Physico-chemical characterization of Anopheles gambiae s.l. breeding sites and kdr mutations in urban areas of Cotonou and Natitingou, Benin.

BACKGROUND: This study aimed to investigate the relationship between the physicochemical characteristics of An. gambiae s.s. and An. coluzzii breeding sites, the susceptibility profiles to commonly used insecticides in public health, and the underlying insecticide resistance mechanisms. METHODS: Anopheles breeding sites surveys were conducted in Cotonou and Natitingou in September 2020, January and August 2021. Physicochemical properties and bacterial loads were determined in individual breeding sites. The WHO susceptibility assays were carried out using the female of the emerging adult mosquitoes. Anopheles species were identified through PCR techniques. Kdr L1014F/S, N1575Y and G119S mutations were investigated using TaqMan genotyping assays. RESULTS: Molecular analysis showed that all mosquitoes analyzed in Cotonou were Anopheles coluzzii, while those of Natitingou were Anopheles gambiae s.s. Fecal coliforms were identified as playing a role in this distribution through their significant influence on the presence of An. coluzzii larvae. WHO susceptibility assay indicated a high level of resistance to deltamethrin in the two cities. The resistance levels to deltamethrin were higher in Cotonou (X2 = 31.689; DF = 1; P < 0.0001). There was a suspected resistance to bendiocarb in Cotonou, whereas the mosquito population in Natitingou was resistant. The kdr L1014F mutation was highly observed in both mosquito populations (frequence: 86-91%), while the Ace-1 mutation was found in a small proportion of mosquitoes. In Cotonou, salinity was the only recorded physicochemical parameter that significantly correlated with the resistance of Anopheles mosquitoes to deltamethrin (P < 0.05). In Natitingou, significant correlations were observed between the allelic frequencies of the kdr L1014F mutation and pH, conductivity, and TDS. CONCLUSION: These results indicate a high level of pyrethroid resistance in the anopheles populations of both Cotonou and Natitingou. Moreover, this study report the involvement of abiotic factors influencing Anopheles susceptibility profile.

Repellent activity against Anopheles gambiae of the leaves of nesting trees in the Sebitoli chimpanzee community of Kibale National Park, Uganda.

BACKGROUND: Every evening, chimpanzees (Pan troglodytes) build a sleeping platform so called "nest" by intertwining branches of tree. Most of chimpanzees' communities studied have a preference for tree species in which they nest. As female mosquitoes are feeding on the blood of their host at nighttime, chimpanzees may prevent being disturbed and bitten by mosquitoes by selecting tree species having properties to repel them. METHODS: To test the hypothesis that chimpanzees choose tree species for their aromatic properties, data related to 1,081 nesting trees built between 2017 and 2019 in the Sebitoli community of Kibale National Park (Uganda) were analysed. The 10 most used trees were compared to the 10 most common trees in the habitat that were not preferred for nesting. Leaves from the 20 trees species were collected and hydro-distillated to obtain essential oils and one of the by-products for behavioural bioassays against females of the African mosquito, Anopheles gambiae. RESULTS: Sebitoli chimpanzees showed tree preferences: 10 species correspond to more than 80% of the nesting trees. Out of the essential oil obtained from the 10 nesting trees, 7 extracts for at least one concentration tested showed spatial repellency, 7 were irritant by contact and none were toxic. In the other hand, for the abundant trees in their habitat not used by chimpanzees, only 3 were repellent and 5 irritants. DISCUSSION AND CONCLUSION: This study contributes to evidence that chimpanzees, to avoid annoying mosquitoes, may select their nesting trees according to their repellent properties (linked to chemical parameters), a potential inspiration for human health.

Spatiotemporal multiple insecticide resistance in Aedes aegypti populations in French Guiana: need for alternative vector control.

