Search

Knowledge of insect dispersal is relevant to the control of agricultural pests, vector-borne transmission of human and veterinary pathogens, and insect biodiversity. Previous studies in a malaria endemic area of the Sahel region in West Africa revealed high-altitude, long-distance migration of insects and various mosquito species. The objective of the current study was to assess whether similar behavior is exhibited by mosquitoes and other insects around the Lake Victoria basin region of Kenya in East Africa. Insects were sampled monthly from dusk to dawn over 1 year using sticky nets suspended on a tethered helium-filled balloon. A total of 17,883 insects were caught on nets tethered at 90, 120, and 160 m above ground level; 818 insects were caught in control nets. Small insects (0.5 cm, n = 2,334) and mosquitoes (n = 299). Seven orders were identified; dipteran was the most common. Barcoding molecular assays of 184 mosquitoes identified 7 genera, with Culex being the most common (65.8%) and Anopheles being the least common (5.4%). The survival rate of mosquitoes, experimentally exposed to high-altitude overnight, was significantly lower than controls maintained in the laboratory (19% vs. 85%). There were no significant differences in mosquito survival and oviposition rate according to capture height. These data suggest that windborne dispersal activity of mosquito vectors of malaria and other diseases occurs on a broad scale in sub-Saharan Africa.

Irrigation not only helps to improve food security but also creates numerous water bodies for mosquito production. This study assessed the effect of irrigation on malaria vector bionomics and transmission in a semi-arid site with ongoing malaria vector control program. The effectiveness of CDC light traps in the surveillance of malaria vectors was also evaluated relative to the human landing catches (HLCs) method. Adult mosquitoes were sampled in two study sites representing irrigated and non-irrigated agroecosystems in western Kenya using a variety of trapping methods. The mosquito samples were identified to species and assayed for host blood meal source and Plasmodium spp. sporozoite infection using polymerase chain reaction. Anopheles arabiensis was the dominant malaria vector in the two study sites and occurred in significantly higher densities in irrigated study site compared to the non-irrigated study site. The difference in indoor resting density of An. arabiensis during the dry and wet seasons was not significant. Other species, including An. funestus, An. coustani, and An. pharoensis, were collected. The An. funestus indoor resting density was 0.23 in irrigated study site while almost none of this species was collected in the non-irrigated study site. The human blood index (HBI) for An. arabiensis in the irrigated study site was 3.44% and significantly higher than 0.00% for the non-irrigated study site. In the irrigated study site, the HBI of An. arabiensis was 3.90% and 5.20% indoor and outdoor, respectively. The HBI of An. funestus was 49.43% and significantly higher compared to 3.44% for An. arabiensis in the irrigated study site. The annual entomologic inoculation rate for An. arabiensis in the irrigated study site was 0.41 and 0.30 infective bites/person/year indoor and outdoor, respectively, whereas no transmission was observed in the non-irrigated study site. The CDC light trap performed consistently with HLC in terms of vector density. These findings demonstrate that irrigated agriculture may increase the risk of malaria transmission in irrigated areas compared to the non-irrigated areas and highlight the need to complement the existing malaria vector interventions with novel tools targeting the larvae and both indoor and outdoor biting vector populations. Graphical abstract

Background Irrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia. Methods Comparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package. Results A total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P Conclusions Significantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area. Graphical abstract

Background Malaria in western Kenya is currently characterized by sustained high Plasmodial transmission and infection resurgence, despite positive responses in some areas following intensified malaria control interventions since 2006. This study aimed to evaluate long-term changes in malaria transmission profiles and to assess patterns of asymptomatic malaria infections in school children aged 5–15 years at three sites in western Kenya with heterogeneous malaria transmission and simultaneous malaria control interventions. Methods The study was conducted from 2018 to 2019 and is based on data taken every third year from 2005 to 2014 during a longitudinal parasitological and mosquito adult surveillance and malaria control programme that was initiated in 2002 in the villages of Kombewa, Iguhu, and Marani. Plasmodium spp. infections were determined using microscopy. Mosquito samples were identified to species and host blood meal source and sporozoite infections were assayed using polymerase chain reaction. Results Plasmodium falciparum was the only malaria parasite evaluated during this study (2018–2019). Asymptomatic malaria parasite prevalence in school children decreased in all sites from 2005 to 2008. However, since 2011, parasite prevalence has resurged by > 40% in Kombewa and Marani. Malaria vector densities showed similar reductions from 2005 to 2008 in all sites, rose steadily until 2014, and decreased again. Overall, Kombewa had a higher risk of infection compared to Iguhu (χ2 = 552.52, df = 1, P χ2 = 1127.99, df = 1, P χ2 = 617.59, df = 2, P χ2 = 27.17, df = 1, P Conclusions Malaria prevalence in western Kenya remains high and has resurged in some sites despite continuous intervention efforts. Targeting malaria interventions to those with asymptomatic infections who serve as human reservoirs might decrease malaria transmission and prevent resurgences. Longitudinal monitoring enables detection of changes in parasitological and entomological profiles and provides core baseline data for the evaluation of vector interventions and guidance for future planning of malaria control. Graphical abstract

Background Identification and characterization of larval habitats, documentation of Anopheles spp. composition and abundance, and Plasmodium spp. infection burden are critical components of integrated vector management. The present study aimed to investigate the effect of landscape heterogeneity on entomological and parasitological indices of malaria in western Kenya. Methods A cross-sectional entomological and parasitological survey was conducted along an altitudinal transect in three eco-epidemiological zones: lakeshore along the lakeside, hillside, and highland plateau during the wet and dry seasons in 2020 in Kisumu County, Kenya. Larval habitats for Anopheles mosquitoes were identified and characterized. Adult mosquitoes were sampled using pyrethrum spray catches (PSC). Finger prick blood samples were taken from residents and examined for malaria parasites by real-time PCR (RT-PCR). Results Increased risk of Plasmodium falciparum infection was associated with residency in the lakeshore zone, school-age children, rainy season, and no ITNs (χ2 = 41.201, df = 9, P Anopheles spp. abundance. However, house structures such as wall type and whether the eave spaces were closed or open, as well as the use of ITNs, did not affect Anopheles spp. densities in the homes (χ2 = 38.695, df = 7, P Anopheles funestus (41.8%) and An. arabiensis (29.1%) were the most abundant vectors in all zones. Sporozoite prevalence was 5.6% and 3.2% in the two species respectively. The lakeshore zone had the highest sporozoite prevalence (4.4%, 7/160) and inoculation rates (135.2 infective bites/person/year). High larval densities were significantly associated with lakeshore zone and hillside zones, animal hoof prints and tire truck larval habitats, wetland and pasture land, and the wet season. The larval habitat types differed significantly across the landscape zones and seasonality (χ2 = 1453.044, df = 298, P Conclusion The empirical evidence on the impact of landscape heterogeneity and seasonality on vector densities, parasite transmission, and Plasmodium infections in humans emphasizes the importance of tailoring specific adaptive environmental management interventions to specific landscape attributes to have a significant impact on transmission reduction. Graphical abstract

Malaria risk factor assessment is a critical step in determining cost-effective intervention strategies and operational plans in a regional setting. We develop a multi-indicator multistep approach to model the malaria risks at the population level in western Kenya. We used a combination of cross-sectional seasonal malaria infection prevalence, vector density, and cohort surveillance of malaria incidence at the village level to classify villages into malaria risk groups through unsupervised classification. Generalized boosted multinomial logistics regression analysis was performed to determine village-level risk factors using environmental, biological, socioeconomic, and climatic features. Thirty-six villages in western Kenya were first classified into two to five operational groups based on different combinations of malaria risk indicators. Risk assessment indicated that altitude accounted for 45–65% of all importance value relative to all other factors; all other variable importance values were < 6% in all models. After adjusting by altitude, villages were classified into three groups within distinct geographic areas regardless of the combination of risk indicators. Risk analysis based on altitude-adjusted classification indicated that factors related to larval habitat abundance accounted for 63% of all importance value, followed by geographic features related to the ponding effect (17%), vegetation cover or greenness (15%), and the number of bed nets combined with February temperature (5%). These results suggest that altitude is the intrinsic factor in determining malaria transmission risk in western Kenya. Malaria vector larval habitat management, such as habitat reduction and larviciding, may be an important supplement to the current first-line vector control tools in the study area.

Several sub-Saharan African countries rely on irrigation for food production. This study examined the impact of environmental modifications resulting from irrigation on the ecology of aquatic stages of malaria vectors in a semi-arid region of western Kenya. Mosquito larvae were collected from irrigated and non-irrigated ecosystems during seasonal cross-sectional and monthly longitudinal studies to assess habitat availability, stability, and productivity of anophelines in temporary, semipermanent, and permanent habitats during the dry and wet seasons. The duration of habitat stability was also compared between selected habitats. Emergence traps were used to determine the daily production of female adult mosquitoes from different habitat types. Malaria vectors were morphologically identified and sibling species subjected to molecular analysis. Data was statistically compared between the two ecosystems. After aggregating the data, the overall malaria vector productivity for habitats in the two ecosystems was estimated. Immatures of the malaria vector (Anopheles arabiensis) Patton (Diptera: Culicidae) comprised 98.3% of the Anopheles in both the irrigated and non-irrigated habitats. The irrigated ecosystem had the most habitats, higher larval densities, and produced 85.8% of emerged adult females. These results showed that irrigation provided conditions that increased habitat availability, stability, and diversity, consequently increasing the An. arabiensis production and potential risk of malaria transmission throughout the year. The irrigated ecosystems increased the number of habitats suitable for Anopheles breeding by about 3-fold compared to non-irrigated ecosystems. These results suggest that water management in the irrigation systems of western Kenya would serve as an effective method for malaria vector control.

