Background: Designing, implementing, and upscaling of effective malaria vector control strategies necessitates an understanding of when and where transmission occurs. This study assessed the biting patterns of potentially infectious malaria vectors at various hours, locations, and associated human behaviors in different ecological settings in western Kenya. Methods: Hourly indoor and outdoor catches of human-biting mosquitoes were sampled from 19:00 to 07:00 for four consecutive nights in four houses per village. The human behavior study was conducted via questionnaire surveys and observations. Species within the Anopheles gambiae complex and Anopheles funestus group were distinguished by polymerase chain reaction (PCR) and the presence of Plasmodium falciparum circumsporozoite proteins (CSP) determined by enzyme-linked immunosorbent assay (ELISA). Results: Altogether, 2037 adult female anophelines were collected comprising the An. funestus group (76.7%), An. gambiae sensu lato (22.8%), and Anopheles coustani (0.5%). PCR results revealed that Anopheles arabiensis constituted 80.5% and 79% of the An. gambiae s.l. samples analyzed from the lowland sites (Ahero and Kisian, respectively). Anopheles gambiae sensu stricto (hereafter An. gambiae) (98.1%) was the dominant species in the highland site (Kimaeti). All the An. funestus s.l. analyzed belonged to An. funestus s.s. (hereafter An. funestus). Indoor biting densities of An. gambiae s.l. and An. funestus exceeded the outdoor biting densities in all sites. The peak biting occurred in early morning between 04:30 and 06:30 in the lowlands for An. funestus both indoors and outdoors. In the highlands, the peak biting of An. gambiae occurred between 01:00 and 02:00 indoors. Over 50% of the study population stayed outdoors from 18:00 to 22:00 and woke up at 05:00, coinciding with the times when the highest numbers of vectors were collected. The sporozoite rate was higher in vectors collected outdoors, with An. funestus being the main malaria vector in the lowlands and An. gambiae in the highlands. Conclusion: This study shows heterogeneity of anopheline distribution, high outdoor malaria transmission, and early morning peak biting activity of An. funestus when humans are not protected by bednets in the lowland sites. Additional vector control efforts targeting the behaviors of these vectors, such as the use of non-pyrethroids for indoor residual spraying and spatial repellents outdoors, are needed. Graphical

The directional selection for insecticide resistance due to indiscriminate use of insecticides in public health and agricultural system favors an increase in the frequency of insecticide-resistant alleles in the natural populations. Similarly, removal of selection pressure generally leads to decay in resistance. Past investigations on the emergence of insecticide resistance in mosquitoes mostly relied on field survey of resistance in vector populations that typically had a complex history of exposure to various public health and agricultural pest control insecticides in nature, and thus the effect of specific insecticides on rate of resistance emergency or resistance decay rate is not known. This study examined the phenotypic, genotypic, and biochemical changes that had occurred during the process of selection for pyrethroid resistance in Anopheles gambiae, the most important malaria vector in Africa. In parallel, we also examined these changes in resistant populations when there is no selection pressure applied. Through repeated deltamethrin selection in adult mosquitoes from a field population collected in western Kenya for 12 generations, we obtained three independent and highly pyrethroid-resistant An. gambiae populations. Three susceptible populations from the same parental population were generated by removing selection pressure. These two lines of mosquito populations differed significantly in monooxygenase and beta-esterase activities, but not in Vgsc gene mutation frequency, suggesting metabolic detoxification mechanism plays a major role in generating moderate-intensity resistance or high-intensity resistance. Pre-exposure to the synergist piperonyl butoxide restored the susceptibility to insecticide among the highly resistant mosquitoes, confirming the role of monooxygenases in pyrethroid resistance. The rate of resistance decay to become fully susceptible from moderate-intensity resistance took 15 generations, supporting at least 2-years interval is needed when the rotational use of insecticides with different modes of action is considered for resistance management.

