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.

Invasive hematophagous arthropods and associated diseases in a changing world.

Biological invasions have increased significantly with the tremendous growth of international trade and transport. Hematophagous arthropods can be vectors of infectious and potentially lethal pathogens and parasites, thus constituting a growing threat to humans-especially when associated with biological invasions. Today, several major vector-borne diseases, currently described as emerging or re-emerging, are expanding in a world dominated by climate change, land-use change and intensive transportation of humans and goods. In this review, we retrace the historical trajectory of these invasions to better understand their ecological, physiological and genetic drivers and their impacts on ecosystems and human health. We also discuss arthropod management strategies to mitigate future risks by harnessing ecology, public health, economics and social-ethnological considerations. Trade and transport of goods and materials, including vertebrate introductions and worn tires, have historically been important introduction pathways for the most prominent invasive hematophagous arthropods, but sources and pathways are likely to diversify with future globalization. Burgeoning urbanization, climate change and the urban heat island effect are likely to interact to favor invasive hematophagous arthropods and the diseases they can vector. To mitigate future invasions of hematophagous arthropods and novel disease outbreaks, stronger preventative monitoring and transboundary surveillance measures are urgently required. Proactive approaches, such as the use of monitoring and increased engagement in citizen science, would reduce epidemiological and ecological risks and could save millions of lives and billions of dollars spent on arthropod control and disease management. Last, our capacities to manage invasive hematophagous arthropods in a sustainable way for worldwide ecosystems can be improved by promoting interactions among experts of the health sector, stakeholders in environmental issues and policymakers (e.g. the One Health approach) while considering wider social perceptions.

Laboratory study of an innovative concept to control aphid pests and mosquito vectors of pathogens to humans.

BACKGROUND: A great number of areas favourable for the proliferation of mosquitoes are found in farmland, the most favourable being market gardens and rice paddies. The only means of limiting crop pests (AGRI) and mosquito vectors of human pathogens (AC = antivectorial control) in agricultural environments consists of incorporating systemic or translaminar insecticides into fertilizers (AGRIAC). The plant used in this study was the chili pepper (Capsicum chinensis). Experiments were carried out with Myzus persicae aphid pests and Aedes albopictus mosquitoes. Fertilizers were applied in association with thiacloprid, cyromazine, azadirachtin and Bacillus thuringiensis var. israelensis (Bti) + Bacillus sphaericus (Bs) insecticides. Chili pepper seedlings were dried to assess the amount of insecticide in their tissues. RESULTS: NPK + thiacloprid and NPK + azadirachtin remained efficient against aphids for 28 days, whereas Bti + Bs had to be added to both mixtures to make them efficient against Ae. albopictus larvae. NPK + cyromazine remained active against both aphids and mosquitoes for 15 days. The search for insecticide residues in chili pepper showed that untreated plants displayed natural toxicity to Ae. albopictus larvae. The toxic effects induced by the capsaicinoid compounds inside the plant add up to the insecticide action of thiacloprid, cyromazine and azadiractin. CONCLUSION: The AGRIAC concept allows for an innovative vector control method that can manage aphid pests and mosquitoes while providing plants with the needed fertilizer.

The bio-interactions between plants, insecticides and fertilizers: an innovative approach for the research of xenobiotic substances.

In this experiment carried out on Caribbean chili pepper plants (Capsicum chinensis), the bio-insecticide azadirachtin in combination with an NPK fertilizer proved to have a greater lethal impact on the larvae of Aedes albopictus than each substance on its own. This synergistic effect is noticeably important when both inputs are sprayed directly on the leaves of the plant (foliar application). While the plants treated with azadirachtin or NPK alone cause a 33.6% and 36.4% mortality respectively of the Ae. albopictus larvae, the combination of the two inputs induces a 74.4% mortality on the mosquito larvae. To account for this synergistic effect phenomenon inside the plant, the azadirachtin + NPK combination most likely interacts with the capsaicinoid compounds naturally produced by the plant. Not only does this study carried out on azadirachtin reveal major results but the methodology itself offers a most interesting approach on how to boost the agricultural inputs within the plants. As a matter of fact, this research axis demands developing since the control of pests harmful to men has been dramatically lacking insecticide molecules acting on new targets over the past three decades.

Combining piperonyl butoxide and dinotefuran restores the efficacy of deltamethrin mosquito nets against resistant Anopheles gambiae (Diptera: Culicidae).

