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.

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.

A novel highly divergent protein family identified from a viviparous insect by RNA-seq analysis: a potential target for tsetse fly-specific abortifacients.

In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4-10). The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.

Tea (Camellia sinensis) infusions ameliorate cancer in 4TI metastatic breast cancer model.

BACKGROUND: Tea (Camellia sinensis) infusions are widely consumed beverages with numerous health benefits. However, physiological and molecular responses mediating these activities are poorly understood. METHOD: Three replicates of 4TI cancer cell suspension (2.0 × 105 cells/ml) were challenged in vitro with various concentrations of green, black and purple tea infusions to asseses their cytoxicity and associated differentially expressed genes in the cells. Inhibitory activity was tested by using serial dilutions of respective tea infusions in a 96 well ELISA plate. RESULTS: Green tea had the highest inhibition on 4TI cells proliferation at a concentration of IC50 = 13.12 µg/ml. Further analysis of the 4TI cancer cell line treated with tea using 454 pyrosequencing generated 425,696 reads with an input mean length of 286.54. Trimmed sequences were imported on a CLC genomic workbench v7.03 and annotated on a reference mouse genome (Mus musculus strain C57BL/6 J). Results revealed a differential expression of apoptosis related genes in the transcriptome. Casp8, Casp9, Casp3, Casp6, Casp8AP2, Aifm1, Aifm2 and Apopt1 genes were significantly upregulated indicating the process of apoptosis was initiated and executed. CONCLUSION: These findings on caspases offer valuable information on the mechanism of tea as an anticancer agent and will contribute to further research in future novel treatments.

High-resolution melting analysis reveals low Plasmodium parasitaemia infections among microscopically negative febrile patients in western Kenya.

BACKGROUND: Microscopy and rapid diagnostic tests (RDTs) are common tools for diagnosing malaria, but are deficient in detecting low Plasmodium parasitaemia. A novel molecular diagnostic tool (nPCR-HRM) that combines the sensitivity and specificity of nested PCR (nPCR) and direct PCR-high resolution melting analysis (dPCR-HRM) was developed. To evaluate patterns of anti-malarial drug administration when no parasites are detected, nPCR-HRM was employed to screen blood samples for low parasitaemia from febrile patients without microscopically detectable Plasmodium infections in a rural malaria-endemic setting. METHODS: Blood samples (n = 197) were collected in two islands of Lake Victoria, Kenya, from febrile patients without Plasmodium detectable by microscopy or RDTs. 18S rRNA gene sequences were amplified from extracted DNA by nPCR-HRM, nPCR, and dPCR-HRM to detect and differentiate Plasmodium parasites. The limits of detection (LoD) were compared using serial dilutions of the WHO International Standard for P. falciparum DNA. Data on administration of anti-malarials were collected to estimate prescription of anti-malarial drugs to patients with and without low parasitaemia Plasmodium infections. RESULTS: The coupled nPCR-HRM assay detected Plasmodium parasites with greater sensitivity (LoD = 236 parasites/mL) than either nPCR (LoD = 4,700 parasites/mL) or dPCR-HRM (LoD = 1,490 parasites/mL). Moreover, nPCR-HRM detected and differentiated low-parasitaemia infections in significantly greater proportions of patients than did either nPCR or dPCR-HRM (p-value <0.001). Among these low-parasitaemia infections, 67.7% of patients were treated with anti-malarials, whereas 81.5% of patients not infected with Plasmodium parasites were treated with anti-malarials. CONCLUSIONS: The enhanced sensitivity of nPCR-HRM demonstrates limitations of differential febrile illness diagnostics in rural malaria endemic settings that confound epidemiological estimates of malaria, and lead to inadvertent misadministration of anti-malarial drugs. This is the first study that employs low-parasitaemia Plasmodium diagnostics to quantify the prescription of anti-malarial drugs to both non-malaria febrile patients and patients with low-parasitaemia Plasmodium infections. nPCR-HRM enhances low-parasitaemia malaria diagnosis and can potentially surmount the deficiencies of microscopy and RDT-based results in determining low-parasitaemia Plasmodium infection rates for evaluating malaria elimination efforts. The findings highlight the need for improved differential diagnostics of febrile illness in remote malaria endemic regions.

