary and obligate Wigglesworthia glossinidia, the commensal Sodalis glossinidius, and Wolbachia pipientis. In addition, tsetse populations often carry a pathogenic DNA virus, the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) that hinders tsetse fertility and fecundity. Interactions between symbionts and pathogens might affect the performance of the insect host. METHODS: In the present study, we assessed associations of GpSGHV and tsetse endosymbionts under field conditions to decipher the possible bidirectional interactions in different Glossina species. We determined the co-infection pattern of GpSGHV and Wolbachia in natural tsetse populations. We further analyzed the interaction of both Wolbachia and GpSGHV infections with Sodalis and Wigglesworthia density using qPCR. RESULTS: The results indicated that the co-infection of GpSGHV and Wolbachia was most prevalent in Glossina austeni and Glossina morsitans morsitans, with an explicit significant negative correlation between GpSGHV and Wigglesworthia density. GpSGHV infection levels > 103.31 seem to be absent when Wolbachia infection is present at high density (> 107.36), suggesting a potential protective role of Wolbachia against GpSGHV. CONCLUSION: The result indicates that Wolbachia infection might interact (with an undefined mechanism) antagonistically with SGHV infection protecting tsetse fly against GpSGHV, and the interactions between the tsetse host and its associated microbes are dynamic and likely species specific; significant differences may exist between laboratory and field conditions.

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Wigglesworthia glossinidia is an obligate, maternally transmitted endosymbiont of tsetse flies. The ancient association between these two organisms accounts for many of their unique physiological adaptations. Similar to other obligate mutualists, Wigglesworthia's genome is dramatically reduced in size, yet it has retained the capacity to produce many B-vitamins that are found at inadequate quantities in the fly's vertebrate blood-specific diet. These Wigglesworthia-derived B-vitamins play essential nutritional roles to maintain tsetse's physiological homeostasis as well as that of other members of the fly's microbiota. In addition to its nutritional role, Wigglesworthia contributes towards the development of tsetse's immune system during the larval period. Tsetse produce amidases that degrade symbiotic peptidoglycans and prevent activation of antimicrobial responses that can damage Wigglesworthia. These amidases in turn exhibit antiparasitic activity and decrease tsetse's ability to be colonized with parasitic trypanosomes, which reduce host fitness. Thus, the Wigglesworthia symbiosis represents a fine-tuned association in which both partners actively contribute towards achieving optimal fitness outcomes.

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Tsetse flies have socioeconomic significance as the obligate vector of multiple Trypanosoma parasites, the causative agents of Human and Animal African Trypanosomiases. Like many animals subsisting on a limited diet, microbial symbiosis is key to supplementing nutrient deficiencies necessary for metabolic, reproductive, and immune functions. Extensive studies on the microbiota in parallel to tsetse biology have unraveled the many dependencies partners have for one another. But far less is known mechanistically on how products are swapped between partners and how these metabolic exchanges are regulated, especially to address changing physiological needs. More specifically, how do metabolites contributed by one partner get to the right place at the right time and in the right amounts to the other partner? Epigenetics is the study of molecules and mechanisms that regulate the inheritance, gene activity and expression of traits that are not due to DNA sequence alone. The roles that epigenetics provide as a mechanistic link between host phenotype, metabolism and microbiota (both in composition and activity) is relatively unknown and represents a frontier of exploration. Here, we take a closer look at blood feeding insects with emphasis on the tsetse fly, to specifically propose roles for microRNAs (miRNA) and DNA methylation, in maintaining insect-microbiota functional homeostasis. We provide empirical details to addressing these hypotheses and advancing these studies. Deciphering how microbiota and host activity are harmonized may foster multiple applications toward manipulating host health, including identifying novel targets for innovative vector control strategies to counter insidious pests such as tsetse.

