september 2011 , mosquito larvae were collected in irrigated rice fields surrounding tiassal , southern cte divoire ( 55247n ; 44948w ) and reared to adults in insectaries on a diet of mikromin ( tetra , melle , germany ) fish food .
mosquitoes , 35 days of age , were exposed to 1 of 5 insecticides ( 0.1% bendiocarb , 1.0% fenitrothion , 0.75% permethrin , 0.05% deltamethrin , 4% ddt ) or a control papers for 1 hour , according to standard who procedures ( 7 ) .
dna was extracted from individual mosquitoes according to the livak method ( 8) , and a subsample of 500 mosquitoes were all found to be the m molecular form of an .
the target site mutation g119s in the ace-1 gene ( ace-1 ) and l1014f and l1014s kdr mutations were screened by using restriction fragment length polymorphism ( 10 ) or taqman assays ( 11 ) , respectively . according to who criteria , an .
gambiae mosquitoes from tiassal are resistant to all insecticide classes , and resistance is extremely prevalent ; more than two thirds of mosquitoes survived the diagnostic dose for 4 of the 5 insecticides tested ( table 1 ) .
to assess the level of resistance , we exposed the tiassal population and a susceptible laboratory population of an .
gambiae ( kisumu ) mosquitoes to the pyrethroid deltamethrin or the carbamate bendiocarb for a range of exposure times and assessed deaths 24 hours later ( technical appendix ) .
we found an unexpectedly strong resistance phenotype to the 2 insecticides ( figure 1 , figure 2 ) .
for deltamethrin , 4 hours of exposure were required to kill 50% ( median lethal time , [ lt50 ] ) ; in comparison , the lt50 for the susceptible kisumu strain was < 2 minutes ( resistance ratio = 138 ) ( technical appendix ) .
similarly , the lt50 for bendiocarb was nearly 5 hours for the tiassal strain yet < 12 minutes for the susceptible strain ( resistance ratio  =  24 ) ( technical appendix ) .
all mosquitoes were resistant according to world health organization classification ( < 80% dead ) ( 7 ) .
time - mortality curve for wild - caught anopheles gambiae mosquitoes from tiassal , southern cte divoire , exposed to deltamethrin ( median time to death =  248 minutes ) .
logistic regression line was fitted to time - response data by using sigmaplot version 11.0 ( www.sigmaplot.com ) .
time - mortality curve for wild - caught anopheles gambiae mosquitoes from tiassal , southern cte divoire , exposed to bendiocarb ( median time to death  =  286 minutes ) .
logistic regression line was fitted to time - response data by using sigmaplot version 11.0 ( www.sigmaplot.com ) .
to investigate the causes of this resistance , we screened a subset of mosquitoes for the target site mutations , kdr 1014f and 1014s . only the 1014f kdr mutation was detected , and this resistance allele was found at high frequency ( 83% ) .
there was a significant association between presence of the 1014f kdr allele and ability to survive exposure to ddt but not to either pyrethroid ( table 2 ) .
in contrast , the ace-1 allele was strongly associated with survival after exposure to bendiocarb and fenitrothion ( table 2 ) . *
f and l represent mutant resistant alleles ( phenylalanine ) and wild - type alleles ( leucine ) , respectively ; s and g represent mutant resistant alleles ( serine ) and wild - type alleles ( glycine ) , respectively .
no resistant homozygotes gg were found among the 186 mosquitoes genotyped for ace-1 by restriction fragment length polymorphism ( a subset of 48 was further screened by using the taqman assay ; congruence between the 2 methods was 100% ) . 
the frequencies were calculated for each insecticide and mosquito status ( alive / dead ) after exposure . 
genotypic odds ratios ( ors ) are shown because these exceed allelic ors for ddt ( recessive model ) , bendiocarb , and fenitrothion ( both overdominant models ) , and are similar for permethrin and deltamethrin .
for bendiocarb and fenitrothion absence of gg genotypes in the  alive  group means that ors are infinity , therefore ors are shown if one gg was present .
f and l represent mutant resistant alleles ( phenylalanine ) and wild - type alleles ( leucine ) , respectively ; s and g represent mutant resistant alleles ( serine ) and wild - type alleles . 
gambiae mosquitoes was first reported from cte divoire in 1993 ( 12 ) ; carbamate resistance was detected in the 1990s ( 13 ) .
nevertheless , 2 decades later , it is surprising and worrying to find complete resistance to all insecticides tested , particularly  for deltamethrin and bendiocarb  at such high levels .
ace-1 is strongly associated with organophosphate and carbamate resistance , and the absence of 119s homozygotes might be attributable to the high fitness cost of the ace-1 allele in the absence of insecticide ( 14 ) .
presence of the 1014f kdr allele alone does not confer the ability to survive diagnostic doses of pyrethroids ; thus , alternative mechanisms must be responsible for the high - level pyrethroid resistance in this population .
the selective pressures responsible for this intense multiple - insecticide resistance in tiassal mosquitoes are unclear .
there is a high coverage of insecticide - treated bed nets , but this coverage does not differ from that in other parts of the continent , and indoor residual spraying has not been conducted in this region .
this use is perhaps the most likely explanation in this district of intense commercial production of rice , cocoa , and coffee .
whatever the cause , the implications of this resistance scenario for malaria control are severe . with no new classes of insecticides for malaria control anticipated until 2020 at the earliest ( 15 ) ,
assessing the effect of pyrethroid resistance on the efficacy of insecticide - treated bed nets is complex because of the poorly understood associations between net integrity , insecticide content , net usage , and net efficacy .
nevertheless , resistance levels , such as those reported here , combined with continual selection pressure will inevitably lead to suboptimal mosquito control by use of insecticide - treated bed nets and indoor residual spraying .
if unchecked , this resistance could spread rapidly and threaten the fragile gains that have been made in reducing malaria across africa .
time - mortality curve for anopheles gambiae mosquitoes , kisumu strain , exposed to deltamethrin and bendiocarb , and time - death data for adult female a. gambiae s.s . mosquitoes ,
tiassal strain , and standard susceptible colony kisumu 24 hours after exposure to bendiocarb or deltamethrin .
 preventing malaria used to seem as simple as killing the vector , the mosquito ; however , a recent study shows that this concept is now anything but simple . 
 the highly effective use of insecticide - treated bed nets and indoor insecticide spraying is being challenged by mosquito resistance to insecticides . in west africa , 
 populations of this mosquito vector are now resistant to all 4 classes of insecticide approved for this use . and 
 no new classes of insecticide are anticipated until 2020 , at the earliest . 
 development of newer classes of insecticide is crucial because if resistance continues unchecked , the hard - earned progress in malaria control in africa could be quickly reversed . 
The Study
Conclusions
None