Larvicidal activity of selected aloe species against Aedes aegypti (Diptera: Culiciade).

Management of mosquito vectors by current classes of mosquitocides is relatively ineffective and necessitates prospecting for novel insecticides with different modes of action. Larvicidal activities of 15 crude extracts from three geographically isolated Aloe ngongensis (Christian), Aloe turkanensis (Christian), and Aloe fibrosa (Lavranos & L.E.Newton) (Xanthorrhoeaceae) species (five each) were evaluated against Aedes aegypti (Linnaeus in Hasselquist) (Diptera: Culiciade L.) yellow fever mosquito. Freshly collected leaves were separately shade-dried to constant weight at room temperature (25 ± 2°C) and powdered. Each powder was macerated in solvents of increasing polarity (hexane, chloroform, ethyl acetate, acetone, and methanol) for 72 h and subsequently filtered. Third-instar larvae (n = 25) of the mosquito were exposed to the extracts at different concentrations for 24 h to establish dose response relationships. All the fractions of A. ngongensis were active below 1 mg/ml except A. fibrosa and A. turkanensis. The highest activity (LC50) mg/ml was obtained with extracts of A. fibrosa hexane (0.05 [0.04-0.06]), followed by A. ngongensis hexane (0.11 [0.08-0.15]) and A. turkanensis ethyl acetate (0.11 [0.09-0.12]). The activities are apparently Aloe species specific and extraction solvent dependent. These findings suggest that extracts from selected Aloe species have mosquitocidal principles that can be exploited in development of new insecticides.

Putative bloodmeal sources in Glossina austeni tsetse fly of Arabuko Sokoke National Reserve in Kenya.

Tsetse flies, the sole biological vectors of trypanosomiasis, are predominantly controlled using visual traps and targets baited with attractant lures. Formulation of the lures is informed by compositions of odors from vertebrate hosts preferred by specific tsetse species. However, there are no effective lures for Glossina austeni, a major vector of trypanosomiasis along eastern-coastal region of Africa. Formulation of the lure can be informed by knowledge of G. austeni, preferred vertebrate hosts. We thus sought to understand these hosts by assessment of putative bloodmeal sources of this tsetse fly in Arabuko Sokoke National Reserve where this species is naturally present. We sampled tsetse flies using NGU traps, isolated non-teneral G. austeni flies based on their feeding status, and identified vertebrate source of bloodmeals in their midgut contents using vertebrate 16S rRNA-PCR High-Resolution Melting analysis. We analyzed the relative vertebrate species frequencies in the bloodmeals using Fisher's exact tests. Overall, we trapped 122 flies, most of which (66.39%) were non-teneral, among which we successfully identified the vertebrate bloodmeals in 30 samples. Specifically, we detected putative suni antelope (Neotragus moschatus), harnessed bushbuck (Tragelaphus scriptus), buffalo (Syncerus caffer) and cattle (Bos taurus) derived bloodmeals. Putative suni antelope bloodmeals were significantly more frequent (63.22%), than those of the harnessed bushbuck (23.33%), buffalo (10.00%) or cattle (3.33%) (p < 0.05 Fisher's exact tests) among the samples analyzed. Suni antelope thus appears to predominate vertebrate bloodmeal source for G. austeni in the reserve, coincident with findings reported elsewhere, and is therefore a viable candidate for bioprospecting for G. austeni responsive attractants.

Sex-specific induction of CYP6 cytochrome P450 genes in cadmium and lead tolerant Anopheles gambiae.

