The pathogenicity of blood stream and central nervous system forms of Trypanosoma brucei rhodesiense trypanosomes in laboratory mice: a comparative study.

Background: Human African trypanosomiasis (HAT) develops in two stages namely early stage when trypanosomes are found in the blood and late stage when trypanosomes are found in the central nervous system (CNS). The two environments are different with CNS environment reported as being hostile to the trypanosomes than the blood environment. The clinical symptoms manifested by the disease in the two environments are different. Information on whether blood stream are pathologically different from CNS trypanosomes is lacking. This study undertook to compare the inter-isolate pathological differences caused by bloodstream forms (BSF) and central nervous system (CNS) of five Trypanosoma brucei rhodesiense ( Tbr) isolates in Swiss white mice. Methods: Donor mice infected with each of the five isolates were euthanized at 21 days post infection (DPI) for recovery of BSF trypanosomes in heart blood and CNS trypanosomes in brain supernatants. Groups of Swiss white mice (n = 10) were then infected with BSF or CNS forms of each isolate and monitored for parasitaemia, packed cell volume (PCV), body weight, survivorship, trypanosome length, gross and histopathology characteristics. Results: Amplification of SRA gene prior to trypanosome morphology and pathogenicity studies confirmed all isolates as T. b. rhodesiense. At 21 DPI, CNS trypanosomes were predominantly long slender (LS) while BSF were a mixture of short stumpy and intermediate forms. The density of BSF trypanosomes was on average 2-3 log-scales greater than that of CNS trypanosomes with isolate KETRI 2656 having the highest CNS trypanosome density. Conclusions: The pathogenicity study revealed clear differences in the virulence/pathogenicity of the five (5) isolates but no distinct and consistent differences between CNS and BSF forms of the same isolate. We also identified KETRI 2656 as a suitable isolate for acute menigo- encephalitic studies.

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.

Characterization of a composite with enhanced attraction to savannah tsetse flies from constituents or analogues of tsetse refractory waterbuck (Kobus defassa) body odor.

Savannah tsetse flies avoid flying toward tsetse fly-refractory waterbuck (Kobus defassa) mediated by a repellent blend of volatile compounds in their body odor comprised of δ-octalactone, geranyl acetone, phenols (guaiacol and carvacrol), and homologues of carboxylic acids (C5-C10) and 2-alkanones (C8-C13). However, although the blends of carboxylic acids and that of 2-alkanones contributed incrementally to the repellency of the waterbuck odor to savannah tsetse flies, some waterbuck constituents (particularly, nonanoic acid and 2-nonanone) showed significant attractive properties. In another study, increasing the ring size of δ-octalactone from six to seven membered ring changed the activity of the resulting molecule (ε-nonalactone) on the savannah tsetse flies from repellency to attraction. In the present study, we first compared the effect of blending ε-nonalactone, nonanoic acid and 2-nonanone in 1:1 binary and 1:1:1 ternary combination on responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies in a two-choice wind tunnel. The compounds showed clear synergistic effects in the blends, with the ternary blend demonstrating higher attraction than the binary blends and individual compounds. Our follow up laboratory comparisons of tsetse fly responses to ternary combinations with different relative proportions of the three components showed that the blend in 1:3:2 proportion was most attractive relative to fermented cow urine (FCU) to both tsetse species. In our field experiments at Shimba Hills game reserve in Kenya, where G. pallidipes are dominant, the pattern of tsetse catches we obtained with different proportions of the three compounds were similar to those we observed in the laboratory. Interestingly, the three-component blend in 1:3:2 proportion when released at optimized rate of 13.71mg/h was 235% more attractive to G. pallidipes than a combination of POCA (3-n-Propylphenol, 1-Octen-3-ol, 4-Cresol, and Acetone) and fermented cattle urine (FCU). This constitutes a novel finding with potential for downstream deployment in bait technologies for more effective control of G. pallidipes, G. m. morsitans, and perhaps other savannah tsetse fly species, in 'pull' and 'pull-push' tactics.

