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Background: Malaria is one of the most devastating tropical diseases, resulting in loss of lives each year, especially in children under the age of 5 years. Malaria burden, related deaths and stall in the progress against malaria transmission is evident, particularly in countries that have moderate or high malaria transmission. Hence, mitigating malaria spread requires information on the distribution of vectors and the drivers of insecticide resistance (IR). However, owing to the impracticality in establishing the critical need for real-world information at every location, modelling provides an informed best guess for such information. Therefore, this review examines the various methodologies used to model spatial, temporal and spatio-temporal patterns of IR within populations of malaria vectors, incorporating pest-biology parameters, adopted ecological principles, and the associated modelling challenges., Methods: The review focused on the period ending March 2023 without imposing restrictions on the initial year of publication, and included articles sourced from PubMed, Web of Science, and Scopus. It was also limited to publications that deal with modelling of IR distribution across spatial and temporal dimensions and excluded articles solely focusing on insecticide susceptibility tests or articles not published in English. After rigorous selection, 33 articles met the review's elibility criteria and were subjected to full-text screening., Results: Results show the popularity of Bayesian geostatistical approaches, and logistic and static models, with limited adoption of dynamic modelling approaches for spatial and temporal IR modelling. Furthermore, our review identifies the availability of surveillance data and scarcity of comprehensive information on the potential drivers of IR as major impediments to developing holistic models of IR evolution., Conclusions: The review notes that incorporating pest-biology parameters, and ecological principles into IR models, in tandem with fundamental ecological concepts, potentially offers crucial insights into the evolution of IR. The results extend our knowledge of IR models that provide potentially accurate results, which can be translated into policy recommendations to combat the challenge of IR in malaria control., (© 2024. The Author(s).)

Background: The demonstration that the recently discovered Anopheles symbiont Microsporidia MB blocks malaria transmission in Anopheles arabiensis and undergoes vertical and horizontal transmission suggests that it is a promising candidate for the development of a symbiont-based malaria transmission-blocking strategy. The infection prevalence and characteristics of Microsporidia MB in Anopheles gambiae sensu stricto (s.s.), another primary vector species of malaria in Kenya, were investigated., Methods: Field-collected females were confirmed to be Microsporidia MB-positive after oviposition. Egg counts of Microsporidia MB-infected and non-infected individuals were used to infer the effects of Microsporidia MB on fecundity. The time to pupation, adult sex ratio and survival were used to determine if Microsporidia MB infection has similar characteristics in the host mosquitoes An. gambiae and An. arabiensis. The intensity of Microsporidia MB infection in tissues of the midgut and gonads, and in carcasses, was determined by quantitative polymerase chain reaction. To investigate horizontal transmission, virgin males and females that were either Microsporidia MB-infected or non-infected were placed in standard cages for 48 h and allowed to mate; transmission was confirmed by quantitative polymerase chain reaction targeting Microsporidia MB genes., Results: Microsporidia MB was found to naturally occur at a low prevalence in An. gambiae s.s. collected in western Kenya. Microsporidia MB shortened the development time from larva to pupa, but other fitness parameters such as fecundity, sex ratio, and adult survival did not differ between Microsporidia MB-infected and non-infected hosts. Microsporidia MB intensities were high in the male gonadal tissues. Transmission experiments indicated that Microsporidia MB undergoes both maternal and horizontal transmission in An. gambiae s.s., Conclusions: The findings that Microsporidia MB naturally infects, undergoes maternal and horizontal transmission, and is avirulent in An. gambiae s.s. indicate that many of the characteristics of its infection in An. arabiensis hold true for the former. The results of the present study indicate that Microsporidia MB could be developed as a tool for the transmission-blocking of malaria across different Anopheles species., (© 2023. BioMed Central Ltd., part of Springer Nature.)

An integrated approach to innovatively counter the transmission of various arthropod-borne diseases to humans would benefit from strategies that sustainably limit onward passage of infective life cycle stages of pathogens and parasites to the insect vectors and vice versa. Aiming to accelerate the impetus towards a disease-free world amid the challenges posed by climate change, discovery, mindful exploitation and integration of active natural products in design of pathogen transmission-blocking interventions is of high priority. Herein, we provide a review of natural compounds endowed with blockade potential against transmissible forms of human pathogens reported in the last 2 decades from 2000 to 2021. Finally, we propose various translational strategies that can exploit these pathogen transmission-blocking natural products into design of novel and sustainable disease control interventions. In summary, tapping these compounds will potentially aid in integrated combat mission to reduce disease transmission trends., (© 2022. The Author(s).)

