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Control of African animal trypanosomiasis (AAT) is hampered by limited diagnostics, inappropriate trypanocide use, poor drug quality, and drug resistance.\ud The scope and quality of current literature on AAT incidence, control, and resistance does not allow for robust comparisons or assessment of the validity of extrapolating to other populations.\ud A united effort is needed to address AAT at local, national, and international settings to ensure a greater chance for success.\ud AAT control programmes must be sustainable through funding, cross-sectoral engagement, and fostering sustainable behavioural change through incentives and accountability.\ud Trypanocides are a key control component of African animal trypanosomiasis (AAT) in tsetse-infested areas of sub-Saharan Africa. While farmers are dependent upon trypanocides, recent research highlights their inappropriate and ineffective use, problems with drug quality, and treatment failure. There are currently gaps in knowledge and investment in inexpensive AAT diagnostics, understanding of drug resistance, and the effective use of trypanocides in the field. Without this important knowledge it is difficult to develop best practice and policy for existing drugs or to inform development and use of new drugs. There needs to be better understanding of the drivers and behavioural practices around trypanocide use so that they can be incorporated into sustainable solutions needed for the development of effective control of AAT.

Background Riverine species of tsetse (Glossina) transmit Trypanosoma brucei gambiense, which causes Gambian human African trypanosomiasis (gHAT), a neglected tropical disease. Uganda aims to eliminate gHAT as a public health problem through detection and treatment of human cases and vector control. The latter is being achieved through the deployment of ‘Tiny Targets’, insecticide-impregnated panels of material which attract and kill tsetse. We analysed the spatial and temporal distribution of cases of gHAT in Uganda during the period 2010–2019 to assess whether Tiny Targets have had an impact on disease incidence. Methods To quantify the deployment of Tiny Targets, we mapped the rivers and their associated watersheds in the intervention area. We then categorised each of these on a scale of 0–3 according to whether Tiny Targets were absent (0), present only in neighbouring watersheds (1), present in the watersheds but not all neighbours (2), or present in the watershed and all neighbours (3). We overlaid all cases that were diagnosed between 2000 and 2020 and assessed whether the probability of finding cases in a watershed changed following the deployment of targets. We also estimated the number of cases averted through tsetse control. Results We found that following the deployment of Tiny Targets in a watershed, there were fewer cases of HAT, with a sampled error probability of 0.007. We estimate that during the intervention period 2012–2019 we should have expected 48 cases (95% confidence intervals = 40–57) compared to the 36 cases observed. The results are robust to a range of sensitivity analyses. Conclusions Tiny Targets have reduced the incidence of gHAT by 25% in north-western Uganda. Graphical abstract

Sleeping sickness is still prevalent in Campo, southern Cameroon, despite the efforts of World Health Organization and the National Control Programme in screening and treating cases. Reducing disease incidence still further may need the control of tsetse vectors. We update entomological and parasitological parameters necessary to guide tsetse control in Campo. Tsetse flies were trapped, their apparent densities were evaluated as the number of flies captured per trap per day and mapped using GIS tools. Polymerase chain reaction based methods were used to identify their trypanosome infection rates. Glossina palpalis palpalis was the dominant vector species representing 93.42% and 92.85% of flies captured respectively during the heavy and light dry seasons. This species presented high densities, that is, 3.87, 95% CI [3.84-3.91], and 2.51, 95% CI [2.49-2.53] flies/trap/day in the two seasons. Moreover, 16.79% (of 1054) and 20.23% (of 1132 flies) were found infected with at least 1 trypanosome species for the 2 seasons respectively, Trypanosoma congolense being the most prevalent species, and Trypanosoma. brucei gambiense identified in 4 samples. Tsetse flies are abundant in Campo and present high trypanosome infection rates. The detection of tsetse infected with human trypanosomes near the newly created palm grove show workers' exposition. Tsetse densities maps built will guide vector control with 'Tiny Targets'.

Despite the ethical issues concerning the continued use of Human Landing Catches (HLC) to monitor the Simulium damnosum complex for epidemiological monitoring of onchocericasis, few attempts to develop alternatives have been reported. In studies on a wild population of S. damnosum in Burkina Faso, we tested visual targets (different sizes and shapes) and olfactory stimuli (CO2, and POCA and BG‐lure® odour blends) for their ability to attract and collect host seeking blackflies. At each trap, blackflies were caught with appropriately sized electrocuting grids and results from Latin square design tests were compared. Throughout, HLCs captured more blackflies than the targets. Of the traps tested, small targets (0.0625 and 0.5 m2) were the most efficient visual lure in terms of the number of S. damnosum captured per unit area 1.7–5× more than larger targets. Overall, results suggested that sticky black targets of horizontal rectangular shape (0.125–0.5 m2) and baited with a POCA and/or CO2 mixture could provide a cheap practical field alternative to HLC for onchocerciasis xenomonitoring, subject to confirmation that the design has no inherent bias for certain members of the S. damnosum species complex.

In Coˆte d’Ivoire, rubber cultivation has more than doubled since 2010. These mass agricultural areas require a large workforce with little information on how this environment might impact risk of mosquitoborne diseases. The objective of this study was to assess the larval ecology of mosquitoes in rubber areas of Dabou, Coˆ te d’Ivoire. From January to June 2017, an entomological survey was conducted of mature (MP) and immature (IP) rubber plantations, as well as in villages surrounded by rubber plantations (SV) and remote from rubber plantations (RV). The number and type of potential and positive breeding sites were recorded, and mosquito larval densities and diversity were estimated. Seven genera divided into 31 species including major vector such as Anopheles gambiae s.l. and Aedes aegypti were identified. A total of 1,660 waterbodies were identified with a larvae positivity rate of 63.1%. A majority of waterbodies were identified in SV (N = 875, 53.4% positivity rate), followed by MP (N = 422, 81.8% positivity rate), IP (N = 194, 72.2% positivity rate) and least in RV (N = 169, 57.4% positivity rate). The most important breeding sites for disease vectors were leaf axils in IP (N = 108, 77.1%), latex collection cups in MP (N = 332, 96.2%) and the containers abandoned in the SV (N = 242, 51.8%) as well as in the RV (N = 59, 60.8%). All these results allow us to affirm that the cultivation of rubber trees has an impact on the larval ecology by increasing the number of available sites and favoring a high larval density and diversity.

The tsetse flies, biological vectors of African trypanosomes, harbour a variety of bacteria involved in their vector competence that may help in developing novel vector control tools. This study provides an inventory of tsetse bacterial communities in Cameroon and explores their possible associations with trypanosome establishment in Glossina palpalis palpalis. High throughput sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene, with subsequent metagenomic, multivariate, and association analyses, were used to investigate the levels and patterns of microbial diversity in four tsetse species. Overall, 31 bacterial genera and four phyla were identified. The primary symbiont Wigglesworthia dominated almost all the samples, with an overall relative abundance of 47.29%, and seemed to be replaced by Serratia or Burkholderia in some G. tachinoides flies. Globally, significant differences were observed in the microbiome diversity and composition among tsetse species and between teneral and non-teneral flies, or between flies displaying or not displaying mature trypanosome infections. In addition, differential abundance testing showed some OTUs, or some bacteria taxa, associated with trypanosome maturation in tsetse flies. These bacteria could be further investigated for an understanding of their mechanism of action and alternatively, transformed and used to block trypanosome development in tsetse flies.

The peritrophic matrix of blood-feeding insects is a chitinous structure that forms a protective barrier against oral pathogens and abrasive particles1. Tsetse flies transmit Trypanosoma brucei, which is the parasite that causes human sleeping sickness and is also partially responsible for animal trypanosomiasis in Sub-Saharan Africa. For this parasite to establish an infection in flies, it must first colonize the area between the peritrophic matrix and gut epithelium called the ectoperitrophic space. Although unproven, it is generally accepted that trypanosomes reach the ectoperitrophic space by penetrating the peritrophic matrix in the anterior midgut2,3,4. Here, we revisited this event using fluorescence- and electron-microscopy methodologies. We show that trypanosomes penetrate the ectoperitrophic space in which the newly made peritrophic matrix is synthesized by the proventriculus. Our model describes how these proventriculus-colonizing parasites can either migrate to the ectoperitrophic space or become trapped within peritrophic matrix layers to form cyst-like bodies that are passively pushed along the gut as the matrix gets remodelled. Furthermore, early proventricular colonization seems to be promoted by factors in trypanosome-infected blood that cause higher salivary gland infections and potentially increase parasite transmission.