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model.

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

Blending studies with selected waterbuck odor constituents or analogues in the development of a potent repellent blend against savannah tsetse.

Previous comparison of the body odors of tsetse-refractory waterbuck and those of tsetse-attractive ox and buffalo showed that a blend of 15 EAG-active compounds specific to waterbuck, including C5-C10 straight chain carboxylic acid homologues, methyl ketones (C8-C12 straight chain homologues and geranyl acetone), phenols (guaiacol and carvacrol) and δ-octalactone, was repellent to tsetse. A blend of four components selected from each class of compounds (δ-octalactone, pentanoic acid, guaiacol, and geranylacetone) showed repellence that is comparable to that of the 15 components blend and can provide substantial protection to cattle (more than 80%) from tsetse bites and trypanosome infections. Structure-activity studies with the lactone and phenol analogues showed that δ-nonalactone and 4-methylguaiacol are significantly more repellent than δ-octalactone and guaiacol, respectively. In the present study, we compared the responses of Glossina pallidipes and Glossina morsitans to i) blends comprising of various combinations of the most active analogues from each class of compounds, and ii) a four-component blend of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranyl acetone in different ratios in a two-choice wind-tunnel, followed by a field study with G. pallidipes population in a completely randomized Latin Square Design set ups. In the wind tunnel experiments, the blend of the four compounds in 6:4:2:1 ratio was found to be significantly more repellent (94.53%) than that in 1:1:1:1 proportion and those in other ratios. G. m. morsitans also showed a similar pattern of results. In field experiments with G. pallidipes population, the 6:4:2:1 blend of the four compounds also gave similar results. The results lay down useful groundwork in the large-scale development of more effective 'push' and 'push-pull' control tactics of the tsetse flies.

Expansions of chemosensory gene orthologs among selected tsetse fly species and their expressions in Glossina morsitans morsitans tsetse fly.

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7) expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d, Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b, GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes in the head were associated with vision, muscle activity and neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments (>90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators.

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 in vitro transportation of pentamidine across the blood-brain barrier using polycaprolactone nanoparticles and phosphatidylcholine liposomes.

Nanoparticles (NPs) have gained importance in addressing drug delivery challenges across biological barriers. Here, we reformulated pentamidine, a drug used to treat Human African Trypanosomiasis (HAT) in polymer based nanoparticles and liposomes and compared their capability to enhance pentamidine penetration across blood brain barrier (BBB). Size, polydispersity index, zeta potential, morphology, pentamidine loading and drug release profiles were determined by various methods. Cytotoxicity was tested against the immortalized mouse brain endothelioma cells over 96 h. Moreover, cells monolayer integrity and transportation ability were examined for 24 h. Pentamidine-loaded polycaprolactone (PCL) nanoparticles had a mean size of 267.58, PDI of 0.25 and zeta potential of -28.1 mV and pentamidine-loaded liposomes had a mean size of 119.61 nm, PDI of 0.25 and zeta potential 11.78. Pentamidine loading was 0.16 µg/mg (w/w) and 0.17 µg/mg (w/w) in PCL NPs and liposomes respectively. PCL nanoparticles and liposomes released 12.13% and 22.21% of pentamidine respectively after 24 h. Liposomes transported 87% of the dose, PCL NPs 66% of the dose and free pentamidine penetration was 63% of the dose. These results suggest that liposomes are comparatively promising nanocarriers for transportation of pentamidine across BBB.

Analysis of the gut-specific microbiome from field-captured tsetse flies, and its potential relevance to host trypanosome vector competence.