BACKGROUND: Aedes aegypti is the sole vector of urban arboviruses in French Guiana. Overtime, the species has been responsible for the transmission of viruses during yellow fever, dengue, chikungunya and Zika outbreaks. Decades of vector control have produced resistant populations to deltamethrin, the sole molecule available to control adult mosquitoes in this French Territory. OBJECTIVES: Our surveillance aimed to provide public health authorities with data on insecticide resistance in Ae. aegypti populations and other species of interest in French Guiana. Monitoring resistance to the insecticide used for vector control and to other molecule is a key component to develop an insecticide resistance management plan. METHODS: In 2009, we started to monitor resistance phenotypes to deltamethrin and target-site mechanisms in Ae. aegypti populations across the territory using the WHO impregnated paper test and allelic discrimination assay. FINDINGS: Eight years surveillance revealed well-installed resistance and the dramatic increase of alleles on the sodium voltage-gated gene, known to confer resistance to pyrethroids (PY). In addition, we observed that populations were resistant to malathion (organophosphorous, OP) and alpha-cypermethrin (PY). Some resistance was also detected to molecules from the carbamate family. Finally, those populations somehow recovered susceptibility against fenitrothion (OP). In addition, other species distributed in urban areas revealed to be also resistant to pyrethroids. CONCLUSION: The resistance level can jeopardize the efficiency of chemical adult control in absence of other alternatives and conducts to strongly rely on larval control measures to reduce mosquito burden. Vector control strategies need to evolve to maintain or regain efficacy during epidemics.

Minimal tillage and intermittent flooding farming systems show a potential reduction in the proliferation of Anopheles mosquito larvae in a rice field in Malanville, Northern Benin.

BACKGROUND: Irrigation systems have been identified as one of the factors promoting malaria disease around agricultural farms in sub-Saharan Africa. However, if improved water management strategy is adopted during rice cultivation, it may help to reduce malaria cases among human population living around rice fields. This study aimed to assess the impact of the different irrigation practices on malaria transmission, as well as to evaluate the water management system that will best mitigate malaria transmission in Malanville, Benin. METHODS: Knowledge, Attitude and Practice (KAP) study was conducted on 104 households staying on and around the rice fields in Malanville. The study focused on the frequency of mosquito bites and preventive measures against malaria as well as soil preparation and rice planting methods. Mosquito larvae density was assessed in different water management system: continuous flooding (CF) or intermittent flooding (IF), deep tillage (DT) or minimal tillage (MT) and normal levelling (NL) or abnormal levelling (AL) in an experimental hut set-up. Larvae were collected using dipping methods and their density was determined. RESULTS: Three tillage systems, which include the use of tiller, plow and hoe, were identified on the rice field. Continuous flooding was the only irrigation system used by farmers. Retrospective data from Malanville Health Centre revealed higher malaria cases during rice production season, which was also confirmed by field participants. The density of Anopheles larvae was reduced by 80.8%, 30.8% and 40.7% (P = 0.000) during transplanting, tillering and maturation periods, respectively with intermittent flooding compared to continuous flooding. In addition, a clear reduction of larva density was observed with both intermittent flooding systems applied to minimal tillage (MT + IF + NL) and intermittent flooding applied to deep tillage (DT + IF + AL), showing that intermittent flooding could reduce the abundance of malaria vector in rice fields. CONCLUSION: Recommending intermittent flooding technology for rice cultivation may not only be useful for water management but could also be an intentional strategy to control mosquitoes vector-borne diseases around rice farms.

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing.

The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

Chemical Composition, Antimicrobial and Insecticidal Activities of Flowers Essential Oils of Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. from Martinique Island.

Alpinia species, used as ornamental plants and flavoring agents, are renowned for their therapeutic properties and their subsequent use in traditional medicines throughout the world. Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. is the most common Alpinia species encountered in Martinique. Several essential oils (EOs) obtained by hydrodistillation of A. zerumbet flowers collected in various locations on the island at different seasons were analysed to evaluate the influence of the collection period and area on the EOs' chemical compositions and to assess their bioactivity. A combination of GC-FID and GC/MS techniques was used to examine the volatile constituents, leading to the identification of a total of 71 components accounting for 97.8 - 99.3% of the respective total GC-FID areas: among them, nineteen compounds were characterized for the first time in A. zerumbet EOs. The antimicrobial activity of these EOs was assessed against eight bacterial, yeast and fungal strains and two multi-resistant strains: some significant bacteriostatic and fungistatic activities of A. zerumbet flower oils were evidenced. Finally, an interesting insecticidal activity of the flower EO was highlighted for the first time against Aedes aegypti.

