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.

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.

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 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.

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.

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.

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.

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.

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.

Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management.

BACKGROUND: Mayotte, a small island in the Indian Ocean, has been affected for many years by vector-borne diseases. Malaria, Bancroftian filariasis, dengue, chikungunya and Rift Valley fever have circulated or still circulate on the island. They are all transmitted by Culicidae mosquitoes. To limit the impact of these diseases on human health, vector control has been implemented for more than 60 years on Mayotte. In this study, we assessed the resistance levels of four major vector species (Anopheles gambiae, Culex pipiens quinquefasciatus, Aedes aegypti and Aedes albopictus) to two types of insecticides: i) the locally currently-used insecticides (organophosphates, pyrethroids) and ii) alternative molecules that are promising for vector control and come from different insecticide families (bacterial toxins or insect growth regulators). When some resistance was found to one of these insecticides, we characterized the mechanisms involved. METHODS: Larval and adult bioassays were used to evaluate the level of resistance. When resistance was found, we tested for the presence of metabolic resistance through detoxifying enzyme activity assays, or for target-site mutations through molecular identification of known resistance alleles. RESULTS: Resistance to currently-used insecticides varied greatly between the four vector species. While no resistance to any insecticides was found in the two Aedes species, bioassays confirmed multiple resistance in Cx. p. quinquefasciatus (temephos: ~ 20 fold and deltamethrin: only 10% mortality after 24 hours). In An. gambiae, resistance was scarce: only a moderate resistance to temephos was found (~5 fold). This resistance appears to be due only to carboxyl-esterase overexpression and not to target modification. Finally, and comfortingly, none of the four species showed resistance to any of the new insecticides. CONCLUSIONS: The low resistance observed in Mayotte's main disease vectors is particularly interesting, because it leaves a range of tools useable by vector control services. Together with the relative isolation of the island (thus limited immigration of mosquitoes), it provides us with a unique place to implement an integrated vector management plan, including all the good practices learned from previous experiences.

Multiple insecticide resistances in the disease vector Culex p. quinquefasciatus from Western Indian Ocean.

Several mosquito-borne diseases affect the Western Indian Ocean islands. Culex pipiens quinquefasciatus is one of these vectors and transmits filariasis, Rift Valley and West Nile viruses and the Japanese encephalitis. To limit the impact of these diseases on public health, considerable vector control efforts have been implemented since the 50s, mainly through the use of neurotoxic insecticides belonging to Organochlorines (OC), Organophosphates (OP) and pyrethroids (PYR) families. However, mosquito control failures have been reported on site, and they were probably due to the selection of resistant individuals in response to insecticide exposure. In this study, we used different approaches to establish a first regional assessment of the levels and mechanisms of resistance to various insecticides. Bioassays were used to evaluate resistance to various insecticides, enzyme activity was measured to assess the presence of metabolic resistances through elevated detoxification, and molecular identification of known resistance alleles was investigated to determine the frequency of target-site mutations. These complementary approaches showed that resistance to the most used insecticides families (OC, OP and PYR) is widespread at a regional scale. However, the distribution of the different resistance genes is quite heterogeneous among the islands, some being found at high frequencies everywhere, others being frequent in some islands and absent in others. Moreover, two resistance alleles displayed clinal distributions in Mayotte and La Réunion, probably as a result of a heterogeneous selection due to local treatment practices. These widespread and diverse resistance mechanisms reduce the capacity of resistance management through classical strategies (e.g. insecticide rotation). In case of a disease outbreak, it could undermine the efforts of the vector control services, as only few compounds could be used. It thus becomes urgent to find alternatives to control populations of Cx. p. quinquefasciatus in the Indian Ocean.

Efficacy of six neonicotinoid insecticides alone and in combination with deltamethrin and piperonyl butoxide against pyrethroid-resistant Aedes aegypti and Anopheles gambiae (Diptera: Culicidae).

BACKGROUND: In this study, carried out on pyrethroid-resistant Aedes aegypti and Anopheles gambiae, different combinations containing pyrethroid deltamethrin, piperonyl butoxide (PBO) synergist and six neonicotinoid insecticides (acetamiprid, nitenpyram, thiamethoxam, clothianidin, imidacloprid and thiacloprid) were tested. The bioassays were carried out by tarsal contact on impregnated paper on Ae. aegypti and with impregnated pieces of netting on An. gambiae. RESULTS: The combination of deltamethrin and PBO was synergistic against pyrethroid-resistant Ae. aegypti (LHP) and An. gambiae (VKPR) strains, with 43 and 57% mortality respectively, versus 17 and 12% with deltamethrin alone. On LHP Ae. aegypti, the deltamethrin + PBO + neonicotinoid mixtures generated insecticide efficacies that were either greater (group 1) or lower (group 2) than the deltamethrin + PBO mixture. The tricomponent mixtures made with thiamethoxam, nitenpyram and thiacloprid (70.7, 64.9 and 55.9% mortality respectively) belong to group 1. Group 2 consists of mixtures made with imidacloprid, clothianidin and acetamiprid (28.9, 32.9 and 34.3% mortality respectively). The nettings impregnated with the three tricomponent mixtures of group 1 confirmed the excellent efficacy of these mixtures against An. gambiae VKPR (87-89% mortality). CONCLUSION: Deltamethrin + PBO + neonicotinoid (group 1) mixtures induce interesting, efficient synergies, most valuable for the management of mosquito resistance to insecticides.