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Many insects rely on intracellular bacterial symbionts to supplement their specialized diets with micronutrients. Using data from diverse and well-studied insect systems, we propose three lines of evidence suggesting that hosts have tight control over the density of their obligate, intracellular bacterial partners. First, empirical studies have demonstrated that the within-host symbiont density varies depending on the nutritional and developmental requirements of the host. Second, symbiont genomes are highly reduced and have limited capacity for self-replication or transcriptional regulation. Third, several mechanisms exist for hosts to tolerate, regulate and remove symbionts including physical compartmentalization and autophagy. We then consider whether such regulation is adaptive, by discussing the relationship between symbiont density and host fitness. We discuss current limitations of empirical studies for exploring fitness effects in host-symbiont relationships, and emphasize the potential for using mathematical models to formalize evolutionary hypotheses and to generate testable predictions for future work.

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BACKGROUND: Tsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species. RESULTS: An active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, 'translation, ribosomal structure and biogenesis' followed by 'coenzyme transport and metabolism' were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways. CONCLUSIONS: These transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

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Tsetse flies are the sole cyclic vector for trypanosomosis, the causative agent for human African trypanosomosis or sleeping sickness and African animal trypanosomosis or nagana. Tsetse population control is the most efficient strategy for animal trypanosomosis control. Among all tsetse control methods, the Sterile Insect Technique (SIT) is one of the most powerful control tactics to suppress or eradicate tsetse flies. However, one of the challenges for the implementation of SIT is the mass production of target species. Tsetse flies have a highly regulated and defined microbial fauna composed of three bacterial symbionts (Wigglesworthia, Sodalis and Wolbachia) and a pathogenic Glossina pallidipes Salivary Gland Hypertrophy Virus (GpSGHV) which causes reproduction alterations such as testicular degeneration and ovarian abnormalities with reduced fertility and fecundity. Interactions between symbionts and GpSGHV might affect the performance of the insect host. In the present study, we assessed the possible impact of GpSGHV on the prevalence of tsetse endosymbionts under laboratory conditions to decipher the bidirectional interactions on six Glossina laboratory species. The results indicate that tsetse symbiont densities increased over time in tsetse colonies with no clear impact of the GpSGHV infection on symbionts density. However, a positive correlation between the GpSGHV and Sodalis density was observed in Glossina fuscipes species. In contrast, a negative correlation between the GpSGHV density and symbionts density was observed in the other taxa. It is worth noting that the lowest Wigglesworthia density was observed in G. pallidipes, the species which suffers most from GpSGHV infection. In conclusion, the interactions between GpSGHV infection and tsetse symbiont infections seems complicated and affected by the host and the infection density of the GpSGHV and tsetse symbionts.

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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.

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BACKGROUND: The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentally acquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes reside within infected flies. Tsetse's bacterial microbiota impacts many aspects of the fly's physiology. However, little is known about the structure of tsetse's midgut-associated bacterial communities as they relate to geographically distinct fly habitats in east Africa and their contributions to parasite infection outcomes. We utilized culture dependent and independent methods to characterize the taxonomic structure and density of bacterial communities that reside within the midgut of tsetse flies collected at geographically distinct locations in Kenya and Uganda. RESULTS: Using culture dependent methods, we isolated 34 strains of bacteria from four different tsetse species (G. pallidipes, G. brevipalpis, G. fuscipes and G. fuscipleuris) captured at three distinct locations in Kenya. To increase the depth of this study, we deep sequenced midguts from individual uninfected and trypanosome infected G. pallidipes captured at two distinct locations in Kenya and one in Uganda. We found that tsetse's obligate endosymbiont, Wigglesworthia, was the most abundant bacterium present in the midgut of G. pallidipes, and the density of this bacterium remained largely consistent regardless of whether or not its tsetse host was infected with trypanosomes. These fly populations also housed the commensal symbiont Sodalis, which was found at significantly higher densities in trypanosome infected compared to uninfected flies. Finally, midguts of field-captured G. pallidipes were colonized with distinct, low density communities of environmentally acquired microbes that differed in taxonomic structure depending on parasite infection status and the geographic location from which the flies were collected. CONCLUSIONS: The results of this study will enhance our understanding of the tripartite relationship between tsetse, its microbiota and trypanosome vector competence. This information may be useful for developing novel disease control strategies or enhancing the efficacy of those already in use.

