Tsetse flies are the sole vectors of human African trypanosomiasis throughout sub-Saharan Africa. Both sexes of adult tsetse feed exclusively on blood and contribute to disease transmission. Notable differences between tsetse and other disease vectors include obligate microbial symbioses, viviparous reproduction, and lactation. Here, we describe the sequence and annotation of the 366-megabase Glossina morsitans morsitans genome. Analysis of the genome and the 12,308 predicted protein–encoding genes led to multiple discoveries, including chromosomal integrations of bacterial (Wolbachia) genome sequences, a family of lactation-specific proteins, reduced complement of host pathogen recognition proteins, and reduced olfaction/chemosensory associated genes. These genome data provide a foundation for research into trypanosomiasis prevention and yield important insights with broad implications for multiple aspects of tsetse biology.

Ancient endosymbionts have been associated with extreme genome structural stability with little differentiation in gene inventory between sister species. Tsetse flies (Diptera: Glossinidae) harbor an obligate endosymbiont, Wigglesworthia, which has coevolved with the Glossina radiation. We report on the ~720-kb Wigglesworthia genome and its associated plasmid from Glossina morsitans morsitans and compare them to those of the symbiont from Glossina brevipalpis. While there was overall high synteny between the two genomes, a large inversion was noted. Furthermore, symbiont transcriptional analyses demonstrated host tissue and development-specific gene expression supporting robust transcriptional regulation in Wigglesworthia, an unprecedented observation in other obligate mutualist endosymbionts. Expression and immunohistochemistry confirmed the role of flagella during the vertical transmission process from mother to intrauterine progeny. The expression of nutrient provisioning genes (thiC and hemH) suggests that Wigglesworthia may function in dietary supplementation tailored toward host development. Furthermore, despite extensive conservation, unique genes were identified within both symbiont genomes that may result in distinct metabolomes impacting host physiology. One of these differences involves the chorismate, phenylalanine, and folate biosynthetic pathways, which are uniquely present in Wigglesworthia morsitans. Interestingly, African trypanosomes are auxotrophs for phenylalanine and folate and salvage both exogenously. It is possible that W. morsitans contributes to the higher parasite susceptibility of its host species.

Host-associated microbial interactions may involve genome complementation, driving-enhanced communal efficiency and stability. The tsetse fly (Diptera: Glossinidae), the obligate vector of African trypanosomes (Trypanosoma brucei subspp.), harbours two enteric Gammaproteobacteria symbionts: Wigglesworthia glossinidia and Sodalis glossinidius. Host coevolution has streamlined the Wigglesworthia genome to complement the exclusively sanguivorous tsetse lifestyle. Comparative genomics reveal that the Sodalis genome contains the majority of Wigglesworthia genes. This significant genomic overlap calls into question why tsetse maintains the coresidence of both symbionts and, furthermore, how symbiont homeostasis is maintained. One of the few distinctions between the Wigglesworthia and Sodalis genomes lies in thiamine biosynthesis. While Wigglesworthia can synthesize thiamine, Sodalis lacks this capability but retains a thiamine ABC transporter (tbpAthi PQ) believed to salvage thiamine. This genetic complementation may represent the early convergence of metabolic pathways that may act to retain Wigglesworthia and evade species antagonism. We show that thiamine monophosphate, the specific thiamine derivative putatively synthesized by Wigglesworthia, impacts Sodalis thiamine transporter expression, proliferation and intracellular localization. A greater understanding of tsetse symbiont interactions may generate alternative control strategies for this significant medical and agricultural pest, while also providing insight into the evolution of microbial associations within hosts.

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (Trypanosoma spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium Wigglesworthia. Extensive concordant evolution with associated Wigglesworthia species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between Wigglesworthia genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between "Wigglesworthia morsitans" (harbored within Glossina morsitans bacteriomes) and the basal species Wigglesworthia glossinidia (harbored within Glossina brevipalpis bacteriomes) include the retention of chorismate and downstream folate (vitamin B 9 ) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these W. morsitans pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by W. morsitans is demonstrated to be pivotal for G. morsitans sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the Wigglesworthia-tsetse mutualism may also provide novel and specific avenues for vector control. Bacteria adapt to specific environments, including host-associated niches, through the retention, rearrangement, gain, or decay of functional capabilities. Research on ancient obligate host associations (i.e., in which bacteria display an extensive concordant evolution with their host) has demonstrated that microbial symbiont genome evolution can be influenced by microbial community dynamics (1-6), in addition to host physiology and ecology (7-9). One extreme case has been described within the mealybug, where dual symbiont species and the host have retained complementary loci that, only when integrated as a symbiotic system (i.e., a holobiont) and not as individual species, are capable of producing specific requisite nutrients (3, 9). Extensive gene purging is characteristic among ancient bacterial symbionts, as they challenge the lower limits of genome size (10, 11). These symbionts typically exhibit tremendous genomic stasis between strains and species, likely due to their environmental isolation, as their genomes encode only those capabilities necessary for the maintenance of the mutualism (reviewed in reference 10). In contrast, the genomes of free-living bacteria allow the bacteria to adapt to their surroundings by encoding a plethora of strain-specific loci, known as dispensable genes (12), contributing to increased habitat occupancy and their successful viability with...

The obligate mutualist Wigglesworthia morsitans provisions nutrients to tsetse flies. The symbiont's response to thiamine (B 1 ) supplementation of blood meals, specifically towards the regulation of thiamine biosynthesis and population density, is described. Despite an ancient symbiosis and associated genome tailoring, Wigglesworthia responds to nutrient availability, potentially accommodating a decreased need.