Resistance to deltamethrin and knockdown resistance mutation in Aedes albopictus from Jiangsu Province

doi:10.12140/j.issn.1000-7423.2022.04.008

Abstract

Abstract:

Objective To investigate the resistance of deltamethrin in Aedes albopictus in Jiangsu Province, and explore the association with knockdown resistance (kdr) mutation, to provide a theoretical basis for scientific control of Ae. albopictus. Methods The diagnostic doses were determined by preparing test bottles containing deltamethrin of 1, 2, 4, 6 or 8 μg/bottle and control bottles without deltamethrin using the CDC bottle bioassay with 10-25 susceptible laboratory-cultured Ae. albopictus in each bottle. Ae. albopictus larvae and pupae were collected from Suining County in Xuzhou City, Huaiyin District in Huai’an City, and Yixing City in Wuxi City, in Jiangsu Province during August and September 2020 and reared to the first filial generation (F1 generation). The mortality rate of Ae. albopictus was calculated by using 10-25 F1 generation female mosquitoes for exposure to a diagnostic dose of deltamethrin in the test bottle and the blank in the control bottle for 30 min to evaluate the resistance level. The DNA was extracted from the mosquitoes after bioassay, and the gene coding region of voltage-gated sodium channel (VGSC) transmembrane structural domains Ⅱ, Ⅲ and Ⅳwere amplified by PCR and sequenced. Bioedit and Seqman software were used for statistical analysis of the kdr mutations, genotypes and mutation frequency. A Chi-square test was used to compare the kdr mutation frequency in the resistant and susceptible phenotype mosquitoes. Results The diagnostic doses of deltamethrin to Ae. albopictus was 4 μg/bottle. A total of 149 Ae. albopictus were collected from Suining County, Huaiyin District and Yixing City. The corrected mean mortality rate of Suining County was (28.2 ± 1.4)%, and the mean mortality rates in Huaiyin District and Yixing City were (64.4 ± 5.5)% and (56.3 ± 17.7)%, respectively. Ae. albopictus collected from all three counties’ (cities and districts) were resistant to deltamethrin. There were 447 DNA sequences obtained, and the sequencing results showed that the kdr mutations existed in the gene coding region at codons 1532 and 1534 of the VGSC structural domain Ⅳ of Ae. albopictus in three counties (cities and districts). Two alleles at codon 1532 were wild-type ATC/I (279/298, 93.6%) and mutant ACC/T (19/298, 6.4%); three genotypes were the wild-type homozygote I/I (133/149, 89.3%), wild/mutant heterozygote I/T (13/149, 8.7%), and mutant homozygote T/T (3/149, 2.0%). Four alleles at codon 1534 were wildtype TTC/F (275/298, 92.3%), mutant TCC/S (5/298, 1.7%), mutant TTA/L (14/298, 4.7%), and mutant TTG/L (4/298, 1.3%); four genotypes were the wild-type homozygote F/F (128/149, 85.9%), wild/mutant heterozygote F/S (5/149, 3.4%), wild/mutant heterozygote F/L (14/149, 9.4%), and mutant homozygote I/I (2/149, 1.3%). Among the 72 resistant mosquitoes phenotyped, 87.5% (63/72) and 86.1% (62/72) were wild-type at codon 1532 and 1534, respectively, and 12.5% (9/72) and 13.9% (10/72) were kdr mutant, respectively. Among the 77 sensitive adults, 90.9% (70/77) and 85.7% (66/77) were wild type at codon 1532 and 1534, respectively, and 9.2% (7/77) and 14.3% (11/77) were kdr mutant, respectively. Chi-square test revealed no significant difference between kdr mutant genotypes at codon 1532 and 1534 with resistant and susceptible phenotypes Ae. albopictus( χ² = 0.168, 4.111, P > 0.05). Conclusion In Suining County, Huaiyin District and Yixing City of Jiangsu Province, Ae. albopictus mosquitoes were found resistant to deltamethrin. The frequencies of kdr mutations at codon 1532 and 1534 in Ae. albopictus was low. No significant correlation was found between the resistance phenotype and kdr mutations at codon 1532 and 1534.

Key words: Aedes albopictus, Deltamethrin, CDC bottle bioassay, Insecticide resistance, Knockdown resistance mutation

CLC Number:

R384.113

WANG Guan-xi, LI Ya-shu, LI Yue-yue, CAO Yuan-yuan, YANG Meng-meng, ZHANG Mei-hua, WU Jing-yao, LIANG Cheng, LI Ju-lin, ZHOU Hua-yun, TANG Jian-xia, ZHU Guo-ding. Resistance to deltamethrin and knockdown resistance mutation in Aedes albopictus from Jiangsu Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(4): 468-474.

