Resistant Mosquitoes Research Articles

Mosquito Mycobiota: An Overview of Non-Entomopathogenic Fungal Interactions.

The growing expansion of mosquito vectors leads to the emergence of vector-borne diseases in new geographic areas and causes major public health concerns. In the absence of effective preventive treatments against most pathogens transmitted, vector control remains one of the most suitable strategies to prevent mosquito-borne diseases. Insecticide overuse raises mosquito resistance and deleterious impacts on the environment and non-target species. Growing knowledge of mosquito biology has allowed the development of alternative control methods. Following the concept of holobiont, mosquito-microbiota interactions play an important role in mosquito biology. Associated microbiota is known to influence many aspects of mosquito biology such as development, survival, immunity or even vector competence. Mosquito-associated microbiota is composed of bacteria, fungi, protists, viruses and nematodes. While an increasing number of studies have focused on bacteria, other microbial partners like fungi have been largely neglected despite their huge diversity. A better knowledge of mosquito-mycobiota interactions offers new opportunities to develop innovative mosquito control strategies. Here, we review the recent advances concerning the impact of mosquito-associated fungi, and particularly nonpathogenic fungi, on life-history traits (development, survival, reproduction), vector competence and behavior of mosquitoes by focusing on Culex, Aedes and Anopheles species.

Genomic Variant Analyses in Pyrethroid Resistant and Susceptible Malaria Vector, Anopheles sinensis.

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.

Insecticide Resistance of Aedes albopictus in Zhejiang Province, China.

From 2003 until 2018, a total of 12 outbreaks with 1,654 confirmed dengue cases have been reported in Zhejiang Province. The emergence of insecticide resistance in mosquitoes will affect the control of dengue. Our study aims to investigate the current situation of insecticide resistance of Ae. albopictus in Zhejiang Province and compares it with the situation in 2016. Ae. albopictus were collected from 12 Zhejiang Province cities in 2019. Resistance to three major categories of insecticides, including 8 commonly used insecticides, was evaluated according to the tube test protocol recommended by China CDC. Ae. albopictus in all cities, except Hangzhou, Wenzhou, Lishui and Shaoxing, showed decreased susceptibility to beta-cypermethrin, deltamethrin and permethrin. For malathion, 3 cities Ae. albopictus have developed resistance, 3 cities Ae. albopictus have decreased susceptibility. For propoxur, in 3 cities Ae. albopictus showed decreased susceptibility with mortality ranging from 94.24% to 96.67%. The resistance to alpha-cypermethrin, lambda-cyhalothrin and fenitrothion is rare in Ae. albopictus in that only Zhoushan's mosquitoes showed decreased susceptibility to alpha-cypermethrin. The resistance to beta-cypermethrin, deltamethrin and permethrin was significantly correlated with each other. Compared to the situation in 2016, the insecticide resistance of Ae. albopictus in Zhejiang Province has become more common in 2019. In the emergency preparedness for future mosquito-borne diseases, two things should be done: 1) the selection of insecticides should be made based on information from insecticide resistance surveillance 2) the use of insecticide should follow scientific guidance.

Toxicological Evaluation of Novel Butenolide Pesticide Flupyradifurone Against Culex quinquefasciatus (Diptera: Culicidae) Mosquitoes.

The impact of increasing resistance of mosquitoes to conventional pesticides has led to investigate various unique tools and pest control strategies. Herein, we assessed the potency of flupyradifurone, a novel pesticide, on fourth instar larvae of Culex quinquefasciatus Say. Further, we evaluated the synergistic action of piperonyl butoxide (PBO) and the octopamine receptor agonists (OR agonists) chlordimeform (CDM) and amitraz (AMZ) on the toxicity of flupyradifurone in comparison with sulfoxaflor and nitenpyram to increase their toxicity on Cx. quinquefasciatus. Results demonstrated that flupyradifurone was the most potent pesticide followed by sulfoxaflor and nitenpyram. Further, the synergetic effect of PBO, CDM, and AMZ was significant for all selected pesticides especially flupyradifurone. However, AMZ had the most significant effect in combination with the selected pesticides followed by CDM and PBO. The toxicity of the pesticides was time-dependent and increased over time from 24, 48, to 72 h of exposure in all experiments. The results indicate that flupyradifurone is a promising component in future mosquito control programs.