Background In recent years, a programme of vector control, screening and treatment of gambiense human African trypanosomiasis (gHAT) infections led to a rapid decline in cases in the Mandoul focus of Chad. To represent the biology of transmission between humans and tsetse, we previously developed a mechanistic transmission model, fitted to data between 2000 and 2013 which suggested that transmission was interrupted by 2015. The present study outlines refinements to the model to: (1) Assess whether elimination of transmission has already been achieved despite low-level case reporting; (2) quantify the role of intensified interventions in transmission reduction; and (3) predict the trajectory of gHAT in Mandoul for the next decade under different strategies. Method Our previous gHAT transmission model for Mandoul was updated using human case data (2000–2019) and a series of model refinements. These include how diagnostic specificity is incorporated into the model and improvements to the fitting method (increased variance in observed case reporting and how underreporting and improvements to passive screening are captured). A side-by-side comparison of fitting to case data was performed between the models. Results We estimated that passive detection rates have increased due to improvements in diagnostic availability in fixed health facilities since 2015, by 2.1-fold for stage 1 detection, and 1.5-fold for stage 2. We find that whilst the diagnostic algorithm for active screening is estimated to be highly specific (95% credible interval (CI) 99.9–100%, Specificity = 99.9%), the high screening and low infection levels mean that some recently reported cases with no parasitological confirmation might be false positives. We also find that the focus-wide tsetse reduction estimated through model fitting (95% CI 96.1–99.6%, Reduction = 99.1%) is comparable to the reduction previously measured by the decline in tsetse catches from monitoring traps. In line with previous results, the model suggests that transmission was interrupted in 2015 due to intensified interventions. Conclusions We recommend that additional confirmatory testing is performed in Mandoul to ensure the endgame can be carefully monitored. More specific measurement of cases, would better inform when it is safe to stop active screening and vector control, provided there is a strong passive surveillance system in place. Graphical Abstract

The single-celled parasite Trypanosoma brucei is transmitted by hematophagous tsetse flies. Life cycle progression from mammalian bloodstream form to tsetse midgut form and, subsequently, infective salivary gland form depends on complex developmental steps and migration within different fly tissues. As the parasite colonizes the glucose-poor insect midgut, ATP production is thought to depend on activation of mitochondrial amino acid catabolism via oxidative phosphorylation (OXPHOS). This process involves respiratory chain complexes and F1Fo-ATP synthase and requires protein subunits of these complexes that are encoded in the parasite's mitochondrial DNA (kDNA). Here, we show that progressive loss of kDNA-encoded functions correlates with a decreasing ability to initiate and complete development in the tsetse. First, parasites with a mutated F1Fo-ATP synthase with reduced capacity for OXPHOS can initiate differentiation from bloodstream to insect form, but they are unable to proliferate in vitro. Unexpectedly, these cells can still colonize the tsetse midgut. However, these parasites exhibit a motility defect and are severely impaired in colonizing or migrating to subsequent tsetse tissues. Second, parasites with a fully disrupted F1Fo-ATP synthase complex that is completely unable to produce ATP by OXPHOS can still differentiate to the first insect stage in vitro but die within a few days and cannot establish a midgut infection in vivo. Third, parasites lacking kDNA entirely can initiate differentiation but die soon after. Together, these scenarios suggest that efficient ATP production via OXPHOS is not essential for initial colonization of the tsetse vector but is required to power trypanosome migration within the fly.\ud \ud IMPORTANCE African trypanosomes cause disease in humans and their livestock and are transmitted by tsetse flies. The insect ingests these parasites with its blood meal, but to be transmitted to another mammal, the trypanosome must undergo complex development within the tsetse fly and migrate from the insect's gut to its salivary glands. Crucially, the parasite must switch from a sugar-based diet while in the mammal to a diet based primarily on amino acids when it develops in the insect. Here, we show that efficient energy production by an organelle called the mitochondrion is critical for the trypanosome's ability to swim and to migrate through the tsetse fly. Surprisingly, trypanosomes with impaired mitochondrial energy production are only mildly compromised in their ability to colonize the tsetse fly midgut. Our study adds a new perspective to the emerging view that infection of tsetse flies by trypanosomes is more complex than previously thought.

The National Programme for the control of human African trypanosomiasis in Democratic Republic of Congo includes a large-scale vector control operation using Tiny Targets. These are small panels of insecticide-impregnated cloth that are deployed in riverine habitat where tsetse flies concentrate. The effectiveness of Tiny Targets depends partly on acceptance by local communities. In 2018, we conducted research to explore the perception and acceptability of Tiny Targets in two different village clusters where Tiny Targets had been deployed by the local community or external teams. We conducted fourteen focus group discussions and seven semistructured interviews in three villages from each cluster in the Yasa Bonga health zone. Our findings showed that acceptability was better in the cluster where communities were involved in the deployment of Tiny Targets. Also in this cluster, awareness about Tiny Targets was satisfactory and the project was implemented within local customs, which promoted a positive perception of Tiny Targets and their benefits. In the cluster where external teams deployed Tiny Targets, a lack of information and communication, stereotypes applied by communities towards the deployment teams and the impression of inadequate respect for local customs led to anxiety and a misleading interpretation of the purpose of Tiny Targets and negatively influenced acceptability. This study highlights the importance of involving communities for programme acceptance. Our research underlined how awareness campaigns and communication are essential, but also how working within the scope of community social norms and customs are equally important. Prospects for the successful use of Tiny Targets are greater when communities are involved because the use can be adapted to social norms.

Background Work to control the gambiense form of human African trypanosomiasis (gHAT), or sleeping sickness, is now directed towards ending transmission of the parasite by 2030. In order to supplement gHAT case-finding and treatment, since 2011 tsetse control has been implemented using Tiny Targets in a number of gHAT foci. As this intervention is extended to new foci, it is vital to understand the costs involved. Costs have already been analysed for the foci of Arua in Uganda and Mandoul in Chad. This paper examines the costs of controlling Glossina palpalis palpalis in the focus of Bonon in Côte d’Ivoire from 2016 to 2017. Methodology/Principal findings Some 2000 targets were placed throughout the main gHAT transmission area of 130 km2 at a density of 14.9 per km2. The average annual cost was USD 0.5 per person protected, USD 31.6 per target deployed of which 12% was the cost of the target itself, or USD 471.2 per km2 protected. Broken down by activity, 54% was for deployment and maintenance of targets, 34% for tsetse surveys/monitoring and 12% for sensitising populations. Conclusions/Significance The cost of tsetse control per km2 of the gHAT focus protected in Bonon was more expensive than in Chad or Uganda, while the cost per km2 treated, that is the area where the targets were actually deployed, was cheaper. Per person protected, the Bonon cost fell between the two, with Uganda cheaper and Chad more expensive. In Bonon, targets were deployed throughout the protected area, because G. p. palpalis was present everywhere, whereas in Chad and Uganda G. fuscipes fuscipes was found only the riverine fringing vegetation. Thus, differences between gHAT foci, in terms of tsetse ecology and human geography, impact on the cost-effectiveness of tsetse control. It also demonstrates the need to take into account both the area treated and protected alongside other impact indicators, such as the cost per person protected., Author summary Sleeping sickness is a fatal disease in Africa caused by trypanosomes transmitted by the bite of infected tsetse flies. The World Health Organization has set the absence of new infections as a goal for 2030. To achieve this, screening and treatment of patients is supplemented by tsetse control. Tiny Targets are small insecticide-impregnated panels of blue cloth and black netting which attract and kill tsetse. To maintain the momentum in these last stages of eliminating the disease, it is important to understand the costs of vector control. This paper presents the results of two years’ cost monitoring in Bonon, an area covering 130 km2, home to 120,000 people. Since 2016, ~2,000 Tiny Targets have been deployed annually at a cost of USD 471 per km2 per year. Bonon is a degraded forested area and a relatively high number of targets was required as tsetse are present throughout. Nevertheless, the cost comes to only USD 0.5 per person protected per year. Compared to operations using Tiny Targets in Uganda and Chad, the cost was higher per km2 protected, reflecting the need to place targets throughout the area, but low per person protected compared to Chad, highlighting the need to use a range of comparators.

Background Tsetse flies are the major vectors of human trypanosomiasis of the form Trypanosoma brucei rhodesiense and T.b.gambiense. They are widely spread across the sub-Saharan Africa and rendering a lot of challenges to both human and animal health. This stresses effective agricultural production and productivity in Africa. Delimiting the extent and magnitude of tsetse coverage has been a challenge over decades due to limited resources and unsatisfactory technology. In a bid to overcome these limitations, this study attempted to explore modelling skills that can be applied to spatially estimate tsetse abundance in the country using limited tsetse data and a set of remote-sensed environmental variables. Methodology Entomological data for the period 2008–2018 as used in the model were obtained from various sources and systematically assembled using a structured protocol. Data harmonisation for the purposes of responsiveness and matching was carried out. The key tool for tsetse trapping was itemized as pyramidal trap in many instances and biconical trap in others. Based on the spatially explicit assembled data, we ran two regression models; standard Poisson and Zero-Inflated Poisson (ZIP), to explore the associations between tsetse abundance in Uganda and several environmental and climatic covariates. The covariate data were constituted largely by satellite sensor data in form of meteorological and vegetation surrogates in association with elevation and land cover data. We finally used the Zero-Inflated Poisson (ZIP) regression model to predict tsetse abundance due to its superiority over the standard Poisson after model fitting and testing using the Vuong Non-Nested statistic. Results A total of 1,187 tsetse sampling points were identified and considered as representative for the country. The model results indicated the significance and level of responsiveness of each covariate in influencing tsetse abundance across the study area. Woodland vegetation, elevation, temperature, rainfall, and dry season normalised difference vegetation index (NDVI) were important in determining tsetse abundance and spatial distribution at varied scales. The resultant prediction map shows scaled tsetse abundance with estimated fitted numbers ranging from 0 to 59 flies per trap per day (FTD). Tsetse abundance was found to be largest at low elevations, in areas of high vegetative activity, in game parks, forests and shrubs during the dry season. There was very limited responsiveness of selected predictors to tsetse abundance during the wet season, matching the known fact that tsetse disperse most significantly during wet season. Conclusions A methodology was advanced to enable compilation of entomological data for 10 years, which supported the generation of tsetse abundance maps for Uganda through modelling. Our findings indicate the spatial distribution of the G. f. fuscipes as; low 0–5 FTD (48%), medium 5.1–35 FTD (18%) and high 35.1–60 FTD (34%) grounded on seasonality. This approach, amidst entomological data shortages due to limited resources and absence of expertise, can be adopted to enable mapping of the vector to provide better decision support towards designing and implementing targeted tsetse and tsetse-transmitted African trypanosomiasis control strategies., Author summary Tsetse flies are the major vectors of human and animal trypanosomiasis. To support control efforts, there is need to understand the location of these vectors in terms of distribution and abundance. Due to limited resources and unsatisfactory technology, delimiting the extent and magnitude of tsetse coverage remains a challenge. In a bid to overcome these limitations, this study attempted to explore modelling skills that can be applied to inform decision makers about the status of tsetse abundance using limited historical tsetse data and a set of remote-sensed environmental variables. Two regression models; standard Poisson and Zero-Inflated Poisson (ZIP) were fitted and evaluated for superiority. The results indicated the level of responsiveness of each covariate in influencing the vector across the study area. Tsetse abundance was found to be largest at low elevations in areas of high vegetative activity, in game parks, forests and permanently flooded shrubs during the dry season. This approach can be adopted to enable mapping of any tsetse species to provide better decision support towards designing and implementing targeted tsetse and tsetse-transmitted African trypanosomiasis control strategies amidst entomological data shortages due to limited resources and absence of expertise.

