Loyce M. Okedi

Molecular phylogenetics of tsetse flies (Diptera: Glossinidae) based on mitochondrial (COI, 16S, ND2) and nuclear ribosomal DNA sequences, with an emphasis on the palpalis group

Relationships of 13 species of the genus Glossina (tsetse flies) were inferred from mitochondrial (cytochrome oxidase 1, NADH dehydrogenase 2 and 16S) and nuclear (internal transcribed spacer 1 of rDNA) sequences. The resulting phylogeny confirms the monophyly of the morphologically defined fusca, morsitans and palpalis subgenera. Genetic distances between palpalis and morsitans subspecies suggest that their status needs revision. In particular, cytochrome oxidase 1 sequences showed large geographical differences within G. palpalis palpalis, suggesting the existence of cryptic species within this subspecies. The morphology of palpalis group female genital plates was examined, and individuals were found varying outside the ranges specified by the standard identification keys, making definitive morphological classification impossible. A diagnostic PCR to distinguish G. palpalis palpalis, G. tachinoides and G. palpalis gambiensis based on length differences of internal transcribed spacer 1 sequences is presented.

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

Background Glossina fuscipes fuscipes, a riverine species of tsetse, is the main vector of both human and animal trypanosomiasis in Uganda. Successful implementation of vector control will require establishing an appropriate geographical scale for these activities. Population genetics can help to resolve this issue by characterizing the extent of linkage among apparently isolated groups of tsetse. Methodology/Principal Findings We conducted genetic analyses on mitochondrial and microsatellite data accumulated from approximately 1000 individual tsetse captured in Uganda and neighboring regions of Kenya and Sudan. Phylogeographic analyses suggested that the largest scale genetic structure in G. f. fuscipes arose from an historical event that divided two divergent mitochondrial lineages. These lineages are currently partitioned to northern and southern Uganda and co-occur only in a narrow zone of contact extending across central Uganda. Bayesian assignment tests, which provided evidence for admixture between northern and southern flies at the zone of contact and evidence for northerly gene flow across the zone of contact, indicated that this structure may be impermanent. On the other hand, microsatellite structure within the southern lineage indicated that gene flow is currently limited between populations in western and southeastern Uganda. Within regions, the average FST between populations separated by less than 100 km was less than ∼0.1. Significant tests of isolation by distance suggested that gene flow is ongoing between neighboring populations and that island populations are not uniformly more isolated than mainland populations. Conclusions/Significance Despite the presence of population structure arising from historical colonization events, our results have revealed strong signals of current gene flow within regions that should be accounted for when planning tsetse control in Uganda. Populations in southeastern Uganda appeared to receive little gene flow from populations in western or northern Uganda, supporting the feasibility of area wide control in the Lake Victoria region by the Pan African Tsetse and Trypanosomiasis Eradication Campaign.

Glossina fuscipes populations provide insights for Human African Trypanosomiasis transmission in Uganda

Uganda has both forms of human African trypanosomiasis (HAT): the chronic gambiense disease in the northwest and the acute rhodesiense disease in the south. The recent spread of rhodesiense into central Uganda has raised concerns given the different control strategies the two diseases require. We present knowledge on the population genetics of the major vector species Glossina fuscipes fuscipes in Uganda with a focus on population structure, measures of gene flow between populations, and the occurrence of polyandry. The microbiome composition and diversity is discussed, focusing on their potential role on trypanosome infection outcomes. We discuss the implications of these findings for large-scale tsetse control programs, including suppression or eradication, being undertaken in Uganda, and potential future genetic applications.

Implications of microfauna-host interactions for trypanosome transmission dynamics in Glossina fuscipes fuscipes in Uganda.

ABSTRACT Tsetse flies (Diptera: Glossinidae) are vectors for African trypanosomes (Euglenozoa: kinetoplastida), protozoan parasites that cause African trypanosomiasis in humans (HAT) and nagana in livestock. In addition to trypanosomes, two symbiotic bacteria (Wigglesworthia glossinidia and Sodalis glossinidius) and two parasitic microbes, Wolbachia and a salivary gland hypertrophy virus (SGHV), have been described in tsetse. Here we determined the prevalence of and coinfection dynamics between Wolbachia, trypanosomes, and SGHV in Glossina fuscipes fuscipes in Uganda over a large geographical scale spanning the range of host genetic and spatial diversity. Using a multivariate analysis approach, we uncovered complex coinfection dynamics between the pathogens and statistically significant associations between host genetic groups and pathogen prevalence. It is important to note that these coinfection dynamics and associations with the host were not apparent by univariate analysis. These associations between host genotype and pathogen are particularly evident for Wolbachia and SGHV where host groups are inversely correlated for Wolbachia and SGHV prevalence. On the other hand, trypanosome infection prevalence is more complex and covaries with the presence of the other two pathogens, highlighting the importance of examining multiple pathogens simultaneously before making generalizations about infection and spatial patterns. It is imperative to note that these novel findings would have been missed if we had employed the standard univariate analysis used in previous studies. Our results are discussed in the context of disease epidemiology and vector control.