Indoor thermal fogging of the mixture of flupyradifurone and transflutrin on Aedes aegypti mosquitoes susceptible and resistant to pyrethroids in southern Mexico

To evaluate the efficacy of thermal fogging of a mixture of flupyrafirudone (26.3 g/L) and transfluthrin (52.5 g/L) against dengue, Zika y chikungunya Aedes mosquito vectors. Groups of 15 caged Ae. aegypti (susceptible and pyrethroid resistant) mosquitoes were placed in living room, kitchen and bedroom inside houses, after which a dose of 2 and 4 mg/m3 of flupyradifurone and transfluthrine, respectively, was applied as thermal fog. After one hour of exposure mosquitoes were transferred to the laboratory and mortality was recorded after 24 h. The mixture killed 97 to 100% of mosquitoes from the strains and the efficacy was similar independently of their place within the premises. The mixture of flupyrafirudone and transfluthrin applied as thermal fog is a promising tool to control Aedes mosquito populations independently of the pyrethroid-insecticide resistance status.

Use of pyriproxyfen in control of Aedes mosquitoes: Asystematic review.

Dengue is the most rapidly spreading arboviral disease in the world. The current lack of fully protective vaccines and clinical therapeutics creates an urgent need to identify more effective means of controlling Aedes mosquitos, principally Aedes aegypti, as the main vector of dengue. Pyriproxyfen (PPF) is an increasingly used hormone analogue that prevents juvenile Aedes mosquitoes from becoming adults and being incapable of transmitting dengue. The objectives of the review were to (1) Determine the effect of PPF on endpoints including percentage inhibition of emergence to adulthood, larval mortality, and resistance ratios; and (2) Determine the different uses, strengths, and limitations of PPF in control of Aedes. A systematic search was applied to Pubmed, EMBASE, Web of Science, LILACS, Global Health, and the Cochrane database of Systematic Reviews. Out of 1,369 records, 90 studies met the inclusion criteria. Nearly all fit in one of the following four categories 1) Efficacy of granules, 2) Auto-dissemination/horizontal transfer, 3) use of ultra-low volume thermal fogging (ULV), thermal fogging (TF), or fumigant technologies, and 4) assessing mosquito resistance. PPF granules had consistently efficacious results of 90-100% inhibition of emergence for up to 90 days. The evidence is less robust but promising regarding PPF dust for auto-dissemination and the use of PPF in ULV, TF and fumigants. Several studies also found that while mosquito populations were still susceptible to PPF, the lethal concentrations increased among temephos-resistant mosquitoes compared to reference strains. The evidence is strong that PPF does increase immature mortality and adult inhibition in settings represented in the included studies, however future research should focus on areas where there is less evidence (e.g. auto-dissemination, sprays) and new use cases for PPF. A better understanding of the biological mechanisms of cross-resistance between PPF, temephos, and other insecticides will allow control programs to make better informed decisions.

Pemetaan Resistensi Nyamuk Aedes sp Terhadap Malation dan Kepadatan Vektor Demam Berdarah Dengue (DBD) di Sulawesi Utara

The event of mosquito immunity or resistance to malation is one of the main inhibitors of chemical eradication of vectors with insecticides. Vector resistance to insecticides is a global phenomenon, especially for managers of vector-borne control programs in Indonesia. The aims of this study was to map the resistance of Aedes sp mosquitoes to the malation and vector density of Dengue Hemorrhagic Fever (Aedes sp) in North Sulawesi Province in 2019. This research was descriptive in order to obtain a description of pre-adult vector density and to map the status of Dengue Hemorrhagic Fever vector resistance (Aedes sp) against malation. Resistance status data using data from 2015-2018 research. The population in this study are houses in North Minahasa Regency, Minahasa Regency, Tomohon City, Bitung City and Manado City. The sample for vector density is a larva survey of 100 resident houses which includes case houses and houses around DHF cases. North Minahasa Regency (Wautumou Village), Minahasa Regency (Papakelan Village), Tomohon City (Rurukan Village), Bitung City (Girian Permai Village), Manado City (Malalayang Village 2). Determination of the sample based on the highest case in each district in the city during the DHF vector resistance test in 2015-2018. The results showed that the average density of HI vectors (31.%), BI (17.5) and CI (34.8%) with resistance status in the four districts of the mosquito vector city of DHF were resistant and one district (North Minahasa) with a vulnerable status. Resistance and density vectors are digitally mapped.