Simple Summary Diverse strains of Wolbachia bacteria, carried by many arthropods, as well as some nematodes, interact in many different ways with their hosts. These include male killing, reproductive incompatibility, nutritional supplementation and suppression or enhancement of the transmission of diseases such as dengue and malaria. Consequently, Wolbachia have an important role to play in novel strategies to control human and livestock diseases and their vectors. Similarly, cell lines derived from insect hosts of Wolbachia constitute valuable research tools in this field. During the generation of novel cell lines from mosquito and sand fly vectors, we isolated two strains of Wolbachia and demonstrated their infectivity for cells from a range of other insects and ticks. These new insect cell lines and Wolbachia strains will aid in the fight against mosquitoes, sand flies and, potentially, ticks and the diseases that these arthropods transmit to humans and their domestic animals. Abstract Endosymbiotic intracellular bacteria of the genus Wolbachia are harboured by many species of invertebrates. They display a wide range of developmental, metabolic and nutritional interactions with their hosts and may impact the transmission of arboviruses and protozoan parasites. Wolbachia have occasionally been isolated during insect cell line generation. Here, we report the isolation of two strains of Wolbachia, wPip and wPap, during cell line generation from their respective hosts, the mosquito Culex pipiens and the sand fly Phlebotomus papatasi. wPip was pathogenic for both new C. pipiens cell lines, CPE/LULS50 and CLP/LULS56, requiring tetracycline treatment to rescue the lines. In contrast, wPap was tolerated by the P. papatasi cell line PPL/LULS49, although tetracycline treatment was applied to generate a Wolbachia-free subline. Both Wolbachia strains were infective for a panel of heterologous insect and tick cell lines, including two novel lines generated from the sand fly Lutzomyia longipalpis, LLE/LULS45 and LLL/LULS52. In all cases, wPip was more pathogenic for the host cells than wPap. These newly isolated Wolbachia strains, and the novel mosquito and sand fly cell lines reported here, will add to the resources available for research on host–endosymbiont relationships, as well as on C. pipiens, P. papatasi, L. longipalpis and the pathogens that they transmit.

Many vector-borne diseases are controlled by methods that kill the insect vectors responsible for disease transmission. Recording the age structure of vector populations provides information on mortality rates and vectorial capacity, and should form part of the detailed monitoring that occurs in the wake of control programmes, yet tools for obtaining estimates of individual age remain limited. We investigate the potential of using markers of gene expression to predict age in tsetse flies, which are the vectors of deadly and economically damaging African trypanosomiases. We use RNAseq to identify candidate expression markers, and test these markers using qPCR in laboratory-reared Glossina morsitans morsitans of known age. Measuring the expression of six genes was sufficient to obtain a prediction of age with root mean squared error of less than 8 days, while just two genes were sufficient to classify flies into age categories of ≤15 and >15 days old. Further testing of these markers in field-caught samples and in other species will determine the accuracy of these markers in the field.

Background Gambian human African trypanosomiasis (gHAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies (Glossina). In Côte d’Ivoire, Bonon is the most important focus of gHAT, with 325 cases diagnosed from 2000 to 2015 and efforts against gHAT have relied largely on mass screening and treatment of human cases. We assessed whether the addition of tsetse control by deploying Tiny Targets offers benefit to sole reliance on the screen-and-treat strategy. Methodology and principal findings In 2015, we performed a census of the human population of the Bonon focus, followed by an exhaustive entomological survey at 278 sites. After a public sensitization campaign, ~2000 Tiny Targets were deployed across an area of 130 km2 in February of 2016, deployment was repeated annually in the same month of 2017 and 2018. The intervention’s impact on tsetse was evaluated using a network of 30 traps which were operated for 48 hours at three-month intervals from March 2016 to December 2018. A second comprehensive entomological survey was performed in December 2018 with traps deployed at 274 of the sites used in 2015. Sub-samples of tsetse were dissected and examined microscopically for presence of trypanosomes. The census recorded 26,697 inhabitants residing in 331 settlements. Prior to the deployment of targets, the mean catch of tsetse from the 30 monitoring traps was 12.75 tsetse/trap (5.047–32.203, 95%CI), i.e. 6.4 tsetse/trap/day. Following the deployment of Tiny Targets, mean catches ranged between 0.06 (0.016–0.260, 95%CI) and 0.55 (0.166–1.794, 95%CI) tsetse/trap, i.e. 0.03–0.28 tsetse/trap/day. During the final extensive survey performed in December 2018, 52 tsetse were caught compared to 1,909 in 2015, with 11.6% (5/43) and 23.1% (101/437) infected with Trypanosoma respectively. Conclusions The annual deployment of Tiny Targets in the gHAT focus of Bonon reduced the density of Glossina palpalis palpalis by >95%. Tiny Targets offer a powerful addition to current strategies towards eliminating gHAT from Côte d’Ivoire., Author summary Gambian sleeping sickness (Gambian human African trypanosomiasis, gHAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies. Currently, Bonon is the focus which provides most cases of gHAT in Côte d’Ivoire. Screening and treatment of human cases has reduced the incidence of gHAT from 262 cases diagnosed between 2000 and 2004 to 24 cases during 2010–2015. We carried out a trial to assess whether Tiny Targets, insecticide-treated targets that attract and kill tsetse, could control Glossina palpalis palpalis, the most important vector of gHAT in Côte d’Ivoire. In 2015, we mapped human settlements, livestock, tracks, rivers and relict forest in Bonon and identified sites where humans may be bitten by tsetse. Monoconical (“Vavoua”) traps were deployed at these sites to provide an estimate of the abundance of tsetse. Between 2016 and 2018, ~2,000 Tiny Targets were deployed annually across Bonon and the impact of Tiny Targets was evaluated by changes in the numbers of tsetse caught by a network of 30 monitoring traps operated quarterly. In 2015, before deployment of Tiny Targets, the mean daily catch from the 30 monitoring traps was 6.4 tsetse/trap/day. Following deployment of targets, catches declined to 95% reduction in tsetse abundance. Between February 2016 and December 2018, no recent (Stage 1) cases of gHAT have been reported in Bonon. Our results demonstrate that Tiny Targets can contribute to the elimination of gHAT through tsetse control. Tiny Targets have been adopted as an important tool in Côte d’Ivoire’s national strategy to eliminate gHAT.

Vector-borne disease control relies on efficient vector surveillance, mostly carried out using traps whose number and locations are often determined by expert opinion rather than a rigorous quantitative sampling design. In this work we propose a framework for ecological sampling design which in its preliminary stages can take into account environmental conditions obtained from open data (i.e. remote sensing and meteorological stations) not necessarily designed for ecological analysis. These environmental data are used to delimit the area into ecologically homogeneous strata. By employing Bayesian statistics within a model-based sampling design, the traps are deployed among the strata using a mixture of random and grid locations which allows balancing predictions and model-fitting accuracies. Sample sizes and the effect of ecological strata on sample sizes are estimated from previous mosquito sampling campaigns open data. Notably, we found that a configuration of 30 locations with four households each (120 samples) will have a similar accuracy in the predictions of mosquito abundance as 200 random samples. In addition, we show that random sampling independently from ecological strata, produces biased estimates of the mosquito abundance. Finally, we propose standardizing reporting of sampling designs to allow transparency and repetition/re-use in subsequent sampling campaigns.

