Developing and quality testing of microsatellite loci for four species of Glossina.

Microsatellite loci still represent valuable resources for the study of the population biology of non-model organisms. Discovering or adapting new suitable microsatellite markers in species of interest still represents a useful task, especially so for non-model organisms as tsetse flies (genus Glossina), which remain a serious threat to the health of humans and animals in sub-Saharan Africa. In this paper, we present the development of new microsatellite loci for four species of Glossina: two from the Morsitans group, G. morsitans morsitans (Gmm) from Zimbabwe, G. pallidipes (Gpalli) from Tanzania; and the other two from the Palpalis group, G. fuscipes fuscipes (Gff) from Chad, and G. palpalis gambiensis (Gpg) from Guinea. We found frequent short allele dominance and null alleles. Stuttering could also be found and amended when possible. Cryptic species seemed to occur frequently in all taxa but Gff. This explains why it may be difficult finding ecumenical primers, which thus need adaptation according to each taxonomic and geographic context. Amplification problems occurred more often in published old markers, and Gmm and Gpg were the most affected (stronger heterozygote deficits). Trinucleotide markers displayed selection signature in some instances (Gmm). Combining old and new loci, for Gmm, eight loci can be safely used (with correction for null alleles); and five seem particularly promising; for Gpalli, only five to three loci worked well, depending on the clade, which means that the use of loci from other species (four morsitans loci seemed to work well), or other new primers will need to be used; for Gff, 14 loci behaved well, but with null alleles, seven of which worked very well; and for G. palpalis sl, only four loci, needing null allele and stuttering corrections seem to work well, and other loci from the literature are thus needed, including X-linked markers, five of which seem to work rather well (in females only), but new markers will probably be needed. Finally, the high proportion of X-linked markers (around 30%) was explained by the non-Y DNA quantity and chromosome structure of tsetse flies studied so far.

Developing a national atlas to support the progressive control of tsetse-transmitted animal trypanosomosis in Kenya.

BACKGROUND: African animal trypanosomosis (AAT) is a major livestock disease in Kenya. Even though, over the years various organizations have collected a vast amount of field data on tsetse and AAT in different parts of the country, recent national-level maps are lacking. To address this gap, a national atlas of tsetse and AAT distribution is being developed by the Kenya Tsetse and Trypanosomosis Eradication Council (KENTTEC) and partners. METHODS: All data collected by KENTTEC from 2006 to 2019 were systematically assembled, georeferenced and harmonized. A comprehensive data repository and a spatially-explicit database were created. Input data were collected mainly in the context of control activities, and include both baseline surveys (i.e. pre-intervention) and the subsequent monitoring during and after interventions. Surveys were carried out in four regions (i.e. Western, Rift Valley, Central and Coast), and in 21 of the 47 counties in Kenya. Various devices were used for entomological data collection (i.e. biconical, NGU and H traps, and sticky panels), while the buffy-coat technique was the method used to detect AAT. RESULTS: Tsetse trapping was carried out in approximately 5000 locations, and flies (> 71,000) were caught in all four investigated regions. Six species of Glossina were detected: G. pallidipes (87% of the catches); G. brevipalpis (8%); G. fuscipes fuscipes (4%); G. longipennis (< 1%); G. austeni (< 1%); and G. swynnertoni (< 1%). A total of 49,785 animals (98% of which cattle) were tested for AAT in approximately 500 locations. Of these, 914 animals were found to be infected. AAT was confirmed in all study regions, in particular caused by Trypanosoma vivax (48% of infections) and T. congolense (42%). Fewer cases of T. brucei were found. CONCLUSIONS: The development and regular update of a comprehensive national database of tsetse and AAT is crucial to guide decision making for the progressive control of the disease. This first version of the atlas based on KENTTEC data has achieved a remarkable level of geographical coverage, but temporal and spatial gaps still exist. Other stakeholders at the national and international level will contribute to the initiative, thus improving the completeness of the atlas.

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.

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.

Genetic diversity of trypanosome species in tsetse flies (Glossina spp.) in Nigeria.

