BACKGROUND
every year,  <dig> to  <dig> million dengue infections world-wide causing from  <dig>  to  <dig>  deaths from dengue and hemorrhagic fever are recorded  <cit> .
as there is still no medicine and efficient vaccine available, vector control by the recourse of environmental management, educational programs and the use of chemical and biological agents, remains the only method to reduce the risk of dengue virus transmission  <cit> .
unfortunately, most of dengue vector control programs implemented worldwide are facing operational challenges with the emergence and development of insecticide resistance in ae.
aegypti  <cit>  and ae.
albopictus  <cit> .
resistance of ae.
aegypti to insecticides has been reported in many regions including south east asia  <cit> , latin america  <cit>  and the caribbean  <cit> .
inherited resistance to chemical insecticides in mosquitoes is mainly the consequence of two distinct mechanisms: the alteration of target sites inducing insensitivity to the insecticide  and/or an increased metabolism of the insecticide   <cit> .
metabolic-based resistance involves the bio-transformation of the insecticide molecule by enzymes and is now considered as a key resistance mechanism of insects to chemical insecticides  <cit> .
this mechanism may result from two distinct but additive genetic events: i) a mutation of the enzyme protein sequence leading to a better metabolism of the insecticide, and/or ii) a mutation in a non-coding regulatory region leading to the over-production of an enzyme capable of metabolizing the insecticide.
so far, only the second mechanism has been clearly associated with the resistant phenotype in mosquitoes.
three large enzyme families, the cytochrome p <dig> monooxygenases , glutathione s-transferases  and carboxy/cholinesterases  have been implicated in the metabolism of insecticides  <cit> .
the rapid expansion and diversification of these so-called 'detoxification enzymes' in insects is likely to be the consequence of their adaptation to a broad range of natural xenobiotics found in their environment such as plant toxins  <cit> .
these enzymes have also been involved in mosquito response to various anthropogenic xenobiotics such as heavy metals, organic pollutants and chemical insecticides  <cit> .
although identifying metabolic resistance is possible by toxicological and biochemical techniques, the large panel of enzymes potentially involved together with their important genetic and functional diversity makes the understanding of the molecular mechanisms and the role of particular genes a challenging task.
as more mosquito genomes have been sequenced and annotated  <cit> , the genetic diversity of genes encoding mosquito detoxification enzymes has been unravelled and new molecular tools such as the aedes and anopheles 'detox chip' microarrays allowing the analysis of the expression pattern of all detoxification genes simultaneously have been developed  <cit> .
these specific microarrays were successfully used to identify detoxification genes putatively involved in metabolic resistance in various laboratory and field-collected mosquito populations resistant to insecticides  <cit> .
in latin america and the caribbean, several ae.
aegypti populations show strong resistance to pyrethroid, carbamate and organophosphate insecticides correlated with elevated activities of at least one detoxification enzyme family  <cit> .
in addition, several points of non-synonymous mutations in the gene encoding the trans-membrane voltage-gated sodium channel  have been described and showed to confer resistance to pyrethroids and ddt  <cit> .
several questions remain concerning the impact of insecticide resistance on the efficacy of vector control operations.
in martinique , high levels of resistance to the organophosphate temephos and the pyrethroid deltamethrin were reported.
this resistance was characterized by an important reduction of both mosquito knock-down and mortality levels after thermal-fogging with deltamethrin and p450-inhibitor synergized pyrethroids, indicating that resistance was negatively impacting on control programmes and that this resistance was conferred, at least in part, by elevated cytochrome p <dig> activity  <cit> .
in this study, we explored the mechanisms conferring insecticide resistance in an ae.
aegypti population from martinique island.
larval bioassays and adult topical applications were used to determine the current resistance level of this population to insecticides.
the presence of metabolic-based resistance mechanisms was investigated by exposing mosquitoes to enzyme inhibitors prior to bioassays with insecticides and by measuring representative enzyme activities of each detoxification enzyme family.
at the molecular level, the frequency of the target-site kdr mutation was investigated and a microarray approach followed by quantitative real-time rt-pcr validation was used to identify detoxification genes putatively involved in metabolic resistance.
results from this study will help to implement more effective resistance management strategies in this major disease vector in the future.
