The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts

Three different bacterial species are regularly described from tsetse flies. However, no broad screens have been performed to investigate the existence of other bacteria in this medically and agriculturally important vector insect. Utilising both culture dependent and independent methods we show that Kenyan populations of Glossina fuscipes fuscipes harbour a surprising diversity of bacteria. Bacteria were isolated from 72% of flies with 23 different bacterial species identified. The Firmicutes phylum dominated with 16 species of which seven belong to the genus Bacillus. The tsetse fly primary symbiont, Wigglesworthia glossinidia, was identified by the culture independent pathway. However, neither the secondary symbiont Sodalis nor Wolbachia was detected with either of the methods used. Two other bacterial species were identified with the DNA based method, Bacillus subtilis and Serratia marcescens. Further studies are needed to determine how tsetse flies, which only ever feed on vertebrate blood, pick up bacteria and to investigate the possible impact of these bacteria on Glossina longevity and vector competence.     

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.     

The population genomics of multiple tsetse fly (Glossina fuscipes fuscipes) admixture zones in Uganda

Understanding the mechanisms that enforce, maintain or reverse the process of speciation is an important challenge in evolutionary biology. This study investigates the patterns of divergence and discusses the processes that form and maintain divergent lineages of the tsetse fly Glossina fuscipes fuscipes in Uganda. We sampled 251 flies from 18 sites spanning known genetic lineages and the four admixture zones between them. We apply population genomics, hybrid zone and approximate Bayesian computation to the analysis of three types of genetic markers: 55,267 double‐digest restriction site‐associated DNA (ddRAD) SNPs to assess genome‐wide admixture, 16 microsatellites to provide continuity with published data and accurate biogeographic modelling, and a 491‐bp fragment of mitochondrial cytochrome oxidase I and II to infer maternal inheritance patterns. Admixture zones correspond with regions impacted by the reorganization of Uganda's river networks that occurred during the formation of the West African Rift system over the last several hundred thousand years. Because tsetse fly population distributions are defined by rivers, admixture zones likely represent both old and new regions of secondary contact. Our results indicate that older hybrid zones contain mostly parental types, while younger zones contain variable hybrid types resulting from multiple generations of interbreeding. These findings suggest that reproductive barriers are nearly complete in the older admixture zones, while nearly absent in the younger admixture zones. Findings are consistent with predictions of hybrid zone theory: Populations in zones of secondary contact transition rapidly from early to late stages of speciation or collapse all together.     

Polymorphic microsatellite markers for the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae), a vector of human African trypanosomiasis

Our understanding of Glossina fuscipes fuscipes, a major vector of sleeping sickness, has been severely constrained by a lack of genetic markers for mapping and population genetic studies. Here we present 10 newly developed microsatellite loci for this tsetse species. Heterozygosity levels in Moyo, an Ugandan population, averaged 0.57, with only two loci showing very low heterozygosity. Five loci carried more than six alleles. Together with five recently published microsatellite loci, this brings the number of available microsatellite loci for this species to 15. Their availability will greatly facilitate future studies on the genetics of this important human disease vector.     

Macrogeographic population structure of the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Tsetse flies are confined to sub-Saharan Africa where they occupy discontinuous habitats. In anticipation of area-wide control programmes, estimates of gene flow among tsetse populations are necessary. Genetic diversities were partitioned at eight microsatellite loci and five mitochondrial loci in 21 Glossina pallidipes Austin populations. At microsatellite loci, Nei's unbiased gene diversity averaged over loci was 0.659 and the total number of alleles was 214, only four of which were shared among all populations. The mean number of alleles per locus was 26.8. Random mating was observed within but not among populations (fixation index FST=0.18) and 81% of the genetic variance was within populations. Thirty-nine mitochondrial variants were detected. Mitochondrial diversities in populations varied from 0 to 0.85 and averaged 0.42, and FST=0.51. High levels of genetic differentiation were characteristic, extending even to subpopulations separated by tens and hundreds of kilometres, and indicating low rates of gene flow.     

