Key gene modules and hub genes associated with pyrethroid and organophosphate resistance in Anopheles mosquitoes: a systems biology approach.

Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

Non-contact detection of pyrethroids widely used in vector control by Anopheles mosquitoes.

Pyrethroids are the most widely used insecticides to control vector borne diseases including malaria. Physiological resistance mechanisms to these insecticides have been well described, whereas those for behavioral resistance remain overlooked. Field data suggest the presence of spatial sensory detection by Anopheles mosquitoes of the pyrethroid molecules used in insecticide-based control tools, such as long-lasting insecticide nets or insecticide residual spraying. This opens the way to the emergence of a wide range of behavioral adaptations among malaria vectors. However, the spatial sensory detection of these molecules is controversial and needs to be demonstrated. The goal of this study was to behaviorally characterize the non-contact detection of three of the most common pyrethroids used for malaria vector control: permethrin, deltamethrin an ⍺-cypermethrin. To reach this goal, we recorded the behavior (takeoff response) of Anopheles gambiae pyrethroid-sensitive and resistant laboratory strains, as well as field collected mosquitoes from the Gambiae Complex, when exposed to the headspace of bottles containing different doses of the insecticides at 25 and 35°C, in order to represent a range of laboratory and field temperatures. We found the proportion of laboratory susceptible and resistant female mosquitoes that took off was, in all treatments, dose and the temperature dependent. Sensitive mosquitoes were significantly more prone to take off only in the presence of ⍺-cypermethrin, whereas sensitive and resistant mosquitoes showed similar responses to permethrin and deltamethrin. Field-collected mosquitoes of the Gambiae Complex were also responsive to permethrin, independently of the species identity (An. gambiae, An. coluzzii and An. arabiensis) or their genotypes for the kdr mutation, known to confer resistance to pyrethroids. The observed ability of Anopheles spp. mosquitoes to detect insecticides without contact could favor the evolution of behavioral modifications that may allow them to avoid or reduce the adverse effect of insecticides and thus, the development of behavioral resistance.

Efficacy of Interceptor G2, Royal Guard and PermaNet 3.0 against pyrethroid-resistant Anopheles gambiae s.l. from Za-Kpota, southern Benin: an experimental hut trial.

BACKGROUND: The widespread use of insecticide-treated nets (ITNs) has significantly contributed to the reduction in malaria cases and deaths observed across Africa. Unfortunately, this control strategy is threatened by the rapid spread of pyrethroid resistance in malaria vectors. Dual-active-ingredient insecticidal nets are now available to mitigate the impact of pyrethroid resistance. To facilitate evidence-based decisions regarding product selection in specific use settings, data are needed on the efficacy of these different nets against local mosquito populations. METHODS: Two experimental hut trials were performed in Za-Kpota, southern Benin in 2021 to evaluate the performance of Interceptor G2 (BASF), Royal Guard (Disease Control Technologies) and PermaNet 3.0 (Vestergaard Frandsen), all dual-active-ingredient bednets, in comparison to untreated or standard pyrethroid-treated bednets, against free-flying wild Anopheles gambiae mosquitoes. The performance of some of these next-generation nets was compared to the same type of nets that have been in use for up to 2 years. Mosquitoes collected in the huts were followed up after exposure to assess the sublethal effects of treatments on certain life-history traits. RESULTS: The predominant species in the study site was Anopheles gambiae sensu stricto (An. gambiae s.s.). Both Anopheles coluzzii and An. gambiae s.s. were resistant to pyrethroids (deltamethrin susceptibility was restored by piperonyl butoxide pre-exposure). In the experimental hut trials, the highest blood-feeding inhibition (5.56%) was recorded for the Royal Guard net, relative to the standard PermaNet 2.0 net (44.44% inhibition). The highest 72-h mortality rate (90.11%) was recorded for the Interceptor G2 net compared to the PermaNet 2.0 net (56.04%). After exposure, the risk of death of An. gambiae sensu lato (An. gambiae s.l.) was 6.5-fold higher with the Interceptor G2 net and 4.4-fold higher with the PermaNet 3.0 net compared to the respective untreated net. Lower mosquito mortality was recorded with an aged Interceptor G2 net compared to a new Interceptor G2 net. Oviposition rates were lower in mosquitoes collected from huts containing ITNs compared to those of untreated controls. None of the mosquitoes collected from huts equipped with Royal Guard nets laid any eggs. CONCLUSIONS: The Royal Guard and Interceptor G2 nets showed a potential to significantly improve the control of malaria-transmitting vectors. However, the PermaNet 3.0 net remains effective in pyrethroid-resistant areas.