Target Site Insensitivity Detection in Deltamethrin Resistant Culex pipiens Complex in Iran

Some mosquito species which belong to the Culex. pipiens complex are primary vectors for West Nile virus, Sindbis, Dirofilaria immitis, and many arboviruses. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene of Cx. pipiens that is inherited, is one of the important threats for the efficacy of pyrethroids insecticides. Knockdown resistance (kdr) mutation, L1014F, is a well-defined mechanism of resistance to pyrethroids and DDT in many insect species. The aim of study was to determine the mechanisms of Insecticide resistance in this species. Specimens of Cx. pipiens, the major vector of West Nile virus, were obtained in Tehran, Iran by collecting larvae from polluted wastewater in Qarchak of Tehran. In 2016 Insecticide susceptibility tests were performed according to WHO methods with deltamethrin 0.05%. We focused on determination of this point mutation in the VGSC gene of Cx. pipiens by Real-time PCR. Our results revealed high levels of resistance to deltamethrin 0.05%. The lethal times i.e. LT50 and LT90 for deltamethrin were 2.1530 and 8.5117 h respectively. The result of Real-time PCR confirmed the presence of resistant genotype in all the members of tested population. This study is the first report on kdr genotyping of Cx. pipiens from Tehran and our results on the VGSC gene in position L1014F confirmed the TTA to TTT nucleotide change. This finding will provide a clue for management of insecticide resistance in mosquito which are vectors of arboviruses and decision for replacement of novel approach for vector control.

Effect of Gut Microbes from Eyprepocnemis alacris alacris (Serv. 1838) against Culex quinquefasciatus Say - An Ecofriendly Approach

Mosquitoes are on focus worldwide because of its role as vectors for devastating parasites and pathogens causing a threat to millions of people worldwide. Vector-borne diseases in mosquitoes are transmitted by females. However, ineffectiveness of insecticides increases the resistance in mosquitoes. The administration of new vector control strategies against the mosquitoes needs the implementation of microbial insecticides. In this study was commenced to determine the mortality of larvae using gut bacteria as of Eyprepocnemis alacris alacris (Serville, 1838) conflict to larvae laboratory conditions to identify an effective bacteria. The results indicated the existence of larvicidal activity in 106 isolates and were found to appear strongly effective activity in three isolates against Cx. quinquefasciatus. Cent per cent mortality was observed from each of the secondary metabolites. The recorded values of LC50 were 6.66, 7.13 and 7.64ppm and LC90 has been 115.90, 205.73 and 242.59 ppm respectively. The sequencing and phylogenetic analysis confirmed that these 3 isolates were identified as Klebsiella pneumoniae, Enterobacter xiangfangensis and Ochrobactrum intermedium respectively. Our results showed high larvicidal potential effect against to these bacteria. Outcome of these work highlight the alternative use of synthetic chemical pesticides to control mosquitoes.

