[Show abstract][Hide abstract] ABSTRACT: Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.
[Show abstract][Hide abstract] ABSTRACT: Glossina fuscipes fuscipes is the main vector of African Trypanosomiasis affecting both humans and livestock in Uganda. The human disease (sleeping sickness) manifests itself in two forms: acute and chronic. The Lake Victoria basin in Uganda has the acute form and a history of tsetse re-emergence despite concerted efforts to control tsetse. The government of Uganda has targeted the basin for tsetse eradication. To provide empirical data for this initiative, we screened tsetse flies from the basin for genetic variation at the mitochondrial DNA cytochrome oxidase II (mtDNA COII) gene with the goal of investigating genetic diversity and gene flow among tsetse, tsetse demographic history; and compare these results with results from a previous study based on microsatellite loci data in the same area.
We collected 429 Gff tsetse fly samples from 14 localities in the entire Ugandan portion of the Lake Victoria coast, covering 40,000 km 2 . We performed genetic analyses on them and added data collected for 56 Gff individuals from 4 additional sampling sites in the basin. The 529pb partial mitochondrial DNA cytochrome oxidase II (mtDNA COII) sequences totaling 485 were analysed for genetic differentiation, structuring and demographic history. The results were compared with findings from a previous study based on microsatellite loci data from the basin.
The differences within sampling sites explained a significant proportion of the genetic variation. We found three very closely related mtDNA population clusters, which co-occurred in multiple sites. Although Φ ST (0 – 0.592; P < 0.05) and Bayesian analyses suggest some level of weak genetic differentiation, there is no correlation between genetic divergence and geographic distance (r = 0.109, P = 0.185), and demographic tests provide evidence of locality-based demographic history.
The mtDNA data analysed here complement inferences made in a previous study based on microsatellite data. Given the differences in mutation rates, mtDNA afforded a look further back in time than microsatellites and revealed that Gff populations were more connected in the past. Microsatellite data revealed more genetic structuring than mtDNA. The differences in connectedness and structuring over time could be related to vector control efforts. Tsetse re-emergence after control interventions may be due to re-invasions from outside the treated areas, which emphasizes the need for an integrated area-wide tsetse eradication strategy for sustainable removal of the tsetse and trypanosomiasis problem from this area.
[Show abstract][Hide abstract] ABSTRACT: While Human African Trypanosomiasis (HAT) is in decline on the continent of Africa, the disease still remains a major health problem in Uganda. There are recurrent sporadic outbreaks in the traditionally endemic areas in south-east Uganda, and continued spread to new unaffected areas in central Uganda. We evaluated the evolutionary dynamics underpinning the origin of new foci and the impact of host species on parasite genetic diversity in Uganda. We genotyped 269 Trypanosoma brucei isolates collected from different regions in Uganda and southwestern Kenya at 17 microsatellite loci, and checked for the presence of the SRA gene that confers human infectivity to T. b. rhodesiense.
Both Bayesian clustering methods and Discriminant Analysis of Principal Components partition Trypanosoma brucei isolates obtained from Uganda and southwestern Kenya into three distinct genetic clusters. Clusters 1 and 3 include isolates from central and southern Uganda, while cluster 2 contains mostly isolates from southwestern Kenya. These three clusters are not sorted by subspecies designation (T. b. brucei vs T. b. rhodesiense), host or date of collection. The analyses also show evidence of genetic admixture among the three genetic clusters and long-range dispersal, suggesting recent and possibly on-going gene flow between them.
Our results show that the expansion of the disease to the new foci in central Uganda occurred from the northward spread of T. b. rhodesiense (Tbr). They also confirm the emergence of the human infective strains (Tbr) from non-infective T. b. brucei (Tbb) strains of different genetic backgrounds, and the importance of cattle as Tbr reservoir, as confounders that shape the epidemiology of sleeping sickness in the region.