One strategy suggested for the management of mosquito insecticide resistance consists of combining a pyrethroid with an insecticide that has a different mode of action. To restore the efficacy of deltamethrin (pyrethroid) against pyrethroid-resistant strain of Anopheles gambiae Giles (VKPR: homozygous Kdr), deltamethrin was combined with the neonicotinoid insecticide dinotefuran and piperonyl butoxide (PBO). Bednets impregnated with deltamethrin, dinotefuran, and PBO alone and in combination were tested in the laboratory. Knockdown (KD) and mortality were measured using WHO cone tests on susceptible and pyrethroid-resistant adult mosquitoes. The combination of deltamethrin and PBO was synergistic against resistant female An. gambiae (58.2% mortality). Both mortality and knockdown time (KDt(50/95) values) of the tricomponent mixture on the VKPR strain were similar to the insecticidal activity of deltamethrin on the pyrethroid-susceptible KIS strain (98.8 and 100% mortality, respectively). The three-compound mixture of deltamethrin + PBO + dinotefuran showed an insecticidal efficacy greater than the deltamethrin + PBO mixture to the extent of completely restoring the efficacy of deltamethrin on pyrethroid-resistant An. gambiae.

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies).

BACKGROUND: The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. RESULTS: The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR. CONCLUSION: These results suggest that the high level of insecticide resistance found in Ae. aegypti mosquitoes from Martinique island is the consequence of both target-site and metabolic based resistance mechanisms. Insecticide resistance levels and associated mechanisms are discussed in relation with the environmental context of Martinique Island. These finding have important implications for dengue vector control in Martinique and emphasizes the need to develop new tools and strategies for maintaining an effective control of Aedes mosquito populations worldwide.

The combinatory effect of Cyt1Aa flexibility and specificity against dipteran larvae improves the toxicity of Bacillus thuringensis kurstaki toxins.

Cyt1A98 is a novel cytolytic protein, from BUPM98 Bacillus thuringiensis strain, characterized by its synergistic activity with B. thuringiensis kurstaki toxins against lepidopteran larvae. In this study, we evidenced that Cyt1A98 improves the toxicity of B. thuringiensis kurstaki toxins against Aedes aegypti larvae. In fact, the strain BNS3pHTcyt1A98 exhibited a larvicidal activity of about 849-fold of that of BNS3pHTBlue against A. aegypti. The molecular and biochemical characterizations, of cyt1A98 gene and its product, were achieved. Cyt1A98 had an LC50 value of about 126.56 mg l-1 against A. aegypti larvae. Compared to Cyt1Aa of B. thuringiensis israelensis, Cyt1A98 amino acid sequence harbours three substitutions of three conserved amino acids among Cyt1Aa family members (Ser42Pro, Pro82Ala, Met188Thr). The Cyt1A98 protein structural analysis evidenced more flexibility than Cyt1Aa. According to the high fluctuation observed for the residue Pro42, the amino acid at position 42 is implicated in the flexibility property of Cyt1Aa especially for the αC and αD helices, involved in the penetration into the cell membrane. The toxicity improvement could be probably due to the higher flexibility combined with the specific affinity toward dipteran larvae. The Cyt1A/B. thuringiensis kurstaki Cry toxins model provides a potential molecular genetic strategy for an efficient bioinsecticide.

[Attractive properties and physicochemical modifications of water following colonization by Aedes aegypti larvae (Diptera: Culicidae)]. / Propriétés attractives et modifications physicochimiques des eaux de gîtes colonisées par des larves de Aedes aegypti (Diptera : Culicidae).

The authors investigated in this paper the attractiveness of a water colonized ('positive' breeding site) or not ('negative' breeding site) by larvae of Aedes aegypti on gravid females and then characterized their physicochemical properties to identify and quantify the principal components. The results showed that the water that sheltered several generations of larvae was more attractive to gravid females than water that had never been colonized. The water in a 'positive' breeding site contains 2.5 times more ammonium ions (NH(+)(4)), four times more nitrite ions (NO(-)(2)) and 20 times more nitrate ions (NO(-)(3)) than 'negative' breeding water. The authors suggest that in the 'positive' breeding sites, where the larvae absorb the organic matter, the turbidity of water decreases, which makes it possible for organic nitrogen to be converted into nitrites and nitrates via the nitrifying activity of the bacteria (NH(+)(4)-->NO(-)(2)-->NO(-)(3)). It is likely that chitin, while accumulating and decomposing at the bottom of the breeding site, may increase the percentage of organic carbon and ammonium ions, then reinforcing the nitrification process. Conversely, in the 'negative' breeding site where the conditions of evolution of the nitrifying bacteria are more restrictive, the nitrogen remains primarily in its organic form.

