Principal Malaria Vector Research Articles

Discovery of Knock-Down Resistance in the Major African Malaria Vector Anopheles funestus.

A major insecticide resistance mechanism in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa, with resistance mainly being conferred by detoxification enzymes. In a parallel study, we monitored 10 populations of An. funestus in Tanzania for insecticide resistance unexpectedly identified resistance to a banned insecticide, DDT, in the Morogoro region. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found eight novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (equivalent to L995F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region and were accompanied by weak signatures of a selective sweep, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). The WHO prequalifies no DDT products for vector control, and the chemical is banned in Tanzania. Widespread DDT contamination and a legacy of extensive countrywide stockpiles may have selected for this mutation. Continued monitoring is necessary to understand the origin of kdr in An. funestus, and the threat posed to insecticide-based vector control in Africa.

Eliminating malaria vectors with precision-guided sterile males

Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Discovery of knock-down resistance in the major African malaria vector Anopheles funestus.

A major mechanism of insecticide resistance in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa. While monitoring 10 populations of An. funestus in Tanzania, we unexpectedly found resistance to DDT, a banned insecticide, in one location. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found 8 novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (L1014F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to the exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). Further study is necessary to identify the origin and spread of kdr in An. funestus, and the potential threat to current insecticide-based vector control in Africa.

Mosquito odour-baited mass trapping reduced malaria transmission intensity: a result from a controlled before-and-after intervention study

BackgroundConventional vector control strategies have significantly reduced the malaria burden. The sustainability of these methods is currently challenged. Odour-based traps are emerging technologies that can complement the existing tools. Implementation of odour-based traps for mass trapping is limited due to the restricted range of vectors caught with available carbon dioxide-dependent lures, and the lack of comprehensive field studies. The objective of this study was to assess the impact of odour-mediated mass trapping targeting outdoor vectors, using a synthetic cattle urine lure that attracts a wide range of vector species in a variety of physiological states, on malaria prevalence and entomological parameters to determine malaria transmission intensities.MethodsA controlled before-and-after study was conducted in two rural communities in southern Ethiopia. Baseline monthly entomological and seasonal cross-sectional malaria prevalence surveys were conducted in both communities for a year. Then, mass trapping of mosquitoes was conducted in one of the villages, while the monthly entomological surveillance and seasonal malaria prevalence surveys continued in both villages. Generalised linear mixed models were constructed and tested to determine which factors were significantly affected by the intervention.ResultsMass trapping contributed to the reduction of the population of the principal malaria vector, Anopheles arabiensis, and the associated entomological indicators, the human bite rate (HBR) and the entomological inoculation rate (EIR), in the intervention village compared to the control village. The intervention village had an average HBR by An. arabiensis of 3.0 (95% CI 1.4–4.6) during the peak malaria transmission season, compared to 10.5 (95% CI − 0.5–21.5; P < 0.0001) in the control village. The intervention village (mean 0.02, 95% CI − 0.05–0.4.8) had a daily EIR eight times lower than the control village (mean 0.17, 95% CI), which likely contributed to the reduced malaria prevalence in the intervention community following its introduction by ca. 60% (95% CI 55–63).ConclusionsThe combined use of odour-based mass trapping and conventional control strategies coincided with a reduction of human-vector contact and malaria prevalence, providing support for odour-baited technologies as a viable option for next-generation vector control tools. Further cluster-randomised control studies are recommended in different eco-epidemiological settings with varying malaria transmission intensities.

Is Anopheles gambiae (sensu stricto), the principal malaria vector in Africa prone to resistance development against new insecticides? Outcomes from laboratory exposure of An. gambiae (s.s.) to sub-lethal concentrations of chlorfenapyr and clothianidin

