Annotations of novel antennae-expressed genes in male Glossina morsitans morsitans tsetse flies.

Tsetse flies use antennal expressed genes to navigate their environment. While most canonical genes associated with chemoreception are annotated, potential gaps with important antennal genes are uncharacterized in Glossina morsitans morsitans. We generated antennae-specific transcriptomes from adult male G. m. morsitans flies fed/unfed on bloodmeal and/or exposed to an attractant (ε-nonalactone), a repellant (δ-nonalactone) or paraffin diluent. Using bioinformatics approach, we mapped raw reads onto G. m. morsitans gene-set from VectorBase and collected un-mapped reads (constituting the gaps in annotation). We de novo assembled these reads (un-mapped) into transcript and identified corresponding genes of the transcripts in G. m. morsitans gene-set and protein homologs in UniProt protein database to further annotate the gaps. We predicted potential protein-coding gene regions associated with these transcripts in G. m. morsitans genome, annotated/curated these genes and identified their putative annotated orthologs/homologs in Drosophila melanogaster, Musca domestica or Anopheles gambiae genomes. We finally evaluated differential expression of the novel genes in relation to odor exposures relative to no-odor control (unfed flies). About 45.21% of the sequenced reads had no corresponding transcripts within G. m. morsitans gene-set, corresponding to the gap in existing annotation of the tsetse fly genome. The total reads assembled into 72,428 unique transcripts, most (74.43%) of which had no corresponding genes in the UniProt database. We annotated/curated 592 genes from these transcripts, among which 202 were novel while 390 were improvements of existing genes in the G. m. morsitans genome. Among the novel genes, 94 had orthologs in D. melanogaster, M. domestica or An. gambiae while 88 had homologs in UniProt. These orthologs were putatively associated with oxidative regulation, protein synthesis, transcriptional and/or translational regulation, detoxification and metal ion binding, thus providing insight into their specific roles in antennal physiological processes in male G. m. morsitans. A novel gene (GMOY014237.R1396) was differentially expressed in response to the attractant. We thus established significant gaps in G. m. morsitans genome annotation and identified novel male antennae-expressed genes in the genome, among which > 53% (108) are potentially G. m. morsitans specific.

A machine learning approach to integrating genetic and ecological data in tsetse flies (Glossina pallidipes) for spatially explicit vector control planning.

Vector control is an effective strategy for reducing vector-borne disease transmission, but requires knowledge of vector habitat use and dispersal patterns. Our goal was to improve this knowledge for the tsetse species Glossina pallidipes, a vector of human and animal African trypanosomiasis, which are diseases that pose serious health and socioeconomic burdens across sub-Saharan Africa. We used random forest regression to (i) build and integrate models of G. pallidipes habitat suitability and genetic connectivity across Kenya and northern Tanzania and (ii) provide novel vector control recommendations. Inputs for the models included field survey records from 349 trap locations, genetic data from 11 microsatellite loci from 659 flies and 29 sampling sites, and remotely sensed environmental data. The suitability and connectivity models explained approximately 80% and 67% of the variance in the occurrence and genetic data and exhibited high accuracy based on cross-validation. The bivariate map showed that suitability and connectivity vary independently across the landscape and was used to inform our vector control recommendations. Post hoc analyses show spatial variation in the correlations between the most important environmental predictors from our models and each response variable (e.g., suitability and connectivity) as well as heterogeneity in expected future climatic change of these predictors. The bivariate map suggests that vector control is most likely to be successful in the Lake Victoria Basin and supports the previous recommendation that G. pallidipes from most of eastern Kenya should be managed as a single unit. We further recommend that future monitoring efforts should focus on tracking potential changes in vector presence and dispersal around the Serengeti and the Lake Victoria Basin based on projected local climatic shifts. The strong performance of the spatial models suggests potential for our integrative methodology to be used to understand future impacts of climate change in this and other vector systems.

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda.

Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense (Tbr) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease. Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive (Tbr EATRO-232) and non-responsive (Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo. We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr.

Population Structure and Migration Patterns of the Tsetse Fly Glossina fuscipes in Congo-Brazzaville.