RESULTS
larval bioassays  showed that the vauclin strain is far less affected by temephos than the susceptible bora-bora strain .
in the susceptible strain, temephos toxicity was not significantly increased in the presence of detoxification enzyme inhibitors .
by contrast, the level of resistance to temephos of the vauclin strain was significantly reduced in the presence of pbo, def and dmc  indicating the involvement of p450s, cces and in a lesser extent gsts in the resistance of larvae to temephos.
resistant ratios rr <dig> and rr <dig> were obtained by calculating the ratio between the lc <dig> and lc <dig> between vauclin and bora-bora strains; synergism ratios sr <dig> and sr <dig> were obtained by calculating the ratio between lc <dig> and lc <dig> with and without enzyme inhibitor.
: confidence interval.
significant rr and sr are shown in bold.
topical applications of the pyrethroid insecticide deltamethrin on adults of each strain  revealed that the vauclin strain is also highly resistant to deltamethrin .
in both strains, the toxicity of deltamethrin increased significantly in the presence of detoxification enzyme inhibitors, however only pbo and dmc induced higher synergistic effects in the vauclin strain than in the susceptible bora-bora strain .
in the vauclin strain, pbo and dmc significantly reduced the resistance level , indicating a significant role of p450s and gsts in the resistance of adults to deltamethrin.
resistant ratios rr <dig> and rr <dig> were obtained by calculating the ratio between the ld <dig> and ld <dig> between vauclin and bora-bora strains; synergism ratios sr <dig> and sr <dig> were obtained by calculating the ratio between ld <dig> and ld <dig> with and without enzyme inhibitor.
: confidence interval.
significant rr and sr are shown in bold.
comparison of constitutive detoxification enzyme activities between the susceptible strain bora-bora and the insecticide-resistant vauclin strain revealed significant differences at both larval and adult stages .
p <dig> activities were elevated in both larvae and adults of the vauclin strain .
similarly, gst activities were found elevated in larvae and adults of the vauclin strain .
finally, α- and β-carboxylesterase activities were also found slightly elevated in the vauclin strain in larvae  and adults .
sequencing of the voltage-gated sodium channel gene conducted on the vauclin strain showed the presence of the kdr mutation at position  <dig>  leading to the replacement of valine by an isoleucine  at a high allelic frequency  =  <dig> , n = 24) with rr =  <dig>  rs =  <dig> and ss =  <dig>  conversely, no kdr resistant allele was detected in the susceptible bora-bora strain .
we used the microarray 'aedes detox chip'  to compare the transcription levels of all ae.
aegypti detoxification genes between the insecticide-resistant strain vauclin and the susceptible strain bora-bora in larvae and adults.
overall,  <dig> and  <dig> probes out of  <dig> were detected consistently in at least  <dig> hybridisations out of  <dig> in larvae and adults respectively.
among them,  <dig> detoxification genes were significantly differentially transcribed  in larvae or adults .
most of these genes encode p450s  with  <dig> of them being differentially transcribed in the vauclin strain at both life stages .
in larvae,  <dig> genes  were found significantly differentially transcribed between the insecticide-resistant strain vauclin and the susceptible strain bora-bora .
among them,  <dig> genes were over-transcribed in the vauclin strain while only  <dig> genes were under-transcribed.
most over-transcribed genes were represented by cyp genes with a majority belonging to the cyp <dig> subfamily .
three cyp9s were also over-transcribed in larvae of the vauclin strain  with a strong over-transcription of cyp9j <dig>  together with  <dig> cyp4s .
among other over-transcribed genes,  <dig> carboxy/cholinesterases  and  <dig> glutathione s-transferase  were slightly over-transcribed in the vauclin strain.
lastly,  <dig> cyps  were under-transcribed in insecticide-resistant larvae comparatively to susceptible larvae.
in adults,  <dig> genes  were found differentially transcribed in the insecticide-resistant strain vauclin comparatively to the susceptible strain bora-bora .