Laboratory evaluation of cyromazine against insecticide-susceptible and -resistant mosquito larvae.

In this study, the activity of cyromazine was evaluated following WHO standard procedures against susceptible and resistant mosquito strains of Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti. The dose for 50% and 90% inhibition of adult emergence (IE50 and IE90) ranged from 0.028 mg/liter to 0.17 mg/liter and from 0.075 mg/liter to 0.42 mg/liter, respectively. The effects of cyromazine were closer to that of chitin synthesis inhibitors rather than that of juvenile hormone analogues, with only 10-20% pupal mortality. The toxicity of cyromazine was not strongly affected by the presence of common resistance mechanism, i.e., Kdr mutation and Ace.1(R) (resistance ratio from 0.5 to 2.3). The absence of cross resistance with common insecticides (pyrethroids, carbamates, organophosphates) makes cyromazine a potential candidate for disease vector control, especially for the management of insecticide resistance.

Synergistic Effect of Fertilizer and Plant Material Combinations on the Development of Aedes aegypti (Diptera: Culicidae) and Anopheles gambiae (Diptera: Culicidae) Mosquitoes.

Chemical fertilizers are used everywhere, which often pollute the breeding sites of mosquitoes. In this laboratory study, the consequences on Aedes aegypti (L.) (Diptera: Culicidae) and Anopheles gambiae (Giles) (Diptera: Culicidae) of water-containing plant matter (PM) alone, or in association with an NPK type of fertilizer (PM+NPK), were evaluated. To obtain a 20% imaginal emergence of An. gambiae (IEt20), the bioassays carried out with PM have evidenced that its larvae need four times as much food as for Ae. aegypti larvae. The PM+NPK combinations significantly improve the survival rates of both mosquitoes multiplying the percentages of imaginal emergence by 1.7-3 (synergistic effect). The log-probit analysis of the adult emergence also reveals that the environments containing fertilizers accelerates by two to four times the development of the mosquito larvae.

The One Health Concept: 10 Years Old and a Long Road Ahead.

Over the past decade, a significant increase in the circulation of infectious agents was observed. With the spread and emergence of epizootics, zoonoses, and epidemics, the risks of pandemics became more and more critical. Human and animal health has also been threatened by antimicrobial resistance, environmental pollution, and the development of multifactorial and chronic diseases. This highlighted the increasing globalization of health risks and the importance of the human-animal-ecosystem interface in the evolution and emergence of pathogens. A better knowledge of causes and consequences of certain human activities, lifestyles, and behaviors in ecosystems is crucial for a rigorous interpretation of disease dynamics and to drive public policies. As a global good, health security must be understood on a global scale and from a global and crosscutting perspective, integrating human health, animal health, plant health, ecosystems health, and biodiversity. In this study, we discuss how crucial it is to consider ecological, evolutionary, and environmental sciences in understanding the emergence and re-emergence of infectious diseases and in facing the challenges of antimicrobial resistance. We also discuss the application of the "One Health" concept to non-communicable chronic diseases linked to exposure to multiple stresses, including toxic stress, and new lifestyles. Finally, we draw up a list of barriers that need removing and the ambitions that we must nurture for the effective application of the "One Health" concept. We conclude that the success of this One Health concept now requires breaking down the interdisciplinary barriers that still separate human and veterinary medicine from ecological, evolutionary, and environmental sciences. The development of integrative approaches should be promoted by linking the study of factors underlying stress responses to their consequences on ecosystem functioning and evolution. This knowledge is required for the development of novel control strategies inspired by environmental mechanisms leading to desired equilibrium and dynamics in healthy ecosystems and must provide in the near future a framework for more integrated operational initiatives.

Efficacy of bifenthrin-impregnated bednets against Anopheles funestus and pyrethroid-resistant Anopheles gambiae in North Cameroon.