Background: Providing protection from malaria vector bites, both indoors and outdoors, is crucial to curbing malaria parasite transmission. Screening of house entry points, especially with incorporated insecticides, confers significant protection but remains a costly and labour-intensive application. Use of spatial repellents has shown promise in creating areas of protection in peri-domestic areas., Methods: This study aimed at comparing the protection provided by transfluthrin-treated and untreated complete screens over open eave gaps with incomplete transfluthrin-treated eave strips as a potential replacement for a full screen. Human landing catches were implemented independently inside and outside an experimental hut under controlled semi-field conditions, with insectary-reared Anopheles arabiensis mosquitoes., Results: The odds of a female mosquito finding a human volunteer indoors and attempting to bite were similar whether the eaves were completely open or there was an untreated fabric strip fixed around the eaves. However, when the eave gap was completely screened without insecticide, the odds of receiving a bite indoors were reduced by 70% (OR 0.30, 95% CI 0.20-0.47). Adding transfluthrin to the full screen, further increased the protection indoors, with the odds of receiving a bite reduced by 92% (0.08, 95% CI 0.04-0.16) compared to the untreated screen. Importantly, the same protection was conferred when only a narrow transfluthrin-treated fabric strip was loosely fixed around the eave gap (OR 0.07, 95% CI 0.04-0.13). The impact of the transfluthrin treatment on outdoor biting was correlated with evening temperatures during the experiments. At lower evening temperatures, a transfluthrin-treated, complete screen provided moderate and variable protection from bites (OR 0.62, 95% CI 0.37-1.03), whilst at higher evening temperatures the odds of receiving a bite outdoors was over four times lower in the presence of transfluthrin, on either a full screen (OR 0.22 95% 0.12-0.38) or a fabric strip (OR 0.25, 95% 0.15-0.42), than when no treatment was present., Conclusion: The findings suggest that transfluthrin-treated fabric strips can provide a substitute for complete eave screens. They are a simple, easy-to-handle tool for protecting people from malaria mosquito bites indoors and potentially around the house in climatic areas where evening and night-time temperatures are relatively high., (© 2022. The Author(s).)

Background: African trypanosomiasis, which is mainly transmitted by tsetse flies (Glossina spp.), is a threat to public health and a significant hindrance to animal production. Tools that can reduce tsetse densities and interrupt disease transmission exist, but their large-scale deployment is limited by high implementation costs. This is in part limited by the absence of knowledge of breeding sites and dispersal data, and tools that can predict these in the absence of ground-truthing., Methods: In Kenya, tsetse collections were carried out in 261 randomized points within Shimba Hills National Reserve (SHNR) and villages up to 5 km from the reserve boundary between 2017 and 2019. Considering their limited dispersal rate, we used in situ observations of newly emerged flies that had not had a blood meal (teneral) as a proxy for active breeding locations. We fitted commonly used species distribution models linking teneral and non-teneral tsetse presence with satellite-derived vegetation cover type fractions, greenness, temperature, and soil texture and moisture indices separately for the wet and dry season. Model performance was assessed with area under curve (AUC) statistics, while the maximum sum of sensitivity and specificity was used to classify suitable breeding or foraging sites., Results: Glossina pallidipes flies were caught in 47% of the 261 traps, with teneral flies accounting for 37% of these traps. Fitted models were more accurate for the teneral flies (AUC = 0.83) as compared to the non-teneral (AUC = 0.73). The probability of teneral fly occurrence increased with woodland fractions but decreased with cropland fractions. During the wet season, the likelihood of teneral flies occurring decreased as silt content increased. Adult tsetse flies were less likely to be trapped in areas with average land surface temperatures below 24 °C. The models predicted that 63% of the potential tsetse breeding area was within the SHNR, but also indicated potential breeding pockets outside the reserve., Conclusion: Modelling tsetse occurrence data disaggregated by life stages with time series of satellite-derived variables enabled the spatial characterization of potential breeding and foraging sites for G. pallidipes. Our models provide insight into tsetse bionomics and aid in characterising tsetse infestations and thus prioritizing control areas., (© 2021. The Author(s).)