Indiscriminate use of pesticides in the public health and agriculture sectors has contributed to the development of resistance in malaria vectors following exposure to sub-lethal concentrations. To preserve the efficacy of vector control tools and prevent resistance from spreading, early resistance detection is urgently needed to inform management strategies. The introduction of new insecticides for controlling malaria vectors such as clothianidin and chlorfenapyr requires research to identify early markers of resistance which could be used in routine surveillance. This study investigated phenotypic resistance of Anopheles gambiae (sensu stricto) Muleba-Kis strain using both WHO bottle and tube assays following chlorfenapyr, clothianidin, and alpha-cypermethrin selection against larvae and adults under laboratory conditions. High mortality rates were recorded for both chlorfenapyr-selected mosquitoes that were consistently maintained for 10 generations (24-h mortality of 92–100% and 72-h mortality of 98–100% for selected larvae; and 24-h mortality of 95–100% and 72-h mortality of 98–100% for selected adults). Selection with clothianidin at larval and adult stages showed a wide range of mortality (18–91%) compared to unselected progeny where mortality was approximately 99%. On the contrary, mosquitoes selected with alpha-cypermethrin from the adult selection maintained low mortality (28% at Generation 2 and 23% at Generation 4) against discrimination concentration compared to unselected progeny where average mortality was 51%. The observed resistance in the clothianidin-selected mosquitoes needs further investigation to determine the underlying resistance mechanism against this insecticide class. Additionally, further investigation is recommended to develop molecular markers for observed clothianidin phenotypic resistance.

Insecticide susceptibility status of Anopheles gambiae (s.l.) in and surrounding areas of Lake Tana, northwest Ethiopia

BackgroundVector control is the most effective malaria control and prevention measure. Among these, IRS and LLINs are the most important chemical insecticide interventions used in malaria prevention and control strategies in Ethiopia. However, the long-term effectiveness of these strategies is under threat due to the emergency and spread of insecticide resistance in the principal malaria vector. Therefore, this study was carried out, under standardized laboratory conditions to assess the killing effect of some insecticides against An. gambiae s.l.MethodsMosquitoes in late instar larvae and pupae stages were collected from different breeding habitats of the study sites using a soup ladle (350 ml capacity). The immature was reared to adults at optimum temperature and humidity in a field insectary using the WHO protocol. Four insecticides representing three chemical classes were used against adult mosquitoes. These were permethrin, deltamethrin, pirimiphos-methyl and bendiocarb. Susceptibility tests were carried out from September to December 2021 using the WHO standard procedures. Mortality rate, variation, interaction effect and knockdown times (KDT50 and KDT95%) were computed using descriptive statistics, multivariate analysis of variance and log-probit regression model using SPSS version 20 software.ResultsTotally, 1300 Anopheles gambiae s.l. were tested to determine the susceptibility status to the four insecticides. Among these, 90.7% of them were susceptible to insecticides, whereas the remaining 9.3% of specimens were resistant to the insecticides. The results of the analysis of variance showed that mortality significantly varied between insecticides (F = 26.06, DF = 3, P < .0001), but not between study locations (F = 1.56, DF = 3, P = 0.212). On the other hand, the mean comparison of dead mosquitoes showed some signs of interaction between bendiocarb and locations, but not other insecticides and locations.ConclusionsThis study revealed that the knockdown times and effectiveness of different insecticides varied in different study sites. Therefore, insecticide resistance information is very essential for concerned bodies to make informed and evidence-based decisions on vector control.

Countrywide insecticide resistance monitoring and first report of the presence of the L1014S knock down resistance in Niger, West Africa

BackgroundMass distribution of insecticide-treated nets (ITNs) is the principal malaria vector control strategy adopted by Niger. To better inform on the most appropriate ITN to distribute, the National Malaria Control Programme (NMCP) of Niger and its partners, conducted insecticide resistance monitoring in selected sites across the country.MethodsThe susceptibility of Anopheles gambiae sensu lato (s.l.) to chlorfenapyr and pyrethroid insecticides was investigated in a total of sixteen sites in 2019 and 2020, using 2–5-day-old adults reared from wild collected larvae per site. The susceptibility status, pyrethroid resistance intensity at 5 and 10 times the diagnostic concentrations, and piperonyl butoxide (PBO) synergism with diagnostic concentrations of deltamethrin, permethrin and alpha-cypermethrin were assessed using WHO bioassays. Two doses (100 and 200 µg/bottle) of chlorfenapyr were tested using the CDC bottle assay method. Species composition and allele frequencies for knock-down resistance (kdr-L1014F and L1014S) and acetylcholinesterase (ace-1 G119S) mutations were further characterized using polymerase chain reaction (PCR).ResultsHigh resistance intensity to all pyrethroids tested was observed in all sites except for alpha-cypermethrin in Gaya and Tessaoua and permethrin in Gaya in 2019 recording moderate resistance intensity. Similarly, Balleyara, Keita and Tillabery yielded moderate resistance intensity for alpha-cypermethrin and deltamethrin, and Niamey V low resistance intensity against deltamethrin and permethrin in 2020. Pre-exposure to PBO substantially increased susceptibility with average increases in mortality between 0 and 70% for tested pyrethroids. Susceptibility to chlorfenapyr (100 µg/bottle) was recorded in all sites except in Tessaoua and Magaria where susceptibility was recorded at the dose of 200 µg/bottle. Anopheles coluzzii was the predominant malaria vector species in most of the sites followed by An. gambiae sensu stricto (s.s.) and Anopheles arabiensis. The kdr-L1014S allele, investigated for the first time, was detected in the country. Both kdr-L1014F (frequencies [0.46–0.81]) and L1014S (frequencies [0.41–0.87]) were present in all sites while the ace-1 G119S was between 0.08 and 0.20.ConclusionThe data collected will guide the NMCP in making evidence-based decisions to better adapt vector control strategies and insecticide resistance management in Niger, starting with mass distribution of new generation ITNs such as interceptor G2 and PBO ITNs.