Background Myanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region. Methods During October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested. Results A total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen. Conclusions This study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management. Graphical Abstract

Background Evolutionary pressures lead to the selection of efficient malaria vectors either resistant or susceptible to Plasmodium parasites. These forces may favour the introduction of species genotypes that adapt to new breeding habitats, potentially having an impact on malaria transmission. Thioester-containing protein 1 (TEP1) of Anopheles gambiae complex plays an important role in innate immune defenses against parasites. This study aims to characterize the distribution pattern of TEP1 polymorphisms among populations of An. gambiae sensu lato (s.l.) in western Kenya. Methods Anopheles gambiae adult and larvae were collected using pyrethrum spray catches (PSC) and plastic dippers respectively from Homa Bay, Kakamega, Bungoma, and Kisumu counties between 2017 and 2020. Collected adults and larvae reared to the adult stage were morphologically identified and then identified to sibling species by PCR. TEP1 alleles were determined in 627 anopheles mosquitoes using restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR) and to validate the TEP1 genotyping results, a representative sample of the alleles was sequenced. Results Two TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) were identified. TEP1*S1 and TEP1*R2 with their corresponding genotypes, homozygous *S1/S1 and heterozygous *R2/S1 were widely distributed across all sites with allele frequencies of approximately 80% and 20%, respectively both in Anopheles gambiae and Anopheles arabiensis. There was no significant difference detected among the populations and between the two mosquito species in TEP1 allele frequency and genotype frequency. The overall low levels in population structure (FST = 0.019) across all sites corresponded to an effective migration index (Nm = 12.571) and low Nei’s genetic distance values ( Conclusion Genotyping TEP1 has identified two common TEP1 alleles (TEP1*S1 and TEP1*R2) and three corresponding genotypes (*S1/S1, *R2/S1, and *R2/R2) in An. gambiae s.l. The TEP1 allele genetic diversity and population structure are low in western Kenya.

Food insecurity, recurrent famine, and poverty threaten the health of millions of African residents. Construction of dams and rural irrigation schemes is key to solving these problems. The sub-Saharan Africa International Center of Excellence for Malaria Research addresses major knowledge gaps and challenges in Plasmodium falciparum and Plasmodium vivax malaria control and elimination in malaria-endemic areas of Kenya and Ethiopia where major investments in water resource development are taking place. This article highlights progress of the International Center of Excellence for Malaria Research in malaria vector ecology and behavior, epidemiology, and pathogenesis since its inception in 2017. Studies conducted in four field sites in Kenya and Ethiopia show that dams and irrigation increased the abundance, stability, and productivity of larval habitats, resulting in increased malaria transmission and a greater disease burden. These field studies, together with hydrological and malaria transmission modeling, enhance the ability to predict the impact of water resource development projects on vector larval ecology and malaria risks, thereby facilitating the development of optimal water and environmental management practices in the context of malaria control efforts. Intersectoral collaborations and community engagement are crucial to develop and implement cost-effective malaria control strategies that meet food security needs while controlling malaria burden in local communities.

Irrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia.Comparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package.A total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [tSignificantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area.

Piperonyl butoxide (PBO)-synergized pyrethroid products are widely available for the control of pyrethroid-resistant mosquitoes. To date, no study has formally examined mosquito resistance to PBO-synergized insecticides. We used Culex quinquefasciatus as a model mosquito examined the insecticide resistance mechanisms of mosquitoes to PBO-synergized pyrethroid using modified World Health Organization tube bioassays and biochemical analysis of metabolic enzyme expressions prior- and post-PBO exposure. We measured mosquito mortalities and metabolic enzyme expressions in mosquitoes with/without pre-exposure to different PBO concentrations and exposure durations. We found that field Culex quinquefasciatus mosquitoes were resistant to all insecticides tested, including PBO-synergized pyrethroids (mortality ranged from 3.7±4.7% to 66.7±7.7%), except malathion. Field mosquitoes had elevated levels of carboxylesterase (COE, 3.8-fold) and monooxygenase (P450, 2.1-fold) but not glutathione S-transferase (GST) compared to susceptible mosquitoes. When the field mosquitoes were pre-exposed to 4% PBO, the 50% lethal concentration of deltamethrin was reduced from 0.22% to 0.10%, compare to 0.02% for susceptible mosquitoes. Knockdown resistance gene mutation (L1014F) rate was 62% in field mosquitoes. PBO pre-exposure suppressed P450 enzyme expression levels by 25∼34%, GST by 11%, and had no impact on COE enzyme expression. Even with the optimal PBO concentration and exposure duration, field mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to laboratory controls. These results demonstrate that PBO alone may not be enough to control highly pyrethroid resistant mosquitoes due to the multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored.Authors’ SummaryMosquitoes are vectors of many major infectious diseases globally. Insecticides and related products are widely used for mosquito controls and disease preventions. Over time and following repeated use, mosquitoes (including Aedes, Anopheles and Culex) have developed very high resistance to multiple insecticides all over the world. Target site insensitivity due to mutations in the voltage-gated sodium channel gene and overproduction of metabolic detoxification enzymes such as cytochrome P450 (CYP) monooxygenases play critical role in insecticide resistance in mosquitoes. To enhance the killing power of insecticides, synergized insecticides were developed by mixing insecticide synergists with pyrethroids. Discovered in the 1940s, piperonyl butoxide (PBO) is one of the earliest and most commonly used insecticide synergists. Field application of PBO-synergized insecticides performed far better than mono-pyrethroids. PBO-treated long-lasting insecticidal nets (PBO-LLINs), which also use pyrethroids, outperformed regular LLIN for malaria control in many African countries. PBO-LLIN is soon to be rolled out on a large scale for malaria control in Africa. One important question regarding the use of synergized insecticides is whether they will select for vector population resistance to synergized insecticide products, in other words, are PBO-synergized pyrethroids effective against highly insecticide-resistant mosquitoes? To date, no study has formally examined mosquito resistance to PBO-synergized insecticides. Here, we used Culex quinquefasciatus as a model mosquito, we examined its resistance status to different insecticides including PBO-synergized pyrethrins and tested how PBO exposure affect mosquito mortality and the expressions of metabolic enzymes. We found that field Culex quinquefasciatus mosquitoes were resistant to multiple insecticides tested, including PBO-synergized pyrethroids. Field mosquitoes had elevated levels of carboxylesterase (COE) and monooxygenase (P450) but not glutathione S-transferase (GST) enzyme expressions compared to susceptible mosquitoes. Even with optimal PBO concentration and exposure duration, field mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to laboratory controls, and PBO exposure had no impact on COE enzyme expressions. The phenomena of the insecticide-resistant mosquitoes’ insensitivity to PBO exposure or PBO-synergized insecticides and multiple-resistance mechanisms have also been reported from Aedes and Anopheles mosquitoes in different countries. These results demonstrate that PBO alone is not enough to control highly pyrethroid resistant mosquitoes due to multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored

There are a number of available and emerging malaria intervention tools that require innovative trial designs to find the optimal combinations at given epidemiologic settings. We simulated intervention strategies based on adaptive interventions, which included long-lasting insecticidal nets (LLINs), piperonyl butoxide–treated LLINs (PBO-LLINs), indoor residual spraying (IRS), and long-lasting microbial larviciding (LLML). The aims were to determine if PBO-LLINs or LLIN+IRS combination is more effective for initial interventions than LLINs and to identify the most effective intervention. We used a clustered, randomized adaptive trial design with malaria infection prevalence (MIP) as the outcome variable. The results indicate that during the initial stage of interventions, compared with regular LLINs, PBO-LLINs (relative reduction [RR]: 29.3%) and LLIN plus IRS with alternative-insecticide (RR: 26.8%) significantly reduced MIP. In the subsequent interventions, adding alternative insecticide IRS (RR: 23.8%) or LLML (RR: 31.2%) to existing PBO-LLIN was effective in further reducing MIP. During the next stage of interventions, adding LLML on top of PBO-LLIN+IRS (with alternative insecticides) had a significant impact on MIP (RR: 39.2%). However, adding IRS (with alternative insecticides) on top of PBO-LLIN+LLML did not significantly reduce MIP (11.6%). Overall, in clusters initiated with PBO-LLIN, adding LLML would be the most effective strategy in reducing MIP; in clusters initiated with LLIN+IRS, replacing LLIN+IRS with PBO-LLIN and LLML would be the most effective in reducing MIP. This study provides a new pathway for informing the optimal integrated malaria vector interventions, and the new strategy can be tested in field trials.