Background: Glossina (tsetse flies) biologically transmit trypanosomes that infect both humans and animals. Knowledge of their distribution patterns is a key element to better understand the transmission dynamics of trypanosomosis. Tsetse distribution in Rwanda has not been well enough documented, and little is known on their current distribution. This study determined the current spatial distribution, abundance, diversity, and seasonal variations of tsetse flies in and around the Akagera National Park., Methods: A longitudinal stratified sampling following the seasons was used. Biconical traps were deployed in 55 sites for 6 consecutive days of each study month from May 2018 to June 2019 and emptied every 48 h. Flies were identified using FAO keys, and the number of flies per trap day (FTD) was used to determine the apparent density. Pearson chi-square (χ2) and parametrical tests (t-test and ANOVA) were used to determine the variations between the variables. The significance (p < 0.05) at 95% confidence interval was considered. Logistic regression was used to determine the association between tsetse occurrence and the associated predictors., Results: A total of 39,516 tsetse flies were collected, of which 73.4 and 26.6% were from inside Akagera NP and the interface area, respectively. Female flies accounted for 61.3 while 38.7% were males. Two species were identified, i.e. G. pallidipes [n = 29,121, 7.4 flies/trap/day (FTD)] and G. morsitans centralis (n = 10,395; 2.6 FTD). The statistical difference in numbers was significant between the two species (p = 0.000). The flies were more abundant during the wet season (15.8 FTD) than the dry season (4.2 FTD). Large numbers of flies were trapped around the swamp areas (69.1 FTD) inside the park and in Nyagatare District (11.2 FTD) at the interface. Glossina morsitans was 0.218 times less likely to occur outside the park. The chance of co-existing between the two species reduced outside the protected area (0.021 times)., Conclusions: The occurrence of Glossina seems to be limited to the protected Akagera NP and a narrow band of its surrounding areas. This finding will be crucial to design appropriate control strategies. Glossina pallidipes was found in higher numbers and therefore is conceivably the most important vector of trypanosomosis. Regional coordinated control and regular monitoring of Glossina distribution are recommended.

Background: Trypanosoma brucei species are motile protozoan parasites that are cyclically transmitted by tsetse fly (genus Glossina) causing human sleeping sickness and nagana in livestock in sub-Saharan Africa. African trypanosomes display digenetic life cycle stages in the tsetse fly vector and in their mammalian host. Experimental work on insect-stage trypanosomes is challenging because of the difficulty in setting up successful in vitro cultures. Therefore, there is limited knowledge on the trypanosome biology during its development in the tsetse fly. Consequently, this limits the development of new strategies for blocking parasite transmission in the tsetse fly., Methods: In this study, RNA-Seq data of insect-stage trypanosomes were used to construct a T. brucei gene co-expression network using the weighted gene co-expression analysis (WGCNA) method. The study identified significant enriched modules for genes that play key roles during the parasite's development in tsetse fly. Furthermore, potential 3' untranslated region (UTR) regulatory elements for genes that clustered in the same module were identified using the Finding Informative Regulatory Elements (FIRE) tool., Results: A fraction of gene modules (12 out of 27 modules) in the constructed network were found to be enriched in functional roles associated with the cell division, protein biosynthesis, mitochondrion, and cell surface. Additionally, 12 hub genes encoding proteins such as RNA-binding protein 6 (RBP6), arginine kinase 1 (AK1), brucei alanine-rich protein (BARP), among others, were identified for the 12 significantly enriched gene modules. In addition, the potential regulatory elements located in the 3' untranslated regions of genes within the same module were predicted., Conclusions: The constructed gene co-expression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. This will enhance understanding of the molecular mechanisms that underlie important biological processes during parasite's development in tsetse fly. Ultimately, these findings will be key in the identification of potential molecular targets for disease control.

Background: Semi-field experiments with human landing catch (HLC) measure as the outcome are an important step in the development of novel vector control interventions against outdoor transmission of malaria since they provide good estimates of personal protection. However, it is often infeasible to determine whether the reduction in HLC counts is due to mosquito mortality or repellency, especially considering that spatial repellents based on volatile pyrethroids might induce both. Due to the vastly different impact of repellency and mortality on transmission, the community-level impact of spatial repellents can not be estimated from such semi-field experiments., Methods: We present a new stochastic model that is able to estimate for any product inhibiting outdoor biting, its repelling effect versus its killing and disarming (preventing host-seeking until the next night) effects, based only on time-stratified HLC data from controlled semi-field experiments. For parameter inference, a Bayesian hierarchical model is used to account for nightly variation of semi-field experimental conditions. We estimate the impact of the products on the vectorial capacity of the given Anopheles species using an existing mathematical model. With this methodology, we analysed data from recent semi-field studies in Kenya and Tanzania on the impact of transfluthrin-treated eave ribbons, the odour-baited Suna trap and their combination (push-pull system) on HLC of Anopheles arabiensis in the peridomestic area., Results: Complementing previous analyses of personal protection, we found that the transfluthrin-treated eave ribbons act mainly by killing or disarming mosquitoes. Depending on the actual ratio of disarming versus killing, the vectorial capacity of An. arabiensis is reduced by 41 to 96% at 70% coverage with the transfluthrin-treated eave ribbons and by 38 to 82% at the same coverage with the push-pull system, under the assumption of a similar impact on biting indoors compared to outdoors., Conclusions: The results of this analysis of semi-field data suggest that transfluthrin-treated eave ribbons are a promising tool against malaria transmission by An. arabiensis in the peridomestic area, since they provide both personal and community protection. Our modelling framework can estimate the community-level impact of any tool intervening during the mosquito host-seeking state using data from only semi-field experiments with time-stratified HLC.