BACKGROUND: Trypanosomes cause disease in humans and livestock in sub-Saharan Africa and rely on tsetse flies as their main insect vector. Nigeria is the most populous country in Africa; however, only limited information about the occurrence and diversity of trypanosomes circulating in the country is available. METHODS: Tsetse flies were collected from five different locations in or adjacent to protected areas, i.e. national parks and game reserves, in Nigeria. Proboscis and gut samples were analysed for trypanosome DNA by molecular amplification of the internal transcribed spacer 1 (ITS1) region and part of the trypanosome specific glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. RESULTS: The most abundant Trypanosoma species found in the tsetse gut was T. grayi, a trypanosome infecting crocodiles. It was ubiquitously distributed throughout the country, accounting for over 90% of all cases involving trypanosomes. Trypanosoma congolense was detected in gut samples from all locations except Cross River National Park, but not in the proboscis, while T. brucei (sensu lato) was not detected at all. In proboscis samples, T. vivax was the most prominent. The sequence diversity of gGAPDH suggests that T. vivax and T. grayi represent genetically diverse species clusters. This implies that they are highly dynamic populations. CONCLUSIONS: The prevalence of animal pathogenic trypanosomes throughout Nigeria emphasises the role of protected areas as reservoirs for livestock trypanosomes. The genetic diversity observed within T. vivax and T. grayi populations might be an indication for changing pathogenicity or host range and the origin and consequences of this diversity has to be further investigated.

A national atlas of tsetse and African animal trypanosomosis in Mali.

BACKGROUND: Tsetse-transmitted trypanosomosis is a deadly, neglected tropical disease and a major challenge for mixed crop-livestock agriculture in sub-Saharan Africa. It is caused by several species of the genus Trypanosoma. Information on the occurrence of tsetse flies and African animal trypanosomosis (AAT) is available for different areas of Mali. However, these data have never been harmonized and centralized, which prevents the development of comprehensive epidemiological maps and constrains an evidence-based planning of control actions. To address this challenge, we created a dynamic geo-spatial database of tsetse and AAT distribution in Mali. METHODS: A digital repository containing epidemiological data collected between 2000 and 2018 was assembled. In addition to scientific publications, the repository includes field datasheets, technical reports and other grey literature. The data were verified, harmonized, georeferenced and integrated into a single spatially-explicit database. RESULTS: For the tsetse component, approximately 19,000 trapping records, corresponding to 6000 distinct trapping locations and 38,000 flies were included in the database. Glossina palpalis gambiensis was the most widespread and abundant species, and it was found in the southern, southern-central and western parts of the country. Glossina tachinoides was only found in the South. Only a few specimens of Glossina morsitans submorsitans were detected. For the AAT component, approximately 1000 survey records were included, corresponding to 450 distinct survey sites and 37,000 tested bovines. AAT was found in all surveyed regions, although data for the tsetse-free North and North-East are lacking. Trypanosoma vivax and Trypanosoma congolense were the dominant species, while Trypanosoma brucei infections were much less numerous. CONCLUSIONS: The atlas of tsetse and AAT in Mali provides a synoptic view of the vector and disease situation at the national level. Still, major geographical gaps affect the North, the North-East and the West, and there is also a severe lack of data over the past five years. Trypanosomosis remains a major animal health problem in Mali. However, despite its prevalence and distribution, monitoring and control activities are presently very limited. Efforts should be made to strengthen the progressive control of AAT in Mali, and the atlas provides a new tool to identify priority areas for intervention.

Detecting Wahlund effects together with amplification problems: Cryptic species, null alleles and short allele dominance in Glossina pallidipes populations from Tanzania.

Population genetics is a convenient tool to study the population biology of non-model and hard to sample species. This is particularly true for parasites and vectors. Heterozygote deficits and/or linkage disequilibrium often occur in such studies and detecting the origin of those (Wahlund effect, reproductive system or amplification problems) is uneasy. We used new tools (correlation between the number of times a locus is found in significant linkage disequilibrium and its genetic diversity, correlations between Wright's FIS and FST , FIS and number of missing data, FIT and allele size and standard errors comparisons) for the first time on a real data set of tsetse flies, a vector of dangerous diseases to humans and domestic animals in sub-Saharan Africa. With these new tools, and cleaning data from null allele, temporal heterogeneity and short allele dominance effects, we unveiled the coexistence of two highly divergent cryptic clades in the same sites. These results are in line with other studies suggesting that the biodiversity of many taxa still largely remain undescribed, in particular pathogenic agents and their vectors. Our results also advocate that including individuals from different cohorts tends to bias subdivision measures and that keeping loci with short allele dominance and/or too frequent missing data seriously jeopardize parameter's estimations. Finally, separated analyses of the two clades suggest very small tsetse densities and relatively large dispersal.