Malaria prevalence and transmission in the Zakpota sub-district of central Benin: baseline characteristics for a community randomised trial of a new insecticide for indoor residual spraying.

BACKGROUND: Malaria transmission is known to be perennial and heterogeneous in Benin. Studies assessing local malaria prevalence, transmission levels and vector characteristics are critical for designing, monitoring and evaluating new vector control interventions in community trials. We conducted a study in the Zakpota sub-district of central Benin to collect baseline data on household characteristics, malaria prevalence, vector characteristics and transmission dynamics in preparation for a randomised controlled trial to evaluate the community impact of VECTRON™ T500, a new broflanilide indoor residual spraying (IRS) product. METHODS: A total of 480 children under 5 years of age from the 15 villages of the sub-district were tested for malaria by rapid diagnostic tests (RDTs). Mosquitoes were collected by human landing catches (HLCs), pyrethrum spray catches (PSCs) and Centers for Disease Control and Prevention miniature light traps (CDC-LTs) in selected houses in each village to assess vector density, composition, vector infectivity and prevalence of insecticide resistance markers. Bioassays were performed to detect vector susceptibility to pyrethroids, broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle). RESULTS: A total of 9080 households were enumerated in the 15 study villages. Insecticide-treated net (ITN) usage was > 90%, with 1-2 ITNs owned per household. Houses were constructed mainly with cement (44%) and mud (38%) substrates or a mixture of cement and mud (18%), and 60% of them had open eaves. The overall prevalence of P. falciparum infection was 19% among surveyed children: 20% among females and 18% among males. The haemoglobin rate showed an anaemia (< 11 g/dl) prevalence of 66%. Anopheles coluzzii and An. gambiae sensu stricto (s.s.) were the two vector species present at an overall proportion of 46% versus 54%, respectively. The human biting rate was 2.3 bites per person per night (b/p/n) and biting occurred mostly indoors compared with outdoors (IRR = 0.776; P = 0.001). The overall proportion of outdoor biting was 44% and exceeded indoor biting in three villages. The sporozoite rate was 2% with a combined yearly entomological inoculation rate (EIR) of 16.1 infected bites per person per year (ib/p/y). There was great variability in malaria transmission risk across the villages, with EIR ranging from 0 to 29.3 ib/p/y. The vector population showed a high intensity of resistance to pyrethroids across the study villages but was largely susceptible to broflanilide and clothianidin. CONCLUSIONS: This study found high levels of malaria prevalence, vector density and transmission in the Zakpota sub-district despite the wide use of insecticide-treated nets. The vector population was mostly indoor resting and showed a high intensity of pyrethroid resistance but was generally fully susceptible to broflanilide. These findings demonstrated the suitability of the study area for the assessment of VECTRON™ T500 in a community randomised trial.

Community end user perceptions of hessian fabric transfluthrin vapour emanators for protecting against mosquitoes under conditions of routine use in Port-au-Prince, Haiti.