Background: Visceral leishmaniasis (VL) and zoonotic cutaneous leishmaniasis (ZCL) are of public health concern in Merti sub-County, Kenya, but epidemiological data on transmission, vector abundance, distribution, and reservoir hosts remain limited. To better understand the disease and inform control measures to reduce transmission, we investigated the abundance and distribution of sand fly species responsible for Leishmania transmission in the sub-County and their blood-meal hosts., Methods: We conducted an entomological survey in five villages with reported cases of VL in Merti sub-County, Kenya, using CDC miniature light traps and castor oil sticky papers. Sand flies were dissected and identified to the species level using standard taxonomic keys and PCR analysis of the cytochrome c oxidase subunit 1 (cox1) gene. Leishmania parasites were detected and identified by PCR and sequencing of internal transcribed spacer 1 (ITS1) genes. Blood-meal sources of engorged females were identified by high-resolution melting analysis of vertebrate cytochrome b (cyt-b) gene PCR products., Results: We sampled 526 sand flies consisting of 8 species, Phlebotomus orientalis (1.52%; n = 8), and 7 Sergentomyia spp. Sergentomyia squamipleuris was the most abundant sand fly species (78.71%; n = 414) followed by Sergentomyia clydei (10.46%; n = 55). Leishmania major, Leishmania donovani, and Trypanosoma DNA were detected in S. squamipleuris specimens. Humans were the main sources of sand fly blood meals. However, we also detected mixed blood meals; one S. squamipleuris specimen had fed on both human and mouse (Mus musculus) blood, while two Ph. orientalis specimens fed on human, hyrax (Procavia capensis), and mouse (Mus musculus) blood., Conclusions: Our findings implicate the potential involvement of S. squamipleuris in the transmission of Leishmania and question the dogma that human leishmaniases in the Old World are exclusively transmitted by sand flies of the Phlebotomus genus. The presence of Trypanosoma spp. may indicate mechanical transmission, whose efficiency should be investigated. Host preference analysis revealed the possibility of zoonotic transmission of leishmaniasis and other pathogens in the sub-County. Leishmania major and L. donovani are known to cause ZCL and VL, respectively. However, the reservoir status of the parasites is not uniform. Further studies are needed to determine the reservoir hosts of Leishmania spp. in the area.

Background: Novel malaria vector control approaches aim to combine tools for maximum protection. This study aimed to evaluate novel and re-evaluate existing putative repellent 'push' and attractive 'pull' components for manipulating the odour orientation of malaria vectors in the peri-domestic space., Methods: Anopheles arabiensis outdoor human landing catches and trap comparisons were implemented in large semi-field systems to (i) test the efficacy of Citriodiol ® or transfluthrin-treated fabric strips positioned in house eave gaps as push components for preventing bites; (ii) understand the efficacy of MB5-baited Suna-traps in attracting vectors in the presence of a human being; (iii) assess 2-butanone as a CO 2 replacement for trapping; (iv) determine the protection provided by a full push-pull set up. The air concentrations of the chemical constituents of the push-pull set-up were quantified., Results: Microencapsulated Citriodiol ® eave strips did not provide outdoor protection against host-seeking An. arabiensis. Transfluthrin-treated strips reduced the odds of a mosquito landing on the human volunteer (OR 0.17; 95% CI 0.12-0.23). This impact was lower (OR 0.59; 95% CI 0.52-0.66) during the push-pull experiment, which was associated with low nighttime temperatures likely affecting the transfluthrin vaporisation. The MB5-baited Suna trap supplemented with CO 2 attracted only a third of the released mosquitoes in the absence of a human being; however, with a human volunteer in the same system, the trap caught < 1% of all released mosquitoes. The volunteer consistently attracted over two-thirds of all mosquitoes released. This was the case in the absence ('pull' only) and in the presence of a spatial repellent ('push-pull'), indicating that in its current configuration the tested 'pull' does not provide a valuable addition to a spatial repellent. The chemical 2-butanone was ineffective in replacing CO 2 . Transfluthrin was detectable in the air space but with a strong linear reduction in concentrations over 5 m from release. The MB5 constituent chemicals were only irregularly detected, potentially suggesting insufficient release and concentration in the air for attraction., Conclusion: This step-by-step evaluation of the selected 'push' and 'pull' components led to a better understanding of their ability to affect host-seeking behaviours of the malaria vector An. arabiensis in the peri-domestic space and helps to gauge the impact such tools would have when used in the field for monitoring or control.