Background Malaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya. Methods The study was carried out in 2018–2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area. Results Anopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8–84% to 83.3–78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1–16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection. Conclusion Anopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides. Graphic abstract

Plasmodium falciparum parasites have evolved genetic adaptations to overcome immune responses mounted by diverse Anopheles vectors hindering malaria control efforts. Plasmodium falciparum surface protein Pfs47 is critical in the parasite’s survival by manipulating the vector’s immune system hence a promising target for blocking transmission in the mosquito. This study aimed to examine the genetic diversity, haplotype distribution, and population structure of Pfs47 and its implications on malaria infections in endemic lowlands in Western Kenya. Cross-sectional mass blood screening was conducted in malaria endemic regions in the lowlands of Western Kenya: Homa Bay, Kombewa, and Chulaimbo. Dried blood spots and slide smears were simultaneously collected in 2018 and 2019. DNA was extracted using Chelex method from microscopic Plasmodium falciparum positive samples and used to genotype Pfs47 using polymerase chain reaction (PCR) and DNA sequencing. Thirteen observed haplotypes of the Pfs47 gene were circulating in Western Kenya. Population-wise, haplotype diversity ranged from 0.69 to 0.77 and the nucleotide diversity 0.10 to 0.12 across all sites. All the study sites displayed negative Tajima’s D values although not significant. However, the negative and significant Fu’s Fs statistical values were observed across all the study sites, suggesting population expansion or positive selection. Overall genetic differentiation index was not significant (FST = -0.00891, P > 0.05) among parasite populations. All Nm values revealed a considerable gene flow in these populations. These results could have important implications for the persistence of high levels of malaria transmission and should be considered when designing potential targeted control interventions.

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.

A thorough understanding of malaria vector species composition and their bionomic characteristics is crucial to devise effective and efficient vector control interventions to reduce malaria transmission. It has been well documented in Africa that malaria interventions in the past decade have resulted in major changes in species composition from endophilic Anopheles gambiae to exophilic An. arabiensis. However, the role of cryptic rare mosquito species in malaria transmission is not well known. This study examined the species composition and distribution, with a particular focus on malaria transmission potential of novel, uncharacterized Anopheles cryptic species in western Kenya. Phylogenetic analysis based on ITS2 and COX1 genes revealed 21 Anopheles mosquito species, including two previously unreported novel species. Unusually high rates of Plasmodium sporozoite infections were detected in An. funestus, An. gambiae and eight cryptic rare species. Plasmodium falciparum, P. malariae and P. ovale sporozoite infections were identified with large proportion of mixed species infections in these vectors. This study, for the first time, reports extensive new Anopheles cryptic species involved in the malaria transmission in western Kenya. These findings underscore the importance of non-common Anopheles species in malaria transmission and the need to target them in routine vector control and surveillance efforts.

Background Mosquitoes are vectors of many tropical diseases. Understanding the ecology of local mosquito vectors, such as species composition, distributions, population dynamics, and species diversity is important for designing the optimal strategy to control the mosquito-borne diseases. Methods Entomological surveillance of adult mosquitoes was conducted in five sites representing different ecological settings across Hainan Island from January to December of 2018 using BG Sentinel (BGS) traps and Centers for Disease Prevention and Control (CDC) light traps. In each site, we selected three areas representing urban, suburban and rural settings. Eighteen trap-days were sampled in each setting at each site, and CDC light traps and BGS traps were setup simultaneously. Mosquito species composition, distribution, population dynamics, and species diversity were analyzed. Mosquito densities were compared between different study sites and between different settings. Results Nine species of mosquitoes belonging to four genera were identified. Culex quinquefasciatus (80.8%), Armigeres subalbatus (13.0%) and Anopheles sinensis (3.1%) were the top three species collected by CDC light traps; Cx. quinquefasciatus (91.9%), Ae. albopictus (5.1%), and Ar. subalbatus (2.8%) were the top three species collected by BGS traps. Predominant species varied among study sites. The population dynamics of Ae. albopictus, An. sinensis and Cx. quinquefasciatus showed clear seasonal variation regardless of study sites with a varied peak season for different species. Mosquito abundance of all species showed significant differences among different study sites and among urban, suburban and rural areas. Danzhou had the highest mosquito biodiversity, with an α, β, and Gini-Simpson biodiversity index of 8, 1.13 and 0.42, respectively. BGS traps captured Aedes mosquito at a higher efficiency than CDC light traps, whereas CDC light traps captured significantly more Anopheles and Armigeres mosquitoes than BGS traps. Conclusions Mosquitoes were abundant on Hainan Island with clear seasonality and spatial heterogeneity. Population density, species composition, distribution, and species diversity were strongly affected by the natural environment. Different tools are required for the surveillance of different mosquito species.

Background In the past two decades, the massive scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has led to significant reductions in malaria mortality and morbidity. Nonetheless, the malaria burden remains high, and a dozen countries in Africa show a trend of increasing malaria incidence over the past several years. This underscores the need to improve the effectiveness of interventions by optimizing first-line intervention tools and integrating newly approved products into control programs. Because transmission settings and vector ecologies vary from place to place, malaria interventions should be adapted and readapted over time in response to evolving malaria risks. An adaptive approach based on local malaria epidemiology and vector ecology may lead to significant reductions in malaria incidence and transmission risk. Methods/design This study will use a longitudinal block-cluster sequential multiple assignment randomized trial (SMART) design with longitudinal outcome measures for a period of 3 years to develop an adaptive intervention for malaria control in western Kenya, the first adaptive trial for malaria control. The primary outcome is clinical malaria incidence rate. This will be a two-stage trial with 36 clusters for the initial trial. At the beginning of stage 1, all clusters will be randomized with equal probability to either LLIN, piperonyl butoxide-treated LLIN (PBO Nets), or LLIN + IRS by block randomization based on their respective malaria risks. Intervention effectiveness will be evaluated with 12 months of follow-up monitoring. At the end of the 12-month follow-up, clusters will be assessed for “response” versus “non-response” to PBO Nets or LLIN + IRS based on the change in clinical malaria incidence rate and a pre-defined threshold value of cost-effectiveness set by the Ministry of Health. At the beginning of stage 2, if an intervention was effective in stage 1, then the intervention will be continued. Non-responders to stage 1 PBO Net treatment will be randomized equally to either PBO Nets + LSM (larval source management) or an intervention determined by an enhanced reinforcement learning method. Similarly, non-responders to stage 1 LLIN + IRS treatment will be randomized equally to either LLIN + IRS + LSM or PBO Nets + IRS. There will be an 18-month evaluation follow-up period for stage 2 interventions. We will monitor indoor and outdoor vector abundance using light traps. Clinical malaria will be monitored through active case surveillance. Cost-effectiveness of the interventions will be assessed using Q-learning. Discussion This novel adaptive intervention strategy will optimize existing malaria vector control tools while allowing for the integration of new control products and approaches in the future to find the most cost-effective malaria control strategies in different settings. Given the urgent global need for optimization of malaria control tools, this study can have far-reaching implications for malaria control and elimination. Trial registration US National Institutes of Health, study ID NCT04182126 . Registered on 26 November 2019.

Anopheles sinensis is a major malaria vector in Southeast Asia. Resistance to pyrethroid insecticides in this species has impeded malaria control in the region. Previous studies found that An. sinensis populations from Yunnan Province, China were highly resistant to deltamethrin and did not carry mutations in the voltage-gated sodium channel gene that cause knockdown resistance. In this study, we tested the hypothesis that other genomic variants are associated with the resistance phenotype. Using paired-end whole genome sequencing (DNA-seq), we generated 108 Gb of DNA sequence from deltamethrin -resistant and -susceptible mosquito pools with an average coverage of 83.3× depth. Using a stringent filtering method, we identified a total of 916,926 single nucleotide variants (SNVs), including 32,240 non-synonymous mutations. A total of 958 SNVs differed significantly in allele frequency between deltamethrin -resistant and -susceptible mosquitoes. Of these, 43 SNVs were present within 37 genes that code for immunity, detoxification, cuticular, and odorant proteins. A subset of 12 SNVs were randomly selected for genotyping of individual mosquitoes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and showed consistent allele frequencies with the pooled DNA-seq derived allele frequencies. In addition, copy number variations (CNVs) were detected in 56 genes, including 33 that contained amplification alleles and 23 that contained deletion alleles in resistant mosquitoes compared to susceptible mosquitoes. The genomic variants described here provide a useful resource for future studies on the genetic mechanism of insecticide resistance in this important malaria vector species.