BACKGROUND: The tsetse fly (Glossina sp.) midgut is colonized by maternally transmitted and environmentally acquired bacteria. Additionally, the midgut serves as a niche in which pathogenic African trypanosomes reside within infected flies. Tsetse's bacterial microbiota impacts many aspects of the fly's physiology. However, little is known about the structure of tsetse's midgut-associated bacterial communities as they relate to geographically distinct fly habitats in east Africa and their contributions to parasite infection outcomes. We utilized culture dependent and independent methods to characterize the taxonomic structure and density of bacterial communities that reside within the midgut of tsetse flies collected at geographically distinct locations in Kenya and Uganda. RESULTS: Using culture dependent methods, we isolated 34 strains of bacteria from four different tsetse species (G. pallidipes, G. brevipalpis, G. fuscipes and G. fuscipleuris) captured at three distinct locations in Kenya. To increase the depth of this study, we deep sequenced midguts from individual uninfected and trypanosome infected G. pallidipes captured at two distinct locations in Kenya and one in Uganda. We found that tsetse's obligate endosymbiont, Wigglesworthia, was the most abundant bacterium present in the midgut of G. pallidipes, and the density of this bacterium remained largely consistent regardless of whether or not its tsetse host was infected with trypanosomes. These fly populations also housed the commensal symbiont Sodalis, which was found at significantly higher densities in trypanosome infected compared to uninfected flies. Finally, midguts of field-captured G. pallidipes were colonized with distinct, low density communities of environmentally acquired microbes that differed in taxonomic structure depending on parasite infection status and the geographic location from which the flies were collected. CONCLUSIONS: The results of this study will enhance our understanding of the tripartite relationship between tsetse, its microbiota and trypanosome vector competence. This information may be useful for developing novel disease control strategies or enhancing the efficacy of those already in use.

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.

Expression profiling of Trypanosoma congolense genes during development in the tsetse fly vector Glossina morsitans morsitans.

BACKGROUND: The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense's developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies. METHODS: In this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370). RESULTS: Consistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse's cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 'GARP', 'iii' and 'i' subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse's cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans' proboscis. CONCLUSION: Collectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse's proboscis. We discuss our results in terms of their relevance to disease transmission processes.

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.

Molecular characterization of tsetse's proboscis and its response to Trypanosoma congolense infection.

Tsetse flies (Glossina spp.) transmit parasitic African trypanosomes (Trypanosoma spp.), including Trypanosoma congolense, which causes animal African trypanosomiasis (AAT). AAT detrimentally affects agricultural activities in sub-Saharan Africa and has negative impacts on the livelihood and nutrient availability for the affected communities. After tsetse ingests an infectious blood meal, T. congolense sequentially colonizes the fly's gut and proboscis (PB) organs before being transmitted to new mammalian hosts during subsequent feedings. Despite the importance of PB in blood feeding and disease transmission, little is known about its molecular composition, function and response to trypanosome infection. To bridge this gap, we used RNA-seq analysis to determine its molecular characteristics and responses to trypanosome infection. By comparing the PB transcriptome to whole head and midgut transcriptomes, we identified 668 PB-enriched transcripts that encoded proteins associated with muscle tissue, organ development, chemosensation and chitin-cuticle structure development. Moreover, transcripts encoding putative mechanoreceptors that monitor blood flow during tsetse feeding and interact with trypanosomes were also expressed in the PB. Microscopic analysis of the PB revealed cellular structures associated with muscles and cells. Infection with T. congolense resulted in increased and decreased expression of 38 and 88 transcripts, respectively. Twelve of these differentially expressed transcripts were PB-enriched. Among the transcripts induced upon infection were those encoding putative proteins associated with cell division function(s), suggesting enhanced tissue renewal, while those suppressed were associated with metabolic processes, extracellular matrix and ATP-binding as well as immunity. These results suggest that PB is a muscular organ with chemosensory and mechanosensory capabilities. The mechanoreceptors may be point of PB-trypanosomes interactions. T. congolense infection resulted in reduced metabolic and immune capacity of the PB. The molecular knowledge on the composition and putative functions of PB forms the foundation to identify new targets to disrupt tsetse's ability to feed and parasite transmission.

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.

Multiple evolutionary origins of Trypanosoma evansi in Kenya.