BACKGROUND: A treated fabric device for emanating the volatile pyrethroid transfluthrin was recently developed in Tanzania that protected against night-biting Anopheles and Culex mosquitoes for several months. Here perceptions of community end users provided with such transfluthrin emanators, primarily intended to protect them against day-active Aedes vectors of human arboviruses that often attack people outdoors, were assessed in Port-au-Prince, Haiti. METHODS: Following the distribution of transfluthrin emanators to participating households in poor-to-middle class urban neighbourhoods, questionnaire surveys and in-depth interviews of end-user households were supplemented with conventional and Photovoice-based focus group discussions. Observations were assessed synthetically to evaluate user perceptions of protection and acceptability, and to solicit advice for improving and promoting them in the future. RESULTS: Many participants viewed emanators positively and several outlined various advantages over current alternatives, although some expressed concerns about smell, health hazards, bulkiness, unattractiveness and future cost. Most participants expressed moderate to high satisfaction with protection against mosquitoes, especially indoors. Protection against other arthropod pests was also commonly reported, although satisfaction levels were highly variable. Diverse use practices were reported, some of which probably targeted nocturnal Culex resting indoors, rather than Aedes attacking them outdoors during daylight hours. Perceived durability of protection varied: While many participants noted some slow loss over months, others noted rapid decline within days. A few participants specifically attributed efficacy loss to outdoor use and exposure to wind or moisture. Many expressed stringent expectations of satisfactory protection levels, with even a single mosquito bite considered unsatisfactory. Some participants considered emanators superior to fans, bedsheets, sprays and coils, but it is concerning that several preferred them to bed nets and consequently stopped using the latter. CONCLUSIONS: The perspectives shared by Haitian end-users are consistent with those from similar studies in Brazil and recent epidemiological evidence from Peru that other transfluthrin emanator products can protect against arbovirus infection. While these encouraging sociological observations contrast starkly with evidence of essentially negligible effects upon Aedes landing rates from parallel entomological assessments across Haiti, Tanzania, Brazil and Peru, no other reason to doubt the generally encouraging views expressed herein by Haitian end users could be identified.

In vitro larvicidal efficacy of Lantana camara essential oil and its nanoemulsion and enzyme inhibition kinetics against Anopheles culicifacies.

Mosquitoes are important vectors for the transmission of several infectious diseases that lead to huge morbidity and mortality. The exhaustive use of synthetic insecticides has led to widespread resistance and environmental pollution. Using essential oils and nano-emulsions as novel insecticides is a promising alternative approach for controlling vector borne diseases. In the current study, Lantana camara EO and NE were evaluated for their larvicidal and pupicidal activities against Anopheles culicifacies. The inhibitory effect of EO and NE on AChE, NSE (α/ß), and GST was also evaluated and compared. GC-MS analysis of oil displayed 61 major peaks. The stable nano-emulsion with an observed hydrodynamic diameter of 147.62 nm was formed using the o/w method. The nano-emulsion exhibited good larvicidal (LC50 50.35 ppm and LC90 222.84 ppm) and pupicidal (LC50 54.82 ppm and LC90 174.58 ppm) activities. Biochemical evaluations revealed that LCEO and LCNE inhibited AChE, NSE (α/ß), and GST, displaying LCNE to be a potent binder to AChE and NSE enzyme, whereas LCEO showed higher binding potency towards GST. The nano-emulsion provides us with novel opportunities to target different mosquito enzymes with improved insecticidal efficacy. Due to its natural origin, it can be further developed as a safer and more potent larvicide/insecticide capable of combating emerging insecticide resistance.

Physical and insecticidal durability of Interceptor®, Interceptor® G2, and PermaNet® 3.0 insecticide-treated nets in Burkina Faso: results of durability monitoring in three sites from 2019 to 2022.