as in larvae, most of the over-transcribed genes belong to the cyp <dig> and cyp <dig> subfamilies  with only  <dig> additional cces  being moderately over-transcribed in the vauclin strain.
nine genes were under-transcribed in vauclin adults, including  <dig> cyps ,  <dig> cce ,  <dig> gst  and  <dig> thioredoxin peroxidases .
interestingly cyp304c <dig> and tpx <dig> were both found strongly under-transcribed  in insecticide-resistant adults.
validation of microarray data was performed by real-time quantitative rt-pcr on  <dig> detoxification genes identified as over-transcribed in larvae or adults of the vauclin strain .
the over-transcription of genes identified from microarray experiments were all confirmed by quantitative rt-pcr in both life stages, although expression ratios obtained from rt-pcr were frequently higher than those obtained from microarray experiments.
discussion
the aim of the present study was to investigate insecticide resistance mechanisms of ae.
aegypti mosquitoes from martinique .
toxicological results confirmed the high level of resistance of the vauclin strain from martinique to the organophosphate temephos at the larval stage and to the pyrethroid deltamethrin at the adult stage  <cit> .
the use of specific detoxification enzyme inhibitors suggested that resistance of larvae to temephos is linked to carboxylesterases and to a lesser extent p450s and gsts.
in adults, resistance to deltamethrin appeared principally linked to p450s and gsts.
comparison of global detoxification enzyme activities between the two strains revealed elevated p450s, gsts and in a lesser extent cces activities in the vauclin strain at both life-stages, confirming the importance of metabolic resistance mechanisms in martinique.
carboxylesterases based-resistance mechanism is a major mechanism for organophosphate resistance in insects  <cit> .
several examples of ae.
aegypti resistance to organophosphates in the caribbean linked to elevated carboxylesterases activities have been described  <cit> .
our toxicological and biochemical data confirms these observations despite a moderate elevated level of cces activities in the vauclin strain.
among detoxification enzymes, p450s have been shown to play a major role in pyrethroid resistance in insects  <cit> .
in martinique, marcombe et al.
<cit>  suggested the involvement of p450s in the reduced efficacy of deltamethrin space-spray operations.
elevated gst levels have also been frequently associated with insect resistance to insecticides such as ddt and pyrethroids  <cit> .
our toxicological and biochemical data support the role of p450s and gsts in insecticide resistance in martinique.
at the molecular level, several mutations in the voltage-gated sodium channel gene have been associated with pyrethroid resistance in ae.
aegypti from asian, latin american and caribbean countries  <cit> .
our results revealed a high frequency  of the v1016i kdr mutation in ae.
aegypti populations from the community of vauclin.
the role of this mutation in pyrethroid resistance was clearly demonstrated by genotype-phenotype association studies  <cit> .
the high frequency of the mutation, together with the incomplete effect of enzyme inhibitors in adults, supports a contribution of this kdr mutation in deltamethrin resistance.
acetylcholinesterase  is critical for hydrolysis of acetylcholine at cholinergic nerve synapses and is a target for organophosphate and carbamate insecticides  <cit> .
altered ache is an important resistance mechanism to organophosphates in many insects.
following the methods of alout et al.
<cit>  and bourguet et al.
<cit> , ache activities of vauclin mosquitoes were determined to investigate the presence of the g119s and/or f290v mutations.
no insensitive ache phenotypes were found in any of the mosquitoes tested , suggesting that organophosphate resistance of the vauclin strain is rather due to detoxification enzymes unless other mutations occurred elsewhere in the ace gene.
our microarray screening identified  <dig> and  <dig> over-transcribed detoxification genes in larvae and adults of the vauclin strain respectively.
among them,  <dig> p450s , the glutathione s-transferase gste <dig> and the carboxy/cholinesterase cceae3a were all confirmed to be over-transcribed at both life-stages, supporting their involvement in insecticide-resistance.
other genes appeared more highly over-transcribed in adults  or in larvae , suggesting that particular enzymes might be more specifically involved in resistance to one insecticide during a particular life-stage as argued by paul et al.