IMPACT OF ADAPTIVE MOSQUITO BEHAVIOR AND INSECTICIDE-TREATED NETS ON MALARIA PREVALENCE

Malaria prevalence in sub-Saharan Africa remains high. Kenya for example, records about 3.5 million new cases and 11 thousand deaths each year.1 Most of these cases and deaths are among children under five. The main control method in malaria endemic regions has been through the use of insecticide-treated nets (ITNs). Although this approach has been fairly successful, the gains are threatened by mosquito-resistance to pyrethroids (insecticides on nets), physical and chemical degradation of ITNs that reduce their efficacy, inconsistent and improper use by humans, etc. We present a model to investigate the effects of ITN use and mosquito-resistance and adaptation to pyrethroids used to treat bed nets on malaria prevalence and control in malaria endemic regions. The model captures the development and loss of resistance to insecticides, the effects of ITN use on malaria control in a setting where proper and consistent use is not guaranteed, as well as differentiated biting of human hosts by resistant and sensitive mosquitoes. Important thresholds, including the basic reproduction number [Formula: see text], and two parameter groupings that are important for disease control and for establishing the existence of endemic equilibria to the model are calculated. Furthermore, a global sensitivity analysis is carried out to identify important parameters such as insecticide treated bed-net coverage, ITN, the maximum biting rate of resistant mosquitoes, etc., that drive the system and that can be targeted for disease control. Threshold levels of ITN coverage and ITN efficacy required for containing the disease are identified and shown to depend on the type of insecticide-resistance. For example, when mosquito-resistance to insecticides is not permanent and is acquired only through recruitment and the efficacy of ITNs is [Formula: see text], about [Formula: see text] net coverage is required to contain malaria. However, for the same ITN efficacy, i.e., [Formula: see text], approximately [Formula: see text] net coverage is required to contain the disease when resistance to insecticides is permanent and is acquired through recruitment and mutation in mosquitoes. The model exhibits a backward bifurcation, which implies that simply reducing [Formula: see text] slightly below unity might not be enough to contain the disease. We conclude that appropriate measures to reduce or eliminate mosquito-resistance to insecticides, ensure that more people in endemic areas own and use ITNs properly, and that the efficacy of these nets remain high most of the time, as well as educating populations in malaria endemic areas on how to keep mosquito densities low and minimize mosquito bites are important for containing malaria.

Evaluation of boric acid as toxic sugar bait against resistant Aedes aegypti mosquitoes.

Current methods of broad area application of contact insecticides used in mosquito control are becoming less effective, primarily due to resistance within mosquito populations. New methods that can deliver ingestible insecticides are being investigated as a means to mitigate resistance. This study evaluated insecticide delivery through toxic sugar baits (TSB) and resulting mortality of susceptible and resistant strains of Aedes aegypti. Two Ae. aegypti strains were evaluated using a 1% boric acid TSB: the susceptible Orlando 1952 (ORL) strain and the resistant Puerto Rican (PR) strain. The TSB resulted in high mortality for both ORL and PR strain of Ae. aegypti. Average mortality of female mosquitoes given TSB was 90.8% for PR and 99.3% for ORL. Our study suggests that targeting resistant mosquitoes with ingestible insecticides through TSBs could be a viable alternative to current mosquito control strategies and should be considered when developing an integrated vector management program.

Long-lasting insecticidal nets and the quest for malaria eradication: a mathematical modeling approach.

Recent dramatic declines in global malaria burden and mortality can be largely attributed to the large-scale deployment of insecticidal-based measures, namely long-lasting insecticidal nets (LLINs) and indoor residual spraying. However, the sustainability of these gains, and the feasibility of global malaria eradication by 2040, may be affected by increasing insecticide resistance among the Anopheles malaria vector. We employ a new differential-equations based mathematical model, which incorporates the full, weather-dependent mosquito lifecycle, to assess the population-level impact of the large-scale use of LLINs, under different levels of Anopheles pyrethroid insecticide resistance, on malaria transmission dynamics and control in a community. Moreover, we describe the bednet-mosquito interaction using parameters that can be estimated from the large experimental hut trial literature under varying levels of effective pyrethroid resistance. An expression for the basic reproduction number, [Formula: see text], as a function of population-level bednet coverage, is derived. It is shown, owing to the phenomenon of backward bifurcation, that [Formula: see text] must be pushed appreciably below 1 to eliminate malaria in endemic areas, potentially complicating eradication efforts. Numerical simulations of the model suggest that, when the baseline [Formula: see text] is high (corresponding roughly to holoendemic malaria), very high bednet coverage with highly effective nets is necessary to approach conditions for malaria elimination. Further, while >50% bednet coverage is likely sufficient to strongly control or eliminate malaria from areas with a mesoendemic malaria baseline, pyrethroid resistance could undermine control and elimination efforts even in this setting. Our simulations show that pyrethroid resistance in mosquitoes appreciably reduces bednet effectiveness across parameter space. This modeling study also suggests that increasing pre-bloodmeal deterrence of mosquitoes (deterring them from entry into protected homes) actually hampers elimination efforts, as it may focus mosquito biting onto a smaller unprotected host subpopulation. Finally, we observe that temperature affects malaria potential independently of bednet coverage and pyrethroid-resistance levels, with both climate change and pyrethroid resistance posing future threats to malaria control.