Variations of Glossina sp. and trypanosome species frequency within different habitats in a sleeping sickness focus, Gabon.

INTRODUCTION: Knowledge of the infectious status of the Glossina is an indicator of risk of resurgence of Human African Trypanosomiasis (HAT). Environmental conditions have an impact on the density and diversity of both vector and Trypanosoma. The aim of the study was to determine the diversity and the infection rate of Glossina as well as the diversity of trypanosome species within habitats of an old HAT focus, in Gabon. METHODOLOGY: Glossina were captured in September 2012 in three ecological sites. Vavoua traps were installed for twelve days. All captured flies were identified. Glossina were selected for trypanosome identification by Polymerase Chain Reaction. RESULTS: 1178 Glossina were captured: 55.8% in degraded forest, 28.9% in flood area and 15.4% in secondary forest. Glossina fusca congolensis (37%) and G.palpalis palpalis (36.4%) were the most abundant vector species. G. fusca congolensis was predominant in secondary forest and in flood area, while in degraded forest, it was G.palpalis palpalis. Trypanosoma infection rate was 30.7%, 42% in secondary forest, 32% in degraded forest and 18% in flood area. Trypanosoma congolense savannah was the main species detected (18.7%) followed by T.brucei brucei (10.7%) and T. brucei gambiense (4%). T. congolense savannah type was predominant in the secondary forest and in degraded forest (66.7% versus 55.5%). CONCLUSION: Glossina density and trypanosome infection rate varied according to the habitat within HAT focus. The density of tsetse was the highest in degraded forest while the infection rate was highest in secondary forest. Continuous disease surveillance and control measures are needed.

Nuclear and Wolbachia-based multimarker approach for the rapid and accurate identification of tsetse species.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are solely responsible for the transmission of African trypanosomes, causative agents of sleeping sickness in humans and nagana in livestock. Due to the lack of efficient vaccines and the emergence of drug resistance, vector control approaches such as the sterile insect technique (SIT), remain the most effective way to control disease. SIT is a species-specific approach and therefore requires accurate identification of natural pest populations at the species level. However, the presence of morphologically similar species (species complexes and sub-species) in tsetse flies challenges the successful implementation of SIT-based population control. RESULTS: In this study, we evaluate different molecular tools that can be applied for the delimitation of different Glossina species using tsetse samples derived from laboratory colonies, natural populations and museum specimens. The use of mitochondrial markers, nuclear markers (including internal transcribed spacer 1 (ITS1) and different microsatellites), and bacterial symbiotic markers (Wolbachia infection status) in combination with relatively inexpensive techniques such as PCR, agarose gel electrophoresis, and to some extent sequencing provided a rapid, cost effective, and accurate identification of several tsetse species. CONCLUSIONS: The effectiveness of SIT benefits from the fine resolution of species limits in nature. The present study supports the quick identification of large samples using simple and cost effective universalized protocols, which can be easily applied by countries/laboratories with limited resources and expertise.

Diversity and phylogenetic relationships of Glossina populations in Nigeria and the Cameroonian border region.

BACKGROUND: Tsetse flies are vectors of trypanosomes, parasites that cause devastating disease in humans and livestock. In the course of vector control programmes it is necessary to know about the Glossina species present in the study area, the population dynamics and the genetic exchange between tsetse fly populations. RESULTS: To achieve an overview of the tsetse fly diversity in Nigeria and at the Nigeria-Cameroon border, tsetse flies were trapped and collected between February and March 2014 and December 2016. Species diversity was determined morphologically and by analysis of Cytochrome C Oxidase SU1 (COI) gene sequences. Internal transcribed spacer-1 (ITS-1) sequences were compared to analyse variations within populations. The most dominant species were G. m. submorsitans, G. tachinoides and G. p. palpalis. In Yankari Game Reserve and Kainji Lake National Park, G. submorsitans and G. tachinoides were most frequent, whereas in Old Oyo National Park and Ijah Gwari G. p. palpalis was the dominant species. Interestingly, four unidentified species were recorded during the survey, for which no information on COI or ITS-1 sequences exists. G. p. palpalis populations showed a segregation in two clusters along the Cameroon-Nigerian border. CONCLUSIONS: The improved understanding of the tsetse populations in Nigeria will support decisions on the scale in which vector control is likely to be more effective. In order to understand in more detail how isolated these populations are, it is recommended that further studies on gene flow be carried out using other markers, including microsatellites.

Comparative evaluation of dry and liquid RIME LAMP in detecting trypanosomes in dead tsetse flies.