Gender Bias in Insecticide Susceptibility of Aedes albopictus is Solely Attributable to Size.

Despite an extensive literature on mosquitoes, remarkably little attention has been paid to males. Current interest in control by release of transgenic males begs attention to this bias. It is well known that males are more susceptible to insecticides than females when determined by the standard World Health Organization (WHO) bioassay, and field observations have shown a higher impact of ultra-low-volume (ULV) space sprays. It is generally assumed that these differences are due to the smaller size of males and/or greater physiological susceptibility. We compared susceptibility by WHO bioassay and by topical application. There was a significant difference between the sexes in terms of dose effect and knockdown by the WHO test, but no significant difference by weight-adjusted topical application. We conclude that greater susceptibility of males is solely a function of their size and suggest that a ULV treatment before the release of transgenic males would greatly increase their competitive ratio versus wild mosquitoes and thus their impact as a control measure.

Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

Bacillus thuringiensis is successfully used in pest management strategies as an eco-friendly bioinsecticide. Isolation and identification of new strains with a wide variety of target pests is an ever growing field. In this paper, new B. thuringiensis isolates were investigated to search for original strains active against diptera and able to produce novel toxins that could be used as an alternative for the commercial H14 strain. Biochemical and molecular characterization revealed a remarkable diversity among the studied strains. Using the PCR method, cry4C/Da1, cry30Ea, cry39A, cry40 and cry54 genes were detected in four isolates. Three strains, BLB355, BLB196 and BUPM109, showed feeble activities against Aedes aegypti larvae. Interestingly, spore-crystal mixtures of BLB361, BLB30 and BLB237 were found to be active against Ceratitis capitata with an LC50 value of about 65.375, 51.735 and 42.972 µg cm(-2), respectively. All the studied strains exhibited important mortality levels using culture supernatants against C. capitata larvae. This suggests that these strains produce a wide range of soluble factors active against C. capitata larvae.

Behavioral Response of Bemisia tabaci (Hemiptera: Aleyrodidae) to 20 Plant Extracts.

In the Mediterranean region, the use of small-mesh netting to protect horticultural crops is an effective sustainable tool against pests. But in tropical regions, because of high humidity under the net favoring fungal development, netting with a larger mesh size has to be used, protecting crops against lepidopteran pests but not against small pests such as hemipterans, thrips, and phytophagous mites. A combination of netting with a repellent or irritant product is one possible solution, but the desire to reduce the use of synthetic chemicals and mitigate resistance issues calls for a natural alternative. The objective of this study was to evaluate the repellent, irritant, and toxic effects of nets dipped in 20 different plant extracts on Bemisia tabaci (Gennadius) adults. The repellent effect of volatile compounds was evaluated using a still-air olfactometer. The irritant effect and toxicity were evaluated with a no-choice test in tubes separated into two parts by an impregnated net. Our results showed the seven most irritant and toxic products against B. tabaci were aframomum, cinnamon, geranium, dill, citronella, litsea, and savory. The most repellent were aframomum and lemongrass, although cinnamon, geranium, and savory were also repellent at higher doses. Effects varied with the plant extract and the concentration, and effects were independent of one another, i.e., an essential oil can be irritant but not repellent, suggesting that the repellent mechanism and that behind the irritant or toxic effects is not the same. The use of repellent compounds in combination with netting as new pest control strategy is discussed.

Interplay between Plasmodium infection and resistance to insecticides in vector mosquitoes.

Despite its epidemiological importance, the impact of insecticide resistance on vector-parasite interactions and malaria transmission is poorly understood. Here, we explored the impact of Plasmodium infection on the level of insecticide resistance to dichlorodiphenyltrichloroethane (DDT) in field-caught Anopheles gambiae sensu stricto homozygous for the kdr mutation. Results showed that kdr homozygous mosquitoes that fed on infectious blood were more susceptible to DDT than mosquitoes that fed on noninfectious blood during both ookinete development (day 1 after the blood meal) and oocyst maturation (day 7 after the blood meal) but not during sporozoite invasion of the salivary glands. Plasmodium falciparum infection seemed to impose a fitness cost on mosquitoes by reducing the ability of kdr homozygous A. gambiae sensu stricto to survive exposure to DDT. These results suggest an interaction between Plasmodium infection and the insecticide susceptibility of mosquitoes carrying insecticide-resistant alleles. We discuss this finding in relation to vector control efficacy.