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BACKGROUND: Symbiotic microbes represent a driving force of evolutionary innovation by conferring novel ecological traits to their hosts. Many insects are associated with microbial symbionts that contribute to their host's nutrition, digestion, detoxification, reproduction, immune homeostasis, and defense. In addition, recent studies suggest a microbial involvement in chemical communication and mating behavior, which can ultimately impact reproductive isolation and, hence, speciation. Here we investigated whether a disruption of the microbiota through antibiotic treatment or irradiation affects cuticular hydrocarbon profiles, and possibly mate choice behavior in the tsetse fly, Glossina morsitans morsitans. Four independent experiments that differentially knock down the multiple bacterial symbionts of tsetse flies were conducted by subjecting tsetse flies to ampicillin, tetracycline, or gamma-irradiation and analyzing their cuticular hydrocarbon profiles in comparison to untreated controls by gas chromatography - mass spectrometry. In two of the antibiotic experiments, flies were mass-reared, while individual rearing was done for the third experiment to avoid possible chemical cross-contamination between individual flies. RESULTS: All three antibiotic experiments yielded significant effects of antibiotic treatment (particularly tetracycline) on cuticular hydrocarbon profiles in both female and male G. m. morsitans, while irradiation itself had no effect on the CHC profiles. Importantly, tetracycline treatment reduced relative amounts of 15,19,23-trimethyl-heptatriacontane, a known compound of the female contact sex pheromone, in two of the three experiments, suggesting a possible implication of microbiota disturbance on mate choice decisions. Concordantly, both female and male flies preferred non-treated over tetracycline-treated flies in direct choice assays. CONCLUSIONS: While we cannot exclude the possibility that antibiotic treatment had a directly detrimental effect on fly vigor as we are unable to recolonize antibiotic treated flies with individual symbiont taxa, our results are consistent with an effect of the microbiota, particularly the obligate nutritional endosymbiont Wigglesworthia, on CHC profiles and mate choice behavior. These findings highlight the importance of considering host-microbiota interactions when studying chemical communication and mate choice in insects.

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Tsetse flies (Diptera: Glossinidae) have medical significance as the obligate vectors of African trypanosomes. In addition, tsetse harbor a simple gut microbiota. A predominant gut microbiota member, the Gammaproteobacterium Wigglesworthia spp., has coevolved with tsetse for a significant portion of Glossina radiation proving critical to tsetse fitness. Although multiple roles have been described for Wigglesworthia within colony flies, little research has been dedicated towards functional characterization within wild tsetse. Here, dual RNA-Seq was performed to characterize the tsetse-Wigglesworthia symbiosis within flies captured in Nguruman, Kenya. A significant correlation in Gene Ontology (GO) distribution between tsetse and Wigglesworthia was observed, with homogeneous enrichment in metabolic and transport categories, likely supporting a hallmark of the symbiosis-bidirectional metabolic exchange. Within field flies, highly transcribed Wigglesworthia loci included those involved in B vitamin synthesis and in substrate translocation, including amino acid transporters and multidrug efflux pumps, providing a molecular means for interaction. The universal expression of several Wigglesworthia and G. pallidipes orthologs, putatively involved in nutrient provisioning and resource allocation, was confirmed in sister tsetse species. These transcriptional profiles varied through host age and mating status likely addressing varying symbiont demands and also confirming their global importance within Glossina. This study, not only supports symbiont nutrient provisioning roles, but also serves as a foundation for insight into novel roles and molecular mechanisms associated with vector-microbiota interactions. The role of symbiont B vitamin provisioning towards impacting host epigenetics is discussed. Knowledge of vector-microbiota interactions may lead to the discovery of novel targets in pest control.

