Selection process
In the present study after detection the highest resistance ratio (RR) about pyrethroids insecticides tested, selection process performed on adult and after 3 rd selection a Lambdacyhalothrin resistant population of Cx.pipiens was achieved with 85.75 -fold resistance ratio at LT50 level.
Biochemical assays
Profile activities of α- and ß-esterases, Mixed Function Oxidase (MFO), Glutathione-S-Transferase (GST), were tested for two Cx .pipiens populations with resistance ratio of 85.75 to Lambdacyhalothrin and resistant to DDT population in comparison with Lab strain . The results are summarized in Table.1 which shows the median level of enzymatic activity related to all three populations.
Mixed function oxidase (MFO)
In both DDT and Lambdacyhalothrin resistant populations in comparison to Lab strain, results showed that there are significant difference in MFO activity levels (P<0.05)(Table1)(Fig 2).
Statistical analysis
For comparison of average between two groups T test were used
Glutathione-S-transferase (GST)
Statistical analysis indicated that, GST activity in the two populations was not significantly different from that of the Lab- strain (P> 0.05) (Table 1) (Fig 2).
α and ß-Esterase
In the present study both DDT and Lambdacyhalothrin resistant populations. The median activity levels of α and ß-EST were significantly different from the Lab strain (P<0.05) (Table 1) (Fig 2).
Amplification and sequencing of sodium channel gene fragments in Cx. pipiens
A 521-bp fragment of the sodium channel gene from all three populations by PCR using primers: Cpp1 (5' CCT GCC ACG GTG GAA CTT C3') andCpp2 (5'GGA CAA AAG CAA GGC TAA GAA3') were amplified (Fig 3). From each Cx.pipiens population forty samples of sodium channel gene fragment was sequenced. By using Bioinformatics software such as Clustal W2 and Blast all sequencing results were checked in order to ensure that results are valid. More over as comparing sequencing results of this study with other sequences recorded in Gen Bank by Blast result showed that our sequence is more similar to Culex pipiens pallens (Accession number: GU198941.1). Based on our finding. More investigation of kdr region showed that: L1014F substitution due to changing TTA codon to TTT and L1014S substitution (TTA to TCA) causing resistance in the kdr gene was not observed. More over in all three population in the mentioned region, similar Culex pipiens pallens (Accession number: GU198941.1 ) , TTA codon was exist(highlighted in yellow) (Fig 4). According to ExPASy results related to translating nucleic acids to amino acids ,sequences of Cx.pipiens sodium channel gene were compared with other similar amino acid sequences of mosquitoes were available in the gene bank. By comparing result of current study with The most similar sequences related to Cx .pipiens pallens results revealed that in the region with possibility Kdr mutation at position 1014 in the sodium channel gene due to translation leucine to phenylalanine or serine There is no mutation and this fact ,confirmed lack of Kdr mutation in two Cx .pipiens populations. Except a substitution due to changing TTA codon to TTT in the exonI (highlighted in gray) resulted in single amino acid substitution of leucine to phenylalanine in Culex pipiens pallens ,all other amino acids were quite similar and also These differences, may be considerable to insecticide resistance In the future about this species (Fig. 4).
Table 1. Quantification of enzymatic activity of MFO, GST , α and β-esterase in three populations (resistant to Lambdacyhalothrin, resistant to DD.T and lab-strian) of Culex pipiens
Strains
|
MFO
(EU Cyt.p450/mg)
|
GST
(mM/min/mg)
|
α-esterase
(µM/min/mg)
|
β-esterase
(µM/min/mg)
|
Na
|
Median b
|
Na
|
Median b
|
Na
|
Median b
|
N a
|
Median b
|
Lab
|
40
|
-0.000019
|
40
|
0.06857
|
40
|
0.0001
|
40
|
0.00034
|
Resistant to DDT
|
40
|
0.000021
|
40
|
0.11485
|
40
|
0.00061
|
40
|
0.00062
|
Resistant to Lambdacyhalothrin
|
40
|
0.000036
|
40
|
0.13222
|
40
|
0.00073
|
40
|
0.00048
|
a Number of mosquitoes tested.
b Median value for each enzymatic activity