Insecticide exposure impacts vector-parasite interactions in insecticide-resistant malaria vectors.

Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles-Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions.

Reports of long-lasting insecticidal bed nets catching on fire: a threat to bed net users and to successful malaria control?

BACKGROUND: One of the control tools to reduce malaria transmission is the use of LLINs. However, several studies show that household bed net use is quite low. A study was developed to better understand the cultural factors that might explain these gaps in Benin. One reason mentioned is that bed nets can catch on fire and cause harm. This paper presents a summary of these findings, their analysis and the ensuing issues. METHODS: This anthropological study is based on an inductive qualitative approach, including 91 semi-structured interviews conducted from July 2011 to March 2012 in a health district in Southern Benin. RESULTS: Fifty-six persons stated that bed nets can catch on fire but do not always refer to specific facts. However, 34 of the 56 people narrate specific events they heard or experienced. 39 accounts were geographically located and situated in time, with various details. In 27 situations, people were burned, for which 12 people reportedly died. DISCUSSION: The disparity between these results and the dearth of bibliographic documentation in the initial search prompted a more in-depth literature review: 16 contributions between 1994 and 2013 were found. Bed net fires were noted in 10 countries, but it is impossible to ascertain the frequency of such events. Moreover, bodily harm can be significant, and several cases of death attributed to bed net fires were noted. CONCLUSIONS: Indisputably, the use of bed nets to reduce the impact of this terrible disease is an optimal control method. However, the perception that LLINs have a potentially negative effect hinders the use rate in the real world, at least for some. If some people fear the risk of fires, this possibility must be addressed during information and prevention sessions on malaria, with a communication strategy tailored to specific social contexts. Moreover, all possible measures should be taken to limit the harm suffered by individuals and their families.

Insecticidal activities of bark, leaf and seed extracts of Zanthoxylum heitzii against the African malaria vector Anopheles gambiae.

The olon tree, Zanthoxylum heitzii (syn. Fagara heitzii) is commonly found in the central-west African forests. In the Republic of Congo (Congo-Brazzaville) its bark is anecdotally reported to provide human protection against fleas. Here we assess the insecticidal activities of Z. heitzii stem bark, seed and leaf extracts against Anopheles gambiae s.s, the main malaria vector in Africa. Extracts were obtained by Accelerated Solvent Extraction (ASE) using solvents of different polarity and by classical Soxhlet extraction using hexane as solvent. The insecticidal effects of the crude extracts were evaluated using topical applications of insecticides on mosquitoes of a susceptible reference strain (Kisumu [Kis]), a strain homozygous for the L1014F kdr mutation (kdrKis), and a strain homozygous for the G119S Ace1R allele (AcerKis). The insecticidal activities were measured using LD50 and LD95 and active extracts were characterized by NMR spectroscopy and HPLC chromatography. Results show that the ASE hexane stem bark extract was the most effective compound against An. gambiae (LD50 = 102 ng/mg female), but was not as effective as common synthetic insecticides. Overall, there was no significant difference between the responses of the three mosquito strains to Z. heitzii extracts, indicating no cross resistance with conventional pesticides.

Biological effects of Lippia alba essential oil against Anopheles gambiae and Aedes aegypti.