Background Surveillance is an essential component of global programs to eliminate infectious diseases and avert epidemics of (re-)emerging diseases. As the numbers of cases decline, costs of treatment and control diminish but those for surveillance remain high even after the ‘last’ case. Reducing surveillance may risk missing persistent or (re-)emerging foci of disease. Here, we use a simulation-based approach to determine the minimal number of passive surveillance sites required to ensure maximum coverage of a population at-risk (PAR) of an infectious disease. Methodology and principal findings For this study, we use Gambian human African trypanosomiasis (g-HAT) in north-western Uganda, a neglected tropical disease (NTD) which has been reduced to historically low levels (95% of a total PAR of ~3million individuals living ≤1h from a diagnostic centre, and we demonstrate an approach to best place these facilities, informing a minimal impact scale back. Conclusions Our results highlight that surveillance of g-HAT in north-western Uganda can be scaled back without substantially reducing coverage of the PAR. The methodology described can contribute to cost-effective and equable strategies for the surveillance of NTDs and other infectious diseases approaching elimination or (re-)emergence., Author summary Disease surveillance systems are an essential component of public health practice and are often considered the first line in averting epidemics for (re-)emerging diseases. Regular evaluation of surveillance systems ensures that they remain operating at maximum efficiency; systems that survey diseases of low incidence, such as those within elimination settings, should be simplified to reduce the reporting burden. A lack of guidance on how to optimise disease surveillance in an elimination setting may result in added expense, and/or the underreporting of disease. Here, we propose a framework methodology to determine systematically the optimal number and placement of surveillance sites for the surveillance of infectious diseases approaching elimination. By utilising estimates of geographic accessibility, through the construction of a resistance surface and a simulation approach, we identify that the number of operational diagnostic facilities for Gambian human African trypanosomiasis in north-western Uganda can be reduced by 70% with a minimal reduction in existing coverage, and identify the minimum number of facilities required to meet coverage targets. Our analysis can be used to inform the number and positioning of surveillance sites for diseases within an elimination setting. Passive surveillance becomes increasingly important as cases decline and active surveillance becomes less cost-effective; methods to evaluate how best to engage this passive surveillance capacity given facility capacity and geographic distribution are pertinent for several NTDs where diagnosis is complex. Not only is this a complicated research area for diseases approaching elimination, a well-designed surveillance system is essential for the detection of emerging diseases, with this work being topical in a climate where emerging pathogens are becoming more commonplace.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anticlotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online\ud with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of\ud highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized\ud by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite\ud transmission into the vertebrate host.

Background Tsetse flies (Glossina) transmit Trypanosoma brucei gambiense which causes Gambian human African trypanosomiasis (gHAT) in Central and West Africa. Several countries use Tiny Targets, comprising insecticide-treated panels of material which attract and kill tsetse, as part of their national programmes to eliminate gHAT. We studied how the scale and arrangement of target deployment affected the efficacy of control. Methodology and principal findings Between 2012 and 2016, Tiny Targets were deployed biannually along the larger rivers of Arua, Maracha, Koboko and Yumbe districts in North West Uganda with the aim of reducing the abundance of tsetse to interrupt transmission. The extent of these deployments increased from ~250 km2 in 2012 to ~1600 km2 in 2015. The impact of Tiny Targets on tsetse populations was assessed by analysing catches of tsetse from a network of monitoring traps; sub-samples of captured tsetse were dissected to estimate their age and infection status. In addition, the condition of 780 targets (~195/district) was assessed for up to six months after deployment. In each district, mean daily catches of tsetse (G. fuscipes fuscipes) from monitoring traps declined significantly by >80% following the deployment of targets. The reduction was apparent for several kilometres on adjacent lengths of the same river but not in other rivers a kilometre or so away. Expansion of the operational area did not always produce higher levels of suppression or detectable change in the age structure or infection rates of the population, perhaps due to the failure to treat the smaller streams and/or invasion from adjacent untreated areas. The median effective life of a Tiny Target was 61 (41.8–80.2, 95% CI) days. Conclusions Scaling-up of tsetse control reduced the population of tsetse by >80% across the intervention area. Even better control might be achievable by tackling invasion of flies from infested areas within and outside the current intervention area. This might involve deploying more targets, especially along smaller rivers, and extending the effective life of Tiny Targets.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes., This study shows that tsetse flies, vectors of African trypanosomiasis, are highly susceptible to killing by nitisinone, a tyrosine catabolism inhibitor currently used to treat human metabolic diseases; this environment-friendly drug could facilitate elimination of African trypanosomiasis and other diseases transmitted by blood feeding insects.

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.

While across the animal kingdom offspring are born smaller than their parents, notable exceptions exist. Several dipteran species belonging to the Hippoboscoidea superfamily can produce offspring larger than themselves. In this essay, the blood‐feeding tsetse is focused on. It is suggested that the extreme reproductive strategy of this fly is enabled by feeding solely on highly nutritious blood, and producing larval offspring that are soft and malleable. This immense reproductive expenditure may have evolved to avoid competition with other biting flies. Tsetse also transmit blood‐borne parasites that cause the fatal diseases called African trypanosomiases. It is discussed how tsetse life history and reproductive strategy profoundly influence the type of vector control interventions used to reduce fly populations. In closing, it is argued that the unusual life history of tsetse warrants their preservation in the areas where human and animal health is not threatened.

Background Invasive mosquito species, such as Aedes albopictus in Congo can affect the distribution of native species, changing the vector composition and pattern of disease transmission. Here, we comparatively establish the geographical distribution and larval habitat preference of Ae. aegypti and Ae. albopictus and the risk of arbovirus disease outbreaks using Stegomyia indices in the city of Brazzaville, the capital of the Republic of the Congo. Methods Human dwelling surveys of water-holding containers for immature stages of Aedes was carried out in December 2017 in Brazzaville through a random cluster sampling method. A total of 268 human dwellings distributed in 9 boroughs and 27 neighbourhoods were surveyed across the city. Results Overall, 455 potential larval habitats were surveyed. Both Ae. aegypti and Ae. albopictus were collected across the city with an overall high prevalence of Ae. aegypti (53.1%) compared to Ae. albopictus (46.9%). Geographical distribution analysis showed that Ae. aegypti was more abundant (mean = 6.6 ± 1.4) in neighbourhoods located in downtown, while the abundance of Ae. albopictus was low (mean = 3.5 ± 0.6) in suburbs. Peridomestic containers, especially discarded tanks, were the most strongly colonized productive larval habitat for both mosquito species with the prevalence of 56.4% and 53.1% for Ae. aegypti and Ae. albopictus, respectively. Globally, the house index (HI), Breteau index (BI) and container index (CI) were high for Ae. aegypti (26.6%, 38.4% and 22.6%) and Ae. albopictus (33.3%, 49.6% and 26.6%) compared to the transmission risk threshold (5%, 5% and 20%) established by the WHO/PAHO. Overall, pupae-based indices (the pupae index and the pupae per person index) were not significantly different between Ae. aegypti (273.4% and 23.2%) and Ae. albopictus (228.8% and 19.5%). Conclusions The findings of this study suggest a high risk for transmission of arbovirus diseases in Brazzaville and call for an urgent need to implement vector control strategies against these vectors in the Republic of the Congo.

Many organisms show signs of deterioration with age in terms of survival and reproduction. We tested whether intraspecific variation in such senescence patterns can be driven by resource availability or reproductive history. We did this by manipulating nutritional stress and age at first reproduction and measuring age-dependent reproductive output in tsetse (Glossina morsitans morsitans), a viviparous fly with high maternal allocation. Across all treatments, offspring weight followed a bell-shaped curve with maternal age. Nutritionally stressed females had a higher probability of abortion and produced offspring with lower starvation tolerance. There was no evidence of an increased rate of reproductive senescence in nutritionally stressed females, or a reduced rate due to delayed mating, as measured by patterns of abortion, offspring weight or offspring starvation tolerance. Therefore, although we found evidence of reproductive senescence in tsetse, our results did not indicate that resource allocation trade-offs or costs of reproduction increase the rate of senescence.

Gambiense Human African Trypanosomiasis (g-HAT) is a neglected tropical disease caused by trypanosomes transmitted by tsetse flies. 70% of cases in 2019 (604/863) occurred in the Democratic Republic of Congo (DRC). The national programme for g-HAT elimination in DRC includes a large-scale deployment of Tiny Targets which attract and kill tsetse. This intervention is directed by vector-control specialists with small teams, moving in canoes, deploying Tiny Targets along riverbanks where tsetse concentrate. While the targets are deployed in communal areas, and the method is cheap and easy-to-use, local people have little involvement. This study aimed to evaluate if a community-led vector control programme was feasible in the context of DRC’s g-HAT elimination programme. In 2017, a community-led intervention was implemented in three villages in the Kwilu province of DRC. This intervention was evaluated through an Action Research with qualitative data collected through 21 focus group discussions and 289 hours of observation. Also the geographical location and quality of each Tiny Targets were collected (total number deployed = 2429). This research revealed that community-based approach largely worked: people were motivated and proactive, showed a good application of the acquired knowledge resulting in an effective deployment of Tiny Targets. In addition, our study provided evidence that acceptability of the targets by the community can improve deployment quality by reducing target loss and damage. The approach was feasible in places where canoe-based teams could not reach. Against these advantages, a community-based approach was time-consuming and had to adapt to the seasonal and daily rhythms of the community. A community-based approach for tsetse control is technically feasible and recommended but limits to the speed and scale of the approach restraints its application as a standalone strategy in a large-scale national programme aiming to eliminate g-HAT in a short timeframe., Author summary Sleeping sickness (Human African Trypanosomiasis, HAT) is a neglected disease caused by Trypanosoma brucei gambiense or T.b.rhodesiense both transmitted by tsetse flies. Currently 70% of the global cases of Gambiense HAT (g-HAT) occur in Democratic Republic of Congo (DRC). Over the last 20 years, a national programme of screening and treating humans has driven down the number of cases of g-HAT from >30,000 to