Tsetse flies of the palpalis group, particularly Glossina fuscipes, are the main vectors of human African trypanosomiasis or sleeping sickness in Congo-Brazzaville. They transmit the deadly human parasite, Trypanosoma brucei gambiense and other trypanosomes that cause animal trypanosomiasis. Knowledge on diversity, population structure, population size, and gene flow is a prerequisite for designing effective tsetse control strategies. There is limited published information on these parameters including migration patterns of G. fuscipes in Congo-Brazzaville. We genotyped 288 samples of G. fuscipes from Bomassa (BMSA), Bouemba (BEMB), and Talangai (TLG) locations at 10 microsatellite loci and determined levels of genetic diversity, differentiation, structuring, and gene flow among populations. We observed high genetic diversity in all three localities. Mean expected heterozygosity was 0.77 ± 0.04, and mean allelic richness was 11.2 ± 1.35. Deficiency of heterozygosity was observed in all populations with positive and significant F IS values (0.077-0.149). Structure analysis revealed three clusters with genetic admixtures, evidence of closely related but potentially different taxa within G. fuscipes. Genetic differentiation indices were low but significant (F ST = 0.049, P < 0.05), indicating ongoing gene flow countered with a stronger force of drift. We recorded significant migration from all the three populations, suggesting exchange of genetic information between and among locations. Ne estimates revealed high and infinite population sizes in BEMB and TLG. These critical factors should be considered when planning area-wide tsetse control interventions in the country to prevent resurgence of tsetse from relict populations and/or reinvasion of cleared habitats.

Genetic diversity and phylogenetic relationships of tsetse flies of the palpalis group in Congo Brazzaville based on mitochondrial cox1 gene sequences.

BACKGROUND: Despite the morphological characterization established in the 1950s and 1960s, the identity of extant taxa that make up Glossina fuscipes (s.l.) in the Congo remains questionable. Previous claims of overlap between G. fuscipes (believed to be G. f. quanzensis) and G. palpalis palpalis around Brazzaville city further complicate the taxonomic status and population dynamics of the two taxa. This study aimed to determine the phylogenetic relationships between G. fuscipes (s.l.) and G. p. palpalis and to assess genetic variation among G. fuscipes (s.l.) populations in Congo Brazzaville. METHODS: We collected 263 G. fuscipes (s.l.) from northern and central regions, and 65 G. p. palpalis from southern part of the country. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using taxa-specific primer pairs. Sequence data were analyzed in DnaSP and Arlequin to assess the genetic diversity, differentiation and demographic history of G. fuscipes (s.l.) populations. RESULTS: The general BLAST analysis yielded a similarity of 99% for G. fuscipes (s.l.) and G. p. palpalis. BLASTn analysis for G. fuscipes (s.l.) showed > 98% identity with GenBank sequences for G. fuscipes (s.l.), with BEMB population showing 100% similarity with G. f. fuscipes. Glossina fuscipes (s.l.) populations showed high haplotype diversity (H = 46, Hd = 0.884), moderate nucleotide diversity ( = 0.012) and moderate (FST = 0.072) to high (FST = 0.152) genetic differentiation. Most of the genetic variation (89.73%) was maintained within populations. The mismatch analysis and neutrality tests indicated recent tsetse population expansions. CONCLUSIONS: Phylogenetic analysis revealed minor differences between G. fuscipes (s.l.) and G. p. palpalis. Genetic diversity of G. fuscipes (s.l.) was high in the populations sampled except one. Genetic differentiation ranged from moderate to high among subpopulations. There was a restricted gene flow between G. fuscipes (s.l.) populations in the north and central part of the country. Genetic signatures based on cox1 showed recent expansion and recovery of G. fuscipes (s.l.) populations from previous bottlenecks. To fully understand the species distribution limits, we recommend further studies involving a wider sampling scheme including the swampy Mossaka focus for G. fuscipes (s.l.) and the entire range of G. p. palpalis in South Congo.

Phylogeography and population structure of the tsetse fly Glossina pallidipes in Kenya and the Serengeti ecosystem.

Glossina pallidipes is the main vector of animal African trypanosomiasis and a potential vector of human African trypanosomiasis in eastern Africa where it poses a large economic burden and public health threat. Vector control efforts have succeeded in reducing infection rates, but recent resurgence in tsetse fly population density raises concerns that vector control programs require improved strategic planning over larger geographic and temporal scales. Detailed knowledge of population structure and dispersal patterns can provide the required information to improve planning. To this end, we investigated the phylogeography and population structure of G. pallidipes over a large spatial scale in Kenya and northern Tanzania using 11 microsatellite loci genotyped in 600 individuals. Our results indicate distinct genetic clusters east and west of the Great Rift Valley, and less distinct clustering of the northwest separate from the southwest (Serengeti ecosystem). Estimates of genetic differentiation and first-generation migration indicated high genetic connectivity within genetic clusters even across large geographic distances of more than 300 km in the east, but only occasional migration among clusters. Patterns of connectivity suggest isolation by distance across genetic breaks but not within genetic clusters, and imply a major role for river basins in facilitating gene flow in G. pallidipes. Effective population size (Ne) estimates and results from Approximate Bayesian Computation further support that there has been recent G. pallidipes population size fluctuations in the Serengeti ecosystem and the northwest during the last century, but also suggest that the full extent of differences in genetic diversity and population dynamics between the east and the west was established over evolutionary time periods (tentatively on the order of millions of years). Findings provide further support that the Serengeti ecosystem and northwestern Kenya represent independent tsetse populations. Additionally, we present evidence that three previously recognized populations (the Mbeere-Meru, Central Kenya and Coastal "fly belts") act as a single population and should be considered as a single unit in vector control.