Rapidly increasing pyrethroid insecticide resistance and changes in vector biting and resting behavior pose serious challenges in malaria control. Mosquito repellents, especially spatial repellents, have received much attention from industry. Many of these repellents contain the same or similar chemicals as those used in insecticides. Does resistance to insecticides affect the efficacy of spatial repellents? We attempted to simulate interactions between mosquitoes and repellents using a machine learning method, the self-propelled particle (SPP) model, which we modified to include attractiveness/repellency effects. We simulated a random walk scenario and scenarios with insecticide susceptible/resistant mosquitoes against repellent alone and against repellent plus attractant (to mimic a human host). We assumed attractant odors and repellent chemicals diffused randomly and omnidirectionally, and that mosquitoes were confined in a cubic cage. We modified the velocity and direction components of SPP using attraction/repulsion rates and concentrations. Simulation results indicated that without attractant/repellent, mosquitoes would fly anywhere in the cage at random. With attractant, mosquitoes might or might not fly toward the source (i.e., the human), depending on the simulation time (and thus the attractant concentration distribution). Eventually, however, all mosquitoes were attracted to the source of the odor. With repellent, results depended on the amount of chemical used and the level of mosquito insecticide resistance. All insecticide-susceptible mosquitoes eventually moved to the corner of the cage farthest from the repellent release point. Surprisingly, a high proportion of highly resistant mosquitoes might reach the attractant release point (the human) earlier in the simulation when repellent was present compare to no repellent was present. At fixed concentration, a high proportion of mosquitoes could be able to reach the host when the relative repellency efficacy (compare to attractant efficacy) was 1. This result implies that repellent may not be sufficient against highly physiologically insecticide resistant mosquitoes, since very high concentrations of repellent are neither practically feasible nor cost-effective.

Author(s): Machani, Maxwell G; Ochomo, Eric; Amimo, Fred; Kosgei, Jackline; Munga, Stephen; Zhou, Guofa; Githeko, Andrew K; Yan, Guiyun; Afrane, Yaw A | Abstract: BackgroundUnderstanding the interactions between increased insecticide resistance and resting behaviour patterns of malaria mosquitoes is important for planning of adequate vector control. This study was designed to investigate the resting behavior, host preference and rates of Plasmodium falciparum infection in relation to insecticide resistance of malaria vectors in different ecologies of western Kenya.MethodsAnopheles mosquito collections were carried out during the dry and rainy seasons in Kisian (lowland site) and Bungoma (highland site), both in western Kenya using pyrethrum spray catches (PSC), mechanical aspiration (Prokopack) for indoor collections, clay pots, pit shelter and Prokopack for outdoor collections. WHO tube bioassay was used to determine levels of phenotypic resistance of indoor and outdoor collected mosquitoes to deltamethrin. PCR-based molecular diagnostics were used for mosquito speciation, genotype for knockdown resistance mutations (1014S and 1014F) and to determine specific host blood meal origins. Enzyme-linked Immunosorbent Assay (ELISA) was used to determine mosquito sporozoite infections.ResultsAnopheles gambiae s.l. was the most predominant species (75%, n = 2706) followed by An. funestus s.l. (25%, n = 860). An. gambiae s.s hereafter (An. gambiae) accounted for 91% (95% CI: 89-93) and An. arabiensis 8% (95% CI: 6-9) in Bungoma, while in Kisian, An. arabiensis composition was 60% (95% CI: 55-66) and An. gambiae 39% (95% CI: 34-44). The resting densities of An. gambiae s.l and An. funestus were higher indoors than outdoor in both sites (An. gambiae s.l; F1, 655 = 41.928, p l 0.0001, An. funestus; F1, 655 = 36.555, p l 0.0001). The mortality rate for indoor and outdoor resting An. gambiae s.l F1 progeny was 37% (95% CI: 34-39) vs 67% (95% CI: 62-69) respectively in Bungoma. In Kisian, the mortality rate was 67% (95% CI: 61-73) vs 76% (95% CI: 71-80) respectively. The mortality rate for F1 progeny of An. funestus resting indoors in Bungoma was 32% (95% CI: 28-35). The 1014S mutation was only detected in indoor resitng An. arabiensis. Similarly, the 1014F mutation was present only in indoor resting An. gambiae. The sporozoite rates were highest in An. funestus followed by An. gambiae, and An. arabiensis resting indoors at 11% (34/311), 8% (47/618) and 4% (1/27) respectively in Bungoma. Overall, in Bungoma, the sporozoite rate for indoor resting mosquitoes was 9% (82/956) and 4% (8/190) for outdoors. In Kisian, the sporozoite rate was 1% (1/112) for indoor resting An. gambiae. None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections (n = 73).ConclusionThe study reports high indoor resting densities of An. gambiae and An. funestus, insecticide resistance, and persistence of malaria transmission indoors regardless of the use of long-lasting insecticidal nets (LLINs). These findings underline the difficulties of controlling malaria vectors resting and biting indoors using the current interventions. Supplemental vector control tools and implementation of sustainable insecticide resistance management strategies are needed in western Kenya.

BackgroundUnderstanding the interactions between increased insecticide resistance in field malaria vector populations and the subsequent resting behaviour patterns is important for planning adequate vector control measures in a specific context and sustaining the current vector interventions. The aim of this study was to investigate the resting behavior, host preference and infection with Plasmodium falciparum sporozoites by malaria vectors in different ecological settings of western Kenya with different levels of insecticide resistance.MethodsIndoor and outdoor resting Anopheline mosquitoes were sampled during the dry and rainy seasons in Kisian (lowland site) and Bungoma (highland site), both in western Kenya. WHO tube bioassay was used to determine levels of phenotypic resistance of first generation offspring (F1 progeny) of malaria vectors resting indoors and outdoors to deltamethrin. PCR-based molecular diagnostics were used for mosquito speciation, genotype for resistance mutations and to determine specific host blood meal origins. Enzyme-linked Immunosorbent Assay (ELISA) was used to determine mosquito sporozoite infections.ResultsOverall, 3,566 female Anopheles mosquitoes were collected with Anopheles gambiae s.l [In Bungoma, An. gambiae s.s (90.9%), An arabiensis (7.6%) and in Kisian, An. gambiae s.s (38.9%), An. arabiensis (60.2%)] being the most abundant species (74.7%) followed by An. funestus s.l (25.3%). The majority of An. gambiae s.l (85.4 and 58%) and An. funestus (96.6 and 91.1%) were caught resting indoors in Bungoma and Kisian respectively.Vgsc-1014S was observed at a slightly higher frequency in An. gambiae s.s hereafter(An. gambiae) resting indoor than outdoor (89.7 vs 84.6% and 71.5 vs 61.1%) in Bungoma and Kisian respectively. For An. arabiensis, Vgsc-1014S was 18.2% indoor and outdoor (17.9%) in Kisian. In Bungoma, the Vgsc-1014S was only detected in An. arabiensis resting indoors with a frequency of 10%. The Vgsc-1014F mutation was only present in An. gambiae resting indoors from both sites, but at very low frequencies in Kisian compared to Bungoma (0.8 and 9.2% respectively. In Bungoma, the sporozoite rates for An. funestus, An. gambiae, and An. arabiensis resting indoors were 10.9, 7.6 and 3.4 % respectively. For outdoor resting, An. gambiae and An. arabiensis in Bungoma, the sporozoite rates were 4.7 and 2.9 % respectively.Overall, in Bungoma, the sporozoite rate for indoor resting mosquitoes was 8.6% and 4.2% for outdoors. In Kisian the sporozoite rate was 0.9% for indoor resting An. gambiae. None of the outdoor collected mosquitoes in Kisian tested positive for sporozoite infections.ConclusionThe study reports high densities of insecticide-resistant An. gambiae and An. funestus resting indoors and the persistence of malaria transmission indoors with high entomological inoculation rates (EIR) regardless of the use of Long-lasting insecticidal nets (LLINs). These findings underline the difficulties of controlling malaria vectors resting and biting indoors using the current interventions. Supplemental vector control tools and implementation of sustainable insecticide resistance management strategies are needed in western Kenya.