Beyond eves and cracks: An interdisciplinary study of socio-spatial variation in urban malaria transmission in Ethiopia.

During the past century, the global trend of reduced malaria transmission has been concurrent with increasing urbanization. Although urbanization has traditionally been considered beneficial for vector control, the adaptation of malaria vectors to urban environments has created concerns among scientific communities and national vector control programs. Since urbanization rates in Ethiopia are among the highest in the world, the Ethiopian government developed an initiative focused on building multi-storied units organized in condominium housing. This study aimed to develop an interdisciplinary methodological approach that integrates architecture, landscape urbanism, medical anthropology, and entomology to characterize exposure to malaria vectors in this form of housing in three condominiums in Jimma Town. Mosquitoes were collected using light trap catches (LTCs) both indoor and outdoor during 2019's rainy season. Architectural drawings and ethnographic research were superposed to entomological data to detect critical interactions between uses of the space and settlement conditions potentially affecting malaria vector abundance and distribution. A total of 34 anopheline mosquitoes comprising three species (Anopheles gambiae s.l, An. pharoensis and An. coustani complex) were collected during the three months of mosquito collection. Anopheles gambiae s.l, the principal malaria vector in Ethiopia, was the predominant species of all the anophelines collected. Distribution of mosquito breeding sites across scales (household, settlement, urban landscape) is explained by environmental conditions, socio-cultural practices involving modification of existing spaces, and systemic misfits between built environment and territory. Variations in mosquito abundance and distribution in this study were mainly related to standard building practices that ignore the original logics of the territory, deficiency of water and waste disposal management systems, and adaptations of the space to fit heterogeneous lifestyles of residents. Our results indicate that contextualizing malaria control strategies in relation to vector ecology, social dynamics determining specific uses of the space, as well as building and territorial conditions could strengthen current elimination efforts. Although individual housing remains a critical unit of research for vector control interventions, this study demonstrates the importance of studying housing settlements at communal level to capture systemic interactions impacting transmission at the household level and in outdoor areas.

Dispersion routes of the main vectors of human malaria in the Americas: Genetic evidence from the mitochondrial COI gene.

In America, of the 44 species of Anopheles, nine are main vectors of malaria and, of these, genetic information exists for seven. Hence, this study sought to know the gene flow and diversity of the seven principal vectors of malaria at the Americas level. For the seven species and the sequences of the mitochondrial cytochrome c oxidase I (COI) gene obtained from the GenBank and Bold System, genetic analyzes of populations and genetic structure were performed and haplotype networks and phylogenetic trees were obtained. For the seven species, 1440 sequences were analyzed and 519 haplotypes were detected. The Hd and π values were higher within a continental context than by countries. Neutrality tests indicated positive and negative values with most of these being significant (p < 0.05). Phylogenetic analyses for all the species recovered three clades with no geographic pattern among them. Studies suggest that native species of Anopheles from the Americas have greater haplotype diversity and low genetic differentiation due to the lack of physical barriers to impede gene flow among these populations. Moreover, all the species are interconnected by roadways. This scenario complicates the epidemiological picture of malaria in the Americas.

