Table 1Primers and PCR conditions for the identification of seven Anopheles spp.
Primer |
Species |
Sequence (5′→3′) |
Diagnostic bands |
Forward |
Universal primer |
TGT GAA CTG CAG GAC ACA TGA A |
— |
Reverse |
Anopheles sinensis
|
ATT GTT GTC CAG CCC GCT AAC |
500 bp |
|
Anopheles pulls
|
ATA TCA TGG CTT AAC ACC GCG T |
260 bp |
|
Anopheles lesteri
|
TGC CTA GAA CTT CCG CCA ATC |
300 bp |
|
Anopheles belenrae
|
CAT TTT TCA CGA CTG CGA CGG |
190 bp |
|
Anopheles kleini
|
GCG TCC ATA CTG TCT CAA CGA |
400 bp |
|
Anopheles sineroides
|
ACC GAG TGG CCT CAC TC |
500 bp |
|
Anopheles koreicus
|
GTA TAC ACG CTT TGT ATG TGG GG |
200 bp |
Primer |
Reagent |
Volume |
Reaction mixture |
PCR premix kit (Accupower, Bioneer) |
— |
|
Primer (each 10 pmol/μL) |
3.0 μL (each 0.5 μL) |
|
DNA template (10 ng/μL) |
2.0 μL |
|
Triple deionized distilled water |
15.0 μL |
|
Total |
20.0 μL |
Primer |
Temperature (°C) |
Time (min) |
Cycles |
PCR |
94 |
10 |
1 |
|
94 |
0.5 |
34 |
|
60 |
1 |
|
|
72 |
1 |
|
|
72 |
5 |
1 |
|
4 |
∞ |
∞ |
Table 2Susceptibility of 13 insecticides against Anopheles sinensis s.s. using direct contact diffusion assay with 24-hour exposure
Insecticide |
na
|
Slope (±SE) |
LC50 (ppm) |
95% CL |
χ2
|
RSb
|
Bifenthrin |
450 |
1.0 ± 0.09 |
0.227 |
0.166–0.312 |
5.35 |
1.0 |
Cyfluthrin |
375 |
1.0 ± 0.12 |
0.446 |
0.315–0.692 |
4.69 |
2.0 |
Etofenprox |
450 |
1.0 ± 0.90 |
1.858 |
1.360–2.331 |
6.53 |
8.2 |
Fenthion |
300 |
1.3 ± 0.16 |
2.377 |
1.732–3.120 |
2.65 |
10.5 |
Cypermethrin |
375 |
1.0 ± 1.12 |
2.419 |
1.748–3.217 |
1.83 |
10.7 |
λ-Cyhalothrin |
300 |
1.2 ± 0.15 |
3.220 |
2.410–3.543 |
0.54 |
14.2 |
α-Cypermethrin |
300 |
1.4 ± 0.16 |
3.323 |
2.561–3.689 |
2.70 |
14.6 |
Deltamethrin |
375 |
1.4 ± 0.14 |
4.522 |
3.444–5.216 |
6.03 |
19.9 |
Dichlorvos |
450 |
1.1 ± 0.13 |
4.850 |
3.907–5.224 |
3.57 |
21.4 |
Chlorpyrifos |
375 |
1.2 ± 0.13 |
4.890 |
3.951–5.336 |
3.39 |
21.5 |
Profenofos |
375 |
1.6 ± 0.14 |
5.829 |
4.916–7.361 |
3.89 |
25.7 |
Fenitrothion |
450 |
1.3 ± 0.13 |
7.860 |
6.115–9.124 |
2.99 |
34.6 |
Permethrin |
375 |
1.5 ± 0.16 |
12.485 |
9.799–13.599 |
0.45 |
55.0 |
Table 3Comparison of insecticide susceptibility of larvae of Anopheles sinensis s.s. among 2001 [21], 2008 [10], and 2012 strains collected in Paju, Republic of Korea
Insecticide |
LC50 (ppm)
|
AS01a
|
AS08 |
AS12 |
RR12-01b
|
RR12-08
|
RR08-01
|
Organophosphates
|
|
|
|
|
|
|
Fenthion |
0.03 |
1.1 |
2.377 |
79.23 |
2.16 |
36.67 |
Dichlorvos |
1.9 |
3.5 |
4.850 |
2.55 |
1.39 |
1.84 |
Chlorpyrifos |
3.4 |
4.7 |
4.890 |
1.44 |
1.04 |
1.38 |
Profenofos |
0.3 |
3.7 |
5.829 |
19.43 |
1.58 |
12.33 |
Fenitrothion |
5.0 |
7.7 |
7.860 |
1.57 |
1.02 |
1.54 |
Pyrethroids
|
|
|
|
|
|
|
Bifenthrin |
0.28 |
0.009 |
0.227 |
0.81 |
25.22 |
0.03 |
Cyfluthrin |
0.5 |
0.2 |
0.446 |
0.89 |
2.23 |
0.40 |
Cypermethrin |
4.7 |
1.6 |
2.419 |
0.51 |
1.51 |
0.34 |
λ-Cyhalothrin |
1.3 |
0.08 |
3.220 |
2.48 |
40.25 |
0.06 |
α-Cypermethrin |
0.2 |
0.06 |
3.323 |
16.62 |
55.38 |
0.30 |
Deltamethrin |
0.2 |
0.3 |
4.522 |
22.61 |
15.07 |
1.50 |
Permethrin |
0.8 |
3.1 |
12.485 |
15.61 |
4.03 |
3.88 |