Background Physiological characteristics (age and blood feeding status) of malaria vectors can influence their susceptibility to the current vector control tools that target their feeding and resting behaviour. To ensure the sustainability of the current and future vector control tools an understanding of how physiological characteristics may contribute to insecticide tolerance in the field is fundamental for shaping resistance management strategies and vector control tools. The aim of this study was to determine whether blood meal and mosquito age affect pyrethroid tolerance in field-collected Anopheles gambiae from western Kenya. Methods Wild mosquito larvae were reared to adulthood alongside the pyrethroid-susceptible Kisumu strain. Adult females from the two populations were monitored for deltamethrin resistance when they were young at 2–5 days old and older 14–16 days old and whether fed or unfed for each age group. Metabolic assays were also performed to determine the level of detoxification enzymes. Mosquito specimens were further identified to species level using the polymerase chain reaction (PCR) method. Results Anopheles gambiae sensu stricto was the predominant species comprising 96% of specimens and 2.75% Anopheles arabiensis. Bioassay results showed reduced pyrethroid induced mortality with younger mosquitoes compared to older ones (mortality rates 83% vs. 98%), independently of their feeding status. Reduced mortality was recorded with younger females of which were fed compared to their unfed counterparts of the same age with a mortality rate of 35.5% vs. 83%. Older blood-fed females showed reduced susceptibility after exposure when compared to unfed females of the same age (mortality rates 86% vs. 98%). For the Kisumu susceptible population, mortality was straight 100% regardless of age and blood feeding status. Blood feeding status and mosquito age had an effect on enzyme levels in both populations, with blood fed individuals showing higher enzyme elevations compared to their unfed counterparts (P

Dengue is one of the most serious mosquito-borne infectious diseases in the world. Aedes albopictus is the most invasive mosquito and one of the primary vectors of dengue. Vector control using insecticides is the only viable strategy to prevent dengue virus transmission. In Guangzhou, after the 2014 pandemic, massive insecticides have been implemented. Massive insecticide use may lead to the development of resistance, but few reports are available on the status of insecticide resistance in Guangzhou after 2014. In this study, Ae. albopictus were collected from four districts with varied dengue virus transmission intensity in Guangzhou from 2015 to 2017. Adult Ae. albopictus insecticide susceptibility to deltamethrin (0.03%), permethrin(0.25%), DDT(4%), malathion (0.8%) and bendiocarb (0.1%) was determined by the standard WHO tube test, and larval resistance bioassays were conducted using temephos, Bacillus thuringiensis israelensis (Bti), pyriproxyfen (PPF) and hexaflumuron. Mutations at the voltage-gated sodium channel (VGSC) gene and acetylcholinesterase (AChE) gene were analyzed. The effect of cytochrome P450s on the resistance of Ae. albopictus to deltamethrin was tested using the synergistic agent piperonyl butoxide (PBO). The results showed that Ae. albopictus populations have rapidly developed very high resistances to multiple commonly used insecticides at all study areas except malathion, Bti and hexaflumuron. We found 1534 codon mutations in the VGSC gene that were significantly correlated with the resistance to pyrethroids and DDT, and 11 synonymous mutations were also found in the gene. The resistance to deltamethrin can be significantly reduced by PBO but may generated cross-resistance to PPF. Fast emerging resistance in Ae. albopictus may affect mosquito management and threaten the prevention and control of dengue, similar to the resistance in Anopheles mosquitoes has prevented the elimination of malaria and call for timely and guided insecticide management., Author summary Guangzhou is the most epidemic area of dengue in China. Massive insecticides have been used to control the vector mosquito Ae. albopictus, as no specific vaccines are available for dengue. Regular monitoring of insecticide susceptibility is essential for insecticide resistance management. In this study, the insecticide resistances of Ae. albopictus in Guangzhou were comparatively analyzed from 2015 to 2017. The results displayed that Ae. albopictus had rapidly generated high resistance to the most commonly used adult insecticide pyrethroid (deltamethrin and permethrin) and larvicide organophosphate (temephos). The combination of malathion for adult mosquitoes and Bti or hexaflumuron for larvae might be a better choice for vector control. Resistance to deltamethrin can be significantly reduced by PBO but may generated cross-resistance to PPF. F1534S and F1534L mutations in the VGSC gene were significantly correlated with resistance to pyrethroids. This study indicated that the insecticide resistances had been generated in Ae. albopictus in Guangzhou which was correlated with the dengue epidemic responses.

Background Surveillance of outdoor resting malaria vector populations is crucial to monitor possible changes in vector resting and feeding behaviour following the widespread use of indoor-based vector control interventions. However, it is seldom included in the routine vector surveillance system in Africa due to lack of well standardized and efficient traps. This study was conducted to evaluate the performance of sticky pots for outdoor resting malaria vector surveillance in western Kenya. Methods Mosquito collections were conducted from September 2015 to April 2016 in Ahero and Iguhu sites, western Kenya using sticky pots, pit shelters, clay pots, exit traps, Prokopack aspirator and CDC light traps (outdoor and indoor). Species within Anopheles gambiae (s.l.) were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was used to determine blood meal sources of malaria vectors. Results A total of 23,772 mosquitoes were collected, of which 13,054 were female anophelines comprising An. gambiae (s.l.) (72.9%), An. funestus (13.2%), An. coustani (8.0%) and An. pharoensis (5.9%). Based on PCR assay (n = 672), 98.6% An. arabiensis and 1.4% An. gambiae (s.s.) constituted An. gambiae (s.l.) in Ahero, while this was 87.2% An. gambiae (s.s.) and 12.8% An. arabiensis in Iguhu. The sticky pots and pit shelters showed similar performance with regard to the relative abundance and host blood meal indices of An. gambiae (s.l.) and An. funestus. In terms of density per trap, a pit shelter caught on average 4.02 (95% CI: 3.06–5.27) times as many An. gambiae (s.l.) as a sticky pot, while a sticky pot captured 1.60 (95% CI: 1.19–2.12) times as many An. gambiae (s.l.) as a clay pot. Exit traps yielded a significantly lower number of An. gambiae (s.l.) than all other traps in Ahero, but a higher number of An. gambiae (s.l.) compared to the other outdoor traps in Iguhu. Indoor CDC light traps captured a significantly higher number of An. funestus than other traps. Conclusions Sticky pots could be a useful and complementary tool for outdoor resting malaria vector surveillance, in settings where using pit shelters is not feasible and less productive. The lower vector density in the sticky pots compared to pit shelters suggests that batches of sticky pots (i.e. four per compound) need to be deployed in order to make a direct comparison. This study also highlighted the need to concurrently undertake indoor and outdoor vector surveillance to better understand residual malaria transmission.

Background Chemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands. Methods Three sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers’ recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months. Results Overall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction. Conclusions Both LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application.

Background Identifying asymptomatic reservoirs of malaria parasites using index cases as entry points into the community is potentially a cost-effective way towards achieving malaria elimination. Methods Within 1 year, 1430 confirmed malaria cases were identified in Marani hospital, western Kenya. Fifty cases were followed up, and 108 index case household members and 612 neighbours within a 100 m radius were screened. As controls, samples were collected from 510 individuals matched with index cases and located at a distance of ≥ 500 m from them. Infections were diagnosed by microscopy and PCR while simultaneously collecting malaria vectors indoor using pyrethrum spray catches. Results In the index case and neighbour households, the prevalence of infection was approximately twice as high as in control households (by PCR: index cases households: 28.9%, neighbours: 25.3%, matched controls: 12.9%). In index case households, the indoor vector density (Anopheles gambiae and Anopheles funestus) was higher (0.46 female/house/night) than in neighbouring (0.31 f/h/n) and control houses (0.29 f/h/n). Conclusions Screening index case households and neighbours approximately doubles the chance to detect asymptomatic infections compared to randomly selected households. However, even if all cases were followed up, only a small proportion (˂ 10%) of the asymptomatic reservoir in the population would have been identified. Control programmes need to weigh the increased chance to find cases around index cases vs. the logistical challenges to target this subgroup within the population.

The widespread use of indoor-based malaria vector control interventions has been shown to alter the behaviour of vectors in Africa. There is an increasing concern that such changes could sustain residual transmission. This study was conducted to assess vector species composition, feeding behaviour and their contribution to indoor and outdoor malaria transmission in western Kenya. Anopheles mosquito collections were carried out from September 2015 to April 2016 in Ahero and Iguhu sites, western Kenya using CDC light traps (indoor and outdoor), pyrethrum spray catches (PSCs) (indoor) and pit shelters (outdoor). Species within Anopheles gambiae s.l. and Anopheles funestus s.l. were identified using polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was used to determine mosquito blood meal sources and sporozoite infections. A total of 10,864 female Anopheles mosquitoes comprising An. gambiae s.l. (71.4%), An. funestus s.l. (12.3%), Anopheles coustani (9.2%) and Anopheles pharoensis (7.1%) were collected. The majority (61.8%) of the anopheline mosquitoes were collected outdoors. PCR result (n = 581) revealed that 98.9% An. arabiensis and 1.1% An. gambiae s.s. constituted An. gambiae s.l. in Ahero while this was 87% An. gambiae s.s. and 13% An. arabiensis in Iguhu. Of the 108 An. funestus s.l. analysed by PCR, 98.1% belonged to An. funestus s.s. and 1.9% to Anopheles leesoni. The human blood index (HBI) and bovine blood index (BBI) of An. arabiensis was 2.5 and 73.1%, respectively. Anopheles gambiae s.s. had HBI and BBI of 50 and 28%, respectively. The HBI and BBI of An. funestus was 60 and 22.3%, respectively. Forage ratio estimate revealed that An. arabiensis preferred to feed on cattle, An. gambiae s.s. showed preference for both human and cattle, while An. funestus preferred human over other hosts. In Ahero, the sporozoite rates for An. arabiensis and An. funestus were 0.16 and 1.8%, respectively, whereas in Iguhu, the sporozoite rates for An. gambiae s.s. and An. funestus were 2.3 and 2.4%, respectively. In Ahero, the estimated indoor and outdoor entomological inoculation rate (EIR) was 108.6 infective bites/person/year (79.0 from An. funestus and 29.6 from An. arabiensis) and 43.5 infective bites/person/year (27.9 from An. arabiensis and 15.6 from An. funestus), respectively. In Iguhu, the estimated indoor and outdoor EIR was 24.5 infective bites/person/year (18.8 from An. gambiae s.s. and 5.7 from An. funestus) and 5.5 infective bites/person/year (all from An. gambiae s.s.), respectively. Anopheles gambiae s.s. showed an increasing tendency to feed on cattle. Anopheles arabiensis was highly zoophagic, whereas An. funestus showed anthropophagic behaviour. While the majority of malaria transmission occurred indoor, the magnitude of outdoor transmission was considerably high. Additional control tools that complement the existing interventions are required to control residual transmission.