Current Status of Insecticide Susceptibility in the Principal Malaria Vector, Anopheles gambiae in Three Northern States of Nigeria.

Background:Malaria is a major public health problem in Nigeria with 97% of its population with high morbidity and mortality. Mosquitoes play an important role in the transmission of malaria parasites. This study was conducted to evaluate the current resistance status of Anopheles gambiae to insecticides.Methods:Larvae of An. gambiae was collected from three zones; A, B and C differentiated on the basis of variation in agricultural ecosystems between August and November, 2018 in the northeast and northwestern parts of Nigeria. They were carefully reared to adult stage and insecticidal susceptibility tests were conducted.Results:The mosquitoes tested showed high levels of resistance to all the insecticides used with the exception of malathion. Study zone A, recorded 74% mortality after 24h to deltamethrin compared to 81% from zone B and 82% from zone C, respectively. Mosquitoes from zone B exposed to DDT had the highest level of resistance at 37% compared to 40% and 53% from zones A and C, respectively. Resistant to bendiocarb was also observed, with zone A having the lowest mortality of 44% compared to 48% from zone C and 55% from Zone B, respectively. According to the results of knockdown tests, mosquitoes from Zone A exposed to deltamethrin recorded the lowest knockdown across the study locations while zone B recorded the lowest knockdown for DDT.Conclusion:The results of the study provide an insight into the current status of An. gambiae to four major insecticides in northern Nigeria as guideline for mosquitocontrol.

Residual malaria of Atlantic Forest systems and the influence of anopheline fauna.

In Brazil, the Amazon region comprises 99.5% of the reported malaria cases. However, another hotspot of the disease is the Atlantic Forest regions, with the sporadic occurrence of autochthonous human cases. In such context, this study sought to investigate the role of anopheline mosquitoes (Diptera: Culicidae) in the residual malaria transmission in Atlantic Forest areas. Two rural areas in the Espírito Santo state were the surveyed sites. Mosquitoes were captured using Shannon trap and CDC light traps and identified into species based on morphological characters. Ecological indexes (Shannon-Wiener diversity, Simpson's dominance, Pielou equability, and Sorensen similarity) were the tools used in the anopheline fauna characterization and comparison along with the two explored areas. The assessment of the sampling adequacy in the studied areas was possible through the generation of a species accumulation curve. A correlation test verified the influence of climatic variables on the anopheline species abundance. A total of 1471 female anopheline mosquitoes were collected from May 2019 to April 2020, representing 13 species. The species richness was higher in Valsugana Velha (hypo-endemic) than in Alto Caparaó (non-endemic). There was a significant variation in the species abundance between Valsugana Velha (n = 1438) and Alto Caparaó (n = 33). The most abundant species was Anopheles (Kerteszia) cruzii complex Dyar and Knab, 1908 representing 87% of the total anophelines collected. These results suggest that the Plasmodium spp. circulation in Brazilian Atlantic Forest areas occurs mainly due to the high frequency of Anopheles (K.) cruzii complex, considered the principal vector of simian and human malaria in the region.

The relationship between house height and mosquito house entry: an experimental study in rural Gambia.

Most malaria infections in sub-Saharan Africa are acquired indoors, thus finding effective ways of preventing mosquito house entry should reduce transmission. Since most malaria mosquitoes fly less than 1 m from the ground, we tested whether raising buildings off the ground would prevent the entry of Anopheles gambiae, the principal African malaria vector, in rural Gambia. Nightly collections of mosquitoes were made using light traps from four inhabited experimental huts, each of which could be moved up or down. Mosquito house entry declined with increasing height, with a hut at 3 m reducing An. gambiae house entry by 84% when compared with huts on the ground. A propensity for malaria vectors to fly close to the ground and reduced levels of carbon dioxide, a major mosquito attractant, in elevated huts, may explain our findings. Raised buildings may help reduce malaria transmission in Africa.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon.