ImergardTMWP: A Non-Chemical Alternative for an Indoor Residual Spray, Effective against Pyrethroid-Resistant Anopheles gambiae (s.l.) in Africa

Malaria is the deadliest mosquito-borne disease and kills predominantly people in sub-Saharan Africa (SSA). The now widespread mosquito resistance to pyrethroids, with rapidly growing resistance to other insecticide classes recommended by the World Health Organization (WHO), may overturn the successes gained in mosquito control in recent years. It is of utmost importance to search for new, inexpensive, and safe alternatives, with new modes of action, that might improve the efficacy of current insecticides. The efficacy of a novel mechanical insecticidal mineral derived from volcanic rock, ImergardTMWP, was investigated to determine its efficacy as a stand-alone residual wall spray and as a mixture with deltamethrin (K-Othrine® Polyzone) in experimental huts in Cove, Benin. The evaluation was conducted with susceptible (Kisumu) and wild-type Anopheles gambiae (s.l.). Deltamethrin applied alone demonstrated 40–45% mortality (at 72 h post-exposure) during the first four months, which declined to 25% at six months for wild An. gambiae from Cove. ImergardTMWP alone and mixed with deltamethrin, under the same assay conditions, produced 79–82% and 73–81% mortality, respectively, during the same six-month period. ImergardTMWP met the 80% WHO bio-efficacy threshold for residual activity for the first five months with 78% residual activity at six months. ImergardTMWP can be used as a mixture with chemical insecticides or as a stand-alone pesticide for mosquito control in Africa.

Spatial variation in the frequency of knockdown resistance genotypes in Florida Aedes aegypti populations

BackgroundThe development of insecticide resistance in disease-vectoring mosquito species can lead to vector control failure and disease resurgence. However, insecticide applications remain an essential public health intervention. In Florida, insecticide resistance in Aedes aegypti, an anthropophilic mosquito species capable of transmitting dengue, chikungunya, and Zika virus, is a major concern. Understanding the location, scale, and driving factors of insecticide resistance can enhance the ability of vector control organizations to target populations effectively.MethodsWe used previously collected data on frequencies of mutations that confer resistance to commonly used pyrethroid insecticides in Ae. aegypti specimens from 62 sites distributed across 18 counties in Florida. To determine the scale of clustering for the most resistant variant, we used a Ripley’s K function. We also used a spatial scanning statistic technique to identify locations of clusters where higher than expected frequencies of susceptible or resistant mosquitoes occurred. We then tested for associations between landscape, demographic, and insecticide-use factors using a beta regression modelling approach and evaluated the effect of spatial lag and spatial error terms on overall explanatory power of these models.ResultsThe scale at which maximum clustering of the most resistant variant occurs is approximately 20 kilometers. We identified statistically significant clusters of genotypes associated with resistance in several coastal cities, although some of these clusters were near significant clusters of susceptible mosquitoes, indicating selection pressures vary at the local scale. Vegetation density, distance from roads, and pyrethroid-use by vector control districts were consistently significant predictors of knockdown resistance genotype frequency in the top-performing beta regression models, although pyrethroid use surprisingly had a negatively associated with resistance. The incorporation of spatial lags resulted in improvements to the fit and explanatory power of the models, indicating an underlying diffusion process likely explains some of the spatial patterns observed.ConclusionsThe genetic mutations that confer resistance to pyrethroids in Ae. aegypti mosquitoes in Florida exhibit spatial autocorrelation and patterns that can be partially explained by landscape and insecticide-use factors. Further work at local scales should be able to identify the mechanisms by which these variables influence selection for alleles associated with resistance.

Broad spectrum immunomodulatory effects of Anopheles gambiae microRNAs and their use for transgenic suppression of Plasmodium.

Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium’s infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito’s innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito’s miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs’ influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes’ antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes.