Comparative genomic analysis of six Glossina genomes, vectors of African trypanosomes.

BACKGROUND: Tsetse flies (Glossina sp.) are the vectors of human and animal trypanosomiasis throughout sub-Saharan Africa. Tsetse flies are distinguished from other Diptera by unique adaptations, including lactation and the birthing of live young (obligate viviparity), a vertebrate blood-specific diet by both sexes, and obligate bacterial symbiosis. This work describes the comparative analysis of six Glossina genomes representing three sub-genera: Morsitans (G. morsitans morsitans, G. pallidipes, G. austeni), Palpalis (G. palpalis, G. fuscipes), and Fusca (G. brevipalpis) which represent different habitats, host preferences, and vectorial capacity. RESULTS: Genomic analyses validate established evolutionary relationships and sub-genera. Syntenic analysis of Glossina relative to Drosophila melanogaster shows reduced structural conservation across the sex-linked X chromosome. Sex-linked scaffolds show increased rates of female-specific gene expression and lower evolutionary rates relative to autosome associated genes. Tsetse-specific genes are enriched in protease, odorant-binding, and helicase activities. Lactation-associated genes are conserved across all Glossina species while male seminal proteins are rapidly evolving. Olfactory and gustatory genes are reduced across the genus relative to other insects. Vision-associated Rhodopsin genes show conservation of motion detection/tracking functions and variance in the Rhodopsin detecting colors in the blue wavelength ranges. CONCLUSIONS: Expanded genomic discoveries reveal the genetics underlying Glossina biology and provide a rich body of knowledge for basic science and disease control. They also provide insight into the evolutionary biology underlying novel adaptations and are relevant to applied aspects of vector control such as trap design and discovery of novel pest and disease control strategies.

Genetic Differentiation of Glossina pallidipes Tsetse Flies in Southern Kenya.

The tsetse fly Glossina pallidipes, the major vector of the parasite that causes animal African trypanosomiasis in Kenya, has been subject to intense control measures with only limited success. The G. pallidipes population dynamics and dispersal patterns that underlie limited success in vector control campaigns remain unresolved, and knowledge on genetic connectivity can provide insights, and thereby improve control and monitoring efforts. We therefore investigated the population structure and estimated migration and demographic parameters in G. pallidipes using genotypic data from 11 microsatellite loci scored in 250 tsetse flies collected from eight localities in Kenya. Clustering analysis identified two genetically distinct eastern and western clusters (mean between-cluster F ST = 0.202) separated by the Great Rift Valley. We also found evidence of admixture and migration between the eastern and western clusters, isolation by distance, and a widespread signal of inbreeding. We detected differences in population dynamics and dispersal patterns between the western and eastern clusters. These included lower genetic diversity (allelic richness; 7.48 versus 10.99), higher relatedness (percent related individuals; 21.4% versus 9.1%), and greater genetic differentiation (mean within-cluster F ST; 0.183 versus 0.018) in the western than the eastern cluster. Findings are consistent with the presence of smaller, less well-connected populations in Western relative to eastern Kenya. These data suggest that recent anthropogenic influences such as land use changes and vector control programs have influenced population dynamics in G. pallidipes in Kenya, and that vector control efforts should include some region-specific strategies to effectively control this disease vector.

Differential virulence of camel Trypanosoma evansi isolates in mice.

This study assessed the virulence of Trypanosoma evansi, the causative agent of camel trypanosomiasis (surra), affecting mainly camels among other hosts in Africa, Asia and South America, with high mortality and morbidity. Using Swiss white mice, we assessed virulence of 17 T. evansi isolates collected from surra endemic countries. We determined parasitaemia, live body weight, packed cell volume (PCV) and survivorship in mice, for a period of 60 days' post infection. Based on survivorship, the 17 isolates were classified into three virulence categories; low (31-60 days), moderate (11-30 days) and high (0-10 days). Differences in survivorship, PCV and bodyweights between categories were significant and correlated (P < 0.05). Of the 10 Kenyan isolates, four were of low, five moderate and one (Type B) of high virulence. These findings suggest differential virulence between T. evansi isolates. In conclusion, these results show that the virulence of T. evansi may be region specific, the phenotype of the circulating parasite should be considered in the management of surra. There is also need to collect more isolates from other surra endemic regions to confirm this observation.

Tsetse fly (Glossina pallidipes) midgut responses to Trypanosoma brucei challenge.

