Many insects that rely on a single food source throughout their developmental cycle harbor beneficial microbes that provide nutrients absent from their restricted diet. Tsetse flies, the vectors of African trypanosomes, feed exclusively on blood and rely on one such intracellular microbe for nutritional provisioning and fecundity. As a result of co-evolution with hosts over millions of years, these mutualists have lost the ability to survive outside the sheltered environment of their host insect cells. We present the complete annotated genome of Wigglesworthia glossinidia brevipalpis, which is composed of one chromosome of 697,724 base pairs (bp) and one small plasmid, called pWig1, of 5,200 bp. Genes involved in the biosynthesis of vitamin metabolites, apparently essential for host nutrition and fecundity, have been retained. Unexpectedly, this obligate's genome bears hallmarks of both parasitic and free-living microbes, and the gene encoding the important regulatory protein DnaA is absent.Many arthropods with restricted diets, such as vertebrate blood, plant juice or wood, rely on symbiotic microorganisms to supply nutrients required for viability and fertility 1 . Among insects harboring such symbionts is the tsetse fly (Diptera: Glossinidae)-the vector of African trypanosomes, agents of deadly diseases in humans and animals in sub-Saharan Africa 2 . Tsetse flies harbor two symbiotic microorganisms in gut tissue: the obligate primary-symbiont Wigglesworthia glossinidia and the commensal secondarysymbiont Sodalis glossinidius. Whereas S. glossinidius may be found in various host tissue types, W. glossinidia is housed in differentiated host epithelial cells (bacteriocytes) that form the bacteriome organ 2 . The functional role of obligate symbionts in tsetses has been difficult to study, as their elimination results in retarded growth and a decrease in egg production and fecundity in the aposymbiotic host 3,4 . The ability to reproduce could be partially restored, however, when aposymbiotic flies received supplementation with B-complex vitamins, suggesting that the endosymbionts might have a metabolic role involving these compounds 5 .The phylogenetic characterization of W. glossinidia from distant tsetse species has shown that they form a distinct clade in the Enterobacteriaceae 6 and display concordant evolution with their host species 7 . This finding implies that a tsetse ancestor was infected with a bacterium some 50-100 million years ago, and extant species of tsetse and associated W. glossinidia strains radiated without horizontal transfer of genetic material between species.As a result of their intracellular lifestyle, the genomes of obligate symbionts have undergone massive reductions in comparison with their free-living relatives. The genome size of W. glossinidia has been estimated as 740-770 kilobases 8 (kb), and that of Buchnera sp., the obligate symbiont of the pea aphid (Homoptera:Aphidoidea), as 640,681 bp 9,10 . Both genomes approach the size of the smallest genome reported thus far, that of My...

Sodalis glossinidius is a maternally transmitted endosymbiont of tsetse flies (Glossina spp.), an insect of medical and veterinary significance. Analysis of the complete sequence of Sodalis' chromosome (4,171,146 bp, encoding 2,432 protein coding sequences) indicates a reduced coding capacity of 51%. Furthermore, the chromosome contains 972 pseudogenes, an inordinately high number compared with that of other bacterial species. A high proportion of these pseudogenes are homologs of known proteins that function either in defense or in the transport and metabolism of carbohydrates and inorganic ions, suggesting Sodalis' degenerative adaptations to the immunity and restricted nutritional status of the host. Sodalis possesses three chromosomal symbiosis regions (SSR): SSR-1, SSR-2, and SSR-3, with gene inventories similar to the Type-III secretion system (TTSS) ysa from Yersinia enterolitica and SPI-1 and SPI-2 from Salmonella, respectively. While core components of the needle structure have been conserved, some of the effectors and regulators typically associated with these systems in pathogenic microbes are modified or eliminated in Sodalis. Analysis of SSR-specific invA transcript abundance in Sodalis during host development indicates that the individual symbiosis regions may exhibit different temporal expression profiles. In addition, the Sodalis chromosome encodes a complete flagella structure, key components of which are expressed in immature host developmental stages. These features may be important for the transmission and establishment of symbiont infections in the intra-uterine progeny. The data suggest that Sodalis represents an evolutionary intermediate transitioning from a free-living to a mutualistic lifestyle.

Tsetse flies (Diptera: Glossinidae) are vectors for trypanosome parasites, the agents of the deadly sleeping sickness disease in Africa. Tsetse also harbor two maternally transmitted enteric mutualist endosymbionts: the primary intracellular obligate Wigglesworthia glossinidia and the secondary commensal Sodalis glossinidius. Both endosymbionts are transmitted to the intrauterine progeny through the milk gland secretions of the viviparous female. We administered various antibiotics either continuously by per os supplementation of the host blood meal diet or discretely by hemocoelic injections into fertile females in an effort to selectively eliminate the symbionts to study their individual functions. A symbiont-specific PCR amplification assay and fluorescence in situ hybridization analysis were used to evaluate symbiont infection outcomes. Tetracycline and rifampin treatments eliminated all tsetse symbionts but reduced the fecundity of the treated females. Ampicillin treatments did not affect the intracellular Wigglesworthia localized in the bacteriome organ and retained female fecundity. The resulting progeny of ampicillin-treated females, however, lacked Wigglesworthia but still harbored the commensal Sodalis. Our results confirm the presence of two physiologically distinct Wigglesworthia populations: the bacteriome-localized Wigglesworthia involved with nutritional symbiosis and free-living Wigglesworthia in the milk gland organ responsible for maternal transmission to the progeny. We evaluated the reproductive fitness, longevity, digestion, and vectorial competence of flies that were devoid of Wigglesworthia. The absence of Wigglesworthia completely abolished the fertility of females but not that of males. Both the male and female Wigglesworthia-free adult progeny displayed longevity costs and were significantly compromised in their blood meal digestion ability. Finally, while the vectorial competence of the young newly hatched adults without Wigglesworthia was comparable to that of their wild-type counterparts, older flies displayed higher susceptibility to trypanosome infections, indicating a role for the mutualistic symbiosis in host immunobiology. The ability to rear adult tsetse that lack the obligate Wigglesworthia endosymbionts will now enable functional investigations into this ancient symbiosis.