[Show abstract][Hide abstract] 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 (tsetse's 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 tsetse's 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
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Tsetse flies (Glossina spp.) are the sole vectors of Trypanosoma brucei-the agent of human (HAT) and animal (AAT) trypanosomiasis. Glossina fuscipes fuscipes (Gff) is the main vector species in Uganda-the only country where the two forms of HAT disease (rhodesiense and gambiense) occur, with gambiense limited to the northwest. Gff populations cluster in three genetically distinct groups in northern, southern, and western Uganda, respectively, with a contact zone present in central Uganda. Understanding the dynamics of this contact zone is epidemiologically important as the merger of the two diseases is a major health concern. We used mitochondrial and microsatellite DNA data from Gff samples in the contact zone to understand its spatial extent and temporal stability. We show that this zone is relatively narrow, extending through central Uganda along major rivers with south to north introgression but displaying no sex-biased dispersal. Lack of obvious vicariant barriers suggests that either environmental conditions or reciprocal competitive exclusion could explain the patterns of genetic differentiation observed. Lack of admixture between northern and southern populations may prevent the sympatry of the two forms of HAT disease, although continued control efforts are needed to prevent the recolonization of tsetse-free regions by neighboring populations.
[Show abstract][Hide abstract] ABSTRACT: 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.
Trends in Parasitology 07/2013; 29(8). DOI:10.1016/j.pt.2013.06.005 · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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).
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).
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.
[Show abstract][Hide abstract] ABSTRACT: Background
Glossina 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.
We 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).
We 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.
Our 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Vertical transmission of obligate symbionts generates a predictable evolutionary history of symbionts that reflects that of
their hosts. In insects, evolutionary associations between symbionts and their hosts have been investigated primarily among
species, leaving population-level processes largely unknown. In this study, we investigated the tsetse (Diptera: Glossinidae)
bacterial symbiont, Wigglesworthia glossinidia, to determine whether observed codiversification of symbiont and tsetse host species extends to a single host species (Glossina fuscipes fuscipes) in Uganda. To explore symbiont genetic variation in G. f. fuscipes populations, we screened two variable loci (lon and lepA) from the Wigglesworthia glossinidia bacterium in the host species Glossina fuscipes fuscipes (W. g. fuscipes) and examined phylogeographic and demographic characteristics in multiple host populations. Symbiont genetic variation was
apparent within and among populations. We identified two distinct symbiont lineages, in northern and southern Uganda. Incongruence
length difference (ILD) tests indicated that the two lineages corresponded exactly to northern and southern G. f. fuscipes mitochondrial DNA (mtDNA) haplogroups (P = 1.0). Analysis of molecular variance (AMOVA) confirmed that most variation was partitioned between the northern and southern
lineages defined by host mtDNA (85.44%). However, ILD tests rejected finer-scale congruence within the northern and southern
populations (P = 0.009). This incongruence was potentially due to incomplete lineage sorting that resulted in novel combinations of symbiont
genetic variants and host background. Identifying these novel combinations may have public health significance, since tsetse
is the sole vector of sleeping sickness and Wigglesworthia is known to influence host vector competence. Thus, understanding the adaptive value of these host-symbiont combinations
may afford opportunities to develop vector control methods.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Glossina pallidipes has been implicated in the spread of sleeping sickness from southeastern Uganda into Kenya. Recent studies indicated resurgence of G. pallidipes in Lambwe Valley and southeastern Uganda after what were deemed to be effective control efforts. It is unknown whether the G. pallidipes belt in southeastern Uganda extends into western Kenya. We investigated the genetic diversity and population structure of G. pallidipes in Uganda and western Kenya.
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 G. pallidipes in Lambwe Valley suggest large relict populations remained even after repeated control efforts.
Our research demonstrated that G. pallidipes 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 G. pallidipes warrants further efforts to distinguish the role of historical and modern gene flow and possible sex-biased dispersal in structuring populations.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: http://tomato.biol.trinity.edu/manuscripts/11-3/mer-10-0402.pdf We searched the Glossina morsitans morsitans genome for short sequence repeats (SSR) in order to adapt polymorphic microsatellite markers to other species of Glossina, G. fuscipes fuscipes and G. pallidipes, two major vectors of African trypanosomiasis. We tested 30 loci containing perfect di-, tri-, or tetranucleotide repeats. We identified seven polymorphic loci that amplified across both G.f. fuscipes and G. pallidipes samples, as well as seven additional loci that were variable in just one species. Five of these fourteen loci were homozygous in males of one or both species and are likely to be X-linked. Although the success rate of adapting SSR markers from the G.m. morsitans genome for use in other species was not very high, this process yielded several polymorphic markers that should be useful in future studies of tsetse ecology and evolution.