Analysis of Multiple Tsetse Fly Populations in Uganda Reveals Limited Diversity and Species-Specific Gut Microbiota

ABSTRACT The invertebrate microbiome contributes to multiple aspects of host physiology, including nutrient supplementation and immune maturation processes. We identified and compared gut microbial abundance and diversity in natural tsetse flies from Uganda using five genetically distinct populations of Glossina fuscipes fuscipes and multiple tsetse species (Glossina morsitans morsitans, G. f. fuscipes, and Glossina pallidipes) that occur in sympatry in one location. We used multiple approaches, including deep sequencing of the V4 hypervariable region of the 16S rRNA gene, 16S rRNA gene clone libraries, and bacterium-specific quantitative PCR (qPCR), to investigate the levels and patterns of gut microbial diversity from a total of 151 individuals. Our results show extremely limited diversity in field flies of different tsetse species. The obligate endosymbiont Wigglesworthia dominated all samples (>99%), but we also observed wide prevalence of low-density Sodalis (tsetses commensal endosymbiont) infections (<0.05%). There were also several individuals (22%) with high Sodalis density, which also carried coinfections with Serratia. Albeit in low density, we noted differences in microbiota composition among the genetically distinct G. f. fuscipes flies and between different sympatric species. Interestingly, Wigglesworthia density varied in different species (104 to 106 normalized genomes), with G. f. fuscipes having the highest levels. We describe the factors that may be responsible for the reduced diversity of tsetses gut microbiota compared to those of other insects. Additionally, we discuss the implications of Wigglesworthia and Sodalis density variations as they relate to trypanosome transmission dynamics and vector competence variations associated with different tsetse species.

Temporal stability of Glossina fuscipes fuscipes populations in Uganda

BackgroundGlossina fuscipes, a riverine species of tsetse, is the major vector of human African trypanosomiasis (HAT) in sub-Saharan Africa. Understanding the population dynamics, and specifically the temporal stability, of G. fuscipes will be important for informing vector control activities. We evaluated genetic changes over time in seven populations of the subspecies G. f. fuscipes distributed across southeastern Uganda, including a zone of contact between two historically isolated lineages. A total of 667 tsetse flies were genotyped at 16 microsatellite loci and at one mitochondrial locus.ResultsResults of an AMOVA indicated that time of sampling did not explain a significant proportion of the variance in allele frequencies observed across all samples. Estimates of differentiation between samples from a single population ranged from approximately 0 to 0.019, using Josts DEST. Effective population size estimates using momentum-based and likelihood methods were generally large. We observed significant change in mitochondrial haplotype frequencies in just one population, located along the zone of contact. The change in haplotypes was not accompanied by changes in microsatellite frequencies, raising the possibility of asymmetric mating compatibility in this zone.ConclusionOur results suggest that populations of G. f. fuscipes were stable over the 8-12 generations studied. Future studies should aim to reconcile these data with observed seasonal fluctuations in the apparent density of tsetse.

The population structure of Glossina fuscipes fuscipes in the Lake Victoria basin in Uganda: implications for vector control

BackgroundGlossina fuscipes fuscipes is the primary vector of trypanosomiasis in humans and livestock in Uganda. The Lake Victoria basin has been targeted for tsetse eradication using a rolling carpet initiative, from west to east, with four operational blocks (3 in Uganda and 1 in Kenya), under a Pan-African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). We screened tsetse flies from the three Ugandan PATTEC blocks for genetic diversity at 15 microsatellite loci from continental and offshore populations to provide empirical data to support this initiative.MethodsWe collected tsetse samples from 11 sites across the Lake Victoria basin in Uganda. We performed genetic analyses on 409 of the collected tsetse flies and added data collected for 278 individuals in a previous study. The flies were screened across 15 microsatellite loci and the resulting data were used to assess the temporal stability of populations, to analyze patterns of genetic exchange and structuring, to estimate dispersal rates and evaluate the sex bias in dispersal, as well as to estimate demographic parameters (NE and NC).ResultsWe found that tsetse populations in this region were stable over 4-16 generations and belong to 4 genetic clusters. Two genetic clusters (1 and 2) corresponded approximately to PATTEC blocks 1 and 2, while the other two (3 and 4) fell within PATTEC block 3. Island populations grouped into the same genetic clusters as neighboring mainland sites, suggesting presence of gene flow between these sites. There was no evidence of the stretch of water separating islands from the mainland forming a significant barrier to dispersal. Dispersal rates ranged from 2.5 km per generation in cluster 1 to 14 km per generation in clusters 3 and 4. We found evidence of male-biased dispersal. Few breeders are successfully dispersing over large distances. Effective population size estimates were low (33–310 individuals), while census size estimates ranged from 1200 (cluster 1) to 4100 (clusters 3 and 4). We present here a novel technique that adapts an existing census size estimation method to sampling without replacement, the scheme used in sampling tsetse flies.ConclusionOur study suggests that different control strategies should be implemented for the three PATTEC blocks and that, given the high potential for re-invasion from island sites, mainland and offshore sites in each block should be targeted at the same time.

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 from Ethiopia. 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.

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

Background Glossina fuscipes fuscipes is the main vector of human and animal trypanosomiasis in Africa, particularly in Uganda. Attempts to control/eradicate this species using biological methods require knowledge of its reproductive biology. An important aspect is the number of times a female mates in the wild as this influences the effective population size and may constitute a critical factor in determining the success of control methods. To date, polyandry in G.f. fuscipes has not been investigated in the laboratory or in the wild. Interest in assessing the presence of remating in Ugandan populations is driven by the fact that eradication of this species is at the planning stage in this country. Methodology/Principal Findings Two well established populations, Kabukanga in the West and Buvuma Island in Lake Victoria, were sampled to assess the presence and frequency of female remating. Six informative microsatellite loci were used to estimate the number of matings per female by genotyping sperm preserved in the female spermathecae. The direct count of the minimum number of males that transferred sperm to the spermathecae was compared to Maximum Likelihood and Bayesian probability estimates. The three estimates provided evidence that remating is common in the populations but the frequency is substantially different: 57% in Kabukanga and 33% in Buvuma. Conclusions/Significance The presence of remating, with females maintaining sperm from different mates, may constitute a critical factor in cases of re-infestation of cleared areas and/or of residual populations. Remating may enhance the reproductive potential of re-invading propagules in terms of their effective population size. 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.

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

BackgroundWolbachia 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).ResultsMLST 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).ConclusionsHigh 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.