Yellow-g and Yellow-g2 proteins are required for egg desiccation resistance and temporal pigmentation in the Asian tiger mosquito, Aedes albopictus

Eggs from Aedes mosquitoes exhibit desiccation resistance that helps them to survive and spread as human disease vectors throughout the world. Previous studies have suggested that eggshell/chorion melanization and/or serosal cuticle formation are important for desiccation resistance. In this study, using dsRNAs for target genes, we analyzed the functional importance of two ovary-specific yellow genes, AalY-g and AalY-g2, in the resistance to egg desiccation of the Asian tiger mosquito, Aedes albopictus, a species in which neither the timing of the melanization nor temporal development of the serosal cuticle is correlated with desiccation resistance. Injections of dsAalY-g, dsAalY-g2 or dsAalY-g/g2 (co-injection) into adult females have no effect on their fecundity. However, initial melanization is delayed by 1–2 h with the eggshells eventually becoming black similar to that observed in eggs from dsEGFP-injected control females. In addition, the shape of the eggs from dsAalY-g, -g2 and -g/g2-treated females is abnormally crescent-shaped and the outermost exochorion is more fragile and partially peeled off. dsEGFP control eggs, like those from the wild-type strain, acquire resistance to desiccation between 18 and 24 h after oviposition (HAO). In contrast, ~80% of the 24 HAO dsAalY-g and dsAalY-g2 eggs collapse when they are transferred to a low humidity environment. In addition, there is no electron-dense outer endochorion evident in either dsAalY-g or dsAalY-g2 eggs. These results support the hypothesis that AalY-g and AalY-g2 regulate the timing of eggshell darkening and are required for integrity of the exochorion as well as for rigidity, normal morphology and formation of the outer endochorion, a structure that apparently is critical for desiccation resistance of the Ae. albopictus egg.

20-Hydroxyecdysone Primes Innate Immune Responses That Limit Bacterial and Malarial Parasite Survival in Anopheles gambiae.

Blood feeding is an integral behavior of mosquitoes to acquire nutritional resources needed for reproduction. This requirement also enables mosquitoes to serve as efficient vectors to acquire and potentially transmit a multitude of mosquito-borne diseases, most notably malaria. Recent studies suggest that mosquito immunity is stimulated following a blood meal, independent of infection status. Since blood feeding promotes production of the hormone 20-hydroxyecdysone (20E), we hypothesized that 20E plays an important role in priming the immune response for pathogen challenge. Here, we examine the immunological effects of priming Anopheles gambiae with 20E prior to pathogen infection, demonstrating a significant reduction in bacteria and Plasmodium berghei survival in the mosquito host. Transcriptome sequencing (RNA-seq) analysis following 20E treatment identifies several known 20E-regulated genes, as well as several immune genes with previously reported function in antipathogen defense. Together, these data demonstrate that 20E influences cellular immune function and antipathogen immunity following mosquito blood feeding, arguing the importance of hormones in the regulation of mosquito innate immune function.IMPORTANCE Blood feeding is required to provide nutrients for mosquito egg production and serves as a mechanism to acquire and transmit pathogens. Shortly after a blood meal is taken, there is a peak in the production of 20-hydroxyecdysone (20E), a mosquito hormone that initiates physiological changes, including yolk protein production and mating refractoriness. Here, we examine additional roles of 20E in the regulation of mosquito immunity, demonstrating that priming the immune system with 20E increases mosquito resistance to pathogens. We identify differentially expressed genes in response to 20E treatment, including several involved in innate immune function as well as lipid metabolism and transport. Together, these data argue that 20E stimulates mosquito cellular immune function and innate immunity shortly after blood feeding.