<cit> .
validation of transcription profiles by real-time quantitative rt-pcr was successful for the  <dig> genes tested although expression ratios obtained with rt-pcr were often higher.
the underestimation of transcription ratios obtained from microarray data is likely due to technical issues and has been previously evidenced in other studies  <cit> .
over-transcription of genes encoding p450s has been frequently associated with metabolic-based insecticide resistance mechanisms in insects  <cit> .
in mosquitoes, the cyp6z subfamily has been previously associated with response to pyrethroid, carbamates and organochlorine insecticides.
in ae.
aegypti, cyp6z <dig> has been found 4-fold over-transcribed in a permethrin-resistant strain collected in northern thailand  <cit> .
in two recent studies, cyp6z <dig> was also identified as inducible by permethrin and other pollutants  <cit> .
in an.
gambiae, cyp6zs have been frequently found constitutively over-transcribed in permethrin- and ddt-resistant strains  <cit> .
recent studies demonstrated that the enzyme encoded by an.
gambiae cyp6z <dig> can metabolize the insecticides carbaryl and ddt while cyp6z <dig> with a narrower active site, can only metabolize carbaryl  <cit> .
recently, another an.
gambiae p <dig> , was shown to be able to degrade pyrethroid insecticides  <cit> .
the over-transcription of cyp6z <dig> in the vauclin strain may indicate the involvement of ae.
aegypti cyp6zs in insecticide resistance in martinique.
however, the decisive demonstration of their capability to metabolize insecticides requires further investigations.
the association of cyp6ms with metabolic resistance to pyrethroids has also been previously described in mosquitoes.
in ae.
aegypti larvae, cyp6m <dig> and cyp6m <dig> were found inducible by permethrin and pollutants  <cit> .
although no aedes cyp6ms have been found constitutively over-transcribed in other insecticide-resistant strains, an.
gambiae cyp6m <dig> was found significantly over-transcribed in various strains resistant to pyrethroids  <cit> .
recent studies indicate that cyp6m <dig> is able to metabolize pyrethroid insecticides .
our results suggest that ae.
aegypti cyp6m <dig> and cyp6m <dig>  with protein sequences similar to an.
gambiae cyp6m <dig>  might also be involved in resistance of ae.
aegypti to pyrethroids in martinique.
finally, the glutathione s-transferase gste <dig> and the carboxy/cholinesterase cceae3a were both found over-transcribed in both life-stages of the vauclin strain.
the role gsts in resistance to chemical insecticides has been previously evidenced in insects with the enzyme encoded by an.
gambiae gste <dig> metabolizing ddt  <cit>  and the housefly mdgst6-a metabolizing two organophosphate insecticides  <cit> .
in ae.
aegypti, gste <dig> also metabolises ddt and is over-transcribed in a pyrethroid and ddt-resistant strain from thailand  <cit> .
in  <dig>  strode et al.
<cit>  also revealed the over-transcription of gste <dig> in pyrethroid-resistant mosquitoes.
our results confirm that gste <dig> might have a role in insecticide resistance in ae.
aegypti.
over-production of carboxylesterases has been showed to play an important role in resistance to organophosphate insecticides in mosquitoes  <cit> .
elevated esterase activities conferring resistance to organophosphate insecticides has usually been linked to genomic amplification of specific alleles although gene over-transcription may also be involved  <cit> .
considering the high resistance of larvae of the vauclin strain to temephos, over-transcribed cces represent good candidates for organophosphate metabolism in ae.
aegypti.
it has been suggested that insecticide resistance could be accentuated by the exposure of mosquito populations to pollutants and pesticides used in agriculture  <cit> .
in martinique, bananas, sugar cane, and pineapple represent important cultured surface areas often localized near mosquito breeding sites.
these cultures have been submitted for decades to heavy use of insecticides such as the organochlorates aldrin, dieldrin and chlordecone and herbicides such as the triazine simazine, the pyridines paraquat and glyphosate  <cit> .
this particular situation is likely to have contributed to the high resistance of ae.
aegypti to chemical insecticides and to the selection of particular detoxification genes in martinique.