Background: In a recent study using DNA barcoding, we identified the plants fed upon by four Afro-tropical mosquito species that vector dengue, malaria, and Rift Valley fever. Herein, we have expanded on this study by investigating the role of three of the plants, Pithecellobium dulce (Fabaceae), Leonotis nepetifolia (Lamiaceae), and Opuntia ficus-indica (Cactaceae), on the survival, fecundity, and egg viability of the dengue vector Aedes aegypti., Methods: We tested these effects using females that received (i) an initial three rations of blood meals and (ii) no blood meal at all. Two controls were included: age-matched females fed on glucose solution with or without an initial blood meal and those fed exclusively on blood meals. Data were collected daily over a 30-day period. The amino acid contents of Ae. aegypti guts and their respective diets were detected by coupled liquid chromatography-mass spectrometry., Results: Females fed on P. dulce and an exclusively blood meal diet had a shorter survival than those fed on glucose. On the other hand, females fed on L. nepetifolia survived longer than those fed exclusively on blood meals, whereas those fed on O. ficus-indica had the shortest survival time. With an initial blood meal, females fed on L. nepetifolia laid 1.6-fold more eggs while those fed on the other diets laid fewer eggs compared to those fed exclusively on blood meals. Hatching rates of the eggs laid varied with the diet. Mass spectroscopic analysis of gut contents of mosquitoes exposed to the different diets showed qualitative and quantitative differences in their amino acid levels., Conclusion: Our findings highlight the central role of plant nutrients in the reproductive fitness of dengue vectors, which may impact their disease transmission potential.

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

Background: In insects, oviposition decisions may lead to egg deposition in substrates with different larval density and nutritional levels. Individuals developing in such substrates may present plasticity in their phenotype. Here, we investigated the effect of two factors related to oviposition decisions, namely larval density and substrate quality, on the wing size and wing shape of the stable fly, Stomoxys calcitrans L. (Diptera: Muscidae)., Methods: We reared S. calcitrans larvae at different densities (5, 15 and 25) and on different substrates (camel, cow, donkey and sheep dung). For each fly that emerged, we recorded body weight, and detached, slide-mounted and photographed the right wing. Next, we collected 15 landmarks on each photographed wing, and applied geometric morphometric analysis to assess variation in wing size and wing shape of S. calcitrans across the different larval densities and substrate types., Results: We observed that wing size and wing shape of S. calcitrans were affected by larval density and the nature of the developmental substrate. Flies reared in a group of 5 had larger wing centroid size, wing length, wing width, wing area and wing loading compared with those reared in a group of 25. Also, flies developed in donkey and sheep dung had larger wing centroid size, wing length, wing width, wing area and wing loading in comparison with those grown in camel and cow dung. Canonical variate analysis followed by discriminant analysis revealed significant wing shape variation in S. calcitrans across the different densities and substrates. Wing size had a significant but weak positive effect on wing shape., Conclusions: This study demonstrates the high sensitivity of S. calcitrans wings to variation in larval density and developmental substrate, and that use of landmark-based geometric morphometric analysis could improve our understanding of how flies of veterinary importance respond to environmental variability.

Background: Pyrethroid and organophosphate resistance in the malaria vector Anopheles gambiae has led to the search for not only alternative insecticides, but also repellent chemical compounds. However, little is known about the potential actions of repellents and the cross-resistance risk between insecticide and repellent compounds., Methods: Here we show the action of permethrin, DEET, geraniol, carvacrol, culminaldehyde and cinnamaldehyde against three A. gambiae strains: 'Kis' (Kisumu susceptible strain), 'KdrKis' (pyrethroid resistant strain) and 'AcerKis' (organophosphate resistant strain), the last two differing from the first by a mutation on the kdr and ace1 genes, respectively., Conclusions: Results from the DEET assays show it induced repellency for the resistant KdrKis and AcerKis strains but maintained irritancy for the susceptible strain. More generally, we show resistance genes modify the behavior of An. gambiae, increasing or decreasing the effectiveness of DEET and natural compounds, depending on the mutation. These findings offer a new avenue for research on the target and mechanism of repellent compounds. We discuss these findings in the context of vector control strategies.