We conducted standard insecticide susceptibility testing across western Kenya and found that the Anopheles gambiae mosquito has acquired high resistance to pyrethroids and DDT, patchy resistance to carbamates, but no resistance to organophosphates. Use of non-pyrethroid-based vector control tools may be preferable for malaria prevention in this region.

BackgroundOutdoor malaria transmission is becoming an increasingly important problem in malaria control in Africa. Larval control is a promising intervention as it can target both indoor and outdoor biting mosquitoes. However, the currently available biolarvicide formulations have a short effective duration, and consequently larval control incurs a high operational expense due to the requirement for frequent re-treatment of larval habitats. Formulations of biolarvicides with long-lasting effects is highly desired. A recently developed FourStar® slow-release briquet formulation of Bacillus thuringiensis israelensis and Bacillus sphaericus was evaluated to test its efficacy on malaria vectors.MethodsThe study evaluated FourStar™ briquets 180-days formulation under semi-natural and natural conditions to test their efficacy in reducing the mosquito population in western Kenya. The semi-natural habitats used the formulation dissolved in rainwater with appropriate concentrations, and second-instar larvae of Anopheles gambiae were introduced and the number of surviving larvae and pupae produced was recorded daily as the outcome. The briquets formulation was then tested in natural habitats for efficacy on pupal productivity reduction in highland and lowland sites in western Kenya. The formulation was finally tested for efficacy in reducing adult mosquito populations in randomized clusters in western Kenya highland.ResultsIn semi-natural conditions, the FourStar™ briquets 180-days formulation completely inhibited mosquito pupal production in the first 3months, and then reduced pupal productivity by 87-98% (P&nbsp

Selection pressure caused by long-term intensive use of insecticides is the key driving force in resistance development. Additional parameters such as environmental conditions may affect both the mosquito response to insecticides and the selection of resistance mechanisms. In this context, we analyzed the environmental determinants of kdr prevalence in Anopheles sinensis across China. We collected kdr frequency from 48 sites across central and southern China, together with key environmental factors including long-term climatic data, topographic features, main crops, and land cover types. Trend surface analysis found that the distribution of kdr frequency can be partitioned into three regions, namely central China (kdr frequency >80%), western China (kdr frequency varies from 0% to 60%), and southern China (kdr frequency

Background Many developing countries are experiencing rapid ecological changes such as deforestation and shifting agricultural practices. These environmental changes may have an important consequence on malaria due to their impact on vector survival and reproduction. Despite intensive deforestation and malaria transmission in the China-Myanmar border area, the impact of deforestation on malaria vectors in the border area is unknown. Methods We conducted life table studies on Anopheles minimus larvae to determine the pupation rate and development time in microcosms under deforested, banana plantation, and forested environments. Results The pupation rate of An. minimus was 3.8 % in the forested environment. It was significantly increased to 12.5 % in banana plantations and to 52.5 % in the deforested area. Deforestation reduced larval-to-pupal development time by 1.9–3.3 days. Food supplementation to aquatic habitats in forested environments and banana plantations significantly increased larval survival rate to a similar level as in the deforested environment. Conclusion Deforestation enhanced the survival and development of An. minimus larvae, a major malaria vector in the China-Myanmar border area. Experimental determination of the life table parameters on mosquito larvae under a variety of environmental conditions is valuable to model malaria transmission dynamics and impact by climate and environmental changes. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1611-5) contains supplementary material, which is available to authorized users.

Background Anopheles sinensis is one of the most abundant vectors of malaria and other diseases in Asia. Vector control through the use of insecticides is the front line control method of vector-borne diseases. Pyrethroids are the most commonly used insecticides due to their low toxicity to vertebrates and low repellency. However, the extensive use of insecticides has imposed strong selection pressure on mosquito populations for resistance. High levels of resistance to pyrethroid insecticides and various mutations and haplotypes in the para sodium channel gene that confers knockdown resistance (kdr) have been detected in An. sinensis. Despite the importance of kdr mutations in pyrethroid resistance, the evolutionary origin of the kdr mutations is unknown. This study aims to examine the evolutionary genetics of kdr mutations in relation to spatial population genetic structure of An. sinensis. Methods Adults or larvae of Anopheles sinensis were collected from various geographic locations in China. DNA was extracted from individual mosquitoes. PCR amplification and DNA sequencing of the para-type sodium channel gene were conducted to analyse kdr allele frequency distribution, kdr codon upstream and downstream intron polymorphism, population genetic diversity and kdr codon evolution. The mitochondrial cytochrome c oxidase COI and COII genes were amplified and sequenced to examine population variations, genetic differentiation, spatial population structure, population expansion and gene flow patterns. Results Three non-synonymous mutations (L1014F, L1014C, and L1014S) were detected at the kdr codon L1014 of para-type sodium channel gene. A patchy distribution of kdr mutation allele frequencies from southern to central China was found. Near fixation of kdr mutation was detected in populations from central China, but no kdr mutations were found in populations from southwestern China. More than eight independent mutation events were detected in the three kdr alleles, and at least one of them evolved multiple times subsequent to their first divergence. Based on sequence analysis of the mitochondrial COI and COII genes, significant and large genetic differentiation was detected between populations from southwestern China and central China. The patchy distribution of kdr mutation frequencies is likely a consequence of geographic isolation in the mosquito populations and the long-term insecticide selection. Conclusion Our results indicate multiple origins of the kdr insecticide-resistant alleles in An. sinensis from southern and central China. Local selection related to intense and prolonged use of insecticide for agricultural purposes, as well as frequent migrations among populations are likely the explanations for the patchy distribution of kdr mutations in China. On the contrary, the lack of kdr mutations in Yunnan and Sichuan is likely a consequence of genetic isolation and absence of strong selection pressure. The present study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1513-6) contains supplementary material, which is available to authorized users.

Background The extensive use of pyrethroids for control of malaria vectors, driven by their cost, efficacy and safety, has led to widespread resistance. To favor their sustainable use, the World Health Organization (WHO) formulated an insecticide resistance management plan, which includes the identification of the mechanisms of resistance and resistance surveillance. Recognized physiological mechanisms of resistance include target site mutations in the para voltage-gated sodium channel, metabolic detoxification and penetration resistance. Such understanding of resistance mechanisms has allowed the development of resistance monitoring tools, including genotyping of the kdr mutation L1014F/S in the para gene. Methods The sequence-based technique RNA-seq was applied to study changes in the transcriptome of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from the Western Province of Kenya. The resulting gene expression profiles were compared to data in the most recent literature to derive a list of candidate resistance genes. RNA-seq data were analyzed also to identify sequence polymorphisms linked to resistance. Results A total of five candidate-resistance genes (AGAP04177, AGAP004572, AGAP008840, AGAP007530 and AGAP013036) were identified with altered expression between resistant and susceptible mosquitoes from West and East Africa. A change from G to C at position 36043997 of chromosome 3R resulting in A101G of the sulfotransferase gene AGAP009551 was significantly associated with the resistance phenotype (odds ratio: 5.10). The kdr L1014S mutation was detected at similar frequencies in both phenotypically resistant and susceptible mosquitoes, suggesting it is no longer fully predictive of the resistant phenotype. Conclusions Overall, these results support the conclusion that resistance to pyrethroids is a complex and evolving phenotype, dependent on multiple gene functions including, but not limited to, metabolic detoxification. Functional convergence among metabolic detoxification genes may exist, with the role of each gene being modulated by the life history and selection pressure on mosquito populations. As a consequence, biochemical assays that quantify overall enzyme activity may be a more suitable method for predicting metabolic resistance than gene-based assays. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1083-z) contains supplementary material, which is available to authorized users.

Background Understanding the ecology of malaria vectors such as species composition and population dynamics is essential for developing cost-effective strategies to control mosquito vector populations. Methods Adult mosquitoes (n = 79,567) were collected in five villages along the China-Myanmar border from April 2012 to September 2014 using the CDC light trap without bait method. Mosquito community structure, Anopheles species composition and diversity were analyzed. Results Twenty species of Anopheles mosquitoes were identified, with An. minimus s.l. accounting for 85 % of the total collections. Mosquito densities varied from 0.05 females per trap per night (f/t/n) to 3.00 f/t/n, with strong seasonality in all sites and densities peaked from June to August. An. minimus s.l. was predominant (accounting for 54–91 % of total captures) in four villages, An. maculatus s.l. was predominant (71 %) in the high elevation village of Dao Nong, and An. culicifacies accounted for 15 % of total captures in the peri-urban area of Simsa Lawk. All 20 species have been captured in the Mung Seng Yang village, 18 and 15 species in Ja Htu Kawng and Na Bang respectively, and nine species in both Simsa Lawk and Dao Nong. Species richness peaked from April to August. Species diversity, species dominance index, and species evenness fluctuated substantially from time to time with no clear seasonality, and varied greatly amongst villages. Conclusions Mosquitoes were abundant in the China-Myanmar bordering agricultural area with clear seasonality. Species composition and density were strongly affected by natural environments. The targeted intervention strategy should be developed and implemented so as to achieve cost-effectiveness for malaria control and elimination along the border areas. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1057-1) contains supplementary material, which is available to authorized users.