BACKGROUND: Anopheles gambiae, one of the main Afro-tropical mosquito vector of malaria, has adapted to heavy metals in its natural habitat, and developed resistance to most conventional insecticides. Investigations were conducted to establish an association between tolerance to cadmium or lead-heavy metals, and expression of specific genes for cytochrome p450 enzymes associated with pyrethroid resistance in the mosquito. METHODS: Juvenile aquatic stages of the mosquito were selected for tolerance to cadmiun or lead through chronic exposure of the stages to maximum acceptable toxicant concentrations (MATCs) of the metals. Using real-time quantitative polymerase chain reaction (qPCR), three replicates each of male or female cadmium or lead-tolerant individuals and relevant controls were separately screened for expression of CYP6M2, CYP6P3 and CYP6Z1 genes. The variance in expression levels of the genes amongst the treatments was compared by ANOVA statistical tool. RESULTS: Expressions of all the genes were significantly lower (P <0.05) in females than in males. Within gender, there 1.3 - 2.3 or 3.1-4.2-fold reduction in expression of the genes in cadmium or lead selected than respective control populations. Expression of all the classes of gene was elevated in cadmium selected female populations relative to their respective controls. CONCLUSION: These findings suggest that tolerance to cadmium or lead in the mosquito can influence response in cytochrome p450 genes associated with metabolism of pyrethroids in the mosquito in a sex-specific manner. This can, in turn, affect sensitivity of the mosquito to pyrethroids and other xenobiotics associated with these genes, with potential implications in mosquito vector control operations.

Bloodmeal host identities among sympatric Glossina austeni and Glossina pallidipes tsetse flies in Shimba Hills National Reserve, Kwale, Kenya.

Odor from preferred/non-preferred tsetse fly vertebrate hosts have been exploited in R&D of attractants/repellents of the fly for human and livestock protection. Odors from vertebrate hosts of Glossina austeni and Glossina pallidipes tsetse flies can facilitate formulation of novel attractants effective against G. austeni or improvement of existing attractant blends for G. pallidipes. We compared vertebrate blood meal sources of both fly species at Shimba Hills National Reserve, Kenya, to establish putative preferred host of either species, hence potential source of G. austeni or G. pallidipes specific odors. We trapped sympatric adult flies in 2021 and 2022 using NGU traps/sticky panels baited with POCA, collected their blood meals and characterize the meals using HRM vertebrate 16S rRNA- PCR (for host identification), and compared host profiles using GLM and Fisher's exact tests. We collected 168 and 62 sympatric G. pallidipes and G. austeni with bloodmeal, respectively in 2021 and, 230 and 142 respectively in 2022. In 2021, we identified putative hosts of 65.48 and 69.35 % of the G. pallidipes and G. austeni respectively and 82.61 and 80.28%, respectively in 2022. In 2021, we detected harnessed bushbuck, buffalo, common warthog and cattle putative host bloodmeals, and additionally bushpig and suni antelope bloodmeals in 2022. Putative vertebrate bloodmeal sources were significantly different by tsetse fly species (χ2(1, N=457) = 43.215, p < 0.001) and sampling year (χ2(1, N=457) = 8.044, p = 0.005). Frequency of common warthog bloodmeals was higher in G. pallidipes (65.79 %) than G. austeni (38.60%), and that of suni antelope and harnessed bushbuck putative bloodmeals higher in G. austeni (21.05-28.07%) than in G. pallidipes (6.84 - 17.37%) in 2022. There was an apparent change in putative feeding preference/host choices in both fly species between 2021 and 2022. Host bloodmeals in G. pallidipes or G. austeni predominantly from putative harnessed bushbuck, suni antelope or common warthog reveal these vertebrates with potential odors that can be harnessed and formulated into appropriate attractants for respective species and integrated into routine control regiment for G. pallidipes and/or G. austeni.

Perspectives on Odor-Based Control of Tsetse Flies in Africa.