Background: In Kenya, malaria remains a major public health menace equally affecting the semi-arid to arid ecologies. However, entomologic knowledge of malaria vectors in such areas remains poor., Methods: Morphologically-identified wild-caught Anopheles funestus (s.l.) specimens trapped outdoors from the semi-arid to arid area of Kacheliba, West Pokot County, Kenya, were analysed by PCR and sequencing for species identification, malaria parasite infection and host blood-meal sources., Results: Three hundred and thirty specimens were analysed to identify sibling species of the An. funestus group, none of which amplified using the available primers; two were infected with Plasmodium falciparum and Plasmodium ovale, separately, while 84% (n = 25) of the blood-fed specimens had fed on humans. Mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences of 55 specimens (Plasmodium-positive, blood-fed and Plasmodium-negative) did not match reference sequences, possibly suggesting a previously unreported species, resolving as two clades., Conclusions: Our findings indicate the existence of yet-to-be identified and described anopheline species with a potential as malaria vectors in Kenya.

Background: Anopheles funestus is among the major malaria vectors in Kenya and sub-Saharan Africa and has been recently implicated in persistent malaria transmission. However, its ecology and genetic diversity remain poorly understood in Kenya., Methods: Using 16 microsatellite loci, we examined the genetic structure of An. funestus sampled from 11 locations (n = 426 individuals) across a wide geographical range in Kenya spanning coastal, western and Rift Valley areas., Results: Kenyan An. funestus resolved as three genetically distinct clusters. The largest cluster (FUN1) broadly included samples from western and Rift Valley areas of Kenya with two clusters identified from coastal Kenya (FUN2 and FUN3), not previously reported. Geographical distance had no effect on population differentiation of An. funestus. We found a significant variation in the mean Plasmodium infectivity between the clusters (χ 2 = 12.1, df = 2, P = 0.002) and proportional to the malaria prevalence in the different risk zones of Kenya. Notably, there was variation in estimated effective population sizes between the clusters, suggesting possible differential impact of anti-vector interventions in represented areas., Conclusions: Heterogeneity among Kenyan populations of An. funestus will impact malaria vector control with practical implications for the development of gene-drive technologies. The difference in Plasmodium infectivity and effective population size between the clusters could suggest potential variation in phenotypic characteristics relating to competence or insecticide resistance. This is worth examining in future studies.

Background: Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied., Methods: We analyzed 1149 wild-caught An. funestus (senso lato) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods., Results: The most abundant sibling species collected in all study sites were Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestus had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae. There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum, only one carried P. falciparum. We also detected P. falciparum infection in two non-blood-fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig., Conclusions: Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.

Background: The ixodid tick Rhipicephalus appendiculatus transmits the apicomplexan protozoan parasite Theileria parva, which causes East coast fever (ECF), the most economically important cattle disease in eastern and southern Africa. Recent analysis of micro- and minisatellite markers showed an absence of geographical and host-associated genetic sub-structuring amongst field populations of R. appendiculatus in Kenya. To assess further the phylogenetic relationships between field and laboratory R. appendiculatus tick isolates, this study examined sequence variations at two mitochondrial genes, cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (rRNA), and the nuclear encoded ribosomal internal transcribed spacer 2 (ITS2) of the rRNA gene, respectively., Results: The analysis of 332 COI sequences revealed 30 polymorphic sites, which defined 28 haplotypes that were separated into two distinct haplogroups (A and B). Inclusion of previously published haplotypes in our analysis revealed a high degree of phylogenetic complexity never reported before in haplogroup A. Neither haplogroup however, showed any clustering pattern related to either the geographical sampling location, the type of tick sampled (laboratory stocks vs field populations) or the mammalian host species. This finding was supported by the results obtained from the analysis of 12S rDNA sequences. Analysis of molecular variance (AMOVA) indicated that 90.8 % of the total genetic variation was explained by the two haplogroups, providing further support for their genetic divergence. These results were, however, not replicated by the nuclear transcribed ITS2 sequences likely because of recombination between the nuclear genomes maintaining a high level of genetic sequence conservation., Conclusions: COI and 12S rDNA are better markers than ITS2 for studying intraspecific diversity. Based on these genes, two major genetic groups of R. appendiculatus that have gone through a demographic expansion exist in Kenya. The two groups show no phylogeographic structure or correlation with the type of host species from which the ticks were collected, nor to the evolutionary and breeding history of the species. The two lineages may have a wide geographic distribution range in eastern and southern Africa. The findings of this study may have implications for the spread and control of R. appendiculatus, and indirectly, on the transmission dynamics of ECF.

