The aim of the present study was to investigate insecticide resistance mechanisms of Ae. aegypti mosquitoes from Martinique (French West Indies).
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 [30
]. 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 [12
]. Several examples of Ae. aegypti
resistance to organophosphates in the Caribbean linked to elevated carboxylesterases activities have been described [25
]. 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 [8
]. 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 [33
]. 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 [27
]. Our results revealed a high frequency (71%) 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 [37
]. 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 (AChE) is critical for hydrolysis of acetylcholine at cholinergic nerve synapses and is a target for organophosphate and carbamate insecticides [38
]. 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 (Corbel V., unpublished data), 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 14 and 9 over-transcribed detoxification genes in larvae and adults of the Vauclin strain respectively. Among them, 4 P450s (CYP6M6
), the glutathione S-transferase GSTe7
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 (CYP9J22
) or in larvae (CYP6M6
), 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 10 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 [14
Over-transcription of genes encoding P450s has been frequently associated with metabolic-based insecticide resistance mechanisms in insects [10
]. In mosquitoes, the CYP6Z
subfamily has been previously associated with response to pyrethroid, carbamates and organochlorine insecticides. In Ae. aegypti
has been found 4-fold over-transcribed in a permethrin-resistant strain collected in Northern Thailand [20
]. In two recent studies, CYP6Z8
was also identified as inducible by permethrin and other pollutants [14
]. In An. gambiae
have been frequently found constitutively over-transcribed in permethrin- and DDT-resistant strains [19
]. Recent studies demonstrated that the enzyme encoded by An. gambiae CYP6Z1
can metabolize the insecticides carbaryl and DDT while CYP6Z2
with a narrower active site, can only metabolize carbaryl [44
]. Recently, another An. gambiae
), was shown to be able to degrade pyrethroid insecticides [22
]. The over-transcription of CYP6Z6
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
were found inducible by permethrin and pollutants [14
]. Although no Aedes CYP6Ms
have been found constitutively over-transcribed in other insecticide-resistant strains, An. gambiae CYP6M2
was found significantly over-transcribed in various strains resistant to pyrethroids [21
]. Recent studies indicate that CYP6M2
is able to metabolize pyrethroid insecticides (Stevenson B. personal communication). Our results suggest that Ae. aegypti CYP6M6
, with protein sequences similar to An. gambiae CYP6M2
, might also be involved in resistance of Ae. aegypti
to pyrethroids in Martinique.
Finally, the glutathione S-transferase GSTE7
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 GSTE2
metabolizing DDT [35
] and the housefly MdGST6-A
metabolizing two organophosphate insecticides [49
]. In Ae. aegypti
, GSTE2 also metabolises DDT and is over-transcribed in a pyrethroid and DDT-resistant strain from Thailand [35
]. In 2008, Strode et al.
] also revealed the over-transcription of GSTE7
in pyrethroid-resistant mosquitoes. Our results confirm that GSTE7
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 [50
]. 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 [12
]. 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 [14
]. 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 [56
]. 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.