Tsetse-transmitted trypanosomiases are among the most neglected tropical diseases in sub-Sahara Africa. Although all tsetse species are susceptible to trypanosome infections, their differential attraction/feeding preferences for different wildlife, domestic animals, and/or humans constitute critical determinants of trypanosomes species they predominantly transmit. Artificial bait technologies, based on long-range tsetse olfactory responses to natural cues emitted by preferred hosts and blends of synthetic versions that mimic these cues, have successfully been applied in attractant-odor-based ("pull" tactic) reduction of field populations of some tsetse species. Olfactory attribute associated with active avoidance of tsetse-refractory non-hosts has similarly been exploited in design of repellent-odor-based ("push" tactic) protection of livestock. These tactics have opened possibility of spatially strategic deployment of the two sets of odor baits in "push-pull" tactics. Possibility of developing blends with enhanced attraction and repellence compared with those associated with savannah tsetse fly hosts and non-hosts, respectively, have been explored, where structure activity and blends of different components generated two novel blends. The studies evaluated structure activity and blends of different components. One based on attractive constituents associated with buffalo (Syncerus caffer) comprised of ε-nonalactone, nonanoic acid, 2-nonanone (in 1:3:2 proportion) delivered together with acetone, which showed significantly better attractancy on savannah tsetse fly than the standard blend comprised of 3-propylphenol, octenol, p-cresol, and acetone (POCA). The other blend comprised of δ-nonalactone, heptanoic acid, 4-methylguaiacol and geranylacetone (in 6:4:2:1 proportion) was significantly more repellent than previously characterized blend based on tsetse fly refractory waterbuck (Kobus defassa) constituents (δ-octalactone, pentanoic acid, guaiacol and geranylacetone). So far, no effective attractants or repellents of riverine tsetse fly species have been characterized. Optimized attractant and repellent blends for savannah tsetse flies lay down useful groundwork for future development of the "push-pull" deployment tactic for area-wide control of tsetse flies. Better understanding of the physiological, cellular, and molecular basis of response in the tsetse fly to odors can potentially augment the current tsetse fly-control interventions.

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.

In silico-driven analysis of the Glossina morsitans morsitans antennae transcriptome in response to repellent or attractant compounds.

BACKGROUND: High-throughput sequencing generates large volumes of biological data that must be interpreted to make meaningful inference on the biological function. Problems arise due to the large number of characteristics p (dimensions) that describe each record [n] in the database. Feature selection using a subset of variables extracted from the large datasets is one of the approaches towards solving this problem. METHODOLOGY: In this study we analyzed the transcriptome of Glossina morsitans morsitans (Tsetsefly) antennae after exposure to either a repellant (δ-nonalactone) or an attractant (ε-nonalactone). We identified 308 genes that were upregulated or downregulated due to exposure to a repellant (δ-nonalactone) or an attractant (ε-nonalactone) respectively. Weighted gene coexpression network analysis was used to cluster the genes into 12 modules and filter unconnected genes. Discretized and association rule mining was used to find association between genes thereby predicting the putative function of unannotated genes. RESULTS AND DISCUSSION: Among the significantly expressed chemosensory genes (FDR < 0.05) in response to Ɛ-nonalactone were gustatory receptors (GrIA and Gr28b), ionotrophic receptors (Ir41a and Ir75a), odorant binding proteins (Obp99b, Obp99d, Obp59a and Obp28a) and the odorant receptor (Or67d). Several non-chemosensory genes with no assigned function in the NCBI database were co-expressed with the chemosensory genes. Exposure to a repellent (δ-nonalactone) did not show any significant change between the treatment and control samples. We generated a coexpression network with 276 edges and 130 nodes. Genes CAH3, Ahcy, Ir64a, Or67c, Ir8a and Or67a had node degree values above 11 and therefore could be regarded as the top hub genes in the network. Association rule mining showed a relation between various genes based on their appearance in the same itemsets as consequent and antecedent.