The management of mosquito resistance to chemical insecticides and the biting behaviour of some species are motivating the search for complementary and/or alternative control methods. The use of plants is increasingly considered as a sustainable biological solution for vector control. The aim of this study was to evaluate the biological effects of the essential oil (EO) of Lippia alba harvested in Abidjan (Côte d'Ivoire) against Anopheles gambiae and Aedes aegypti mosquitoes. Phytochemical compounds were identified by GC-MS. Knockdown and mortality were determined according to the WHO test tube protocol. Contact irritancy was assessed by observing the movement of mosquitoes from a treated WHO tube to a second untreated tube. Non-contact repellency was assessed using a standardised high-throughput screening system (HITSS). Blood meal inhibition was assessed using a membrane feeding assay treated with EO. The EO was identified as the citral chemotype. The EO gave 100% KD60 in both species at a concentration of 1%. Mortalities of 100% were recorded with An. gambiae and Ae. aegypti at concentrations of 1% and 5% respectively. The highest proportions of females escaping during the contact irritancy test were 100% for An. gambiae at 1% concentration and 94% for Ae. aegypti at 2.5% concentration. The 1% concentration produced the highest proportions of repelled mosquitoes in the non-contact repellency tests: 76.8% (An. gambiae) and 68.5% (Ae. aegypti). The blood meal inhibition rate at a dose of 10% was 98.4% in Ae. aegypti but only 15.5% in An. gambiae. The citral chemotype of L. alba EO has promising biological effects in both species that make it a potentially good candidate for its use in mosquito control. The results obtained in this study encourage the further evaluation of L. alba EOs from other localities and of different chemotypes, under laboratory and field conditions.

Importance of adequate local spatiotemporal transmission measures in malaria cohort studies: application to the relation between placental malaria and first malaria infection in infants.

According to several studies, infants whose mothers had a malaria-infected placenta (MIP) at delivery are at increased risk of a first malaria infection. Immune tolerance caused by intrauterine contact with the parasite could explain this phenomenon, but it is also known that infants who are highly exposed to Anopheles mosquitoes infected with Plasmodium are at greater risk of contracting malaria. Consequently, local malaria transmission must be taken into account to demonstrate the immune tolerance hypothesis. From data collected between 2007 and 2010 on 545 infants followed from birth to age 18 months in southern Benin, we compared estimates of the effect of MIP on time to first malaria infection obtained through different Cox models. In these models, MIP was adjusted for either 1) "village-like" time-independent exposure variables or 2) spatiotemporal exposure prediction derived from local climatic, environmental, and behavioral factors. Only the use of exposure prediction improved the model's goodness of fit (Bayesian Information Criterion) and led to clear conclusions regarding the effect of placental infection, whereas the models using the village-like variables were less successful than the univariate model. This demonstrated clearly the benefit of adequately taking transmission into account in cohort studies of malaria.

Proposed use of spatial mortality assessments as part of the pesticide evaluation scheme for vector control.

BACKGROUND: The WHO Pesticide Evaluation Scheme to evaluate the efficacy of insecticides does not include the testing of a lethal effect at a distance. A tool was developed to evaluate the spatial mortality of an insecticide product against adult mosquitoes at a distance under laboratory and field conditions. Operational implications are discussed. METHODS: Insecticide paint, Inesfly 5A IGR™, containing two organophosphates (OPs): chlorpyrifos and diazinon, and one insect growth regulator (IGR): pyriproxyfen, was the product tested. Laboratory tests were performed using "distance boxes" with surfaces treated with one layer of control or insecticide paint at a dose of 1 kg/6 sq m. Field tests were conducted up to 12 months in six experimental huts randomly allocated to control or one or two layers of insecticide paint at 1 kg/6 sq m. All distance tests were performed using reference-susceptible strains of Anopheles gambiae and Culex quinquefasciatus left overnight at a distance of 1 m from control or treated surfaces. RESULTS: After an overnight exposition at distances of 1 m, field and laboratory evaluations at 0 months after treatment (T0) yielded 100% mortality rates on surfaces treated with one layer at 1 kg/6 sq m against susceptible strains of An. gambiae and Cx. quinquefasciatus. Testing for long-term efficacy in the field gave mortality rates of 96-100% after an overnight exposition at a distance of 1 m for up to 12 months in huts where a larger volume was treated (walls and ceilings) with one or two layers of insecticide paint. CONCLUSION: A comprehensive evaluation of the full profile of insecticide products, both upon contact and spatially, may help rationalize vector control efforts more efficiently. Treating a large enough volume may extend a product's mortality efficacy in the long-term, which contact tests would fail to assess. It is hereby proposed to explore the development of cost effective methods to assess spatial mortality and to include them as one additional measurement of insecticide efficacy against mosquitoes and other arthropod vectors in WHOPES Phase I and Phase II studies.