Characterization of microsatellite markers in the tsetse fly, Glossina pallidipes (Diptera: Glossinidae)

Glossina pallidipes is a vector of African trypanosomiasis. Here we characterize eight new polymorphic microsatellite loci in 288 G. pallidipes sampled from 12 Kenya populations. The number of alleles per locus ranged from four to 36 with a mean of 20.5 +/- 10.1. Expected single locus heterozygosities varied from 0.044 to 0.829. Heterozygosity averaged 0.616 +/- 0.246. No linkage disequilibrium was found. We also report results in eight other tsetse species estimated by using the primers developed in G. pallidipes. The primers worked best in G. swynnertoni and G. austeni and worst in G. m. morsitans and G. m. submorsitans.     

Maturation of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) in relation to trap-orientated behaviour

ABSTRACT The fat free dry weight or residual dry weight of the thorax (Trdw) increased linearly for the first 10 days of adult life in both sexes of G.pallidipes in the laboratory as their flight muscles developed. Using ovarian dissection to estimate the ages of nulliparous adult females of G.pallidipes , the Trdw was also found to increase linearly for at least 14 days in the field. Significant increases in pteridine fluorescence with age were measured in both laboratory-reared males and females of known chronological age and in wild-caught nulliparous females whose ages were estimated by ovarian dissection. A linear relationship existed between pteridine fluorescence and wing fray category for a wide range of ages of field-caught flies of both sexes. A stationary trap baited with ox odour was selective in that only the hungrier portion of the flies attracted to it actually entered. However, it was not selective in terms of the mean ages of flies caught. Comparisons were made of the age compositions of catches of both sexes of G.pallidipes attracted to a stationary trap baited with synthetic odours or to a mobile electrified net by plotting Trdw values against pteridine fluorescence. Nulliparous females were not attracted to the stationary trap, but were attracted to the mobile bait. Males of all ages appeared to be equally attracted to both. It is concluded that nulliparous females do not respond to host odour stimuli until they are ready to mate, perhaps relying on the energy-conserving strategy of watching for a moving host animal before attempting to feed. Alternatively, synthetic odours may differ from natural host odours in terms of their attractiveness to young females. Males, however, probably exhibit dual sexual and feeding behaviour by responding to an odour-baited stationary trap even when young.     

A cytogenetical study of Glossina fuscipes fuscipes including a comparison of the polytene chromosome maps with those of Glossina austeni (Diptera,Glossinidae)

The male meiotic sequence is described for the tsetse fly Glossina fuscipes fuscipes together with the polytene chromosome maps and all principal cytological markers. The diploid chromosome number is 2n=6 and includes a pair of large submetacentric autosomes (L₁), a shorter pair of metacentric autosomes (L₂), and an X and Y which constitute a heteromorphic pair. Male meiosis is normally achiasmate although evidence is presented which suggests that chiasmata do form in about 1% of males. A detailed comparison between the polytene chromosomes of this species and Glossina austeni indicates that although they must have had a common ancestor, G. austeni is genetically more closely related to morsitans group tsetses.     

A spatial genetics approach to inform vector control of tsetse flies (Glossina fuscipes fuscipes) in Northern Uganda

Tsetse flies (genus Glossina) are the only vector for the parasitic trypanosomes responsible for sleeping sickness and nagana across sub‐Saharan Africa. In Uganda, the tsetse fly Glossina fuscipes fuscipes is responsible for transmission of the parasite in 90% of sleeping sickness cases, and co‐occurrence of both forms of human‐infective trypanosomes makes vector control a priority. We use population genetic data from 38 samples from northern Uganda in a novel methodological pipeline that integrates genetic data, remotely sensed environmental data, and hundreds of field‐survey observations. This methodological pipeline identifies isolated habitat by first identifying environmental parameters correlated with genetic differentiation, second, predicting spatial connectivity using field‐survey observations and the most predictive environmental parameter(s), and third, overlaying the connectivity surface onto a habitat suitability map. Results from this pipeline indicated that net photosynthesis was the strongest predictor of genetic differentiation in G. f. fuscipes in northern Uganda. The resulting connectivity surface identified a large area of well‐connected habitat in northwestern Uganda, and twenty‐four isolated patches on the northeastern margin of the G. f. fuscipes distribution. We tested this novel methodological pipeline by completing an ad hoc sample and genetic screen of G. f. fuscipes samples from a model‐predicted isolated patch, and evaluated whether the ad hoc sample was in fact as genetically isolated as predicted. Results indicated that genetic isolation of the ad hoc sample was as genetically isolated as predicted, with differentiation well above estimates made in samples from within well‐connected habitat separated by similar geographic distances. This work has important practical implications for the control of tsetse and other disease vectors, because it provides a way to identify isolated populations where it will be safer and easier to implement vector control and that should be prioritized as study sites during the development and improvement of vector control methods.     