CONCLUSIONS
we have identified multiple insecticide resistance mechanisms in ae.
aegypti mosquitoes from martinique  significantly reducing the insecticidal activity of insecticides used for their control.
microarray screening identified multiple detoxification genes over-transcribed at both life-stages in resistant mosquitoes, suggesting their possible involvement in insecticide-resistance.
further experimental validation by using enzyme characterization and rna interference will allow confirming the role of these genes in the resistance phenotype.
as previously shown in mosquitoes  <cit> , the epistasis between the kdr mutation and particular p450s genes is likely to contribute to the high level of resistance to pyrethroids in ae.
aegypti from martinique and might seriously threatens the control of dengue vectors in the future.
a better understanding of the genetic basis of insecticide resistance is an essential step to implement more effective vector control strategies in the field in order to minimize dengue outbreaks.

 every year, to million dengue infections world-wide causing from to deaths from dengue and hemorrhagic fever are recorded. as there is still no medicine and efficient vaccine available, vector control by the recourse of environmental management, educational programs and the use of chemical and biological agents, remains the only method to reduce the risk of dengue virus transmission. unfortunately, most of dengue vector control programs implemented worldwide are facing operational challenges with the emergence and development of insecticide resistance in ae. aegypti and ae. albopictus. resistance of ae. aegypti to insecticides has been reported in many regions including south east asia, latin america and the caribbean. inherited resistance to chemical insecticides in mosquitoes is mainly the consequence of two distinct mechanisms: the alteration of target sites inducing insensitivity to the insecticide and/or an increased metabolism of the insecticide. metabolic-based resistance involves the bio-transformation of the insecticide molecule by enzymes and is now considered as a key resistance mechanism of insects to chemical insecticides. this mechanism may result from two distinct but additive genetic events: i) a mutation of the enzyme protein sequence leading to a better metabolism of the insecticide, and/or ii) a mutation in a non-coding regulatory region leading to the over-production of an enzyme capable of metabolizing the insecticide. so far, only the second mechanism has been clearly associated with the resistant phenotype in mosquitoes. three large enzyme families, the cytochrome p monooxygenases, glutathione s-transferases and carboxy/cholinesterases have been implicated in the metabolism of insecticides. the rapid expansion and diversification of these so-called'detoxification enzymes' in insects is likely to be the consequence of their adaptation to a broad range of natural xenobiotics found in their environment such as plant toxins. these enzymes have also been involved in mosquito response to various anthropogenic xenobiotics such as heavy metals, organic pollutants and chemical insecticides. although identifying metabolic resistance is possible by toxicological and biochemical techniques, the large panel of enzymes potentially involved together with their important genetic and functional diversity makes the understanding of the molecular mechanisms and the role of particular genes a challenging task. as more mosquito genomes have been sequenced and annotated, the genetic diversity of genes encoding mosquito detoxification enzymes has been unravelled and new molecular tools such as the aedes and anopheles'detox chip' microarrays allowing the analysis of the expression pattern of all detoxification genes simultaneously have been developed. these specific microarrays were successfully used to identify detoxification genes putatively involved in metabolic resistance in various laboratory and field-collected mosquito populations resistant to insecticides. in latin america and the caribbean, several ae. aegypti populations show strong resistance to pyrethroid, carbamate and organophosphate insecticides correlated with elevated activities of at least one detoxification enzyme family. in addition, several points of non-synonymous mutations in the gene encoding the trans-membrane voltage-gated sodium channel have been described and showed to confer resistance to pyrethroids and ddt. several questions remain concerning the impact of insecticide resistance on the efficacy of vector control operations. in martinique, high levels of resistance to the organophosphate temephos and the pyrethroid deltamethrin were reported. this resistance was characterized by an important reduction of both mosquito knock-down and mortality levels after thermal-fogging with deltamethrin and p450-inhibitor synergized pyrethroids, indicating that resistance was negatively impacting on control programmes and that this resistance was conferred, at least in part, by elevated cytochrome p activity. in this study, we explored the mechanisms conferring insecticide resistance in an ae. aegypti population from martinique island. larval bioassays and adult topical applications were used to determine the current resistance level of this population to insecticides. the presence of metabolic-based resistance mechanisms was investigated by exposing mosquitoes to enzyme inhibitors prior to bioassays with insecticides and by measuring representative enzyme activities of each detoxification enzyme family. at the molecular level, the frequency of the target-site kdr mutation was investigated and a microarray approach followed by quantitative real-time rt-pcr validation was used to identify detoxification genes putatively involved in metabolic resistance. results from this study will help to implement more effective resistance management strategies in this major disease vector in the future.  