Background: Animal African Trypanosomiasis (AAT) is caused by several species of trypanosomes including Trypanosoma congolense, T. vivax, T. godfreyi, T. simiae and T. brucei. Two of the subspecies of T. brucei also cause Human African Trypanosomiasis. Although some of them can be mechanically transmitted by biting flies; these trypanosomes are all transmitted by tsetse flies which are the cyclical vectors of Trypanosoma congolense, T. godfreyi, T. simiae and T. brucei. We present here the first report assessing the prevalence of trypanosomes in tsetse flies in Nigeria using molecular tools., Methods: 488 tsetse flies of three species, Glossina palpalis palpalis, G. tachinoides and G. morsitans submorsitans were collected from Wuya, Niger State and Yankari National Park, Bauchi State in 2012. Trypanosomes were detected and identified using an ITS1 PCR assay on DNA purified from the 'head plus proboscis' (H + P) and abdomen (ABD) parts of each fly., Results: T. vivax and T. congolense Savannah were the major parasites detected. Trypanosomes prevalence was 7.1 % in G. p. palpalis, 11.9 % in G. tachinoides and 13.5 % in G. m. submorsitans. Prevalences of T. congolense Savannah ranged from 2.5 to 6.7 % and of T. vivax were approximately 4.5 %. Trypanosoma congolense Forest, T. godfreyi and T. simiae were also detected in the site of Yankari. The main biological and ecological determinants of trypanosome prevalence were the fly sex, with more trypanosomes found in females than males, and the site, with T. congolense subspp. being more abundant in Yankari than in Wuya. As expected, the trypanosome species diversity was higher in Yankari National Park than in the more agricultural site of Wuya where vertebrate host species diversity is lower., Conclusions: Our results show that T. congolense Savannah and T. vivax are the main species of parasite potentially causing AAT in the two study sites and that Yankari National Park is a potential reservoir of trypanosomes both in terms of parasite abundance and species diversity.

Background: Laboratory and field studies showed that repellent, irritant and toxic actions of common public health insecticides reduce human-vector contact and thereby interrupt disease transmission. One of the more effective strategies to reduce disease risk involves the use of long-lasting treated bednets. However, development of insecticide resistance in mosquito populations makes it imperative to find alternatives to these insecticides. Our previous study identified four essential oils as alternatives to pyrethroids: Thymus vulgaris, Cymbopogon winterianus, Cuminum cyminum, Cinnamomum zeylanicum. The objectives of this study were to identify active compounds of these essential oils, to characterize their biological activity, and to examine their potential as a treatment for bednets., Methods: We evaluated the electrophysiological, behavioural (repellency, irritancy) and toxic effects of the major compounds of these oils against Anopheles gambiae strain 'Kisumu'., Results: Aldehydes elicited the strongest responses and monoterpenes the weakest responses in electroantennogram (EAG) trials. However, EAG responses did not correlate consistently with results of behavioral assays. In behavioral and toxicity studies, several of the single compounds did exhibit repellency, irritancy or toxicity in An. gambiae; however, the activity of essential oils did not always correlate with activity expected from the major components. On the contrary, the biological activity of essential oils appeared complex, suggesting interactions between individual compounds and the insect under study. Data also indicated that the three effects appeared independent, suggesting that repellency mechanism(s) may differ from mechanisms of irritancy and toxicity., Conclusions: Based on the bioassays reported here, some of the compounds merit consideration as alternative bednet treatments.

Background: Persistent monomolecular surface films could benefit larval source management for malaria control by reducing programme costs and managing insecticide resistance. This study evaluated the efficacy of the silicone-based surface film, Aquatain® Mosquito Formulation (AMF), for the control of the Afrotropical malaria vectors, Anopheles gambiae sensu stricto and Anopheles arabiensis in laboratory dose-response assays and standardized field tests., Methods: Tests were carried out following guidelines made by the World Health Organization Pesticide Evaluation Scheme (WHOPES). Sub-lethal effects of AMF were evaluated by measuring egg-laying and hatching of eggs laid by female An. gambiae s.s. that emerged from habitats treated with a dose that resulted in 50% larval mortality in laboratory tests., Results: Both vector species were highly susceptible to AMF. The estimated lethal doses to cause complete larval mortality in dose-response tests in the laboratory were 1.23 (95% confidence interval (CI) 0.99-1.59) ml/m2 for An. gambiae s.s. and 1.35 (95% CI 1.09-1.75) ml/m2 for An. arabiensis. Standardized field tests showed that a single dose of AMF at 1 ml/m2 inhibited emergence by 85% (95% CI 82-88%) for six weeks. Females exposed as larvae to a sub-lethal dose of AMF were 2.2 times less likely (Odds ratio (OR) 0.45, 95% CI 0.26-0.78) to lay eggs compared to those from untreated ponds. However, exposure to sub-lethal doses neither affected the number of eggs laid by females nor the proportion hatching., Conclusion: AMF provided high levels of larval control for a minimum of six weeks, with sub-lethal doses reducing the ability of female mosquitoes to lay eggs. The application of AMF provides a promising novel strategy for larval control interventions against malaria vectors in Africa. Further field studies in different eco-epidemiological settings are justified to determine the persistence of AMF film for mosquito vector control and its potential for inclusion in integrated vector management programmes.