Pyrethroid-treated bed-nets and indoor spray are important components of malaria control strategies in Kenya. Information on resistance to pyrethroid insecticides in Anopheles gambiae and An. arabiensis populations is essential to the selection of appropriate insecticides and the management of insecticide resistance. Monooxygenase activity and knockdown resistance (kdr) allele frequency are biochemical and molecular indicators of mosquito resistance to pyrethroids. This study determined baseline information on monooxygenase activity and kdr allele frequency in anopheline mosquitoes in the western region, the Great Rift Valley-central region, and the coastal region of Kenya. In total, 1,990 field-collected individuals, representing 12 An. gambiae and 22 An. arabiensis populations were analyzed. We found significant among-population variation in monooxygenase activity in An. gambiae and An. arabiensis and substantial variability among individuals within populations. Nine of 12 An. gambiae populations exhibited significantly higher average monooxygenase activity than the susceptible Kisumu reference strain. The kdr alleles (L1014S) were detected in three An. gambiae populations, and one An. arabiensis population in western Kenya, but not in the Rift Valley-central region and the coastal Kenya region. All genotypes with the kdr alleles were heterozygous, and the conservative estimation of kdr allele frequency was below 1% in these four populations. Information on monooxygenase activity and kdr allele frequency reported in this study provided baseline data for monitoring insecticide resistance changes in Kenya during the era when large-scale insecticide-treated bed-net and indoor residual spray campaigns were being implemented.

Information on the spatial relationships between disease vectors and environmental factors is fundamental to vector-borne disease control. Although it is well known that mosquito abundance is associated with the amount of rainfall and thus the number of larval breeding sites, the spatial relationship between larval habitat availability and adult mosquito abundance is not clear. We investigated the impact of environmental heterogeneity and larval habitats on the spatial distribution of Anopheles gambiae s. s. and An. funestus adult mosquitoes, the most important malaria vectors in the highlands of western Kenya. Mosquito sampling was conducted in May, August, and November 2002, and February 2003. Geographic information system layers of larval habitats, land use type, human population distribution, house structure, and hydrologic schemes were overlaid with adult mosquito abundance. Correlography was used to determine the spatial autocorrelation in adult mosquito abundance among houses and the cross-correlation between adult mosquito abundance and environmental factors. Getis’ Gi*(d) index was used to define focal adult mosquito abundance clusters.We found a significant autocorrelation in the vector population and a significant cross-correlation between the vector population and larval habitat availability. The threshold distances of both autocorrelation and cross-correlation were significantly varied among seasons. Focal clustering analysis revealed that the adult vector population was concentrated along the Yala River Valley where most larval habitats were found. Regression analysis found that distance of a house to the Yala River, age of the house, elevation, house structure, and tree canopy coverage significantly affected adult mosquito abundance. Our results suggest that vector control targeted at malaria transmission hotspots and supplemented by larval control may be an effective approach for epidemic malaria control in the western Kenya highlands., American journal of tropical medicine and hygiene 77(1), pp.29-35:2007

© 2016 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In the past decade, developing countries have been experiencing rapid land use and land cover changes, including deforestation and cultivation of previously forested land. However, little is known about the impact of deforestation and land-use changes on the life history of malaria vectors and their effects on malaria transmission. This study examined the effects of deforestation and crop cultivation on the adult survivorship of major malaria mosquitoes, Anopheles sinensis and An. minimus in the China-Myanmar border region. We examined three conditions: indoor, forested, and banana plantation. Mean survival time of An. sinensis in banana plantation environment was significantly longer than those in forested environment, and mosquitoes exhibited the longest longevity in the indoor environment. This pattern held for both males and females, and also for An. minimus. To further test the effect of temperature on mosquito survival, we used two study sites with different elevation and ambient temperatures. Significantly higher survivorship of both species was found in sites with lower elevation and higher ambient temperature. Increased vector survival in the deforested area could have an important impact on malaria transmission in Southeast Asia. Understanding how deforestation impacts vector survivorship can help combat malaria transmission.

© 2015 Ototo et al. Background: Malaria is a great public health burden and Africa suffers the largest share of malaria-attributed deaths. Despite control efforts targeting indoor malaria transmission, such as insecticide-treated bed nets (ITNs) and deployment of indoor residual spraying, transmission of the parasite in western Kenya is still maintained. This study was carried out to determine the impact of ITNs on indoor vector densities and biting behaviour in western Kenya. Methods: Indoor collection of adult mosquitoes was done monthly in six study sites in western Kenya using pyrethrum spray collections from 2012 to 2014. The rotator trap collections were done in July-August in 2013 and May-June in 2014. Mosquitoes were collected every 2 h between 18.00 and 08.00 h. Human behaviour study was conducted via questionnaire surveys. Species within Anopheles gambiae complex was differentiated by PCR and sporozoite infectivity was determined by ELISA. Species distribution was determined and bed net coverage in the study sites was recorded. Results: During the study a total of 5,469 mosquito vectors were collected from both PSC and Rotator traps comprising 3,181 (58.2%) Anopheles gambiae and 2,288 (41.8%) Anopheles funestus. Compared to all the study sites, Rae had the highest density of An. gambiae with a mean of 1.2 (P < 0.001) while Kombewa had the highest density of An. funestus with a mean of 1.08 (P < 0.001). Marani had the lowest density of vectors with 0.06 An. gambiae and 0.17 An. funestus (P < 0.001). Among the 700 PCR confirmed An. gambiae s.l. individuals, An. gambiae s.s. accounted for 49% and An. arabiensis 51%. Over 50% of the study population stayed outdoors between 18.00 and 20.00 and 06.00 and 08.00 which was the time when highest densities of blood fed vectors were collected. Anopheles gambie s.s. was the main malaria parasite vector in the highland sites and An. arabiensis in the lowland sites. Bed net ownership in 2012 averaged 87% across the study sites. Conclusions: This study suggests that mass distribution of ITNs has had a significant impact on vector densities, species distribution and sporozoite rate. However, shift of biting time poses significant threats to the current malaria vector control strategies which heavily rely on indoor controls.

The reasons for the resurgence of malaria in the African highlands have been subject of debate. Because vector abundance is important for malaria transmission, gaining a better understanding of vector biology is a key to understanding the mechanisms of highland malaria. We studied vector productivity in relation to the highland environment and compared productivity between lowland and highland sites. We found lower vector productivity in the highland and in wetlands where the temperature was lower. Immature stage development time was significantly longer in the highland site. Development time was significantly shorter in aquatic habitats in cultivated areas than in wetlands, and survival rate was significantly higher in cultivated areas. Fecundity was significantly lower in the highland site. These findings suggest that changes in local temperature and land use contribute to an increase of malaria vectors in the highland., American journal of tropical medicine and hygiene, 75(3), 2006, pp. 448-453

We studied the oviposition site preference and egg hatchability of Anopheles gambiae Giles with water collected from farmlands, forests, and natural wetlands. Water types significantly affected oviposition preference. Mosquitoes deposited significantly more eggs in rainwater in both the dry and wet seasons than waters from forests and wetlands, suggesting that An. gambiae prefers water with few impurities for oviposition. In the dry season, An. gambiae females also deposited significantly more eggs in waters from farmlands than those from forests and natural wetlands, but these differences were not statistically significant during the wet season. In both indoor and natural conditions, egg mortality in natural wetland habitats was significantly higher than in farmland habitats. The average water temperature in natural wetland habitats was significantly lower than farmland habitats in the natural conditions, but it remained the same under indoor experimental conditions, suggesting that factors other than water temperature play an important role in egg hatchability. Together with the findings from previous studies on the effects of land cover on larval survivorship, our results support the hypothesis that variations in habitat conditions induced by different land cover types contribute to the heterogeneous spatial distribution of An. gambiae larvae in the western Kenya highland.

The distributions of anopheline larval habitats were aggregated in valley bottoms in Kenya in both the rainy and dry seasons, although the degree of aggregation was higher in the dry season than in the rainy season. Larvae of the Anopheles gambiae complex larvae were found more frequently in habitats in farmlands and pastures. However, An. funestus larvae were found more frequently in natural swamps and pastures. Canopy cover was the only variable significantly associated with the occurrence of the An. gambiae complex and An. funestus. The average canopy cover was significantly less in the habitats with the An. gambiae complex and An. funestus larvae than those without the anopheline larvae. Thus, land cover types and topographic features showed important effects on the distribution of anopheline larval habitats. These results suggest that clearing riparian forests would improve growing conditions of the An. gambiae complex and An. funestus larvae in Kenyan highlands.