larval bioassays showed that the vauclin strain is far less affected by temephos than the susceptible bora-bora strain. in the susceptible strain, temephos toxicity was not significantly increased in the presence of detoxification enzyme inhibitors. by contrast, the level of resistance to temephos of the vauclin strain was significantly reduced in the presence of pbo, def and dmc indicating the involvement of p450s, cces and in a lesser extent gsts in the resistance of larvae to temephos. resistant ratios rr and rr were obtained by calculating the ratio between the lc and lc between vauclin and bora-bora strains; synergism ratios sr and sr were obtained by calculating the ratio between lc and lc with and without enzyme inhibitor.: confidence interval. significant rr and sr are shown in bold. topical applications of the pyrethroid insecticide deltamethrin on adults of each strain revealed that the vauclin strain is also highly resistant to deltamethrin. in both strains, the toxicity of deltamethrin increased significantly in the presence of detoxification enzyme inhibitors, however only pbo and dmc induced higher synergistic effects in the vauclin strain than in the susceptible bora-bora strain. in the vauclin strain, pbo and dmc significantly reduced the resistance level, indicating a significant role of p450s and gsts in the resistance of adults to deltamethrin. resistant ratios rr and rr were obtained by calculating the ratio between the ld and ld between vauclin and bora-bora strains; synergism ratios sr and sr were obtained by calculating the ratio between ld and ld with and without enzyme inhibitor.: confidence interval. significant rr and sr are shown in bold. comparison of constitutive detoxification enzyme activities between the susceptible strain bora-bora and the insecticide-resistant vauclin strain revealed significant differences at both larval and adult stages. p activities were elevated in both larvae and adults of the vauclin strain. similarly, gst activities were found elevated in larvae and adults of the vauclin strain. finally, α- and β-carboxylesterase activities were also found slightly elevated in the vauclin strain in larvae and adults. sequencing of the voltage-gated sodium channel gene conducted on the vauclin strain showed the presence of the kdr mutation at position leading to the replacement of valine by an isoleucine at a high allelic frequency =, n = 24) with rr = rs = and ss = conversely, no kdr resistant allele was detected in the susceptible bora-bora strain. we used the microarray'aedes detox chip' to compare the transcription levels of all ae. aegypti detoxification genes between the insecticide-resistant strain vauclin and the susceptible strain bora-bora in larvae and adults. overall, and probes out of were detected consistently in at least hybridisations out of in larvae and adults respectively. among them, detoxification genes were significantly differentially transcribed in larvae or adults. most of these genes encode p450s with of them being differentially transcribed in the vauclin strain at both life stages. in larvae, genes were found significantly differentially transcribed between the insecticide-resistant strain vauclin and the susceptible strain bora-bora. among them, genes were over-transcribed in the vauclin strain while only genes were under-transcribed. most over-transcribed genes were represented by cyp genes with a majority belonging to the cyp subfamily. three cyp9s were also over-transcribed in larvae of the vauclin strain with a strong over-transcription of cyp9j together with cyp4s. among other over-transcribed genes, carboxy/cholinesterases and glutathione s-transferase were slightly over-transcribed in the vauclin strain. lastly, cyps were under-transcribed in insecticide-resistant larvae comparatively to susceptible larvae. in adults, genes were found differentially transcribed in the insecticide-resistant strain vauclin comparatively to the susceptible strain bora-bora. as in larvae, most of the over-transcribed genes belong to the cyp and cyp subfamilies with only additional cces being moderately over-transcribed in the vauclin strain. nine genes were under-transcribed in vauclin adults, including cyps, cce, gst and thioredoxin peroxidases. interestingly cyp304c and tpx were both found strongly under-transcribed in insecticide-resistant adults. validation of microarray data was performed by real-time quantitative rt-pcr on