Insecticide resistance status of Anopheles gambiae s.s population from M'Bé: a WHOPES-labelled experimental hut station, 10 years after the political crisis in Côte d'Ivoire.

BACKGROUND: An experimental hut station built at M'Bé in 1998 was used for many years for the evaluation of insecticidal product for public health until the civil war broke out in 2002. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices and the West African Rice Development Association (WARDA, actually AfricaRice) in a large rice growing area. Ten years after the crisis, bioassays, molecular and biochemical analyses were conducted to update the resistance status and study the evolution of resistance mechanisms of Anopheles gambiae s.s population. METHODS: Anopheles gambiae s.s larvae from M'Bé were collected in breeding sites and reared until emergence. Resistance status of this population to conventional insecticides was assessed using WHO bioassay test kits for adult mosquitoes, with 10 insecticides belonging to pyrethroids, pseudo-pyrethroid, organochlorides, carbamates and organophosphates with and without the inhibitor piperonyl butoxyde (PBO). Molecular and biochemical assays were carried out to identify the L1014F kdr, L1014S kdr and ace-1(R) alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO) level, non-specific esterases (NSE) and glutathione S-transferases (GST) activities. RESULTS AND DISCUSSION: Anopheles gambiae s.s from M'Bé exerted high resistance levels to organochlorides, pyrethroids, and carbamates. Mortalities ranged from 3% to 21% for organochlorides, from 50% to 75% for pyrethroids, 34% for etofenprox, the pseudo-pyrethroid, and from 7% to 80% for carbamates. Tolerance to organophosphates was observed with mortalities ranging from 95% to 98%. Bioassays run with a pre-exposition of mosquitoes to PBO induced very high levels of mortalities compared to the bioassays without PBO, suggesting that the resistance to pyrethroid and carbamate relied largely on detoxifying enzymes' activities. The L1014F kdr allelic frequency was 0.33 in 2012 compared to 0.05 before the crisis in 2002. Neither the L1014S kdr nor ace-1(R) mutations were detected. An increased activity of NSE and level of MFO was found relative to the reference strain Kisumu. This was the first evidence of metabolic resistance based resistance in An. gambiae s.s from M'Bé. CONCLUSION: The An. gambiae s.s population showed very high resistance to organochlorides, pyrethroids and carbamates. This resistance level relied largely on two major types of resistance: metabolic and target-site mutation. This multifactorial resistance offers a unique opportunity to evaluate the impact of both mechanisms and their interaction with the vector control tools currently used or in development.

Changes in Anopheles funestus biting behavior following universal coverage of long-lasting insecticidal nets in Benin.

BACKGROUND: Behavioral modification of malaria vectors in response to vector control methods is of great concern. We investigated whether full coverage of long-lasting insecticide-treated mosquito nets (LLINs) may induce a switch in biting behavior in Anopheles funestus, a major malaria vector in Africa. METHODS: Human-landing collections were conducted indoor and outdoor in 2 villages (Lokohouè and Tokoli) in Benin before and 1 year and 3 years after implementation of universal LLIN coverage. Proportion of outdoor biting (POB) and median catching times (MCT) were compared. The resistance of A. funestus to deltamethrin was monitored using bioassays. RESULTS: MCT of A. funestus switched from 2 AM in Lokohoué and 3 AM in Tokoli to 5 AM after 3 years (Mann-Whitney U test, P < .0001). In Tokoli, POB increased from 45% to 68.1% (odds ratio = 2.55; 95 confidence interval = 1.72-3.78; P < .0001) 1 year after the universal coverage, whereas POB was unchanged in Lokohoué. In Lokohoué, however, the proportion of A. funestus that bites after 6 am was 26%. Bioassays showed no resistance to deltamethrin. CONCLUSIONS: This study provides evidence for a switch in malaria vectors' biting behavior after the implementation of LLIN at universal coverage. These findings might have direct consequences for malaria control in Africa and highlighted the need for alternative strategies for better targeting malaria vectors.