Background Anopheles sinensis is a major malaria vector in China and other Southeast Asian countries, and it is becoming increasingly resistant to the insecticides used for agriculture, net impregnation, and indoor residual spray. Very limited genomic information on this species is available, which has hindered the development of new tools for resistance surveillance and vector control. We used the 454 GS FLX system and generated expressed sequence tag (EST) databases of various life stages of An. sinensis, and we determined the transcriptional differences between deltamethrin resistant and susceptible mosquitoes. Results The 454 GS FLX transcriptome sequencing yielded a total of 624,559 reads (average length of 290 bp) with the pooled An. sinensis mosquitoes across various development stages. The de novo assembly generated 33,411 contigs with average length of 493 bp. A total of 8,057 ESTs were generated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. A total of 2,131 ESTs were differentially expressed between deltamethrin resistant and susceptible mosquitoes collected from the same field site in Jiangsu, China. Among these differentially expressed ESTs, a total of 294 pathways were mapped to the KEGG database, with the predominant ESTs belonging to metabolic pathways. Furthermore, a total of 2,408 microsatellites and 15,496 single nucleotide polymorphisms (SNPs) were identified. Conclusions The annotated EST and transcriptome databases provide a valuable genomic resource for further genetic studies of this important malaria vector species. The differentially expressed ESTs associated with insecticide resistance identified in this study lay an important foundation for further functional analysis. The identified microsatellite and SNP markers will provide useful tools for future population genetic and comparative genomic analyses of malaria vectors. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-448) contains supplementary material, which is available to authorized users.

Anopheles sinensis is the most important vector of malaria in Southeast Asia, including China. Currently, the most effective measure to prevent malaria transmission relies on vector control through the use of insecticides, primarily pyrethroids. Extensive use of insecticides poses strong selection pressure on mosquito populations for resistance. Resistance to insecticides can arise due to mutations in the insecticide target site (target site resistance), which in the case of pyrethroids is the para-type sodium channel gene, and/or the catabolism of the insecticide by detoxification enzymes before it reaches its target (metabolic detoxification resistance). In this study, we examined deltamethrin resistance in An. sinensis from China and investigated the relative importance of target site versus metabolic detoxification mechanisms in resistance. A high frequency (>85%) of nonsynonymous mutations in the para gene was found in populations from central China, but not in populations from southern China. Metabolic detoxification as measured by the activity of monooxygenases and glutathione S-transferases (GSTs) was detected in populations from both central and southern China. Monooxygenase activity levels were significantly higher in the resistant than the susceptible mosquitoes, independently of their geographic origin. Stepwise multiple regression analyses in mosquito populations from central China found that both knockdown resistance (kdr) mutations and monooxygenase activity were significantly associated with deltamethrin resistance, with monooxygenase activity playing a stronger role. These results demonstrate the importance of metabolic detoxification in pyrethroid resistance in An. sinensis, and suggest that different mechanisms of resistance could evolve in geographically different populations. © 2013 Zhong et al.

Anopheline Larval Habitats Seasonality and Species Distribution: A Prerequisite for Effective Targeted Larval Habitats Control Programmes Eliningaya J. Kweka 1,2 * ¤ , Guofa Zhou 3 , Stephen Munga 1 , Ming-Chieh Lee 3 , Harrysone E. Atieli 1 , Mramba Nyindo 2 , Andrew K. Githeko 1 , Guiyun Yan 3 1 Climate and Human Health Research Unit, Kenya Medical Research Institute, Kisumu, Kenya, 2 Kilimanjaro Christian Medical University College, Tumaini University Makumira, Moshi, Tanzania, 3 Program in Public Health, College of Health Sciences, University of California Irvine, Irvine, California, United States of America Abstract Background: Larval control is of paramount importance in the reduction of malaria vector abundance and subsequent disease transmission reduction. Understanding larval habitat succession and its ecology in different land use managements and cropping systems can give an insight for effective larval source management practices. This study investigated larval habitat succession and ecological parameters which influence larval abundance in malaria epidemic prone areas of western Kenya. Methods and Findings: A total of 51 aquatic habitats positive for anopheline larvae were surveyed and visited once a week for a period of 85 weeks in succession. Habitats were selected and identified. Mosquito larval species, physico-chemical parameters, habitat size, grass cover, crop cycle and distance to nearest house were recorded. Polymerase chain reaction revealed that An. gambiae s.l was the most dominant vector species comprised of An.gambiae s.s (77.60%) and An.arabiensis (18.34%), the remaining 4.06% had no amplification by polymerase chain reaction. Physico-chemical parameters and habitat size significantly influenced abundance of An. gambiae s.s (P = 0.024) and An. arabiensis (P = 0.002) larvae. Further, larval species abundance was influenced by crop cycle (P#0.001), grass cover (P#0.001), while distance to nearest houses significantly influenced the abundance of mosquito species larvae (r = 0.920;P#0.001). The number of predator species influenced mosquito larval abundance in different habitat types. Crop weeding significantly influenced with the abundance of An.gambiae s.l (P#0.001) when preceded with fertilizer application. Significantly higher anopheline larval abundance was recorded in habitats in pasture compared to farmland (P = 0.002). When habitat stability and habitat types were considered, hoof print were the most productive followed by disused goldmines. Conclusion: These findings suggest that implementation of effective larval control programme should be targeted with larval habitats succession information when larval habitats are fewer and manageable. Crop cycles and distance from habitats to household should be considered as effective information in planning larval control. Citation: Kweka EJ, Zhou G, Munga S, Lee M-C, Atieli HE, et al. (2012) Anopheline Larval Habitats Seasonality and Species Distribution: A Prerequisite for Effective Targeted Larval Habitats Control Programmes. PLoS ONE 7(12): e52084. doi:10.1371/journal.pone.0052084 Editor: Julio Francisco Turrens, University of South Alabama, United States of America Received December 15, 2011; Accepted November 15, 2012; Published December 18, 2012 Copyright: s 2012 Kweka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work received financial support from American National Institute of Health (D43 TW01505 and R01 AI-50243 to GY). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pat.kweka@gmail.com ¤ Current address: Tropical Pesticides Research Institute, Division of Livestock and Human Diseases Vector Control, Mosquito Section, Arusha, Tanzania for settlement and pasture for grazing livestock. This creates more habitats which are exposed to more sunlight which in turn increases water temperature and shortens the developmental cycle of immature stages of malaria vectors [7,12,13,15,16]. The rise in temperature has increased developmental rate of parasites in adult mosquitoes [1,2]. The main malaria vectors in these highlands of western Kenya are An. gambiae s.s, An. arabiensis and An. funestus Malaria control in this region relies heavily on use of insecticides treated bed nets (ITNs), Indoor residual spray (IRS) and diagnosis and treatment of all active malaria cases [21,22,23,24]. In highlands of western Kenya, larval habitats are concentrated on the valley bottom due to regional topography [7,9,14,25]. The Introduction In most African highlands the natural forest ecology has been changing in the recent past due to human population increase and demand for more agricultural land, thus favoring mosquito survivorship and parasite development [1,2,3]. This has resulted into continuous local transmission and increased risk of malaria epidemics in highlands [4,5,6,7,8]. Malaria transmission in highlands have been fostered with rise in temperature as the output of high rate of deforestation and land use changes [6,9,10,11,12,13,14]. The increased human population has put more pressure on land resources resulting into reclamation of swamps to increase food security and deforestation to create land PLOS ONE | www.plosone.org December 2012 | Volume 7 | Issue 12 | e52084

Background The existing metrics of malaria transmission are limited in sensitivity under low transmission intensity. Robust surveillance systems are needed as interventions to monitor reduced transmission and prevention of rapid reintroduction. Serological tools based on antibody responses to parasite and vector antigens are potential tools for transmission measurements. The current study sought to evaluate antibody responses to Anopheles gambiae salivary gland peptide (gSG6- P1), as a biomarker of human exposure to Anopheles bites, in different transmission settings and seasons. The comparison between anti-MSP-119 IgG immune responders and non-responders allowed exploring the robustness of the gSG6-P1 peptide as a surveillance tool in an area of decreasing malaria transmission. Methods Total IgG levels to gSG6-P1 were measured in an age-stratified cohort (< 5, 5–14 and ≥ 15 years) in a total of 1,366 participants from three localities in western Kenya [Kisii (hypoendemic), Kakamega (mesoendemic), and Kombewa (hyperendemic)] including 607 sera that were additionally tested for MSP-119 specific responses during a low and a high malaria transmission seasons. Antibody prevalence and levels were compared between localities with different transmission intensities. Regression analysis was performed to examine the association between gSG6-P1 and MSP-119 seroprevalence and parasite prevalence. Result Seroprevalence of gSG6-P1 in the uphill population was 36% while it was 50% valley bottom (χ2 = 13.2, df = 1, p