Transcriptomic response of Anopheles gambiae sensu stricto mosquito larvae to Curry tree (Murraya koenigii) phytochemicals.

BACKGROUND: Insect growth regulators (IGRs) can control insect vector populations by disrupting growth and development in juvenile stages of the vectors. We previously identified and described the curry tree (Murraya koenigii (L.) Spreng) phytochemical leaf extract composition (neplanocin A, 3-(1-naphthyl)-L-alanine, lumiflavine, terezine C, agelaspongin and murrayazolinol), which disrupted growth and development in Anopheles gambiae sensu stricto mosquito larvae by inducing morphogenetic abnormalities, reducing locomotion and delaying pupation in the mosquito. Here, we attempted to establish the transcriptional process in the larvae that underpins these phenotypes in the mosquito. METHODS: We first exposed third-fourth instar larvae of the mosquito to the leaf extract and consequently the inherent phytochemicals (and corresponding non-exposed controls) in two independent biological replicates. We collected the larvae for our experiments sampled 24 h before peak pupation, which was 7 and 18 days post-exposure for controls and exposed larvae, respectively. The differences in duration to peak pupation were due to extract-induced growth delay in the larvae. The two study groups (exposed vs control) were consequently not age-matched. We then sequentially (i) isolated RNA (whole larvae) from each replicate treatment, (ii) sequenced the RNA on Illumina HiSeq platform, (iii) performed differential bioinformatics analyses between libraries (exposed vs control) and (iv) independently validated the transcriptome expression profiles through RT-qPCR. RESULTS: Our analyses revealed significant induction of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples. Our analysis also revealed alteration of pathways functionally associated with putrescine metabolism and structural constituents of the cuticle in the extract-exposed larvae relative to the non-exposed control, putatively linked to the exoskeleton and immune response in the larvae. The extract-exposed larvae also appeared to have suppressed pathways functionally associated with molting, cell division and growth in the larvae. However, given the age mismatch between the extract-exposed and non-exposed larvae, we can attribute the modulation of innate immune, detoxification, cuticular and associated transcripts and pathways we observed to effects of age differences among the larvae samples (exposed vs control) and to exposures of the larvae to the extract. CONCLUSIONS: The exposure treatment appears to disrupt cuticular development, immune response and oxidative stress pathways in Anopheles gambiae s.s larvae. These pathways can potentially be targeted in development of more efficacious curry tree phytochemical-based IGRs against An. gambiae s.s mosquito larvae.

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.

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.

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.

Expression of metallothionein and alpha-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae).

Anopheles mosquitoes have been shown to adapt to heavy metals in their natural habitats. In this study we explored the possibility of using Anopheles gambiae sensu stricto as bio-reporters for environmental heavy metal pollution through expressions of their metal-responsive metallothionein and alpha-tubulin genes. The study was undertaken with third instar larvae after selection by cadmium, copper, or lead at LC(30) through five successive generations. Expression levels were determined in the 5th generation by semi-quantitative RT-PCR on the experimental and control populations. The data were analyzed using one-way ANOVA. The highest metallothionein (F(3,11)=4.574, P=0.038) and alpha-tubulin (F(3,11)=12.961, P=0.002) responses were observed in cadmium-tolerant treatments. There was significantly higher expression of metallothionein in cadmium or copper treatments relative to the control (P=0.012), and in cadmium than in lead treatments (P=0.044). Expressions of alpha-tubulin were significantly higher in cadmium than in control treatments (P=0.008). These results demonstrate the capacity of An. gambiae s.s. to develop tolerance to increased levels of heavy metal challenge. The results also confirm the potential of heavy metal-responsive genes in mosquitoes as possible bio-indicators of heavy metal environmental pollution. How the tolerance and expressions relate to An. gambiae s.s. fitness and vectorial capacity in the environment remains to be elucidated.

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.

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.

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.