Xenomonitoring is an important approach in assessing the progress of trypanosomiasis control as well as in estimating the endemicity of trypanosomes in affected areas. One of the major challenges in this approach is the unavailability of sensitive and easy to use xenomonitoring tools that can be used in the remote areas where the disease occurs. One tool that has been used successfully in detecting the parasites in tsetse flies is the repetitive insertion mobile element loop-mediated isothermal amplification (RIME LAMP). This tool has recently been modified from the liquid form to dry form for use in remote areas; however, uptake for use in the field has been slow. Field-collected tsetse flies were used to evaluate the performance of dry RIME LAMP over the conventional liquid RIME LAMP. All the samples were also subjected to internal transcribed spacer 1 (ITS1) ribosomal deoxyribonucleic acid (DNA) polymerase chain reaction (PCR) as a standard. ITS1-PCR-positive samples were further sequenced for confirmation of the species. A total of 86 wild tsetse flies were left to dry at room temperature for 3 months and DNA was extracted subsequently. All 86 flies were Glossina morsitans morsitans. From these, dry RIME LAMP detected 16.3% while liquid RIME LAMP detected 11.6% as infected with trypanosomes. Ten positive samples on ITS1-PCR were sequenced and all were shown to be trypanosomes. The use of dry RIME LAMP in the field for xenomonitoring of trypanosomes in tsetse flies will greatly contribute towards control of this neglected tropical disease as it provides the cheapest, fastest and simplest way to estimate possible human infective trypanosome infection rates in the tsetse fly vectors.

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.

Tsetse fly evolution, genetics and the trypanosomiases - A review.

This reviews work published since 2007. Relative efforts devoted to the agents of African trypanosomiasis and their tsetse fly vectors are given by the numbers of PubMed accessions. In the last 10 years PubMed citations number 3457 for Trypanosoma brucei and 769 for Glossina. The development of simple sequence repeats and single nucleotide polymorphisms afford much higher resolution of Glossina and Trypanosoma population structures than heretofore. Even greater resolution is offered by partial and whole genome sequencing. Reproduction in T. brucei sensu lato is principally clonal although genetic recombination in tsetse salivary glands has been demonstrated in T. b. brucei and T. b. rhodesiense but not in T. b. gambiense. In the past decade most genetic attention was given to the chief human African trypanosomiasis vectors in subgenus Nemorhina e.g., Glossina f. fuscipes, G. p. palpalis, and G. p. gambiense. The chief interest in Nemorhina population genetics seemed to be finding vector populations sufficiently isolated to enable efficient and long-lasting suppression. To this end estimates were made of gene flow, derived from FST and its analogues, and Ne, the size of a hypothetical population equivalent to that under study. Genetic drift was greater, gene flow and Ne typically lesser in savannah inhabiting tsetse (subgenus Glossina) than in riverine forms (Nemorhina). Population stabilities were examined by sequential sampling and genotypic analysis of nuclear and mitochondrial genomes in both groups and found to be stable. Gene frequencies estimated in sequential samplings differed by drift and allowed estimates of effective population numbers that were greater for Nemorhina spp than Glossina spp. Prospects are examined of genetic methods of vector control. The tsetse long generation time (c. 50 d) is a major contraindication to any suggested genetic method of tsetse population manipulation. Ecological and modelling research convincingly show that conventional methods of targeted insecticide applications and traps/targets can achieve cost-effective reduction in tsetse densities.

Remarkable richness of trypanosomes in tsetse flies (Glossina morsitans morsitans and Glossina pallidipes) from the Gorongosa National Park and Niassa National Reserve of Mozambique revealed by fluorescent fragment length barcoding (FFLB).