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Insects with restricted diets rely on obligate microbes to fulfil nutritional requirements essential for biological function. Tsetse flies, vectors of African trypanosome parasites, feed exclusively on vertebrate blood and harbour the obligate endosymbiont Wigglesworthia glossinidia. Without Wigglesworthia, tsetse are unable to reproduce. These symbionts are sheltered within specialized cells (bacteriocytes) that form the midgut-associated bacteriome organ. To decipher the core functions of this symbiosis essential for tsetse's survival, we performed dual-RNA-seq analysis of the bacteriome, coupled with metabolomic analysis of bacteriome and haemolymph collected from normal and symbiont-cured (sterile) females. Bacteriocytes produce immune regulatory peptidoglycan recognition protein (pgrp-lb) that protects Wigglesworthia, and a multivitamin transporter (smvt) that can aid in nutrient dissemination. Wigglesworthia overexpress a molecular chaperone (GroEL) to augment their translational/transport machinery and biosynthesize an abundance of B vitamins (specifically B1-, B2-, B3- and B6-associated metabolites) to supplement the host's nutritionally deficient diet. The absence of Wigglesworthia's contributions disrupts multiple metabolic pathways impacting carbohydrate and amino acid metabolism. These disruptions affect the dependent downstream processes of nucleotide biosynthesis and metabolism and biosynthesis of S-adenosyl methionine (SAM), an essential cofactor. This holistic fundamental knowledge of the symbiotic dialogue highlights new biological targets for the development of innovative vector control methods.

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Tsetse flies (Glossina spp.), vectors of African trypanosomes, are distinguished by their specialized reproductive biology, defined by adenotrophic viviparity (maternal nourishment of progeny by glandular secretions followed by live birth). This trait has evolved infrequently among insects and requires unique reproductive mechanisms. A key event in Glossina reproduction involves the transition between periods of lactation and nonlactation (dry periods). Increased lipolysis, nutrient transfer to the milk gland, and milk-specific protein production characterize lactation, which terminates at the birth of the progeny and is followed by a period of involution. The dry stage coincides with embryogenesis of the progeny, during which lipid reserves accumulate in preparation for the next round of lactation. The obligate bacterial symbiont Wigglesworthia glossinidia is critical to tsetse reproduction and likely provides B vitamins required for metabolic processes underlying lactation and/or progeny development. Here we describe findings that utilized transcriptomics, physiological assays, and RNA interference-based functional analysis to understand different components of adenotrophic viviparity in tsetse flies.

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Tsetse flies (Glossina sp.) that transmit trypanosomes causing human (and animal) African trypanosomiasis (HAT and AAT, respectively) harbor symbiotic microorganisms, including the obligate primary symbiont Wigglesworthia glossinidia. A relationship between Wigglesworthia and tsetse fly infection by trypanosomes has been suggested, as removal of the symbiont results in a higher susceptibility to midgut infection in adult flies. To investigate this relationship and to decipher the role of W. glossinidia in the fly's susceptibility to trypanosome infection, we challenged flies with trypanosomes and subsequently analyzed and compared the transcriptomes of W. glossinidia from susceptible and refractory tsetse flies at three time points (3, 10, and 20 days). More than 200 W. glossinidia genes were found to be differentially expressed between susceptible and refractory flies. The high specificity of these differentially expressed genes makes it possible to distinguish Wigglesworthia inhabiting these two distinct groups of flies. Furthermore, gene expression patterns were observed to evolve during the infection time course, such that very few differentially expressed genes were found in common in Wigglesworthia from the 3-, 10- and 20-day post-feeding fly samples. The overall results clearly demonstrate that the taking up of trypanosomes by flies, regardless of whether flies proceed with the developmental program of Trypanosoma brucei gambiense, strongly alters gene expression in Wigglesworthia. These results therefore provide a novel framework for studies that aim to decrease or even abolish tsetse fly vector competence.

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Tsetse flies are the primary vectors of African trypanosomes, which cause Human and Animal African trypanosomiasis in 36 countries in sub-Saharan Africa. These flies have also established symbiotic associations with bacterial and viral microorganisms. Laboratory-reared tsetse flies harbor up to four vertically transmitted organisms-obligate Wigglesworthia, commensal Sodalis, parasitic Wolbachia and Salivary Gland Hypertrophy Virus (SGHV). Field-captured tsetse can harbor these symbionts as well as environmentally acquired commensal bacteria. This microbial community influences several aspects of tsetse's physiology, including nutrition, fecundity and vector competence. This review provides a detailed description of tsetse's microbiome, and describes the physiology underlying host-microbe, and microbe-microbe, interactions that occur in this fly.

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