<i>Culex pipiens pallens</i> mosquito mortality rate in standard WHO deltamethrin resistance bioassay.

<p><i>Culex pipiens pallens</i> mosquito mortality rate in standard WHO deltamethrin resistance bioassay.</p

Values of 50% lethal concentration (LC₅₀) in Tangkou population of <i>Culex pipiens pallens</i> in response to deltamethrin selection.

<p>*Resistance ratio is the ratio of LC₅₀ of the test population to LC₅₀ of the initial parental (G₀) population.</p

Frequencies (in percentage) of <i>kdr</i> alleles in relation to mosquito survival phenotype determined by the deltamethrin susceptibility bioassay in six <i>Culex pipiens pallens</i> populations from China.

<p>Frequencies (in percentage) of <i>kdr</i> alleles in relation to mosquito survival phenotype determined by the deltamethrin susceptibility bioassay in six <i>Culex pipiens pallens</i> populations from China.</p

Map of China showing the distribution of mosquito sampling sites.

<p>1, Wuxi (31°33′58.47″N, 120°18′9.88″E); 2, Nanjing (32°3′30.11″N, 118°47′47.28″E); 3, Huaibei (33°57′13.67″N, 116°47′56.95″E); 4, Weishan (34°48′25.40″N, 117°7′43.69″E; 5, Tangkou (34°52′34.97″N, 117°22′53.69″E); and 6, Qingdao (36°3′58.85″N, 120°22′57.98″E).</p

Dynamics of <i>kdr</i> allele frequency in response to deltamethrin selection.

<p><b>A</b>: wildtype L1014 allele frequency was significantly decreased under deltamethrin selection, but significantly increased in the absence of selection; <b>B</b>: L1014F allele frequency was significantly increased under selection, but significantly decreased when no insecticide selection pressure was available; and <b>C</b>: L1014S allele frequency was decreased regardless of deltamethrin selection.</p

Relationship between mosquito survivorship in WHO insecticide susceptibility bioassay and frequency of <i>kdr</i> alleles (L1014F and L1014S) combined of <i>Culex pipiens pallens</i>.

<p>Numbers in the figure corresponds to the following sample localities: 1, Nanjing; 2, Wuxi; 3, Weishan; 4, Huaibei; 5, Qingdao; and 6, Tangkou.</p

Allele-Specific PCR (AS-PCR) for <i>kdr</i> genotyping.

<p><b>A</b>: Schematic representation of the primer location and predicted size of PCR products. Cpp1–Cpp5 indicate PCR primers in which sequences are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011681#pone-0011681-t005" target="_blank">Table 5</a>. Primer pair Cpp2 and Cpp3 amplifies a 389 bp fragment for the wildtype susceptible allele (for codon TTA). Primer pair Cpp1/Cpp4 yields a 176 bp fragment for resistant L1014F allele (codon TTT). Similarly, primer pair Cpp1/Cpp5 leads to amplification of a 176 bp fragment diagnostic to the L1014S resistant allele (for codon TCA). <b>B</b>: An example of AS-PCR gel. Two PCR reactions were run in parallel for each specimen (lanes 1 and 2 for specimen 1; lanes 3 and 4 for specimen 2…). Samples in lanes 1, 3, 5, 7, 9 and 11 were amplified with the primers Cpp1, Cpp2, Cpp3 and Cpp4 to detect wildtype L1014 (TTA) and resistant L1014F (TTT) alleles, whereas samples in lanes 2, 4, 6, 8, 10 and 12 were amplified with primers Cpp1, Cpp2, Cpp3 and Cpp5 to detect wildtype L1014 (TTA) and resistant L1014S (TCA) alleles. Lanes 13 and 14 were negative control. Genotype results: specimen 1: TTA/TTA; specimen 2: TTT/TTT; specimen 3: TCA/TCA; specimen 4: TTA/TTT; specimen 5: TTA/TCA; and specimen 6: TTT/TCA.</p

Sensitivity and specificity of allele-specific PCR (AS-PCR) and real-time TaqMan methods in reference to allele sequence data for identification of <i>kdr</i> genotypes in <i>Culex pipiens pallens</i>.

<p>The 97 <i>Cx. pipiens pallens</i> individuals used in this assay were from Wuxi population.</p

PCR primer and TaqMan probe sequences used in the present study.

<p>PCR primer and TaqMan probe sequences used in the present study.</p