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a locality situated 2km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adults. Bioassays were performed to assess resistance profile to the four insecticides classes. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was the most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (<40% mortality). An. gambiae s.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the significant Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Multiple insecticide resistance and Plasmodium infection in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background: Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance, Plasmodium infection rate, insecticide resistance profiles and underlying resistance mechanisms. Methods: Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a locality situated 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F 1 adults. Bioassays were performed to assess resistance profile to the four insecticides classes. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance and Plasmodium infection rates were investigated. Results: Anopheles funestus s.s. was the most abundant species in Elende (85%) followed by Anopheles gambiae s.s. (15%) with both having similar sporozoite rate. Both species exhibited high levels of resistance to the pyrethroids, permethrin and deltamethrin (<40% mortality). An. gambiae s.s. was resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate. An. funestus s.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119S ace-1 resistance allele (15%) were detected in An. funestus s.s. and An. gambiae s.s., respectively . Furthermore, the high pyrethroid/DDT resistances in An. gambiae corresponded with an increase frequency of 1014F kdr allele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression of CYP6P5, CYP6P9a and CYP6P9b. Conclusion: The resistance to multiple insecticide classes observed in these vector populations alongside the significant Plasmodium sporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

Environmental Factors Associated with Larval Habitats of Anopheline Mosquitoes (Diptera: Culicidae) in Metema District, Northwestern Ethiopia.

Background:Malaria is one of the major public health concerns in Ethiopia. Control options available for the management of malaria, include case detection, personal protection and larval source management. Effective control of Anopheles larvae largely depends on understanding of the habitats of the vectors. The aims of this study were to identify the breeding habitats of mosquitoes and characterize the larval habitats in Gende Wuha Town in northwestern Ethiopia.Methods:Different aquatic habitats were sampled and characterized for anopheline larvae from November 2012 to June 2013.Results:In total, 2784 larvae of Anopheles mosquitoes were collected from various breeding habitats. Microscopic identification of the III and IV instars revealed the presence of seven Anopheles species. Of the Anopheles spp, Anopheles gambiae s.l. (80%) was the most predominant species in the study area. Spearman correlation coefficient results also determined that the density of An. gambiae s.l. increased significantly with habitat temperature (r= 0.346, p< 0.01). Significantly higher An. gambiae s.l. larvae were obtained in non-shaded habitats (z= −3.120, p< 0.05) when compared with shaded habitats.Conclusion:The current study demonstrated An. gambiae s.l., the principal malaria vector in the country, is the predominant species in the larval sampling habitats. It was also noted the importance of edge of stream as larva breeding habitats for this species during the dry season of the year. Therefore, attention should be given for this breeding habitat for control of the vector during dry season.

Elevated Plasmodium sporozoite infection and multiple insecticide resistance in the principal malaria vectors Anopheles funestus and Anopheles gambiae in a forested locality close to the Yaoundé airport, Cameroon

Background:Reducing the burden of malaria requires better understanding of vector populations, particularly in forested regions where the incidence remains elevated. Here, we characterized malaria vectors in a locality near the Yaoundé international airport, Cameroon, including species composition, abundance,Plasmodiuminfection rate, insecticide resistance profiles and underlying resistance mechanisms.Methods:Blood-fed adult mosquitoes resting indoors were aspirated from houses in April 2019 at Elende, a village located 2 km from the Yaoundé-Nsimalen airport. Female mosquitoes were forced to lay eggs to generate F1adult progeny. Bioassays were performed to assess resistance profile to insecticides. The threshold of insecticide susceptibility was defined above 98% mortality rate and mortality rates below 90% were indicative of confirmed insecticide resistance. Furthermore, the molecular basis of resistance andPlasmodiuminfection rates were investigated.Results:Anopheles funestuss.s. was most abundant species in Elende (85%) followed byAnopheles gambiaes.s. (15%) with both having a similar sporozoite rate. Both species exhibited high levels of resistance to pyrethroids (&lt;40% mortality).An. gambiaes.s. was also resistant to DDT (9.9% mortality) and bendiocarb (54% mortality) while susceptible to organophosphate.An. funestuss.s. was resistant to dieldrin (1% mortality), DDT (86% mortality) but susceptible to carbamates and organophosphates. The L119F-GSTe2 resistance allele (8%) and G119Sace-1 resistance allele (15%) were detected inAn. funestuss.s. andAn. gambiaes.s., respectively.Furthermore, the high pyrethroid/DDT resistances inAn. gambiaes.s. corresponded with an increase frequency of 1014Fkdrallele (95%). Transcriptional profiling of candidate cytochrome P450 genes reveals the over-expression ofCYP6P5,CYP6P9aandCYP6P9b.Conclusion:The resistance to multiple insecticide classes observed in these vector populations alongside the highPlasmodiumsporozoite rate highlights the challenges that vector control programs encounter in sustaining the regular benefits of contemporary insecticide-based control interventions in forested areas.