Insecticide resistance in the dengue vector Aedes aegypti from Martinique: distribution, mechanisms and relations with environmental factors.

Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the "knock-down resistance" V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.

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.

Pyrethroid resistance reduces the efficacy of space sprays for dengue control on the island of Martinique (Caribbean).

BACKGROUND: Dengue fever is reemerging on the island of Martinique and is a serious threat for the human population. During dengue epidemics, adult Aedes aegypti control with pyrethroid space sprays is implemented in order to rapidly reduce transmission. Unfortunately, vector control programs are facing operational challenges with the emergence of pyrethroid resistant Ae. aegypti populations. METHODOLOGY/PRINCIPAL FINDINGS: To assess the impact of pyrethroid resistance on the efficacy of treatments, applications of deltamethrin and natural pyrethrins were performed with vehicle-mounted thermal foggers in 9 localities of Martinique, where Ae. aegypti populations are strongly resistant to pyrethroids. Efficacy was assessed by monitoring mortality rates of naturally resistant and laboratory susceptible mosquitoes placed in sentinel cages. Before, during and after spraying, larval and adult densities were estimated. Results showed high mortality rates of susceptible sentinel mosquitoes treated with deltamethrin while resistant mosquitoes exhibited very low mortality. There was no reduction of either larval or adult Ae. aegypti population densities after treatments. CONCLUSIONS/SIGNIFICANCE: This is the first documented evidence that pyrethroid resistance impedes dengue vector control using pyrethroid-based treatments. These results emphasize the need for alternative tools and strategies for dengue control programs.

Field efficacy of new larvicide products for control of multi-resistant Aedes aegypti populations in Martinique (French West Indies).

World-wide dengue vector control is hampered by the spread of insecticide resistance in Aedes aegypti. We report the resistance status of a wild Ae. aegypti population from Martinique (Vauclin) to conventional larvicides (Bacillus thuringiensis var israeliensis [Bti] and temephos) and potential alternatives (spinosad, diflubenzuron, and pyriproxyfen). The efficacy and residual activity of these insecticides were evaluated under simulated and field conditions. The Vauclin strain exhibited a high level of resistance to temephos, a tolerance to insect growth regulators, and full susceptibility to spinosad and Bti. In simulated trials, pyriproxyfen and Bti showed long residual activities in permanent breeding containers (28 and 37 weeks), whereas under field conditions they failed to curtail Ae. aegypti populations after four weeks. Conversely, diflubenzuron and spinosad showed a residual efficacy of 16 weeks, suggesting that these chemicals may be promising alternatives to Bti and temephos for controlling insecticide-resistant Ae. aegypti populations.

Field evaluation of pyriproxyfen and spinosad mixture for the control of insecticide resistant Aedes aegypti in Martinique (French West Indies).

BACKGROUND: The resistance of Ae. aegypti to insecticides is already widespread and continues to develop. It represents a serious problem for programmes aimed at the control and prevention of dengue in tropical countries. In the light of this problem measures to control Ae. aegypti are being orientated towards how best to use existing insecticides, notably by combining those that have different modes of action. RESULTS: In this study we evaluated the operational efficiency of a mixture composed of pyriproxyfen (an insect growth regulator) and spinosad (a biopesticide) against a population of Ae. aegypti from Martinique resistant to pyrethroid and organophosphate insecticides. The first step consisted of evaluating the efficacy of pyriproxyfen and spinosad when used alone, or in combination, against Ae. aegypti larvae under simulated conditions. The results showed that the mixture of pyriproxyfen+spinosad remained active for at least 8 months, compared with 3 months for spinosad alone, and 5 months for pyriproxyfen alone. In a second step in containers experiencing natural conditions, pyriproxyfen and spinosad, maintained the rate of adult emergence at 20% for 3 weeks and 3.5 months, respectively. Following the same criteria of evaluation, the mixture pyriproxyfen+spinosad remained effective for 4.5 months, showing that the combination of the two larvicides with different modes of action acted to increase the residual activity of the treatment. CONCLUSION: The mixture of pyriproxyfen and spinosad kills larvae and pupae giving it a broader range of action than either insecticide. This mixture could preserve the utility of both insecticides in public health programs.

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.

Reduced efficacy of pyrethroid space sprays for dengue control in an area of Martinique with pyrethroid resistance.

In the Caribbean, insecticide resistance is widely developed in Aedes aegypti and represents a serious obstacle for dengue vector control. The efficacy of pyrethroid and organophosphate ultra-low volume space sprays was investigated in Martinique where Ae. aegypti has been shown to be resistant to conventional insecticides. In the laboratory, a wild-field caught population showed high levels of resistance to deltamethrin, organophosphate (naled), and pyrethrum. Simulated-field trials showed that this resistance can strongly reduce the knock-down effect and mortality of deltamethrin and synergized pyrethrins when applied by thermal-fogging. Conversely, the efficacy of naled was high against insecticide-resistant mosquitoes. Chemical analyses of nettings exposed to the treatments showed a decrease in residues over distance from release for the pyrethroids, and naled was not detected. This finding has important implications for dengue vector control and emphasizes the need to develop innovative strategies to maintain effective control of resistant Ae. aegypti populations.