Over the past 20 years there has been a >95% reduction in the number of Gambian Human African trypanosomiasis (g-HAT) cases reported globally, largely as a result of large-scale active screening and treatment programmes. There are however still foci where the disease persists, particularly in parts of the Democratic Republic of the Congo (DRC). Additional control efforts such as tsetse control using Tiny Targets may therefore be required to achieve g-HAT elimination goals. The purpose of this study was to evaluate the impact of Tiny Targets within DRC. In 2015–2017, pre- and post-intervention tsetse abundance data were collected from 1,234 locations across three neighbouring Health Zones (Yasa Bonga, Mosango, Masi Manimba). Remotely sensed dry season data were combined with pre-intervention tsetse presence/absence data from 332 locations within a species distribution modelling framework to produce a habitat suitability map. The impact of Tiny Targets on the tsetse population was then evaluated by fitting a generalised linear mixed model to the relative fly abundance data collected from 889 post-intervention monitoring sites within Yasa Bonga, with habitat suitability, proximity to the intervention and intervention duration as covariates. Immediately following the introduction of the intervention, we observe a dramatic reduction in fly catches by > 85% (pre-intervention: 0.78 flies/trap/day, 95% CI 0.676–0.900; 3 month post-intervention: 0.11 flies/trap/day, 95% CI 0.070–0.153) which is sustained throughout the study period. Declines in catches were negatively associated with proximity to Tiny Targets, and while habitat suitability is positively associated with abundance its influence is reduced in the presence of the intervention. This study adds to the body of evidence demonstrating the impact of Tiny Targets on tsetse across a range of ecological settings, and further characterises the factors which modify its impact. The habitat suitability maps have the potential to guide the expansion of tsetse control activities in this area., Author summary There have been large declines in the number of cases of sleeping sickness as a result of programmes that actively screen and treat the at-risk population. Additional control is needed in areas where the disease persists such as parts of the Democratic Republic of Congo (DRC). The disease is transmitted by tsetse flies, and reducing the tsetse population using Tiny Targets has been shown to control the disease in other countries. Extensive tsetse monitoring has been undertaken in one Health Zone in DRC where Tiny Targets have been deployed. We used these data to gain a better understanding of tsetse habitat, to produce habitat suitability maps, and to subsequently measure the impact of Tiny Targets on the tsetse population. We show that tsetse flies are largely found along rivers and surrounding densely vegetated habitat, with there being a positive relationship between habitat suitability and the number of flies caught. Once Tiny Targets were introduced, the number of flies caught in monitoring traps decreased by >85%, with habitat suitability at the trap location, and the proximity of the trap to the nearest Tiny Target influencing the size of the effect of the intervention. This study adds to the body of evidence demonstrating the impact of Tiny Targets on tsetse distribution in addition to providing information that can be used to guide the expansion of tsetse control activities in this area.

BackgroundSince 2012, the World Health Organisation and the countries affected by the Gambian form of human African trypanosomiasis (HAT) have been committed to eliminating the disease, primarily through active case-finding and treatment. To interrupt transmission ofTrypanosoma brucei gambienseand move more rapidly towards elimination, it was decided to add vector control using ‘tiny targets’. Chad’s Mandoul HAT focus extends over 840 km2, with a human population of 39,000 as well as 14,000 cattle and 3000 pigs. Some 2700 tiny targets were deployed annually from 2014 onwards.MethodsA protocol was developed for the routine collection of tsetse control costs during all field missions. This was implemented throughout 2015 and 2016, and combined with the recorded costs of the preliminary survey and sensitisation activities. The objective was to calculate the full costs at local prices in Chad. Costs were adjusted to remove research components and to ensure that items outside the project budget lines were included, such as administrative overheads and a share of staff salaries.ResultsTargets were deployed at about 60 per linear km of riverine tsetse habitat. The average annual cost of the operation was USD 56,113, working out at USD 66.8 per km2 protected and USD 1.4 per person protected. Of this, 12.8% was an annual share of the initial tsetse survey, 40.6% for regular tsetse monitoring undertaken three times a year, 36.8% for target deployment and checking and 9.8% for sensitisation of local populations. Targets accounted for 8.3% of the cost, and the cost of delivering a target was USD 19.0 per target deployed.ConclusionsThis study has confirmed that tiny targets provide a consistently low cost option for controlling tsetse in gambiense HAT foci. Although the study area is remote with a tsetse habitat characterised by wide river marshes, the costs were similar to those of tiny target work in Uganda, with some differences, in particular a higher cost per target delivered. As was the case in Uganda, the cost was between a quarter and a third that of historical target operations using full size targets or traps.

Background Vector control is emerging as an important component of global efforts to control Gambian sleeping sickness (human African trypanosomiasis, HAT). The deployment of insecticide-treated targets (“Tiny Targets”) to attract and kill riverine tsetse, the vectors of Trypanosoma brucei gambiense, has been shown to be particularly cost-effective. As this method of vector control continues to be implemented across larger areas, knowledge of the abundance of tsetse to guide the deployment of “Tiny Targets” will be of increasing value. In this paper, we use a geostatistical modelling framework to produce maps of estimated tsetse abundance under two scenarios: (i) when accurate data on the local river network are available; and (ii) when river information is sparse. Methods Tsetse abundance data were obtained from a pre-intervention survey conducted in northern Uganda in 2010. River network data obtained from either digitised maps or derived from 30 m resolution digital elevation model (DEM) data as a proxy for ground truth data. Other environmental variables were derived from publicly-available resolution remotely sensed data (e.g. Landsat, 30 m resolution). Zero-inflated negative binomial geostatistical models were fitted to the abundance data using an integrated nested Laplace approximation approach, and maps of estimated tsetse abundance were produced. Results Restricting the analysis to traps located within 100 m of any river, positive associations were identified between the length of river and the minimum soil/vegetation moisture content of the surrounding area and daily fly catches, whereas negative associations were identified with elevation and distance to the river. The resulting models could accurately distinguish between traps with high and low fly catches (e.g. < 5 or > 5 flies/day), with a ROC-AUC (receiver-operating characteristic - area under the curve) greater than 0.9. Whilst the precise course of the river was not well approximated using the DEM data, the models fitted using DEM-derived river data performed similarly to those that incorporated the more accurate local river information. Conclusions These models can now be used to assist in the design, implementation and monitoring of tsetse control operations in northern Uganda and further can be used as a framework by which to undertake similar studies in other areas where Glossina fuscipes fuscipes spreads Gambian sleeping sickness. Electronic supplementary material The online version of this article (10.1186/s13071-018-2922-5) contains supplementary material, which is available to authorized users.

The global disruptions caused by the coronavirus disease 2019 crisis posed a threat to the momentum the vector control team at the Liverpool School of Tropical Medicine (LSTM) and the Programme National de Lutte contre la Tryaponosomiase Humaine Africaine (PNLTHA) had built in their efforts to control tsetse fly populations in the Democratic Republic of Congo. But despite the pandemic and global lockdown, field activities did continue and the same impressive results in tsetse fly reduction were observed and the team followed this by completing a round of ‘tiny target’ deployment without any external presence. Such a success was possible due to the investment in vector control capacity strengthening undertaken by the LSTM and PNLTHA.

Gambiense human African trypanosomiasis (g-HAT) is the chronic form of sleeping sickness caused by Trypanosoma brucei gambiense in West and Central Africa, while Trypanosoma brucei rhodesiense causes an acute form in eastern Africa. g-HAT is targeted for elimination as a public health problem by 2020 and 0 transmission by 2030 [1,2]. Control of g-HAT is largely based on identification and treatment of infected individuals, supplemented by control of the tsetse fly vectors [3]. There has been growing evidence that when both tsetse control and case identification activities are carried out simultaneously in the same geographies, elimination of the disease is accelerated [4–6]. Here, we describe how the Trypa-NO! Partnership is using novel and classical tools to drive g-HAT elimination in an integrated approach, progress made, lessons learnt, and future directions.

Introduction Dengue is the most important mosquito-borne diseases worldwide but was considered scarce in West-Central Africa. During the last decade, dengue outbreaks have increasingly been reported in urban foci in this region suggesting major epidemiological changes. However, in Central Africa where both vectors, Aedes aegypti and Aedes albopictus are well established, the role of each species in dengue transmission remains poorly investigated. Methodology/Principal findings Field-collected strains of Ae. aegypti and Ae. albopictus from different ecological settings in Central Africa were experimentally challenged with dengue 2 virus (DENV-2). Mosquitoes were analysed at 14- and 21-days post-infection. Analysis provide evidence that both Ae. aegypti and Ae. albopictus in Central Africa were able to transmit dengue virus with Ae. aegypti exhibiting a higher transmission rate. Unexpectedly, two Ae. aegypti populations from Bénoué and Maroua, in northern Cameroon, were not able to transmit DENV-2. Conclusions/Significance We conclude that both Ae. aegypti and Ae. albopictus are susceptible to DENV-2 and may intervene as active dengue vectors. These findings highlight the urgent need to plan a vector surveillance program and control methods against dengue vectors in Central Africa in order to prevent future outbreaks., Author summary Dengue virus (DENV) is a flavivirus mainly transmitted to humans through the bite of infected mosquitoes notably Aedes aegypti and Aedes albopictus. In Central Africa where both vectors, Ae. aegypti and Ae. albopictus are well established, the role of each species in dengue transmission remains poorly investigated. Here, we assessed the vector competence of Ae. aegypti and Ae. albopictus collected in different ecological settings in Central Africa to transmit dengue 2 virus (DENV-2). We provide evidence that both Ae. aegypti and Ae. albopictus in Central Africa were able to transmit dengue virus with Ae. aegypti exhibiting a higher transmission rate. These findings could increase the risk of dengue outbreak in the region and emphasize the need for a comprehensive vector surveillance program to prevent and preparedness for an intervention in case of outbreaks.