BACKGROUND: National Malaria Programmes (NMPs) monitor the durability of insecticide-treated nets (ITNs) to inform procurement and replacement decisions. This is crucial for new dual active ingredients (AI) ITNs, for which less data is available. Pyrethroid-only ITN (Interceptor®) and dual AI (Interceptor® G2, and PermaNet® 3.0) ITNs were assessed across three health districts over 36 months in southern Burkina Faso to estimate median ITN survival, insecticidal efficacy, and to identify factors contributing to field ITN longevity. METHODS: Durability was monitored through a prospective study of a cohort of nets distributed during the 2019 mass campaign. Three health districts were selected for their similar pyrethroid-resistance, environmental, epidemiological, and population profiles. Households were recruited after the mass campaign, with annual household questionnaire follow-ups over three years. Each round, ITNs were withdrawn for bioassays and chemical residue testing. Key measures were the percentage of cohort ITNs in serviceable condition, insecticidal effectiveness, and chemical residue content against target dose. Cox proportional hazard models were used to identify determinants influencing ITN survival. RESULTS: At endline, the median useful life was 3.2 (95% CI 2.5-4.0) years for PermaNet® 3.0 ITNs in Orodara, 2.6 (95% CI 1.9-3.2) years for Interceptor® G2 ITNs in Banfora and 2.4 (95% CI 1.9-2.9) years for Interceptor® ITNs in Gaoua. Factors associated with ITN survival included cohort ITNs from Orodara (adjusted hazard ratio (aHR) = 0.58, p = 0.026), households seeing less rodents (aHR = 0.66, p = 0.005), female-headed households (aHR = 0.66, p = 0.044), exposure to social behavior change (SBC) messages (aHR = 0.52, ≤ 0.001) and folding nets when not in use (aHR = 0.47, p < 0.001). At endline, PermaNet® 3.0 ITN recorded 24-h mortality of 26% against resistant mosquitos on roof panels, with an 84% reduction in PBO content. Interceptor® G2 ITN 72-h mortality was 51%, with a 67% reduction in chlorfenapyr content. Interceptor® ITN 24-h mortality was 71%, with an 84% reduction in alpha-cypermethrin content. CONCLUSION: Only PermaNet® 3.0 ITNs surpassed the standard three-year survival threshold. Identified protective factors should inform SBC messaging. Significant decreases in chemical content and resulting impact on bioefficacy warrant more research in other countries to better understand dual AI ITN insecticidal performance.

Mosquitocidal effect of ivermectin-treated nettings and sprayed walls on Anopheles gambiae s.s.

Ivermectin (IVM) has been proposed as a new tool for malaria control as it is toxic on vectors feeding on treated humans or cattle. Nevertheless, IVM may have a direct mosquitocidal effect when applied on bed nets or sprayed walls. The potential for IVM application as a new insecticide for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) was tested in this proof-of-concept study in a laboratory and semi-field environment. Laboratory-reared, insecticide-susceptible Kisumu Anopheles gambiae were exposed to IVM on impregnated netting materials and sprayed plastered- and mud walls using cone bioassays. The results showed a direct mosquitocidal effect of IVM on this mosquito strain as all mosquitoes died by 24 h after exposure to IVM. The effect was slower on the IVM-sprayed walls compared to the treated nettings. Further work to evaluate possibility of IVM as a new insecticide formulation in LLINs and IRS will be required.

Structure-based virtual screening study for identification of potent insecticides against Anopheles gambiae to combat the malaria.

BACKGROUND OBJECTIVES: Vector-borne infectious diseases contribute significantly to global mortality, with over 700,000 annual deaths, and malaria alone accounts for more than 400,000 of these fatalities. Anopheles gambiae, a prominent mosquito species, serves as a primary vector for transmitting malaria to humans. To address this issue, researchers have identified the D1-like dopamine receptor (DAR), specifically DOP2, as a promising target for developing new insecticides. METHODS: The three-dimensional structure of DOP2 from A. gambiae was unavailable; in-silico approach was used to model and validate DOP2 structure. The Discovery Studio 2021 program was used to identify potential binding sites on DOP2. Virtual screening of 235 anti-parasitic compounds was performed against DOP2 using PyRx 0.8. RESULTS: The screening demonstrated strong binding and interactions with active site residues of DOP2 for five compounds: Diclazuril, Kaempferol, Deracoxib, Clindamycin, and Diaveridine. These compounds exhibited higher binding affinity values compared to the control (Asenapine). In addition, the predicted physiochemical properties for these compounds were within acceptable ranges and there were no violations in drug-likeness properties. INTERPRETATION CONCLUSION: These compounds show promise as potential new insecticides targeting A. gambiae mosquito by inhibiting the DOP2 protein. However, additional experimental validation is required to optimize their efficacy as DOP2 inhibitors.

Efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr nets on entomological indicators of malaria transmission: third year of a randomised controlled trial in Benin.

The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46-1.88), p = 0.8527; and RR 1.10 (95% CI 0.44-2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37-1.48), p = 0.3946; and RR 1.00 (95% CI 0.40-2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Insufficient duration of insecticidal efficacy of Yahe® insecticide-treated nets in Papua New Guinea.

BACKGROUND: Insecticide-treated nets (ITNs) are the backbone of anti-malarial vector control in Papua New Guinea (PNG). Over recent years the quality and performance of ITNs delivered to PNG decreased, which has likely contributed to the stagnation in the malaria control effort in the country. The present study reports results from the first 24 months of a durability study with the ITN product Yahe LN® in PNG. METHODS: The durability study was conducted in four villages on the northern coast of PNG, in an area with high malaria parasite transmission, following WHO-recommended methodology adapted to the local scenario. A cohort of n = 500 individually identifiable Yahe® ITNs was distributed by the PNG National Malaria Control Programme from October to December 2021. Insecticidal efficacy of the ITNs was tested using cone bioassays with fully pyrethroid susceptible Anopheles farauti colony mosquitoes at baseline and at 6 months intervals, alongside evaluation of physical integrity and the proportion of ITNs lost to follow-up. A questionnaire was used to collect information on ITN end user behaviour, such as the frequency of use and washing. The observations from the durability study were augmented with simulated laboratory wash assays. RESULTS: Gradual uptake and replacement of previous campaign nets by the communities was observed, such that at 6 months 45% of all newly distributed nets were in use in their designated households. Insecticidal efficacy of the Yahe® nets, expressed as the percent 24 h mortality in cone bioassays decreased from 91 to 45% within the first 6 months of distribution, even though > 90% of study nets had never been washed. Insecticidal efficacy decreased further to < 20% after 24 months. ITNs accumulated physical damage (holes) at a rate similar to previous studies, and 35% were classified as 'too torn' by proportional hole index after 24 months. ITNs were lost to follow-up such that 61% of cohort nets were still present after 24 months. Laboratory wash assays indicated a rapid reduction in insecticidal performance with each consecutive wash such that average 24 h mortality was below 20% after 10 washes. CONCLUSION: Yahe® ITNs are not performing as per label claim in an area with fully pyrethroid susceptible vectors, and should be investigated more comprehensively and in other settings for compliance with currently recommended durability and efficacy thresholds. The mass distribution of low quality ITN products with variable performance is one of the major ongoing challenges for global malaria control in the last decade.

Malaria Vector Bionomics: Countrywide Surveillance Study on Implications for Malaria Elimination in India.