detoxification genes identified as over-transcribed in larvae or adults of the vauclin strain. the over-transcription of genes identified from microarray experiments were all confirmed by quantitative rt-pcr in both life stages, although expression ratios obtained from rt-pcr were frequently higher than those obtained from microarray experiments. discussion the aim of the present study was to investigate insecticide resistance mechanisms of ae. aegypti mosquitoes from martinique. toxicological results confirmed the high level of resistance of the vauclin strain from martinique to the organophosphate temephos at the larval stage and to the pyrethroid deltamethrin at the adult stage. the use of specific detoxification enzyme inhibitors suggested that resistance of larvae to temephos is linked to carboxylesterases and to a lesser extent p450s and gsts. in adults, resistance to deltamethrin appeared principally linked to p450s and gsts. comparison of global detoxification enzyme activities between the two strains revealed elevated p450s, gsts and in a lesser extent cces activities in the vauclin strain at both life-stages, confirming the importance of metabolic resistance mechanisms in martinique. carboxylesterases based-resistance mechanism is a major mechanism for organophosphate resistance in insects. several examples of ae. aegypti resistance to organophosphates in the caribbean linked to elevated carboxylesterases activities have been described. our toxicological and biochemical data confirms these observations despite a moderate elevated level of cces activities in the vauclin strain. among detoxification enzymes, p450s have been shown to play a major role in pyrethroid resistance in insects. in martinique, marcombe et al. suggested the involvement of p450s in the reduced efficacy of deltamethrin space-spray operations. elevated gst levels have also been frequently associated with insect resistance to insecticides such as ddt and pyrethroids. our toxicological and biochemical data support the role of p450s and gsts in insecticide resistance in martinique. at the molecular level, several mutations in the voltage-gated sodium channel gene have been associated with pyrethroid resistance in ae. aegypti from asian, latin american and caribbean countries. our results revealed a high frequency of the v1016i kdr mutation in ae. aegypti populations from the community of vauclin. the role of this mutation in pyrethroid resistance was clearly demonstrated by genotype-phenotype association studies. the high frequency of the mutation, together with the incomplete effect of enzyme inhibitors in adults, supports a contribution of this kdr mutation in deltamethrin resistance. acetylcholinesterase is critical for hydrolysis of acetylcholine at cholinergic nerve synapses and is a target for organophosphate and carbamate insecticides. altered ache is an important resistance mechanism to organophosphates in many insects. following the methods of alout et al. and bourguet et al., ache activities of vauclin mosquitoes were determined to investigate the presence of the g119s and/or f290v mutations. no insensitive ache phenotypes were found in any of the mosquitoes tested, suggesting that organophosphate resistance of the vauclin strain is rather due to detoxification enzymes unless other mutations occurred elsewhere in the ace gene. our microarray screening identified and over-transcribed detoxification genes in larvae and adults of the vauclin strain respectively. among them, p450s, the glutathione s-transferase gste and the carboxy/cholinesterase cceae3a were all confirmed to be over-transcribed at both life-stages, supporting their involvement in insecticide-resistance. other genes appeared more highly over-transcribed in adults or in larvae, suggesting that particular enzymes might be more specifically involved in resistance to one insecticide during a particular life-stage as argued by paul et al.. validation of transcription profiles by real-time quantitative rt-pcr was successful for the genes tested although expression ratios obtained with rt-pcr were often higher. the underestimation of transcription ratios obtained from microarray data is likely due to technical issues and has been previously evidenced in other studies. over-transcription of genes encoding p450s has been frequently associated with metabolic-based insecticide resistance mechanisms in insects. in mosquitoes, the cyp6z subfamily has been previously associated with response to pyrethroid, carbamates and organochlorine insecticides. in ae. aegypti, cyp6z has been found 4-fold over-transcribed in a permethrin-resistant strain collected in northern thailand. in two recent studies, cyp6z was also identified as inducible by permethrin and other pollutants. in an. gambiae, cyp6zs have been frequently found constitutively over-transcribed in permethrin- and ddt-resistant strains. recent studies demonstrated that the enzyme encoded by an. gambiae cyp6z can metabolize the insecticides carbaryl and ddt while cyp6z with a narrower active site, can only metabolize carbaryl. recently, another an. gambiae p, was shown to be able to degrade pyrethroid insecticides. the over-transcription of cyp6z in