Trypanosomes of African wild ungulates transmitted by tsetse flies can cause human and livestock diseases. However, trypanosome diversity in wild tsetse flies remains greatly underestimated. We employed FFLB (fluorescent fragment length barcoding) for surveys of trypanosomes in tsetse flies (3086) from the Gorongosa National Park (GNP) and Niassa National Reserve (NNR) in Mozambique (MZ), identified as Glossina morsitans morsitans (GNP/NNR=77.6%/90.5%) and Glossina pallidipes (22.4%/9.5%). Trypanosomes were microscopically detected in 8.3% of tsetse guts. FFLB of gut samples revealed (GNP/NNR): Trypanosoma congolense of Savannah (27%/63%), Kilifi (16.7%/29.7%) and Forest (1.0%/0.3%) genetic groups; T. simiae Tsavo (36.5%/6.1%); T. simiae (22.2%/17.7%); T. godfreyi (18.2%/7.0%); subgenus Trypanozoon (20.2%/25.7%); T. vivax/T. vivax-like (1.5%/5.2%); T. suis/T. suis-like (9.4%/11.9%). Tsetse proboscises exhibited similar species composition, but most prevalent species were (GNP/NNR): T. simiae (21.9%/28%), T. b. brucei (19.2%/31.7%), and T. vivax/T. vivax-like (19.2%/28.6%). Flies harboring mixtures of trypanosomes were common (~ 64%), and combinations of more than four trypanosomes were especially abundant in the pristine NNR. The non-pathogenic T. theileri was found in 2.5% while FFLB profiles of unknown species were detected in 19% of flies examined. This is the first report on molecular diversity of tsetse flies and their trypanosomes in MZ; all trypanosomes pathogenic for ungulates were detected, but no human pathogens were detected. Overall, two species of tsetse flies harbor 12 species/genotypes of trypanosomes. This notable species richness was likely uncovered because flies were captured in wildlife reserves and surveyed using the method of FFLB able to identify, with high sensitivity and accuracy, known and novel trypanosomes. Our findings importantly improve the knowledge on trypanosome diversity in tsetse flies, revealed the greatest species richness so far reported in tsetse fly of any African country, and indicate the existence of a hidden trypanosome diversity to be discovered in African wildlife protected areas.

Challenging the Wigglesworthia, Sodalis, Wolbachia symbiosis dogma in tsetse flies: Spiroplasma is present in both laboratory and natural populations.

Profiling of wild and laboratory tsetse populations using 16S rRNA gene amplicon sequencing allowed us to examine whether the "Wigglesworthia-Sodalis-Wolbachia dogma" operates across species and populations. The most abundant taxa, in wild and laboratory populations, were Wigglesworthia (the primary endosymbiont), Sodalis and Wolbachia as previously characterized. The species richness of the microbiota was greater in wild than laboratory populations. Spiroplasma was identified as a new symbiont exclusively in Glossina fuscipes fuscipes and G. tachinoides, members of the palpalis sub-group, and the infection prevalence in several laboratory and natural populations was surveyed. Multi locus sequencing typing (MLST) analysis identified two strains of tsetse-associated Spiroplasma, present in G. f. fuscipes and G. tachinoides. Spiroplasma density in G. f. fuscipes larva guts was significantly higher than in guts from teneral and 15-day old male and female adults. In gonads of teneral and 15-day old insects, Spiroplasma density was higher in testes than ovaries, and was significantly higher density in live versus prematurely deceased females indicating a potentially mutualistic association. Higher Spiroplasma density in testes than in ovaries was also detected by fluorescent in situ hybridization in G. f. fuscipes.

Genetic diversity of Glossina fuscipes fuscipes along the shores of Lake Victoria in Tanzania and Kenya: implications for management.

BACKGROUND: Tsetse flies (Diptera: Glossinidae) are sole vectors for trypanosomiasis, which affect human health and livestock productivity in Africa. Little is known about the genetic diversity of Glossina fuscipes fuscipes, which is an important species in Tanzania and Kenya. The main objective of the study was to provide baseline data to determine the genetic variability and divergence of G. f. fuscipes in the Lake Victoria basin of Tanzania and Kenya in order to guide future vector control efforts in the region. FINDINGS: Two hundred and seventy five G. f. fuscipes from 8 sites along the shores of Lake Victoria were screened for genetic polymorphisms at 19 microsatellite loci. Samples were collected from two sites in Kenya and six sites in Tanzania. Four of the Tanzanian sites were located in the Rorya district, on the eastern shores of Lake Victoria, while the other two sites were from Ukerewe and Bukoba districts from the southern and western Lake Victoria shores, respectively. Four genetically distinct allopatric clusters were revealed by microsatellite analysis, which sorted the sampling sites according to geography, with sites separated by as little as ~65 km belonging to distinct genetic clusters, while samples located within ~35 km from each other group in the same cluster. CONCLUSION: Our results suggest that there is ongoing genetic admixture within sampling sites located ~35 km from each other, while sites located ~65 km apart are genetically isolated from each other. Similar patterns emerged from a parallel study on G. f. fuscipes analyzed from the Lake Victoria Uganda shores. From a control perspective these results suggest that for sites within the same genetic cluster, control efforts should be carried out in a coordinated fashion in order to avoid re-invasions. Future work should focus on better quantifying the extent and spatial patterns of the observed genetic discontinuities of the G. f. fuscipes populations along the Tanzanian shores. This will aid in their control by providing guidelines on the geographical extent of the area to be treated at the same time.