Background: The demonstration that the recently discovered Anopheles symbiont Microsporidia MB blocks malaria transmission in Anophelesarabiensis 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. Graphical

Background: Malaria vector control has been implemented chiefly through indoor interventions targeting primary vectors resulting in population declines—pointing to a possible greater proportional contribution to transmission by secondary malaria vectors with their predominant exophagic and exophilic traits. With a historical focus on primary vectors, there is paucity of data on secondary malaria vectors in many countries in Africa. This study sought to determine the species compositions and bionomic traits, including proportions infected withPlasmodium falciparumand phenotypic insecticide resistance, of secondary vectors in three sites with high malaria transmission in Kisumu County, western Kenya.Methods: Cross-sectional sampling of adultAnopheleswas conducted using indoor and outdoor CDC light traps (CDC-LT) and animal-baited traps (ABTs) in Kakola-Ombaka and Kisian, while larvae were sampled in Ahero. Secondary vectors captured were exposed to permethrin using WHO bioassays and then analyzed by ELISA to test for proportions infected withP. falciparumsporozoites. AllAnopheleswere identified to species using morphological keys with a subset being molecularly identified using ITS2 andCO1sequencing for species identification.Results: Two morphologically identified secondary vectors captured—An. coustaniandAn. pharoensis—were determined to consist of four species molecularly. These includedAn. christyi,An. sp. 15 BSL-2014,an unidentified member of theAn. coustanicomplex (An. cf. coustani) and a species similar to that ofAn. pharoensisandAn. squamosus(An. cf. pharoensis). Standardized (Anophelesper trap per night) capture rates demonstrate higher proportions of secondary vectors across most trapping methods—with overall indoor and outdoor CDC-LTs and ABT captures composed of 52.2% (n = 93), 78.9% (n = 221) and 58.1% (n = 573) secondary vectors respectively. Secondary vectors were primarily caught outdoors. The overall proportion of secondary vectors withP. falciparumsporozoite was 0.63% (n = 5), with the unidentified speciesAn. cf. pharoensis,determined to carryPlasmodium. Overall secondary vectors were susceptible to permethrin with a > 99% mortality rate.Conclusions: Given their high densities, endophily equivalent to primary vectors, higher exophily andPlasmodium-positive proportions, secondary vectors may contribute substantially to malaria transmission. Unidentified species demonstrate the need for further morphological and molecular identification studies towards further characterization. Continued monitoring is essential for understanding their temporal contributions to transmission, the possible elevation of some to primary vectors and the development of insecticide resistance.Graphical

Background: Long-lasting insecticidal nets are an effective tool in reducing malaria transmission. However, with increasing insecticide resistance little is known about how physiologically resistant malaria vectors behave around a human-occupied bed net, despite their importance in malaria transmission. We used the Mbita bednet trap to assess the host-seeking behavior of insecticide-resistant Anopheles gambiae mosquitoes under semi-field conditions. The trap incorporates a mosquito netting panel which acts as a mechanical barrier that prevents host-seeking mosquitoes from reaching the human host baiting the trap. Methods: Susceptible and pyrethroid-resistant colonies of female Anopheles gambiae mosquitoes aged 3–5 days old were used in this study. The laboratory-bred mosquitoes were color-marked with fluorescent powders and released inside a semi-field environment where a human subject slept inside a bednet trap erected in a traditional African hut. The netting panel inside the trap was either untreated (control) or deltamethrin-impregnated. The mosquitoes were released outside the hut. Only female mosquitoes were used. A window exit trap was installed on the hut to catch mosquitoes exiting the hut. A prokopack aspirator was used to collect indoor and outdoor resting mosquitoes. In addition, clay pots were placed outside the hut to collect outdoor resting mosquitoes. The F1 progeny of wild-caught mosquitoes were also used in these experiments. Results: The mean number of resistant mosquitoes trapped in the deltamethrin-impregnated bed net trap was higher (mean = 50.21± 3.7) compared to susceptible counterparts (mean + 22.4 ± 1.31) (OR = 1.445; P<0.001). More susceptible mosquitoes were trapped in an untreated (mean = 51.9 ± 3.6) compared to a deltamethrin-treated bed net trap (mean = 22.4 ± 1.3) (OR = 2.65; P<0.001). Resistant mosquitoes were less likely to exit the house when a treated bed net was present compared to the susceptible mosquitoes. The number of susceptible mosquitoes caught resting outdoors (mean + 28.6 ± 2.22) when a treated bed net was hanged was higher than when untreated bednet was present inside the hut (mean = 4.6 ± 0.74). The susceptible females were 2.3 times more likely to stay outdoors away from the treated bed net (OR = 2.25; 95% CI = [1.7–2.9]; P<0.001). Conclusion: The results show that deltamethrin-treatment of netting panels inside the bednet trap did not alter the host-seeking behavior of insecticide-resistant female An. gambiae mosquitoes. On the contrary, susceptible females exited the hut and remained outdoors when a treated net was used. However, further investigations of the behavior of resistant mosquitoes under natural conditions should be undertaken to confirm these observations and improve the current intervention which are threatened by insecticide resistance and altered vector behavior.