BACKGROUND: Recent field studies indicated that insecticide-treated bednets (ITNs) maintain their efficacy despite a high frequency of the knock-down resistance (kdr) gene in Anopheles gambiae populations. It was essential to evaluate ITNs efficacy in areas with metabolic-based resistance. METHODS: Bifenthrin was used in this experiment because it is considered a promising candidate for bednets impregnation. Nets were treated at 50 mg/m2, a dose that has high insecticidal activity on kdr mosquitoes and at 5 mg/m2, a dose that kills 95% of susceptible mosquitoes under laboratory conditions with 3 minutes exposure. Bednets were holed to mimic physical damage. The trial was conducted in three experimental huts from Pitoa, North-Cameroon where Anopheles gambiae displays metabolic resistance and cohabits with An. funestus. RESULTS: Bifenthrin at 50 mg/m2 significantly reduced anophelines' entry rate (>80%). This was not observed at 5 mg/m2. Both treatments increased exophily in An. gambiae, and to a lesser extent in An. funestus. With bifenthrin at high dosage, over 60% reduction in blood feeding and 75-90% mortality rates were observed in both vectors. Despite presence of holes, only a single An. gambiae and two An. funestus females were collected inside the treated net, and all were found dead. The same trends were observed with low dosage bifenthrin though in most cases, no significant difference was found with the untreated control net. CONCLUSION: Bifenthrin-impregnated bednets at 50 mg/m2 were efficient in the reduction of human-vector contact in Pitoa. Considerable personal protection was gained against An. funestus and metabolic pyrethroid resistant An. gambiae populations.

Laboratory evaluation of pyriproxyfen and spinosad, alone and in combination, against Aedes aegypti larvae.

In this study, the efficacy of pyriproxyfen and spinosad, alone and in combination, was evaluated against the dengue vector Aedes aegypti (L.). Larval bioassays were carried out on susceptible mosquito larvae to determine the concentration-mortality responses of mosquitoes exposed to each insecticide alone and in mixture. Synergism between pyriproxyfen and spinosad was determined by the calculation of a combination index (CI) by using the isobologram method. For pyriproxyfen, LC50 and LC95 were 1.1 x 10(-4) (1.0 x 10(-4)-1.1 x 10(-4)) and 3.2 x 10(-4) (2.9 x 10(-4)-3.6 x 10(-4)) mg/liter, respectively. Pyriproxyfen acted at very low concentrations by inhibiting the adult emergence of Ae. aegypti (97% inhibition rates at 3.3 x 10(-4) mg/liter). Spinosad activity was -500 times lower than that of pyriproxyfen against the Bora strain, with LC50 and LC95 values estimated at 0.055 (0.047-0.064) and 0.20 (0.15-0.27) mg/liter, respectively. A binary mixture of pyriproxyfen and spinosad was realized at the ratio 1:500 by considering the values of the LC50 obtained for each product. The LC50 and LC95 of the mixture were 0.019 (0.016 - 0.022) and 0.050 (0.040 - 0.065) mg/liter, respectively. The mixture combined both the larvicidal activity of spinosad and the juvenoid action of pyriproxyfen. From the LC70 to LC99 a significant synergism effect was observed between the two insecticides (CI ranged from 0.74 to 0.31). This strong synergism observed at high concentrations allows a reduction by five and nine-fold of pyriproxyfen and spinosad amounts to kill almost 100% mosquitoes. Combination of pyriproxyfen and spinosad may then represent a promising strategy to improve mosquito control in situations with insecticide-resistant Aedes dengue vectors.

An anti-mosquito mixture for domestic use, combining a fertiliser and a chemical or biological larvicide.

