Wolbachia association with the tsetse fly, Glossina fuscipes fuscipes, reveals high levels of genetic diversity and complex evolutionary dynamics

BACKGROUND: Wolbachia pipientis, a diverse group of α-proteobacteria, can alter arthropod host reproduction and confer a reproductive advantage to Wolbachia-infected females (cytoplasmic incompatibility (CI)). This advantage can alter host population genetics because Wolbachia-infected females produce more offspring with their own mitochondrial DNA (mtDNA) haplotypes than uninfected females. Thus, these host haplotypes become common or fixed (selective sweep). Although simulations suggest that for a CI-mediated sweep to occur, there must be a transient phase with repeated initial infections of multiple individual hosts by different Wolbachia strains, this has not been observed empirically. Wolbachia has been found in the tsetse fly, Glossina fuscipes fuscipes, but it is not limited to a single host haplotype, suggesting that CI did not impact its population structure. However, host population genetic differentiation could have been generated if multiple Wolbachia strains interacted in some populations. Here, we investigated Wolbachia genetic variation in G. f. fuscipes populations of known host genetic composition in Uganda. We tested for the presence of multiple Wolbachia strains using Multi-Locus Sequence Typing (MLST) and for an association between geographic region and host mtDNA haplotype using Wolbachia DNA sequence from a variable locus, groEL (heat shock protein 60). RESULTS: MLST demonstrated that some G. f. fuscipes carry Wolbachia strains from two lineages. GroEL revealed high levels of sequence diversity within and between individuals (Haplotype diversity = 0.945). We found Wolbachia associated with 26 host mtDNA haplotypes, an unprecedented result. We observed a geographical association of one Wolbachia lineage with southern host mtDNA haplotypes, but it was non-significant (p = 0.16). Though most Wolbachia-infected host haplotypes were those found in the contact region between host mtDNA groups, this association was non-significant (p = 0.17). CONCLUSIONS: High Wolbachia sequence diversity and the association of Wolbachia with multiple host haplotypes suggest that different Wolbachia strains infected G. f. fuscipes multiple times independently. We suggest that these observations reflect a transient phase in Wolbachia evolution that is influenced by the long gestation and low reproductive output of tsetse. Although G. f. fuscipes is superinfected with Wolbachia, our data does not support that bidirectional CI has influenced host genetic diversity in Uganda

Cryptic diversity within the major trypanosomiasis vector Glossina fuscipes revealed by molecular markers

Background: The tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units. Principal Findings: The nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f.fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled fromEthiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations. Conclusion/Significance: We propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background: The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary haracterisation of the putative resistance mechanisms involved. Methodology/Principal Findings: A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F1 adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG. Conclusion: This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa

Genetic diversity and population structure of Glossina pallidipes in Uganda and western Kenya

<p>Abstract</p> <p>Background</p> <p><it>Glossina pallidipes </it>has been implicated in the spread of sleeping sickness from southeastern Uganda into Kenya. Recent studies indicated resurgence of <it>G. pallidipes </it>in Lambwe Valley and southeastern Uganda after what were deemed to be effective control efforts. It is unknown whether the <it>G. pallidipes </it>belt in southeastern Uganda extends into western Kenya. We investigated the genetic diversity and population structure of <it>G. pallidipes </it>in Uganda and western Kenya.</p> <p>Results</p> <p>AMOVA indicated that differences among sampling sites explained a significant proportion of the genetic variation. Principal component analysis and Bayesian assignment of microsatellite genotypes identified three distinct clusters: western Uganda, southeastern Uganda/Lambwe Valley, and Nguruman in central-southern Kenya. Analyses of mtDNA confirmed the results of microsatellite analysis, except in western Uganda, where Kabunkanga and Murchison Falls populations exhibited haplotypes that differed despite homogeneous microsatellite signatures. To better understand possible causes of the contrast between mitochondrial and nuclear markers we tested for sex-biased dispersal. Mean pairwise relatedness was significantly higher in females than in males within populations, while mean genetic distance was lower and relatedness higher in males than females in between-population comparisons. Two populations sampled on the Kenya/Uganda border, exhibited the lowest levels of genetic diversity. Microsatellite alleles and mtDNA haplotypes in these two populations were a subset of those found in neighboring Lambwe Valley, suggesting that Lambwe was the source population for flies in southeastern Uganda. The relatively high genetic diversity of <it>G. pallidipes </it>in Lambwe Valley suggest large relict populations remained even after repeated control efforts.</p> <p>Conclusion</p> <p>Our research demonstrated that <it>G. pallidipes </it>populations in Kenya and Uganda do not form a contiguous tsetse belt. While Lambwe Valley appears to be a source population for flies colonizing southeastern Uganda, this dispersal does not extend to western Uganda. The complicated phylogeography of <it>G. pallidipes </it>warrants further efforts to distinguish the role of historical and modern gene flow and possible sex-biased dispersal in structuring populations.</p

Polyandry Is a Common Event in Wild Populations of the Tsetse Fly Glossina fuscipes fuscipes and May Impact Population Reduction Measures

Glossina fuscipes fuscipes is the most common tsetse species in Uganda where it is responsible for transmitting Trypanosoma brucei rhodensiense and Trypanosoma brucei gambiense parasites causing sleeping sickness in humans in addition to related trypanosomes that cause Nagana in cattle. An understanding of the reproductive biology of this vector is essential for the application of sustainable control/eradication methods such as Sterile Insect Technique (SIT). We have analysed the number of times a female mates in the wild as this aspect of the reproductive behaviour may affect the stability and size of populations. We provide evidence that remating is a common event in the wild and females store sperm from multiple males, which may potentially be used for insemination. In vector eradication programmes, re-infestation of cleared areas and/or in cases of residual populations, the occurrence of remating may unfortunately enhance the reproductive potential of the re-invading propagules. We suggest that population age structure may influence remating frequency. Considering the seasonal demographic changes that this fly undergoes during the dry and wet seasons, control programmes based on SIT should release large numbers of sterile males, even in residual surviving target populations, in the dry season

Phylogeography and Population Structure of Glossina fuscipes fuscipes in Uganda: Implications for Control of Tsetse

Glossina fuscipes fuscipes is the most common species of tsetse in Uganda, where it transmits human sleeping sickness and nagana, a related disease of cattle. A consortium of African countries dedicated to controlling these diseases is poised to begin area wide control of tsetse, but a critical question remains: What is the most appropriate geographical scale for these activities? To address this question, we used population genetics to determine the extent of linkage between populations of tsetse confined to discrete patches of riverine habitat. Our results suggest that Uganda was colonized by two distinct lineages of G. f. fuscipes, which now co-occur only in a narrow band across central Uganda. Evidence for interbreeding at the zone of contact and movement of genes from the south to the north suggest that this historical genetic structure may dissolve in the future. At smaller scales, we have demonstrated that exchange of genes among neighboring populations via dispersal is at equilibrium with the differentiating force of genetic drift. Our results highlight the need for investment in vector control programs that account for the linkage observed among tsetse populations. Given its genetic isolation and its location at the far edge of G. fuscipes' range, the Lake Victoria region appears to be an appropriate target for area wide control

Genome sequence of the tsetse fly (Glossina morsitans):Vector of African trypanosomiasis

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.IS