Background: The use of gravid mosquitoes as vehicles to auto-disseminate larvicides was recently demonstrated for the transfer of pyriproxyfen (PPF) by container-breeding Aedes mosquitoes and presents an appealing idea to explore for other disease vectors. The success of this approach depends on the female's behaviour, the time of exposure and the amount of PPF that can be carried by an individual. We explore the effect of PPF exposure at seven time points around blood feeding on individual Anopheles gambiae sensu stricto and Culex quinquefasciatus fecundity and ability to transfer in laboratory assays., Method: Mosquitoes were exposed to 2.6 mg PPF per m2 at 48, 24 and 0.5 hours before and after a blood meal and on the day of egg-laying. The proportion of exposed females (N=80-100) laying eggs, the number of eggs laid and hatched was studied. Transfer of PPF to oviposition cups was assessed by introducing 10 late instar insectary-reared An. gambiae s.s. larvae into all the cups and monitored for adult emergence inhibition., Results: Exposure to PPF between 24 hours before and after a blood meal had significant sterilizing effects: females of both species were 6 times less likely (Odds ratio (OR) 0.16, 95% confidence interval (CI) 0.10-0.26) to lay eggs than unexposed females. Of the few eggs laid, the odds of an egg hatching was reduced 17 times (OR 0.06, 95% CI 0.04-0.08) in Anopheles but only 1.2 times (OR 0.82, 95% CI 0.73-0.93) in Culex. Adult emergence inhibition from larvae introduced in the oviposition cups was observed only from cups in which eggs were laid. When females were exposed to PPF close to egg laying they transferred enough PPF to reduce emergence by 65-71% (95% CI 62-74%)., Conclusion: PPF exposure within a day before and after blood feeding affects egg-development in An. gambiae s.s. and Cx. quinquefasciatus and presents a promising opportunity for integrated control of vectors and nuisance mosquitoes. However, sterilized females are unlikely to visit an oviposition site and therefore do not transfer lethal concentrations of PPF to aquatic habitats. This suggests that for successful auto-dissemination the optimum contamination time is close to oviposition.

Background: Electric nets (e-nets) are used to analyse the flight behaviour of insects and have been used extensively to study the host-oriented flight of tsetse flies. Recently we adapted this tool to analyse the oviposition behaviour of gravid malaria vectors, Anopheles gambiae s.s., orienting towards aquatic habitats and traps by surrounding an artificial pond with e-nets and collecting electrocuted mosquitoes on sticky boards on the ground next to the nets. Here we study whether e-nets themselves affect the responses of gravid An. gambiae s.s.., Methods: Dual-choice experiments were carried out in 80 m2 screened semi-field systems where 200 gravid An. gambiae s.s. were released each night for 12 nights per experiment. The numbers of mosquito landing on or approaching an oviposition site were studied by adding detergent to the water in an artificial pond or surrounding the pond with a square of e-nets. We also assessed whether the supporting framework of the nets or the sticky boards used to retain electrocuted mosquitoes influenced the catch., Results: Two similar detergent treated ponds presented in choice tests caught an equal proportion of the mosquitoes released, whereas a pond surrounded by e-nets caught a higher proportion than an open pond (odds ratio (OR) 1.7, 95% confidence interval (CI) 1.1 - 2.7; p<0.017). The separate evaluation of the impact of the square of electric nets and the yellow boards on the approach of gravid females towards a pond suggests that the tower-like construction of the square of electric nets did not restrict the approach of females but the yellow sticky boards on the ground attract gravid females to a source of water (OR 2.7 95% CI 1.7 - 4.3; p<0.001)., Conclusion: The trapping efficiency of the electric nets is increased when large yellow sticky boards are placed on the ground next to the e-nets to collect electrocuted mosquitoes, possibly because of increased visual contrast to the aquatic habitat. It is therefore important when comparing two treatments that the same trapping device is used in both. The importance of contrast around artificial habitats might be exploited to improve collections of An. gambiae s.s. in gravid traps.