Background: The rise of insecticide resistance poses a growing challenge to the effectiveness of vector control tools, particularly in rural areas. However, the urban setting has received comparatively less focus despite its significance in attracting rural to urban migration. Unplanned urbanization, often overlooked, exacerbates insecticide resistance as Anopheles mosquitoes adapt to the polluted environments of rapidly expanding cities. This study aimed to assess the insecticide susceptibility status of malaria vectors and identify potential underlying mechanisms across three distinct ecological settings characterized by differing levels of urbanization in Kisumu County, Kenya. Methods: The study was conducted in 2022–2023 in Kisumu County, western Kenya. Field-derived An. gambiae (s.l.) larvae collected from a long stretch of urban-to-rural continuum were phenotyped as either resistant or susceptible to six different insecticides using the World Health Organization (WHO) susceptibility test. Polymerase chain reaction (PCR) techniques were used to identify the species of the An. gambiae complex and screened for mutations at voltage-gated sodium channels (Vgsc-1014F, Vgsc-1014S, Vgsc-1575Y) and acetylcholinesterase (Ace1) target site mutation 119S. Metabolic enzyme activities (non-specific β-esterases and monooxygenases) were evaluated in mosquitoes not exposed to insecticides using microplate assays. Additionally, during larval sampling, a retrospective questionnaire survey was conducted to determine pesticide usage by the local inhabitants. Results: Anopheles arabiensis dominated in urban (96.2%) and peri-urban (96.8%) areas, while An. gambiae (s.s.) was abundant in rural settings (82.7%). Urban mosquito populations showed high resistance intensity to deltamethrin (Mortality rate: 85.2% at 10x) and suspected resistance to Pirimiphos-methyl and bendiocarb while peri-urban and rural populations exhibited moderate resistance intensity to deltamethrin (mortality rate >98% at 10x). Preexposure of mosquitoes to a synergist piperonyl butoxide (PBO) significantly increased mortality rates: from 40.7% to 88.5% in urban, 51.9% to 90.3% in peri-urban, and 55.4% to 87.6% in rural populations for deltamethrin, and from 41.4% to 78.8% in urban, 43.7% to 90.7% in peri-urban, and 35% to 84.2% in rural populations for permethrin. In contrast, 100% mortality to chlorfenapyr and clothianidin was observed in all the populations tested. The prevalence of L1014F mutation was notably higher in urban An. arabiensis (0.22) unlike the peri-urban (0.11) and rural (0.14) populations while the L1014S mutation was more prevalent in rural An. gambiae (0.93). Additionally, urban An. arabiensis exhibited elevated levels of mixed function oxidases (0.8/mg protein) and non-specific esterases (2.12/mg protein) compared to peri-urban (0.57/mg protein and 1.5/mg protein, respectively) and rural populations (0.6/mg protein and 1.8/mg protein, respectively). Pyrethroids, apart from their use in public health through LLINs, were being highly used for agricultural purposes across all ecological settings (urban 38%, peri-urban 36% and rural 37%) followed by amidine group, with organophosphates, neonicotinoids and carbamates being of secondary importance. Conclusion: These findings show high resistance of An. arabiensis to insecticides commonly used for vector control, linked with increased levels of detoxification enzymes. The observed intensity of resistance underscores the pressing issue of insecticide resistance in urban areas, potentially compromising the effectiveness of vector control measures, especially pyrethroid-treated LLINs. Given the species’ unique behavior and ecology compared to An. gambiae, tailored vector control strategies are needed to address this concern in urban settings.