BACKGROUND: Plant saucers are an important larval habitat for Aedes aegypti and Ae. albopictus in peridomestic situations. Because NPK fertilisers in plant containers tend to enhance the oviposition of these species, we investigated the effects of Bacillus thuringiensis var. israelensis, spinosad, pyriproxyfen and diflubenzuron larvicides in combination with fertiliser on the adult emergence and fecundity of the mosquitoes coming from plant saucers in controlled greenhouse experiments. NPK + larvicide (NPK-LAV) treatments were tested on Ae. aegypti. Each treatment was compared with water and with fertiliser alone on a total of five houseplants and their saucers. The fertilising treatment was renewed every 30-45 days. RESULTS: With less than 5% imaginal emergence, the NPK + spinosad 0.5% treatment remained effective for 30 days. Both NPK + pyriproxyfen 0.1% and NPK + diflubenzuron 0.25% were effective for 45 days. The average number of eggs laid in the three treatments was similar to the NPK treatment, indicating that spinosad, pyriproxyfen and diflubenzuron did not alter the attraction effect of the fertiliser on egg laying. NPK + pyriproxyfen and NPK + diflubenzuron also had ovicidal activity and an important impact on the fecundity of the Ae. aegypti female imagos and the fertility of their eggs. CONCLUSION: The addition of NPK fertiliser to insecticides can increase larval control of Aedes mosquitoes. This innovative measure for personal protection, which is harmless for both humans and animals, would be an additional support for the community-based actions led by the institutional services for vector control. © 2015 Society of Chemical Industry.

Experimental hut evaluation of bednets treated with an organophosphate (chlorpyrifos-methyl) or a pyrethroid (lambdacyhalothrin) alone and in combination against insecticide-resistant Anopheles gambiae and Culex quinquefasciatus mosquitoes.

BACKGROUND: Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments. METHODS: Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1R). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments. RESULTS AND CONCLUSION: All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1R and Ace.1S genes did not differ significantly from mosquitoes that carried only Ace.1S genes on any of the treated nets, indicating that the Ace.1R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.

Spinosad: a new larvicide against insecticide-resistant mosquito larvae.

Among the characteristics of spinosad that are worth noting is its environmentally favorable toxicity profile. It is a compound of biological origin, whose insecticide action is highly effective against a great number of insects. Laboratory larval bioassays of spinosad on Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae (specimens that were either susceptible or resistant to pyrethroids, carbamates, and organophosphates) showed that this product had a lethal action (mortality after 24 h of exposure) regardless of the original status, susceptible or resistant, of the mosquito larvae.

Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis: Interactions and toxicity mechanism against Aedes aegypti.

Individual crystal proteins from Bacillus thuringiensis israelensis exhibit variable levels of insecticidal activities against mosquito larvae. In all cases, they are much less active compared to the whole crystal proteins due to described complex synergistic interactions among them. In the present study we investigated the effects of Cyt1A98 (a Cyt1Aa type protein) on Cry4BLB (a Cry4Ba type toxin) insecticidal activity toward the dengue vector Aedes aegypti. The bioassay analyses demonstrated the ability of Cyt1A98 protein to enhance Cry4BLB toxin larvicidal activity even at a low proportion in the mixture (1%). In vitro interaction assays showed that Cyt1A98 provides supplementary binding sites for Cry4BLB in A. aegypti BBMVs. Moreover, it enhances the formation of Cry4BLB oligomeric structure. These results support that Cyt1A98 protein could act as a membrane-bound receptor fixing Cry4BLB δ-endotoxins and promoting its oligomerization.

Combating malaria vectors in Africa: current directions of research.

Vector control remains an important component of malaria control, particularly in Africa where most infant deaths occur. Among the different methods, insecticide-treated bednets seem to be a suitable way to reduce morbidity and child mortality in endemic areas. To facilitate their large-scale use and to investigate alternative vector control methods, the authors propose these current directions of research that are already being explored in Africa through a collaborating network involving several African countries: (1) vector genetics, (2) insecticide resistance and (3) vector control strategies.

The role of agricultural use of insecticides in resistance to pyrethroids in Anopheles gambiae s.l. in Burkina Faso.

Agricultural use of insecticides is involved in the selection of resistance to these compounds in field populations of mosquitoes in Burkina Faso. Anopheles gambiae s.l. was resistant to permethrin and DDT in cotton-growing and urban areas, but susceptible in areas with limited insecticide selection pressure (rice fields and control areas). Nevertheless, resistance to these insecticides was observed in a village on the outskirts of the rice fields at the end of the rainy season, suggesting that the latter population of mosquitoes had migrated from the surrounding cotton villages into the rice fields. A seasonal variation of resistance observed in the cotton-growing area is related to the distribution of the molecular M and S forms of An. gambiae, since resistance to pyrethroids has so far only been reported in the S form. Pyrethroid resistance in west African An. gambiae was conferred by target site insensitivity through a knockdown resistance (kdr)-like mutation, which was present at high frequencies in mosquitoes in the cotton-growing and urban areas.