A white eye color mutant in the tsetse fly Glossina morsitans submotsitans Newstead (Diptera: Glossinidae).

A spontaneous mutation in Glossina morsitans submorsitans Newstead is described. The mutant, designated wht, has white compound eyes but the ocelli and testes have normal coloration. Mutants have lower than normal amounts of xanthommatin and pteridines in their heads. The lesion occurs late in the tryptophan to xanthommatin pathway, in the storage of xanthommatin in the compound eyes, or, most likely, in the transport of precursors into the compound eyes. The locus wht is on the X chromosome.     

Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts

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 (tbpAthiPQ) 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.     

Regulation of last reproduction steps in tsetse flies (Glossina: Diptera)

Summary

The regulation of ovulation and parturition, in the viviparous tsetse flies, constitutes a very precisely adjusted mechanism, Indeed the process of vitellogenesis takes places, in one oocyte only, during the incubation period when the larva is nourished in utero on milk produced by the mother. Neuropeptides, biogenic amines, neurotransmitters as well as ecdysone, and 20-hydroxyecdysone and also nervous outputs regulate ovulation and parturition. A model is proposed for the mechanisms controlling these processes.     

The salivary secretome of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) infected by salivary gland hypertrophy virus

Background

The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly''s salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs.

Methodology/Principal Findings

After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins.

Conclusions/Significance

SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.     

Activities of kreb's cycle enzymes in the flight muscles of the tsetse fly (Glossina) and the fleshfly (Sarcophaga)

The specific activities of enzymes of the tricarboxylic acid cycle have been measured in flight muscle mitochondria of tsetse flies and fleshflies. Similar levels of those enzymes which are involved in the metabolism of both carbodydrate and proline (2-oxoglutarate dehydrogenase, succinate dehydrogenase and fumarase) were found in the mitochondria of the two insects. Enzymes of the cycle not involved in the oxidation of proline (citrata synthase, aconitase, isocitrate dehydrogenase and malate dehydrogenase) had considerably lower specific activities in the tsetse fly.     

Persistent courtship reduces male and female longevity in captive tsetse flies Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

Where males can increase their mating success by harassing femalesuntil they accept copulation, harassing tactics can be expectedto evolve to a point where they have costs to the longevityof both sexes. By experimentally manipulating the sex ratioin captive groups of tsetse flies Glossina morsitans morsitans,we demonstrated that the longevity of females declines wheresex ratios are biased toward males, while the longevity of malesdeclines where the sex ratio is biased toward females. Neitherirradiation of males nor prevention of copulation by blockingor damaging the external male genitalia increased the longevityof females caged with them, suggesting that female longevitywas reduced by the physical aspects of male harassment ratherthan by components of the ejaculate     

Does reproductive investment change with age in tsetse flies, Glossina morsitans morsitans (Diptera: Glossinidae)?