The savannah tsetse flies, Glossina morsitans morsitans and G. pallidipes, are important vectors of Rhodesian human African trypanosomiasis and animal African trypanosomiasis in East and southern Africa. We tested in Zimbabwe whether robust, synthetic fabrics, and innovative fly’s-eye-view approaches to optimise fabric colour, can improve insecticide-treated targets employed for tsetse control. Flies were caught by electrocution at a standard target comprising a 1m x 1m black cotton cloth panel with 1m x 0.5m black polyester net panels on each side. Catches were subdivided by species and sex. Tsetse catches were unaffected by substitution of the black cotton with a blue polyester produced for riverine tsetse targets. Exchanging the net panels for phthalogen blue cotton to simulate the target routinely used in Zimbabwe significantly reduced catches of female G. m. morsitans (mean catch 0.7 times that at standard), with no effect on other tsetse catches. However, significantly greater proportions of the catch were intercepted at the central panel of the Zimbabwe (means 0.47–0.79) versus standard designs (0.11–0.29). We also engineered a new violet polyester cloth using models of tsetse attraction based upon fly photoreceptor responses. With and without odour lure, catches of females of both species at the violet target were significantly greater than those at standard (means 1.5–1.6 times those at standard), and typical blue polyester targets (means 0.9–1.3 times those at standard). Similar effects were observed for males under some combinations of species and odour treatment. The proportions of catch intercepted at the central panel of the violet target (means 0.08–0.18) were intermediate between those at standard and typical blue polyester. Further, the reflectance spectrum of violet polyester was more stable under field conditions than that of black cotton. Our results demonstrate the effectiveness of photoreceptor-based models as a novel means of improving targets to control tsetse and trypanosomiases.

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

The World Health Organisation has set the goal for elimination of Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei gambiense (gHAT), as a public health problem for 2020 and for the total interruption of transmission to humans for 2030. Targeting human carriers and potential animal reservoir infections will be critical to achieving this ambitious goal. However, there is continuing debate regarding the significance of reservoir host animals, wild and domestic, in different epidemiological contexts, whilst the extent and duration of the asymptomatic human carrier state is similarly undefined.\ud \ud This paper reviews the status of the knowledge of latent infections in wild and domestic animal reservoir hosts towards the goal of better understanding their role in the transmission dynamic of the disease. Focus areas include the transmission cycles in non-human hosts, the infectivity of animal reservoirs to Glossina palpalis s.l., the longevity of infection and the stability of T. b. gambiense biological characteristics in antelopes and domestic animals. There is compelling evidence that T. b. gambiense can establish and persist in experimentally infected antelopes, pigs and dogs for a period of at least two years. In particular, metacyclic transmission of T. b. gambiense has been reported in antelope-G.p.palpalis-antelope and pig-G.p.gambiensis-pig cycles.\ud \ud Experimental studies demonstrate that the infectiveness of latent animal reservoir infections with T. b. gambiense is retained in animal-Glossina-animal cycles (antelopes and pigs) for periods of three years and human infectivity markers (human serum resistance, zymodeme, DNA) are stable in non-human hosts for the same period. These observations shed light on the epidemiological significance of animal reservoir hosts in specific ecosystems characterized by presently active, as well as known “old” HAT foci whilst challenging the concept of total elimination of all transmission by 2030. This target is also compromised by the existence of human asymptomatic carriers of T. b. gambiense often detected outside Africa after having lived outside tsetse infested areas for many years - sometimes decades.\ud \ud Non-tsetse modes of transmission may also play a significant but underestimated role in the maintenance of foci and also preclude the total elimination of transmission - these include mother to child transmission and sexual transmission. Both these modes of transmission have been the subject of case reports yet their frequency in African settings remains to be ascertained when the context of residual foci are discussed yet both challenge the concept of the possibility of the total elimination of transmission.

INTRODUCTION\ud Arboviral diseases including dengue are increasingly spreading in the tropical/subtropical world including Africa. Updated knowledge on the distribution and abundance of the major vectors Aedes aegypti and Aedes albopictus constitutes crucial surveillance action to prepare African countries such as Cameroon for potential arbovirus outbreaks. Here, we present a nationwide survey in Cameroon to assess the current geographical distribution and prevalence of both vectors including a genetic diversity profiling of Ae. albopictus (invasive species) using mitochondrial DNA.\ud METHODS\ud Immature stages of Aedes were collected between March and August 2017 in 29 localities across Cameroon following north-south and east-west transects. Larvae and pupae were collected from several containers in each location, reared to adult and morphologically identified. Genetic diversity of Ae. albopictus from 16 locations were analysed using Cytochrome Oxidase I gene (COI).\ud RESULTS\ud In total, 30,381 immature stages of Aedes with an average of 646.40±414.21 per location were identified across the country comprising 69.3% of Ae. albopictus and 30.7% of Ae. aegypti. Analysis revealed that Ae. aegypti is still distributed nation widely whereas Ae. albopictus is limited to the southern part, around 6°4'N. However, Ae. albopictus is the most prevalent species in all southern locations where both species are sympatric except in Douala where Ae. aegypti is predominant. This suggests that factors such as climate, vegetation, and building density impact the distribution of both species in Cameroon. Mitochondrial DNA analysis revealed a low genetic diversity in Ae. albopictus populations with a major common haplotype resulting in low haplotype diversity ranging from 0.13 to 0.65 and 0.35 for the total sample. Similarly, low nucleotide diversity was also reported varying from 0.0000 to 0.0017 with an overall index of 0.0008. This low genetic polymorphism is consistent with the recent introduction of Ae. albopictus in Cameroon.\ud CONCLUSION\ud This updated distribution of arbovirus vectors across Cameroon will help in planning vector control programme against possible outbreak of arbovirus related diseases in the country.

Background\ud Loiasis is caused by the filarial parasite Loa loa, which is widespread through Central and West Africa and largely confined the tropical equatorial rainforests. The tabanid flies Chrysops silacea and Chrysops dimidiata are the main vectors driving transmission. This study aimed to better define the spatial distribution and ecological niche of the two vectors to help define spatial-temporal risk and target appropriate, timely intervention strategies for filariasis control and elimination programmes.\ud Methods\ud Chrysops spp. distributions were determined by collating information from the published literature into a database, detailing the year, country, locality, latitude/longitude and species collected. Environmental factors including climate, elevation and tree canopy characteristics were summarised for each vector from data obtained from satellite modelled data or imagery, which were also used to identify areas with overt landcover changes. The presence of each Chrysops vector was predicted using a maximum entropy species distribution modelling (MaxEnt) method.\ud Results\ud A total of 313 location-specific data points from 59 published articles were identified across seven loiasis endemic countries. Of these, 186 sites were included in the climate and elevation analysis, and due to overt landcover changes, 83 sites included in tree canopy analysis and MaxEnt model. Overall, C. silacea and C. dimidiata were found to have similar ranges; annual mean temperature (24.6 °C and 24.1 °C, respectively), annual precipitation (1848.6 mm and 1868.8 mm), elevation (368.8 m and 400.6 m), tree canopy cover (61.4% and 66.9%) and tree canopy height (22.4 m and 25.1 m). MaxEnt models found tree canopy coverage was a significant environmental variable for both vectors.\ud Conclusions\ud The Chrysops spp. database and large-scale environmental analysis provides insights into the spatial and ecological parameters of the L. loa vectors driving transmission. These may be used to further delineate loiasis risk, which will be important for implementing filariasis control and elimination programmes in the equatorial rainforest region of Central and West Africa.