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense.

Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains.

African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.). In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6). We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP) periods were 8.4 ± 0.9 (range, 4-11) and 4.5 ± 0.2 (range, 4-6) for T. congolense and T. brucei isolates, respectively (p < 0.01). Despite the longer mean PP, T. congolense-infected mice exhibited a significantly (p < 0.05) shorter survival time than T. brucei-infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection-causing T. congolense EATRO 1829 and chronic infection-causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

Correction: Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species.

[This corrects the article DOI: 10.1371/journal.pntd.0004421.].

Responses of Glossina pallidipes and Glossina morsitans morsitans tsetse flies to analogues of δ-octalactone and selected blends.

Previous studies have shown that δ-octalactone is an important component of the tsetse-refractory waterbuck (Kobus defassa) repellent odour blend. In the present study, structure-activity comparison was undertaken to determine the effects of the length of the side chain and ring size of the lactone on adult Glossina pallidipes and Glossina morsitans morsitans. The responses of the flies to each compound were studied in a two-choice wind tunnel. Increasing the chain length from C3 (δ-octalactone) to C4 (δ-nonalactone) enhanced repellency to both species (G. pallidipes from 60.0 to 72.0%, and G. m. morsitans from 61.3 to 72.6%), while increasing the ring size from six (δ-octalactone) to seven members (ε-nonalactone) changed the activity from repellency to attraction that was comparable to that of the phenolic blend associated with fermented cow urine (p>0.05). Blending δ-nonalactone with 4-methylguaiacol (known tsetse repellent) significantly (p<0.05) raised repellency to 86.7 and 91.7% against G. pallidipes and G. m. morsitans respectively. Follow-up Latin Square Designed field studies (Shimba hills in coastal areas in Kenya) with G. pallidipes populations confirmed the higher repellence of δ-nonalactone (with/without 4-methylguaiacol) compared to δ-octalactone (also, with/without 4-methylguaiacol). The results show that subtle structural changes of olfactory signals can significantly change their interactions with olfactory receptor neurons, and either shift their potency, or change their activity from repellence to attraction. Our results also lay down useful groundwork in the development of more effective control of tsetse by 'push', 'pull' and 'push-pull' tsetse control tactics.

Genome-Wide Comparative Analysis of Chemosensory Gene Families in Five Tsetse Fly Species.

For decades, odour-baited traps have been used for control of tsetse flies (Diptera; Glossinidae), vectors of African trypanosomes. However, differential responses to known attractants have been reported in different Glossina species, hindering establishment of a universal vector control tool. Availability of full genome sequences of five Glossina species offers an opportunity to compare their chemosensory repertoire and enhance our understanding of their biology in relation to chemosensation. Here, we identified and annotated the major chemosensory gene families in Glossina. We identified a total of 118, 115, 124, and 123 chemosensory genes in Glossina austeni, G. brevipalpis, G. f. fuscipes, G. pallidipes, respectively, relative to 127 reported in G. m. morsitans. Our results show that tsetse fly genomes have fewer chemosensory genes when compared to other dipterans such as Musca domestica (n>393), Drosophila melanogaster (n = 246) and Anopheles gambiae (n>247). We also found that Glossina chemosensory genes are dispersed across distantly located scaffolds in their respective genomes, in contrast to other insects like D. melanogaster whose genes occur in clusters. Further, Glossina appears to be devoid of sugar receptors and to have expanded CO2 associated receptors, potentially reflecting Glossina's obligate hematophagy and the need to detect hosts that may be out of sight. We also identified, in all species, homologs of Ir84a; a Drosophila-specific ionotropic receptor that promotes male courtship suggesting that this is a conserved trait in tsetse flies. Notably, our selection analysis revealed that a total of four gene loci (Gr21a, GluRIIA, Gr28b, and Obp83a) were under positive selection, which confers fitness advantage to species. These findings provide a platform for studies to further define the language of communication of tsetse with their environment, and influence development of novel approaches for control.