BACKGROUND: Tsetse flies (Glossina spp.) are the prominent vector of African trypanosome parasites (Trypanosoma spp.) in sub-Saharan Africa, and Glossina pallidipes is the most widely distributed species in Kenya. This species displays strong resistance to infection by parasites, which are typically eliminated in the midgut shortly after acquisition from the mammalian host. Although extensive molecular information on immunity for the related species Glossina morsitans morsitans exists, similar information is scarce for G. pallidipes. METHODS: To determine temporal transcriptional responses of G. pallidipes to Trypanosoma brucei brucei challenge, we conducted Illumina based RNA-seq on midgut organ and carcass from teneral females G. pallidipes at 24 and 48 h post-challenge (hpc) with T. b. brucei relative to their respective controls that received normal blood meals (without the parasite). We used a suite of bioinformatics tools to determine differentially expressed and enriched transcripts between and among tissues, and to identify expanded transcripts in G. pallidipes relative to their orthologs G. m. morsitans. RESULTS: Midgut transcripts induced at 24 hpc encoded proteins were associated with lipid remodelling, proteolysis, collagen metabolism, apoptosis, and cell growth. Midgut transcripts induced at 48 hpc encoded proteins linked to embryonic growth and development, serine endopeptidases and proteosomal degradation of the target protein, mRNA translation and neuronal development. Temporal expression of immune responsive transcripts at 48 relative to 24 hpc was pronounced, indicative of a gradual induction of host immune responses the following challenge. We also searched for G. m. morsitans orthologous groups that may have experienced expansions in the G. pallidipes genome. We identified ten expanded groups in G. pallidipes with putative immunity-related functions, which may play a role in the higher refractoriness exhibited by this species. CONCLUSIONS: There appears to be a lack of strong immune responses elicited by gut epithelia of teneral adults. This in combination with a compromised peritrophic matrix at this stage during the initial phase of T. b. brucei challenge may facilitate the increased parasite infection establishment noted in teneral flies relative to older adults. Although teneral flies are more susceptible than older adults, the majority of tenerals are still able to eliminate parasite infections. Hence, robust responses elicited at a later time point, such as 72 hpc, may clear parasite infections from the majority of flies. The expanded G. m. morsitans orthologous groups in G. pallidipes may also be functionally associated with the enhanced refractoriness to trypanosome infections reported in G. pallidipes relative to G. m. morsitans.

Temporal genetic differentiation in Glossina pallidipes tsetse fly populations in Kenya.

BACKGROUND: Glossina pallidipes is a major vector of both Human and Animal African Trypanosomiasis (HAT and AAT) in Kenya. The disease imposes economic burden on endemic regions in Kenya, including south-western Kenya, which has undergone intense but unsuccessful tsetse fly control measures. We genotyped 387 G. pallidipes flies at 13 microsatellite markers to evaluate levels of temporal genetic variation in two regions that have been subjected to intensive eradication campaigns from the 1960s to the 1980s. One of the regions, Nguruman Escarpment, has been subject to habitat alteration due to human activities, while the other, Ruma National Park, has not. In addition, Nguruman Escarpment is impacted by the movement of grazing animals into the area from neighboring regions during the drought season. We collected our samples from three geographically close sampling sites for each of the two regions. Samples were collected between the years 2003 and 2015, spanning ~96 tsetse fly generations. RESULTS: We established that allelic richness averaged 3.49 and 3.63, and temporal Ne estimates averaged 594 in Nguruman Escarpment and 1120 in Ruma National Park. This suggests that genetic diversity is similar to what was found in previous studies of G. pallidipes in Uganda and Kenya, implying that we could not detect a reduction in genetic diversity following the extensive control efforts during the 1960s to the 1980s. However, we did find differences in temporal patterns of genetic variation between the two regions, indicated by clustering analysis, pairwise FST, and Fisher's exact tests for changes in allele and genotype frequencies. In Nguruman Escarpment, findings indicated differentiation among samples collected in different years, and evidence of local genetic bottlenecks in two locations previous to 2003, and between 2009 and 2015. In contrast, there was no consistent evidence of differentiation among samples collected in different years, and no evidence of local genetic bottlenecks in Ruma National Park. CONCLUSION: Our findings suggest that, despite extensive control measures especially between the 1960s and the 1980s, tsetse flies in these regions persist with levels of genetic diversity similar to that found in populations that did not experience extensive control measures. Our findings also indicate temporal genetic differentiation in Nguruman Escarpment detected at a scale of > 80 generations, and no similar temporal differentiation in Ruma National Park. The different level of temporal differentiation between the two regions indicates that genetic drift is stronger in Nugruman Escarpment, for as-yet unknown reasons, which may include differences in land management. This suggests land management may have an impact on G. pallidipes population genetics, and reinforces the importance of long term monitoring of vector populations in estimates of parameters needed to model and plan effective species-specific control measures.

Genetic diversity and population structure of Trypanosoma brucei in Uganda: implications for the epidemiology of sleeping sickness and Nagana.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.