Background Riverine tsetse (Glossina spp.) transmit Trypanosoma brucei gambiense which causes Gambian Human African Trypanosomiasis. Tiny Targets were developed for cost-effective riverine tsetse control, and comprise panels of insecticide-treated blue polyester fabric and black net that attract and kill tsetse. Versus typical blue polyesters, two putatively more attractive fabrics have been developed: Vestergaard ZeroFly blue, and violet. Violet was most attractive to savannah tsetse using large targets, but neither fabric has been tested for riverine tsetse using Tiny Targets. Methods We measured numbers of G. f. fuscipes attracted to electrified Tiny Targets in Kenya and Uganda. We compared violets, Vestergaard blues, and a typical blue polyester, using three replicated Latin squares experiments. We then employed Bayesian statistical analyses to generate expected catches for future target deployments incorporating uncertainty in model parameters, and prior knowledge from previous experiments. Results Expected catches for average future replicates of violet and Vestergaard blue targets were highly likely to exceed those for typical blue. Accounting for catch variability between replicates, it remained moderately probable (70–86% and 59–84%, respectively) that a given replicate of these targets would have a higher expected catch than typical blue on the same day at the same site. Meanwhile, expected catches for average violet replicates were, in general, moderately likely to exceed those for Vestergaard blue. However, the difference in medians was small, and accounting for catch variability, the probability that the expected catch for a violet replicate would exceed a Vestergaard blue equivalent was marginal (46–71%). Conclusion Violet and Vestergaard ZeroFly blue are expected to outperform typical blue polyester in the Tiny Target configuration. Violet is unlikely to greatly outperform Vestergaard blue deployed in this way, but because violet is highly attractive to both riverine and savannah tsetse using different target designs, it may provide the more suitable general-purpose fabric., Author summary Riverine tsetse flies transmit parasites that cause sleeping sickness, so Tiny Targets have been developed to control them. Tiny Targets comprise adjacent panels of insecticide-coated blue polyester fabric and black net, and they work because tsetse are attracted by the blue fabric, and are killed by the insecticide if they make contact. In order to improve Tiny Targets, new coloured polyester fabrics have been developed with the aim of attracting more tsetse. These include a blue fabric produced by Vestergaard S.A. and currently used to make Tiny Targets, and a violet fabric developed by modelling fly colour vision and effective against savannah tsetse. We recorded the numbers of tsetse attracted to Tiny Targets made from these fabrics, and a typical blue polyester. Based on our data, we predicted the performance of such Tiny Targets in future deployments. It is highly probable that both Vestergaard blue and violet fabrics will attract more tsetse than typical blue. The average catch at a violet Tiny Target is quite likely to exceed that at a Vestergaard blue one, but because the difference is small and catches variable, there is unlikely to be a meaningful performance difference between these fabrics when deployed this way.

Many wilderness areas of East and Southern Africa are foci for Rhodesian sleeping sickness, a fatal zoonotic disease caused by trypanosomes transmitted by tsetse flies. Although transmission in these foci is traditionally driven by wildlife reservoirs, rising human and livestock populations may increase the role of livestock in transmission cycles. Deciphering transmission dynamics at wildlife and livestock interface areas is key to developing appropriate control. Data are lacking for key parameters, including host distributions, tsetse density, and mortality rates, and the relative roles of livestock and wildlife as hosts in fragmented habitats, limiting the development of meaningful models to assist in the assessment and implementation of control strategies.

Background: It has been suggested that attempts to eradicate populations of tsetse (Glossina spp.) using stationary\ud targets might fail because smaller, less mobile individuals are unlikely to be killed by the targets. If true, tsetse\ud caught in stationary traps should be larger than those from mobile baits, which require less mobility on the part\ud of the flies.\ud Results: Sampling tsetse in the Zambezi Valley of Zimbabwe, we found that the number of tsetse caught from\ud stationary traps, as a percent of total numbers from traps plus a mobile vehicle, was ~5% for male G. morsitans\ud morsitans (mean wing length 5.830 mm; 95% CI: 5.800–5.859 mm) and ~10% for females (6.334 mm; 95% CI: 6.329–\ud 6.338 mm); for G. pallidipes the figures were ~50% for males (6.830 mm; 95% CI: 6.821–6.838 mm) and ~75% for\ud females (7.303 mm, 95% CI: 7.302–7.305 mm). As expected, flies of the smaller species (and the smaller sex) were\ud less likely to be captured using stationary, rather than mobile sampling devices. For flies of a given sex and species\ud the situation was more complex. Multivariable analysis showed that, for females of both species, wing lengths\ud changed with ovarian age and the month, year and method of capture. For G. pallidipes, there were statistically\ud significant interactions between ovarian age and capture month, year and method. For G. m. morsitans, there was\ud only a significant interaction between ovarian age and capture month. The effect of capture method was, however,\ud small in absolute terms: for G. pallidipes and G. m. morsitans flies caught on the mobile vehicle had wings only 0.24\ud and 0.48% shorter, respectively, than flies caught in stationary traps. In summary, wing length in field samples of\ud tsetse varies with ovarian age, capture month and year and, weakly, with capture method. Suggestions that a\ud target-based operation against G. f. fuscipes in Kenya caused a shift towards a smaller, less mobile population of\ud tsetse, unavailable to the targets, failed to account for factors other than capture method.\ud Conclusions: The results are consistent with the successful use of targets to eradicate populations of tsetse in\ud Zimbabwe. Until further, more nuanced, studies are conducted, it is premature to conclude that targets alone could\ud not, similarly, be used to eradicate G. f. fuscipes populations in Kenya.

Monitoring abundance is essential for vector management, but it is often only possible in a fraction of managed areas. For vector control programmes, sampling to estimate abundance is usually carried out at a local‐scale (10s km2), while interventions often extend across 100s km2. Geostatistical models have been used to interpolate between points where data are available, but this still requires costly sampling across the entire area of interest. Instead, we used geostatistical models to predict local‐scale spatial variation in the abundance of tsetse—vectors of human and animal African trypanosomes—beyond the spatial extent of data to which models were fitted, in Serengeti, Tanzania.We sampled Glossina swynnertoni and Glossina pallidipes >10 km inside the Serengeti National Park (SNP) and along four transects extending into areas where humans and livestock live. We fitted geostatistical models to data >10 km inside the SNP to produce maps of abundance for the entire region, including unprotected areas.Inside the SNP, the mean number of G. pallidipes caught per trap per day in dense woodland was 166 (± 24 SE), compared to 3 (±1) in grassland. Glossina swynnertoni was more homogenous with respective means of 15 (±3) and 15 (±8). In general, models predicted a decline in abundance from protected to unprotected areas, related to anthropogenic changes to vegetation, which was confirmed during field survey. Synthesis and applications. Our approach allows vector control managers to identify sites predicted to have relatively high tsetse abundance, and therefore to design and implement improved surveillance strategies. In East and Southern Africa, trypanosomiasis is associated with wilderness areas. Our study identified pockets of vegetation which could sustain tsetse populations in farming areas outside the Serengeti National Park. Our method will assist countries in identifying, monitoring and, if necessary, controlling tsetse in trypanosomiasis foci. This has specific application to tsetse, but the approach could also be developed for vectors of other pathogens., Our approach allows vector control managers to identify sites predicted to have relatively high tsetse abundance, and therefore to design and implement improved surveillance strategies. In East and Southern Africa, trypanosomiasis is associated with wilderness areas. Our study identified pockets of vegetation which could sustain tsetse populations in farming areas outside the Serengeti National Park. Our method will assist countries in identifying, monitoring and, if necessary, controlling tsetse in trypanosomiasis foci. This has specific application to tsetse, but the approach could also be developed for vectors of other pathogens.

Innovations in mosquito mass-rearing techniques are essential in the quest to develop SIT (sterile insect technique) methods to fight mosquito vectors of disease. This study reports modifications to the Food and Agriculture Organisation/International Atomic Energy Agency (FAO/IAEA) mass-rearing cage (MRC) for mosquitoes to support the behaviour of adult Anopheles arabiensis Patton (Diptera: Culicidae) and to maximize egg production. The effects of an improved sugar-feeding device, and the addition of resting sites and a black cloth shroud to create an artificial horizon (visual contrast of light vs. dark at the edge) were assessed for their effect on adult longevity and egg production. Egg production of adults resulting from larvae reared in individual free-standing trays vs. those reared in the same trays in the FAO/IAEA larval rearing rack was also compared. Finally, the effect of blood feeding and frequency of egg collection from the MRC on average egg production per batch was investigated. Overall, the modifications to the MRC enhanced adult longevity, and the improved cage prototype allowed the collection of more eggs overall from a cohort of adults than was possible using the original and previous cage prototypes. These stepwise improvements are important for the development of economical and logistically efficient mass-rearing systems for the malaria vector An. arabiensis.