the vauclin strain may indicate the involvement of ae. aegypti cyp6zs in insecticide resistance in martinique. however, the decisive demonstration of their capability to metabolize insecticides requires further investigations. the association of cyp6ms with metabolic resistance to pyrethroids has also been previously described in mosquitoes. in ae. aegypti larvae, cyp6m and cyp6m were found inducible by permethrin and pollutants. although no aedes cyp6ms have been found constitutively over-transcribed in other insecticide-resistant strains, an. gambiae cyp6m was found significantly over-transcribed in various strains resistant to pyrethroids. recent studies indicate that cyp6m is able to metabolize pyrethroid insecticides. our results suggest that ae. aegypti cyp6m and cyp6m with protein sequences similar to an. gambiae cyp6m might also be involved in resistance of ae. aegypti to pyrethroids in martinique. finally, the glutathione s-transferase gste and the carboxy/cholinesterase cceae3a were both found over-transcribed in both life-stages of the vauclin strain. the role gsts in resistance to chemical insecticides has been previously evidenced in insects with the enzyme encoded by an. gambiae gste metabolizing ddt and the housefly mdgst6-a metabolizing two organophosphate insecticides. in ae. aegypti, gste also metabolises ddt and is over-transcribed in a pyrethroid and ddt-resistant strain from thailand. in strode et al. also revealed the over-transcription of gste in pyrethroid-resistant mosquitoes. our results confirm that gste might have a role in insecticide resistance in ae. aegypti. over-production of carboxylesterases has been showed to play an important role in resistance to organophosphate insecticides in mosquitoes. elevated esterase activities conferring resistance to organophosphate insecticides has usually been linked to genomic amplification of specific alleles although gene over-transcription may also be involved. considering the high resistance of larvae of the vauclin strain to temephos, over-transcribed cces represent good candidates for organophosphate metabolism in ae. aegypti. it has been suggested that insecticide resistance could be accentuated by the exposure of mosquito populations to pollutants and pesticides used in agriculture. in martinique, bananas, sugar cane, and pineapple represent important cultured surface areas often localized near mosquito breeding sites. these cultures have been submitted for decades to heavy use of insecticides such as the organochlorates aldrin, dieldrin and chlordecone and herbicides such as the triazine simazine, the pyridines paraquat and glyphosate. this particular situation is likely to have contributed to the high resistance of ae. aegypti to chemical insecticides and to the selection of particular detoxification genes in martinique.  we have identified multiple insecticide resistance mechanisms in ae. aegypti mosquitoes from martinique significantly reducing the insecticidal activity of insecticides used for their control. microarray screening identified multiple detoxification genes over-transcribed at both life-stages in resistant mosquitoes, suggesting their possible involvement in insecticide-resistance. further experimental validation by using enzyme characterization and rna interference will allow confirming the role of these genes in the resistance phenotype. as previously shown in mosquitoes, the epistasis between the kdr mutation and particular p450s genes is likely to contribute to the high level of resistance to pyrethroids in ae. aegypti from martinique and might seriously threatens the control of dengue vectors in the future. a better understanding of the genetic basis of insecticide resistance is an essential step to implement more effective vector control strategies in the field in order to minimize dengue outbreaks. 
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. 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. 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. 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 p 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. 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. bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. the present paper investigates the molecular basis of insecticide resistance in a population of ae. the over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative rt-pcr. molecular screening for common insecticide target-site mutations showed a high frequency of the sodium channel'knock down resistance' mutation. the yellow fever mosquito aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to million dengue infections every year.
 the yellow fever mosquito aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 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.  the present paper investigates the molecular basis of insecticide resistance in a population of ae. aegypti collected in martinique. 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 of the sodium channel'knock down resistance' 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 p 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.  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. 