BACKGROUND: The biological characteristics of mosquito vectors vary, impacting their response to control measures. Thus, having up-to-date information on vector bionomics is essential to maintain the effectiveness of existing control strategies and tools, particularly as India aims for malaria elimination by 2030. OBJECTIVE: This study aims to assess the proportions of vector species resting indoors and outdoors, determine their preference for host biting/feeding, identify transmission sites, and evaluate the susceptibility of vectors to insecticides used in public health programs. METHODS: Mosquito collections were conducted in 13 districts across 8 Indian states from 2017 to 2020 using various methods to estimate their densities. Following morphological identification in the field, sibling species of Anopheles mosquitoes were identified molecularly using polymerase chain reaction (PCR)-specific alleles. Plasmodium falciparum and Plasmodium vivax infections in the vectors were detected using enzyme-linked immunosorbent assay (ELISA) and PCR assays. In addition, we assessed the insecticide susceptibility status of primary malaria vectors following the World Health Organization (WHO) protocol. RESULTS: Anopheles culicifacies, a primary malaria vector, was collected (with a man-hour density ranging from 3.1 to 15.9) from all states of India except those in the northeastern region. Anopheles fluviatilis, another primary vector, was collected from the states of Madhya Pradesh, Maharashtra, Karnataka, and Odisha. In Haryana and Karnataka, An. culicifacies sibling species A predominated, whereas species C and E were predominant in Madhya Pradesh and Maharashtra. An. culicifacies displayed mainly endophilic behavior across all states, except in Madhya Pradesh, where the proportion of semigravid and gravid mosquitoes was nearly half of that of unfed mosquitoes. The human blood index of An. culicifacies ranged from 0.001 to 0.220 across all study sites. The sporozoite rate of An. culicifacies ranged from 0.06 to 4.24, except in Madhya Pradesh, where none of the vector mosquitoes were found to be infected with the Plasmodium parasite. In the study area, An. culicifacies exhibited resistance to DDT (dichlorodiphenyltrichloroethane; with <39% mortality). Moreover, it showed resistance to malathion (with mortality rates ranging from 49% to 78%) in all districts except Angul in Odisha and Palwal in Haryana. In addition, resistance to deltamethrin was observed in districts of Maharashtra, Gujarat, Haryana, and Karnataka. CONCLUSIONS: Our study offers vital insights into the prevalence, resting behavior, and sibling species composition of malaria vectors in India. It is evident from our findings that resistance development in An. culicifacies, the primary vector, to synthetic pyrethroids is on the rise in the country. Furthermore, the results of our study suggest a potential change in the resting behavior of An. culicifacies in Madhya Pradesh, although further studies are required to confirm this shift definitively. These findings are essential for the development of effective vector control strategies in India, aligning with the goal of malaria elimination by 2030.

LLIN Evaluation in Uganda Project (LLINEUP)-effects of a vector control trial on Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395 .

Antimalarial activity of cecropin antimicrobial peptides derived from Anopheles mosquitoes.

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.

Chemical Composition, Repellent, and Oviposition Deterrent Potential of Wild Plant Essential Oils against Three Mosquito Species.

In this study, the chemical composition, repellent, and oviposition deterrent effects of five plant essential oils (EOs) extracted from Lantana camara (Verbenaceae), Schinus terebinthifolia (Anacardiaceae), Callistemon viminalis (Myrtaceae), Helichrysum odoratissimum (Asteraceae), and Hyptis suaveolens (Lamiaceae) were evaluated against Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus. When tested at 33.3 µg/cm2, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum were effective repellents against Ae. aegypti (89%, 91%, 90%, and 51% repellency, respectively), but they were less repellent against An. gambiae (66%, 86%, 59%, and 49% repellency, respectively). Interestingly, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum exhibited 100% repellency against Cx. quinquefasciatus at 33.3 µg/cm2. In time-span bioassays performed at 333 µg/cm2, the EO of L. camara exhibited 100% repellence against Ae. aegypti and An. gambiae for up to 15 min and against Cx. quinquefasciatus for 75 min. The oviposition bioassays revealed that L. camara exhibited the highest activity, showing 85%, 59%, and 89% oviposition deterrence against Ae. aegypti, An. gambiae, and Cx. quinquefasciatus, respectively. The major compounds of L. camara, S. terebinthifolia, and C. viminalis were trans-ß-caryophyllene (16.7%), α-pinene (15.5%), and 1,8-cineole (38.1%), respectively. In conclusion, the L. camara and S. terebinthifolia EOs have the potential to be natural mosquito repellents.

3D video tracking analysis reveals that mosquitoes pass more likely through holes in permethrin-treated than in untreated nets.