Background Gambian sleeping sickness or HAT (human African trypanosomiasis) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by riverine species of tsetse. A global programme aims to eliminate the disease as a public health problem by 2020 and stop transmission by 2030. In the South of Chad, the Mandoul area is a persistent focus of Gambian sleeping sickness where around 100 HAT cases were still diagnosed and treated annually until 2013. Pre-2014, control of HAT relied solely on case detection and treatment, which lead to a gradual decrease in the number of cases of HAT due to annual screening of the population. Methods Because of the persistence of transmission and detection of new cases, we assessed whether the addition of vector control to case detection and treatment could further reduce transmission and consequently, reduce annual incidence of HAT in Mandoul. In particular, we investigated the impact of deploying ‘tiny targets’ which attract and kill tsetse. Before tsetse control commenced, a census of the human population was conducted and their settlements mapped. A pre-intervention survey of tsetse distribution and abundance was implemented in November 2013 and 2600 targets were deployed in the riverine habitats of tsetse in early 2014, 2015 and 2016. Impact on tsetse and on the incidence of sleeping sickness was assessed through nine tsetse monitoring surveys and four medical surveys of the human population in 2014 and 2015. Mathematical modelling was used to assess the relative impact of tsetse control on incidence compared to active and passive screening. Findings The census indicated that a population of 38674 inhabitants lived in the vicinity of the Mandoul focus. Within this focus in November 2013, the vector is Glossina fuscipes fuscipes and the mean catch of tsetse from traps was 0.7 flies/trap/day (range, 0–26). The catch of tsetse from 44 sentinel biconical traps declined after target deployment with only five tsetse being caught in nine surveys giving a mean catch of 0.005 tsetse/trap/day. Modelling indicates that 70.4% (95% CI: 51–95%) of the reduction in reported cases between 2013 and 2015 can be attributed to vector control with the rest due to medical intervention. Similarly tiny targets are estimated to have reduced new infections dramatically with 62.8% (95% CI: 59–66%) of the reduction due to tsetse control, and 8.5% (95% 8–9%) to enhanced passive detection. Model predictions anticipate that elimination as a public health problem could be achieved by 2018 in this focus if vector control and screening continue at the present level and, furthermore, there may have been virtually no transmission since 2015. Conclusion This work shows that tiny targets reduced the numbers of tsetse in this focus in Chad, which may have interrupted transmission and the combination of tsetse control to medical detection and treatment has played a major role in reducing in HAT incidence in 2014 and 2015., Author summary A global programme aims to eliminate Gambian sleeping sickness (Human African Trypanosomiasis, HAT) as a public health problem by 2020. Gambian HAT is a neglected tropical disease caused by trypanosomes spread by tsetse flies and its control has relied largely on detection and treatment of human cases. In the Mandoul focus of southern Chad, regular screening of the human population (~39000 people) between 2002 and 2013 resulted in the detection and treatment of ~100 cases/year. We examined whether even better control might be achieved through the addition of vector control to medical screening. In February 2014, 2600 insecticide-treated targets (‘Tiny Targets’) were deployed in areas where tsetse were present; tsetse are attracted to the targets and on contacting it they pick up a lethal dose of insecticide. Monitoring of the tsetse population, using a network of 44 traps operated regularly between November 2013 and October 2016, showed that the mean daily catch of tsetse declined by 99.99%, from 0.7 tsetse/trap before targets were deployed to 0.005 tsetse/trap. The number of HAT cases detected by a programme of active screening also declined during this period. Mathematical modelling of the number of HAT cases reported during the period 2000–2015 suggests that 70% of the decline in cases during 2014–2015 was due to vector control. The model also suggests that the combination of these interventions may have interrupted transmission and may to lead to the elimination of sleeping sickness in the Mandoul focus by 2020. The results from Mandoul provide further empirical and theoretical evidence that the global elimination of Gambian HAT can be achieved through the integrated use of (i) case detection and treatment and (ii) vector control.

Females of all blood-feeding arthropod vectors must find and feed on a host in order to produce offspring. For tsetse—vectors of the trypanosomes that cause human and animal African trypanosomiasis—the problem is more extreme, since both sexes feed solely on blood. Host location is thus essential both for survival and reproduction. Host population density should therefore be an important driver of population dynamics for haematophagous insects, and particularly for tsetse, but the role of host density is poorly understood. We investigate the issue using data on changes in numbers of tsetse (Glossina morsitans morsitans Westwood) caught during a host elimination experiment in Zimbabwe in the 1960s. During the experiment, numbers of flies caught declined by 95%. We aimed to assess whether models including starvation-dependent mortality could explain observed changes in tsetse numbers as host density declined. An ordinary differential equation model, including starvation-dependent mortality, captured the initial dynamics of the observed tsetse population. However, whereas small numbers of tsetse were caught throughout the host elimination exercise, the modelled population went extinct. Results of a spatially explicit agent-based model suggest that this discrepancy could be explained by immigration of tsetse into the experimental plot. Variation in host density, as a result of natural and anthropogenic factors, may influence tsetse population dynamics in space and time. This has implications for Trypanosoma brucei rhodesiense transmission. Increased tsetse mortality as a consequence of low host density may decrease trypanosome transmission, but hungrier flies may be more inclined to bite humans, thereby increasing the risk of transmission to humans. Our model provides a way of exploring the role of host density on tsetse population dynamics and could be incorporated into models of trypanosome transmission dynamics to better understand how spatio-temporal variation in host density impacts trypanosome prevalence in mammalian hosts., Author summary Tsetse flies are the vectors of human and animal African trypanosomiasis. The flies rely solely on vertebrate blood for food and water and changes in vertebrate host density may therefore influence tsetse survival. We develop models including starvation-dependent mortality to explain changes in numbers of the savannah tsetse Glossina morsitans morsitans caught during a host elimination experiment in Zimbabwe in the 1960s. Our models provide a way of exploring the role of host density on tsetse population dynamics and could be incorporated into models of trypanosome transmission dynamics to better understand how spatial and/or temporal variation in host density impacts prevalence of trypanosome infection in vertebrate hosts.

Background\ud \ud Indoor residual spraying (IRS) with DDT has been the primary strategy for control of the visceral leishmaniasis (VL) vector Phlebotomus argentipes in India but efficacy may be compromised by resistance. Synthetic pyrethroids are now being introduced for IRS, but with a shared target site, the para voltage-gated sodium channel (VGSC), mutations affecting both insecticide classes could provide cross-resistance and represent a threat to sustainable IRS-based disease control.\ud \ud Methodology/Principal findings\ud \ud A region of the Vgsc gene was sequenced in P. argentipes from the VL hotspot of Bihar, India. Two knockdown resistance (kdr) mutations were detected at codon 1014 (L1014F and L1014S), each common in mosquitoes, but previously unknown in phlebotomines. Both kdr mutations appear largely recessive, but as homozygotes (especially 1014F/F) or as 1014F/S heterozygotes exert a strong effect on DDT resistance, and significantly predict survivorship to class II pyrethroids in short-duration bioassays. The mutations are present at high frequency in wild P. argentipes populations from Bihar, with 1014F significantly more common in higher VL areas.\ud \ud Conclusions/Significance\ud \ud The Vgsc mutations detected appear to be a primary mechanism underlying DDT resistance in P. argentipes and a contributory factor in reduced pyrethroid susceptibility, suggesting a potential impact if P. argentipes are subjected to suboptimal levels of pyrethroid exposure, or additional resistance mechanisms evolve. The assays to detect kdr frequency changes provide a sensitive, high-throughput monitoring tool to detecting spatial and temporal variation in resistance in P. argentipes.

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly’s midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

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.

BackgroundIdentifying hosts of blood-feeding insect vectors is crucial in understanding their role in disease transmission. Rhodesian human African trypanosomiasis (rHAT), also known as acute sleeping sickness is caused by Trypanosoma brucei rhodesiense and transmitted by tsetse flies. The disease is commonly associated with wilderness areas of east and southern Africa. Such areas hold a diverse range of species which form communities of hosts for disease maintenance. The relative importance of different wildlife hosts remains unclear. This study quantified tsetse feeding preferences in a wilderness area of great host species richness, Serengeti National Park, Tanzania, assessing tsetse feeding and host density contemporaneously.MethodsGlossina swynnertoni and G. pallidipes were collected from six study sites. Bloodmeal sources were identified through matching Cytochrome B sequences amplified from bloodmeals from recently fed flies to published sequences. Densities of large mammal species in each site were quantified, and feeding indices calculated to assess the relative selection or avoidance of each host species by tsetse.ResultsThe host species most commonly identified in G. swynnertoni bloodmeals, warthog (94/220), buffalo (48/220) and giraffe (46/220), were found at relatively low densities (3-11/km2) and fed on up to 15 times more frequently than expected by their relative density. Wildebeest, zebra, impala and Thomson’s gazelle, found at the highest densities, were never identified in bloodmeals. Commonly identified hosts for G. pallidipes were buffalo (26/46), giraffe (9/46) and elephant (5/46).ConclusionsThis study is the first to quantify tsetse host range by molecular analysis of tsetse diet with simultaneous assessment of host density in a wilderness area. Although G. swynnertoni and G. pallidipes can feed on a range of species, they are highly selective. Many host species are rarely fed on, despite being present in areas where tsetse are abundant. These feeding patterns, along with the ability of key host species to maintain and transmit T. b. rhodesiense, drive the epidemiology of rHAT in wilderness areas.

Recent news headlines claimed that corpses thrown into Syrian streets are causing cutaneous leishmaniasis (CL) outbreaks. However, leishmaniasis is only transmitted by blood-feeding sandflies, not through human remains. High CL prevalence in Syria may instead be attributed to the absence of disease control programs due to the disruption of health services.

BackgroundGenetic surveillance of the human filarial parasite, Onchocerca volvulus, from onchocerciasis endemic regions will ideally focus on genotyping individual infective larval stages collected from their intermediate host, Simuliid blackflies. However, blackflies also transmit other Onchocerca species, including the cattle parasite O. ochengi, which are difficult to distinguish from the human parasite based on morphological characteristics alone. This study describes a versatile approach to discriminate between O. volvulus and O. ochengi that is demonstrated using parasite infective larvae dissected from blackflies.ResultsA speciation assay was designed based on genetic differentiation between O. volvulus and O. ochengi mitochondrial genome sequences that can be performed in highthroughput high-resolution melt (HRM)- or lower throughput conventional restriction fragment length polymorphism (RFLP) analyses. This assay was validated on 185 Onchocerca larvae dissected from blackflies captured from 14 communities in Ghana throughout 2011-13. The frequency of O. ochengi was approximately 67 % of all larvae analysed, which is significantly higher than previously reported in this region. Furthermore, the species distribution was not uniform throughout the study region, with 25 %, 47 % and 93 % of O. volvulus being found in the western-most (Black Volta, Tain and Tombe), the central (Pru) and eastern-most (Daka) river basins, respectively.ConclusionsThis tool provides a simple and cost-effective approach to determine the identity and distribution of two Onchocerca species, and will be valuable for future genetic studies that focus on parasites collected from blackflies. The results presented highlight the need to discriminate Onchocerca species in transmission studies, as the frequency of each species varied significantly between the communities studied.