1. Viviparous insects such as tsetse ( Glossina spp.) provide unusual opportunities to compare age-related changes in the proportion of maternal resources transferred to offspring.
2. In laboratory populations of Glossina morsitans morsitans the survival of females was high for the first 60 days of adult life but declined rapidly thereafter.
3. Average longevity did not differ significantly between mated and unmated females (93·6 and 90 days, respectively).
4. Nutritional state in terms of fat content and residual dry mass did not decline with adult female age.
5. The fecundity of mated females was constant for the first 60 days of adult life and declined only slightly thereafter.
6. Offspring size did not change towards the end of the adult female lifespan and there was no evidence of an increase in the allocation of resources to reproduction in older females.
7. Results contrast with those obtained recently for vertebrates and may indicate that age-related changes in offspring size in Glossina are not adaptive, or that so few females reach old age under natural conditions that there is no selection for a strategy of terminal investment.     

Sulphaquinoxaline in host diet as the cause of reproductive abnormalities in the tsetse fly (Glossina spp.)

Groups of females of Glossina austeni and G. morsitans morsitans fed on rabbits with 75 ppm sulphaquinoxaline and 7.5 ppm pyrimethamine in their diets had markedly lower fecundity than other groups fed on rabbits supplied with additive-free diets. There was little effect on the longevity of female flies. Females of G. austeni produced normal numbers of puparia for one reproductive cycle, large numbers of aborted larvae in succeeding reproductive cycles and many females eventually became sterile. The effect on G. m. morsitans was less marked but when the dose rate of the additives in the rabbit diet was quadrupled this species also showed greatly reduced fecundity, with no effect on longevity. In G. austeni, those puparia which were produced in later reproductive cycles were lighter and less viable than those produced in the first cycle.It is concluded that the toxicant in the rabbit diet was sulphaquinoxaline acting as a systemic insecticide but, at these low dose rates, at a sub-lethal level. The possible mode of action of sulphaquinoxaline and the significance of the results for the laboratory rearing of haematophagous arthropods are discussed.

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Résumé Des groupes de femelles de Glossina austeni et Gl. morsitans morsitans sont nourris sur des lapins, euxmêmes alimentés avec des aliments artificiels de type commercial, qui ont été ou non additionnés de composés chimiques coccidiostatiques, soit 75 ppm de sulfaquinoxaline et 7,5 ppm de pyrimethamine. Quand les glossines sont nourries sur des lapins ayant reçu l'aliment additionné des substances coccidiostatiques, la fécondité de G. austeni (en termes de production de pupes viables par femelle) est réduite de façon nette, bien que la longévité demeure inaffectée; G. m. morsitans se révèle moins sévèrement touchée.Dans les expériences conçues pour étudier l'effet de la sulfaquinoxaline et de la pyrimethamine, les lapins reçoivent un aliment en poudre auquel sont ajoutées des quantités appropriées de ces substances. Des groupes de femelles de G. austeni nourries sur des lapins ayant reçu un aliment contenant 75 ppm de sulfaquinoxaline et 7,5 ppm pyrimethamine se reproduisent normalement pendant un premier cycle reproducteur, après quoi elles engendrent un grand nombre de larves avortées et beaucoup de femelles deviennent éventuellement stériles. Les pupes produites par G. austeni au cours des cycles reproducteurs plus tardifs sont plus légères et moins viables que celles produites lors du ler cycle. Quand le taux des composés coccidiostatiques est quadruplé dans l'aliment des lapins, la fécondité de Gl. m. morsitans est aussi fortement réduite, sans effet sur la longévité. Quand Gl. morsitans est alimentée artificiellement sur du sang contenant 75 et 300 ppm. de sulfaquinoxaline, la fécondité des femelles est sérieusement réduite. Aucune différence ne se décèle quant aux effets relatifs de ces deux doses du composé coccidiostatique; il est possible que les mouches en absorbent une même petite quantité dans les deux cas, en raison de la faible solubilité du produit, qui n'est soluble que dans des solutions alcalines. La sulfaquinoxaline, aux faibles doses absorbées agit comme un insecticide systémique à des doses sub-létales. Ce coccidiostatique peut agir en interférant avec le métabolisme du folate au niveau des ovaires; les effets sur la fécondité deviennent progressivement plus marqués chez les femelles âgées et chez celles qui ont cessé de se reproduire on observe des ovaires atrophiés.L' incidence de ces faits sur les conditions d'élevage des Glossines, et d'autres arthropodes hématophages, est discuté.