Insect symbioses lack the complexity and diversity of those associated with higher eukaryotic hosts. Symbiotic microbiomes are beneficial to their insect hosts in many ways, including dietary supplementation, tolerance to environmental perturbations and maintenance and/or enhancement of host immune system homeostasis. Recent studies have also highlighted the importance of the microbiome in the context of host pathogen transmission processes. Here we provide an overview of the relationship between insect disease vectors, such as tsetse flies and mosquitoes, and their associated microbiome. Several mechanisms are discussed through which symbiotic microbes may influence their host’s ability to transmit pathogens, as well as potential disease control strategies that harness symbiotic microbes to reduce pathogen transmission through an insect vector.

Expression of molecules with antiparasitic activity by genetically transformed symbiotic bacteria of disease-transmitting insects may serve as a powerful approach to control certain arthropod-borne diseases. The endosymbiont of the Chagas disease vector, Rhodnius prolixus, has been transformed to express cecropin A, a peptide lethal to the parasite, Trypanosoma cruzi. In insects carrying the transformed bacteria, cecropin A expression results in elimination or reduction in number of T. cruzi. A method has been devised to spread the transgenic bacteria to populations of R. prolixus, in a manner that mimics their natural coprophagous route of symbiont acquisition.

Tsetse flies are the medically and agriculturally important vectors of African trypanosomes. Information on the molecular and biochemical nature of the tsetse͞trypanosome interaction is lacking. Here we describe three antimicrobial peptide genes, attacin, defensin, and diptericin, from tsetse fat body tissue obtained by subtractive cloning after immune stimulation with Escherichia coli and trypanosomes. Differential regulation of these genes shows the tsetse immune system can discriminate not only between molecular signals specific for bacteria and trypanosome infections but also between different life stages of trypanosomes. The presence of trypanosomes either in the hemolymph or in the gut early in the infection process does not induce transcription of attacin and defensin significantly. After parasite establishment in the gut, however, both antimicrobial genes are expressed at high levels in the fat body, apparently not affecting the viability of parasites in the midgut. Unlike other insect immune systems, the antimicrobial peptide gene diptericin is constitutively expressed in both fat body and gut tissue of normal and immune stimulated flies, possibly reflecting tsetse immune responses to the multiple Gram-negative symbionts it naturally harbors. When flies were immune stimulated with bacteria before receiving a trypanosome containing bloodmeal, their ability to establish infections was severely blocked, indicating that up-regulation of some immune responsive genes early in infection can act to block parasite transmission. The results are discussed in relation to transgenic approaches proposed for modulating vector competence in tsetse.Glossina ͉ insect immunity ͉ vector control ͉ transgenesis ͉ symbiosis T he life cycle of the parasitic African trypanosomes (Euglenozoa: Kinetoplastida) in their insect vector, the tsetse fly (Diptera: Glossinidae), begins when it feeds from an infected mammalian host. For successful transmission, the parasite undergoes two stages of differentiation in the fly: first, establishment in midgut and then maturation in the mouthparts or salivary glands. In the midgut, the mammalian bloodstream parasites rapidly differentiate to procyclic forms and begin to replicate (establishment). Once established in the midgut, trypanosomes migrate forwards to the proventriculus and the mouthparts, where they begin to differentiate into epimastigotes and eventually colonize the proboscis or salivary glands, depending on the parasite species (1). Here they differentiate into metacyclic forms infective to mammals (maturation) and can be transmitted to the next host during blood feeding by the fly (2). It is generally thought that during normal development in the fly, there are no intracellular stages, although reports of intracellular Trypanosoma brucei rhodesiense (3, 4) and Trypanosoma congolense (5) in the anterior midgut cells have been published. It is also thought that during normal infection, trypanosomes do not cross an epithelial barrier to enter the fly, although there are several repo...

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the gamma subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species.

Tsetse flies (Diptera: Glossinidae) harbour three different symbiotic organisms in addition to the pathogenic African trypanosomes they transmit. The two gut-associated symbionts (primary, P; secondary, S) are enteric and are nutritionally required, whereas the third microorganism Wolbachia (family Rickettsiaceae) affects the reproductive biology of the insects it infects. The bacteriome-associated P-symbiont (Wigglesworthia glossinidia) displays a concordant phylogeny with its host tsetse species, whereas midgut S-symbionts characterized from distant tsetse have identical 16S rDNA sequences and therefore may either represent recent independent acquisitions or horizontal transfer between species. The S-symbionts have been cultured in vitro and a genetic transformation system has been developed. Here we report on their density and tissue tropism in different species (G. m. morsitans, G. p. palpalis, G. austeni and G. brevipalpis) and on their maternal route of transmission to tsetse progeny. Using a bacterium-specific PCR-assay, the S-symbionts were found primarily in the midgut, haemolymph, milk gland and in G. palpalis also in salivary glands of teneral flies. In older flies these infections were found to spread to other tissues including muscle, testes and fat body. The S-symbionts were transformed to express the marker gene product, Green Fluorescent Protein (GFP) in vitro. When the recombinant symbionts were introduced into the haemoceal of fertile female flies via intrathoracic microinjection, they were detected in the intrauterine progeny, indicating that haemolymph may provide a possible route for their transmission. The implications of these results for symbiont-host interactions and for transgenic strategies in tsetse are discussed.