A preliminary analysis on the effect of copper on Anopheles coluzzii insecticide resistance in vegetable farms in Benin

The use of agrochemicals in vegetable production could influence the selection for insecticide resistance in malaria vectors. Unfortunately, there is a dearth of information on the potential contribution of agrochemicals to insecticide resistance in Anopheles mosquitoes breeding on vegetable farms in southern Benin. A Knowledge, Attitudes and Practices study was conducted with 75 vegetable farmers from Houeyiho and Seme to determine the main agrochemicals used in vegetable production, and the concentration and frequency of application, among other details. Mosquitoes and breeding water were sampled from the farms for analysis. Bioassays were conducted on mosquitoes, while breeding water was screened for heavy metal and pesticide residue contamination. Lambda-cyhalothrin was the main insecticide (97.5%) used by farmers, and Anopheles coluzzii was the main mosquito identified. This mosquito species was resistant (30–63% mortality rate) to λ-cyhalothrin. It was also observed that 16.7% of the examined breeding sites were contaminated with λ-cyhalothrin residues. Furthermore, copper contamination detected in mosquito breeding sites showed a positive correlation (r = 0.81; P = 0.0017) with mosquito resistance to λ-cyhalothrin. The presence of copper in λ-cyhalothrin-free breeding sites, where mosquitoes have developed resistance to λ-cyhalothrin, suggests the involvement of copper in the insecticide resistance of malaria vectors; this, however, needs further investigation.

Quantifying late-stage host-seeking behaviour of Anopheles gambiae at the insecticidal net interface using a baited-box bioassay

BackgroundInsecticide-treated nets (ITNs) are losing efficacy against pyrethroid-resistant malaria vector populations throughout Africa. Safeguarding bed net efficacy, vital for effective malaria control, requires greater knowledge of mosquito–ITN interactions and how this impacts on the mosquito.MethodsA purpose-built benchtop apparatus with a closed 10 cm cubic chamber (the ‘Baited-box’) was used to video record behaviour of individual free-flying female Anopheles gambiae during approach and blood-feeding on a human hand through untreated nets and ITNs at close range. Time and duration of defined behavioural events, and knockdown and mortality at 1- and 24-h post-exposure respectively, were recorded for pyrethroid susceptible and resistant mosquitoes.ResultsUsing three human volunteers differing in relative attractiveness to mosquitoes, 328 mosquitoes were individually tested. There were no significant differences between response rates to ITNs and untreated nets (P > 0.1) or between resistant (Tiassalé) and susceptible (Kisumu) mosquito strains, at untreated nets (P = 0.39) or PermaNet 2.0 (P = 1). The sequence of behavioural events from host-seeking to completion of blood-feeding was consistent in all tests but duration and start time of events involving net contact were reduced or delayed respectively with ITNs. Blood-feeding durations at untreated nets (means from 4.25 to 8.47 min (95% confidence interval (CI) = 3.39–9.89) at 3 human volunteers) were reduced by 37–50% at PermaNet 2.0, in susceptible (mean 2.59–4.72 min, 95% CI = 1.54–5.5, P = < 0.05) and resistant (mean 4.20 min, 95% CI = 3.42–4.97, P = 0.01) strains. Total accumulated net contact was approximately 50% lower at PermaNet and Olyset ITNs (P < 0.0001) in susceptible (two of the three volunteers) and resistant mosquitoes. Times prior to first net contact were similar at untreated nets and ITNs (P > 0.2), and neither ITN type showed detectable spatial repellency. After initial contact, blood-feeding commenced later at Olyset (mean 2.76 min, 95% CI = 1.74–3.76, P = 0.0009) and PermaNet (mean 2.4 min, 95% CI = 1.52–3.33, P = 0.0058) than untreated netting (mean 0.68 min, 95% CI = 0.42–0.94).ConclusionsThe baited box offers a simple method for detailed characterization of mosquito behavioural responses to insecticidal nets, for comparing entomological modes of action between nets and for defining the behavioural responses of particular mosquito strains or populations. The device has potential as a screening assay in the search for novel net treatments and for investigations into behavioural resistance mechanisms.

Malaria mosquito chemical ecology.

The field of mosquito chemical ecology has shifted focus over the past five years, driven by the recognition that odour-mediated behaviours are regulated by distinct chemical codes, that is, odour blends emanating from the natural environment. As a research community, we have shifted from our anthropocentric focus to include other behaviours, including plant seeking and oviposition site seeking, in order to develop new tools to combat residual malaria in the wake of the increased insecticide and behavioural resistance in mosquitoes across sub-Saharan Africa. In this short review, we will outline the progress made, and the future directions, in understanding blend recognition and chemical parsimony, and their implications for preadaptation of the odour coding system in malaria mosquitoes.