In addition to killing, mosquito nets treated with permethrin have been claimed to repel mosquitoes, reducing their success in passing through a holed net. We have tested this hypothesis by tracking mosquitoes in a modified World Health Organization tunnel test. In the original assay, mosquitoes are released at one end of the tunnel and have to pass through a holed piece of net to reach the bait at the other end. The mosquitoes are left in the tunnel overnight, while mortality and feeding rates are scored the following morning. Since the original test does not reveal how mosquitoes move within the tunnel, we combined the tunnel with a 3D video camera system. We tracked susceptible and permethrin-resistant Anopheles gambiae s.s. as they moved in the tunnel and interacted with an untreated or a permethrin-treated net (Olyset Net®). Surprisingly, while permethrin increased the mortality and reduced blood-feeding rates, twice as many mosquitoes passed through the holes of the permethrin-treated net. The flight trajectories reveal that upon exposure to the permethrin-treated net, both mosquito colonies showed increased 'excitation', thereby augmenting their chance of getting through the holes in the net. The study underlines the complexity of behavioural modes of action of insecticides.

Identifying suitable methods for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors: a comparison of the oviposition and ovary dissection methods.

BACKGROUND: Nets containing pyriproxyfen, an insect growth regulator that sterilizes adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection). METHOD: Laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles (100 µg and 200 µg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties by trained laboratory technicians. RESULTS: The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99-100% vs. 34-59%, P < 0.05) and cone bioassays (99-100% vs. 18-33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid-resistant An. gambiae s.l. Cové, compared to the laboratory-maintained reference susceptible An gambiae sensu stricto Kisumu (18-34% vs. 58-76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 µg) were suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%), but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89-98% vs. 89-100%). Specificity was substantially higher with ovary dissection compared to the oviposition method in both bottle bioassays (99-100% vs. 34-48%) and cone tests (100% vs.18-76%). Ovary dissection was also more sensitive for detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming. Laboratory technicians preferred ovary dissection mostly due to its reliability. CONCLUSION: The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets.

Insecticide susceptibility status of Anopheles albimanus populations in historical malaria foci in Quintana Roo, Mexico.

BACKGROUND: Mexico has experienced a significant reduction in malaria cases over the past two decades. Certification of localities as malaria-free areas (MFAs) has been proposed as a steppingstone before elimination is achieved throughout the country. The Mexican state of Quintana Roo is a candidate for MFA certification. Monitoring the status of insecticide susceptibility of major vectors is crucial for MFA certification. This study describes the susceptibility status of Anopheles albimanus, main malaria vector, from historically important malaria foci in Quintana Roo, using both phenotypic and genotypic approaches. METHODS: Adult mosquito collections were carried out at three localities: Palmar (Municipality of Othon P. Blanco), Buenavista (Bacalar) and Puerto Morelos (Puerto Morelos). Outdoor human-landing catches were performed by pairs of trained staff from 18:00 to 22:00 during 3-night periods at each locality during the rainy season of 2022. Wild-caught female mosquitoes were exposed to diagnostic doses of deltamethrin, permethrin, malathion, pirimiphos-methyl or bendiocarb using CDC bottle bioassays. Mortality was registered at the diagnostic time and recovery was assessed 24 h after exposure. Molecular analyses targeting the Voltage-Gated Sodium Channel (vgsc) gene and acetylcholinesterase (ace-1) gene were used to screen for target site polymorphisms. An SNP analysis was carried out to identify mutations at position 995 in the vgsc gene and at position 280 in the ace-1 gene. RESULTS: A total of 2828 anophelines were collected. The main species identified were Anopheles albimanus (82%) and Anopheles vestitipennis (16%). Mortalities in the CDC bottle bioassay ranged from 99% to 100% for all the insecticides and mosquito species. Sequence analysis was performed on 35 An. albimanus across the three localities; of those, 25 were analysed for vgsc and 10 for ace-1 mutations. All individuals showed wild type alleles. CONCLUSION: The results demonstrated that An. albimanus populations from historical malaria foci in Quintana Roo are susceptible to the main insecticides used by the Ministry of Health.

Lethal and sublethal impacts of membrane-fed ivermectin are concentration dependent in Anopheles coluzzii.