A pilot study to delimit tsetse target populations in Zimbabwe.

BACKGROUND: Tsetse (Glossina sensu stricto) are cyclical vectors of human and animal trypanosomoses, that are presently targeted by the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) coordinated by the African Union. In order to achieve effective control of tsetse, there is need to produce elaborate plans to guide intervention programmes. A model intended to aid in the planning of intervention programmes and assist a fuller understanding of tsetse distribution was applied, in a pilot study in the Masoka area, Mid-Zambezi valley in Zimbabwe, and targeting two savannah species, Glossina morsitans morsitans and Glossina pallidipes. METHODOLOGY/PRINCIPAL FINDINGS: The field study was conducted between March and December 2015 in 105 sites following a standardized grid sampling frame. Presence data were used to study habitat suitability of both species based on climatic and environmental data derived from MODIS and SPOT 5 satellite images. Factors influencing distribution were studied using an Ecological Niche Factor Analysis (ENFA) whilst habitat suitability was predicted using a Maximum Entropy (MaxEnt) model at a spatial resolution of 250 m. Area Under the Curve (AUC), an indicator of model performance, was 0.89 for G. m. morsitans and 0.96 for G. pallidipes. We then used the predicted suitable areas to calculate the probability that flies were really absent from the grid cells where they were not captured during the study based on a probability model using a risk threshold of 0.05. Apart from grid cells where G. m. morsitans and G. pallidipes were captured, there was a high probability of presence in an additional 128 km2 and 144 km2 respectively. CONCLUSIONS/SIGNIFICANCE: The modelling process promised to be useful in optimizing the outputs of presence/absence surveys, allowing the definition of tsetse infested areas with improved accuracy. The methodology proposed here can be extended to all the tsetse infested parts of Zimbabwe and may also be useful for other PATTEC national initiatives in other African countries.

Genetic diversity and population structure of the tsetse fly Glossina fuscipes fuscipes (Diptera: Glossinidae) in Northern Uganda: Implications for vector control.

Uganda is the only country where the chronic and acute forms of human African Trypanosomiasis (HAT) or sleeping sickness both occur and are separated by < 100 km in areas north of Lake Kyoga. In Uganda, Glossina fuscipes fuscipes is the main vector of the Trypanosoma parasites responsible for these diseases as well for the animal African Trypanosomiasis (AAT), or Nagana. We used highly polymorphic microsatellite loci and a mitochondrial DNA (mtDNA) marker to provide fine scale spatial resolution of genetic structure of G. f. fuscipes from 42 sampling sites from the northern region of Uganda where a merger of the two disease belts is feared. Based on microsatellite analyses, we found that G. f. fuscipes in northern Uganda are structured into three distinct genetic clusters with varying degrees of interconnectivity among them. Based on genetic assignment and spatial location, we grouped the sampling sites into four genetic units corresponding to northwestern Uganda in the Albert Nile drainage, northeastern Uganda in the Lake Kyoga drainage, western Uganda in the Victoria Nile drainage, and a transition zone between the two northern genetic clusters characterized by high level of genetic admixture. An analysis using HYBRIDLAB supported a hybrid swarm model as most consistent with tsetse genotypes in these admixed samples. Results of mtDNA analyses revealed the presence of 30 haplotypes representing three main haplogroups, whose location broadly overlaps with the microsatellite defined clusters. Migration analyses based on microsatellites point to moderate migration among the northern units located in the Albert Nile, Achwa River, Okole River, and Lake Kyoga drainages, but not between the northern units and the Victoria Nile drainage in the west. Effective population size estimates were variable with low to moderate sizes in most populations and with evidence of recent population bottlenecks, especially in the northeast unit of the Lake Kyoga drainage. Our microsatellite and mtDNA based analyses indicate that G. f. fuscipes movement along the Achwa and Okole rivers may facilitate northwest expansion of the Rhodesiense disease belt in Uganda. We identified tsetse migration corridors and recommend a rolling carpet approach from south of Lake Kyoga northward to minimize disease dispersal and prevent vector re-colonization. Additionally, our findings highlight the need for continuing tsetse monitoring efforts during and after control.

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.