4524 Fighting Malaria, One Image at a Time: Using Computer Vision to Develop an Automated Vector Speciation Tool

OBJECTIVES/GOALS: Rapid and accurate identification of primary malaria vector species from collected specimens is the most critical aspect of effective vector surveillance and control. This interdisciplinary team of engineers aims to automate identification using a deep learning computer vision algorithm. METHODS/STUDY POPULATION: The team spent August of 2019 observing and participating in control and surveillance activities in Zambia and Uganda. They conducted &gt;65 interviews with key stakeholders across 9 malaria control and surveillance sites, ranging from field and community health workers, to malaria researchers and Ministry of Health employees. Stakeholder feedback validated the need for a more accurate and efficient method of vector identification in order to more effectively deploy targeted malaria interventions. The team set forth in designing and prototyping a portable, automated field tool that could speciate mosquito vectors to the complex level using artificial intelligence. RESULTS/ANTICIPATED RESULTS: The team’s research demonstrated that accuracy, cost effectiveness, and ease of use would be critical to the successful adoption of the tool. Results of initial prototyping, usability studies, and stakeholder surveys were used to determine the tool’s minimal user specifications: 1) the ability to distinguish between Anopheles Gambiae and Anopheles Funestus, the two principal malaria vectors in the countries visited, 2) achieving an identification accuracy of ≥90% to the complex level, and 3) accessibility to the speciation data 3-7 days following vector collection. Next steps include optimizing the tool to deploy a minimal viable product for testing in Kenya by the summer of 2020. DISCUSSION/SIGNIFICANCE OF IMPACT: The accurate, high-quality surveillance enabled by this device would allow malaria control programs to scale surveillance to remote regions where an entomologist may not be available, allowing malaria programs to deploy effective interventions, monitor results, and prevent disease.

Intensity of pyrethroid resistance in Anopheles culicifacies s.l. (Diptera: Culicidae) in Odisha State, India

ABSTRACT Anopheles culicifacies s.l . is the principal malaria vector in India and has recently developed resistance to synthetic pyrethroids. For identifying the possible operational impact, quantitative measure of the intensity of this resistance is required. The purpose of this study was to measure the intensity of pyrethroid resistance in An. culicifacies s.l. The intensity bioassays with wild caught An. culicifacies s.l were carried out in 10 districts of east central India using 5x and 10x diagnostic concentrations of deltamethrin following WHO insecticides susceptibility guidelines. The results showed that the mortality of An. culicifacies s.l. ranged from 70% to 80% while exposed to 1x DC of deltamethrin (0.05%). Further bioassays conducted with 5x concentration of deltamethrin (0.25%) showed that the mortality of An. culicifacies s.l. in all the 10 districts varied from 92% to 97% indicating moderate resistance. While exposing to 10x concentration (0.5%) of this insecticide, 100% mortality was observed in all the districts; thereby confirmed moderate resistance intensity of this species. Since resistance was not confirmed at the 10x concentration, operational failure of this insecticide was unlikely. Hence, it is recommended to continue the use of LLINs for malaria vector control in these areas.

Assessing connectivity despite high diversity in island populations of a malaria mosquito.

Documenting isolation is notoriously difficult for species with vast polymorphic populations. High proportions of shared variation impede estimation of connectivity, even despite leveraging information from many genetic markers. We overcome these impediments by combining classical analysis of neutral variation with assays of the structure of selected variation, demonstrated using populations of the principal African malaria vector Anopheles gambiae. Accurate estimation of mosquito migration is crucial for efforts to combat malaria. Modeling and cage experiments suggest that mosquito gene drive systems will enable malaria eradication, but establishing safety and efficacy requires identification of isolated populations in which to conduct field testing. We assess Lake Victoria islands as candidate sites, finding one island 30 km offshore is as differentiated from mainland samples as populations from across the continent. Collectively, our results suggest sufficient contemporary isolation of these islands to warrant consideration as field‐testing locations and illustrate shared adaptive variation as a useful proxy for connectivity in highly polymorphic species.