BACKGROUND: Ivermectin is a well-tolerated anthelminthic drug with wide clinical and veterinary applications. It also has lethal and sublethal effects on mosquitoes. Mass drug administration with ivermectin has therefore been suggested as an innovative vector control tool in efforts to curb emerging insecticide resistance and reduce residual malaria transition. To support assessments of the feasibility and efficacy of current and future formulations of ivermectin for vector control, we sought to establish the relationship between ivermectin concentration and its lethal and sublethal impacts in a primary malaria vector. METHODS: The in vitro effects of ivermectin on daily mortality and fecundity, measured by egg production, were assessed up to 14 days post-blood feed in a laboratory colony of Anopheles coluzzii. Mosquitoes were fed ivermectin in blood meals delivered by membrane feeding at one of six concentrations: 0 ng/ml (control), 10 ng/ml, 15 ng/ml, 25 ng/ml, 50 ng/ml, 75 ng/ml, and 100 ng/ml. RESULTS: Ivermectin had a significant effect on mosquito survival in a concentration-dependent manner. The LC50 at 7 days was 19.7 ng/ml. The time to median mortality at ≥ 50 ng/ml was ≤ 4 days, compared to 9.6 days for control, and 6.3-7.6 days for ivermectin concentrations between 10 and 25 ng/ml. Fecundity was also affected; no oviposition was observed in surviving females from the two highest concentration treatment groups. While females exposed to 10 to 50 ng/ml of ivermectin did oviposit, significantly fewer did so in the 50 ng/ml treatment group compared to the control, and they also produced significantly fewer eggs. CONCLUSIONS: Our results showed ivermectin reduced mosquito survival in a concentration-dependent manner and at ≥ 50 ng/ml significantly reduced fecundity in An. coluzzii. Results indicate that levels of ivermectin found in human blood following ingestion of a single 150-200 µg/kg dose would be sufficient to achieve 50% mortality across 7 days; however, fecundity in survivors is unlikely to be affected. At higher doses, a substantial impact on both survival and fecundity is likely. Treating human populations with ivermectin could be used as a supplementary malaria vector control method to kill mosquito populations and supress their reproduction; however strategies to safely maintain mosquitocidal blood levels of ivermectin against all Anopheles species require development.

Characteristics of the Western Province, Zambia, trial site for evaluation of attractive targeted sugar baits for malaria vector control.

BACKGROUND: The attractive targeted sugar bait (ATSB) is a novel malaria vector control tool designed to attract and kill mosquitoes using a sugar-based bait, laced with oral toxicant. Western Province, Zambia, was one of three countries selected for a series of phase III cluster randomized controlled trials of the Westham ATSB Sarabi version 1.2. The trial sites in Kenya, Mali, and Zambia were selected to represent a range of different ecologies and malaria transmission settings across sub-Saharan Africa. This case study describes the key characteristics of the ATSB Zambia trial site to allow for interpretation of the results relative to the Kenya and Mali sites. METHODS: This study site characterization incorporates data from the trial baseline epidemiological and mosquito sugar feeding surveys conducted in 2021, as well as relevant literature on the study area. RESULTS: CHARACTERIZATION OF THE TRIAL SITE: The trial site in Zambia was comprised of 70 trial-designed clusters in Kaoma, Nkeyema, and Luampa districts. Population settlements in the trial site were dispersed across a large geographic area with sparsely populated villages. The overall population density in the 70 study clusters was 65.7 people per square kilometre with a total site population of 122,023 people living in a geographic area that covered 1858 square kilometres. However, the study clusters were distributed over a total area of approximately 11,728 square kilometres. The region was tropical with intense and seasonal malaria transmission. An abundance of trees and other plants in the trial site were potential sources of sugar meals for malaria vectors. Fourteen Anopheles species were endemic in the site and Anopheles funestus was the dominant vector, likely accounting for around 95% of all Plasmodium falciparum malaria infections. Despite high coverage of indoor residual spraying and insecticide-treated nets, the baseline malaria prevalence during the peak malaria transmission season was 50% among people ages six months and older. CONCLUSION: Malaria transmission remains high in Western Province, Zambia, despite coverage with vector control tools. New strategies are needed to address the drivers of malaria transmission in this region and other malaria-endemic areas in sub-Saharan Africa.