Figures/Tables 5

Table 1

Fig. 1

Table 2

Table 3

Table 4

Alleles and genotypes at codon 1534 of kdr mutation gene in Aedes albopictus field population

采集地点Sampling site	表型（只） Phenotype (No.)	等位基因数量（%） Number of allele（%）				基因型数量（%） Number of genotype（%）
		野生型Wildtype	突变型 Mutant			野生型纯合子Wildtype homozygote	野生型/突变型杂合子 Wild/mutant heterozygote		突变型纯合子 Mutant homozygote
		TTC/F	TCC/S	TTA/L	TTG/L	F/F	F/S	F/L	L/L
睢宁县 Suining County	抗性Resistance（32）	56（87.5）	2（3.1）	6（9.4）	0（0）	26（81.3）	2（6.3）	2（6.3）	2（6.3）
	敏感 Susceptible（17）	24（70.6）	2（5.9）	8（23.5）	0（0）	7（41.2）	2（11.8）	8（47.1）	0（0）
	小计 Subtotal（49）	80（81.6）	4（4.1）	14（14.3）	0（0）	33（67.3）	4（8.2）	10（20.4）	2（4.1）
淮阴区 Huaiyin District	抗性Resistance（19）	35（92.1）	0（0）	0（0）	3（7.9）	16（84.2）	0（0）	3（15.8）	0（0）
	敏感 Susceptible（36）	71（98.6）	0（0）	0（0）	1（1.4）	35（97.2）	0（0）	1（2.8）	0（0）
	小计 Subtotal（55）	106（96.4）	0（0）	0（0）	4（3.6）	51（92.7）	0（0）	4（7.3）	0（0）
宜兴市 Yixing City	抗性Resistance（21）	41（97.6）	1（2.3）	0（0）	0（0）	20（95.2）	1（4.8）	0（0）	0（0）
	敏感 Susceptible（24）	48（100）	0（0）	0（0）	0（0）	24（100）	0（0）	0（0）	0（0）
	小计 Subtotal（45）	88（98.9）	1（1.1）	0（0）	0（0）	44（97.8）	1（2.2）	0（0）	0（0）
合计 Total	抗性Resistance（72）	132（91.7）	3（2.1）	6（4.2）	3（2.1）	62（86.1）	3（4.2）	5（6.9）	2（2.8）
	敏感 Susceptible（77）	143（92.9）	2（1.3）	8（5.2）	1（0.6）	66（85.7）	2（2.6）	9（11.7）	0（0）
	合计Total（149）	275（92.3）	5（1.7）	14（4.7）	4（1.3）	128（85.9）	5（3.4）	14（9.4）	2（1.3）

Table 4

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扩增片段 Amplicon	引物名称 Primer name	突变位点 Mutation site	引物序列（5′→3′） Primer sequence (5′→3′)	片段长度/bp Fragment length/bp
结构域Ⅱ Domain Ⅱ	aegSCF20	S989、I1011、L1014、V1016	gacaatgtggatcgcttccc	480
结构域Ⅱ Domain Ⅱ	aegSCR21	S989、I1011、L1014、V1016	gcaatctggcttgttaacttg	480
结构域Ⅲ Domain Ⅲ	aegSCF7	I1532、F1534	gagaactcgccgatgaactt	740
结构域Ⅲ Domain Ⅲ	aegSCR7	I1532、F1534	gacgacgaaatcgaacaggt	740
结构域Ⅳ Domain Ⅳ	albSCF6	D1763	tcgagaagtacttcgtgtcg	280
结构域Ⅳ Domain Ⅳ	albSCR8	D1763	aacagcaggatcatgctctg	280

采集地点Sampling site	死亡率/% Mortality/%				平均或校正死亡率/% Mortality/%
采集地点Sampling site	对照组Control group	实验组1 Experimental group 1	实验组2 Experimental group 2	实验组3 Experimental group 3	平均或校正死亡率/% Mortality/%
睢宁县 Suining County	9.1	35.3	35.7	33.3	28.2^a
淮阴区 Huaiyin District	0.0	70.6	60.0	62.5	64.4^b
宜兴市 Yixing City	0.0	64.0	68.8	36.0	56.3^b

采集地点 Sampling site	表型（只） Phenotype (No.)	等位基因数量（%） Number of allele（%）		基因型数量（%） Number of genotype（%）
采集地点 Sampling site	表型（只） Phenotype (No.)	野生型 ATC/I Wildtype ATC/I	突变型 ACC/T Mutant ACC/T	野生型纯合子Wildtype homozygoteI/I	野生型/突变型杂合子 Wild/mutant heterozygoteI/T	突变型纯合子 Mutant homozygote T/T
睢宁县 Suining County	抗性Resistance（32）	56（87.5）	8（12.5）	25（78.1）	6（18.8）	1（3.1）
	敏感 Susceptible （17）	26（76.5）	8（23.5）	11（64.7）	4（23.5）	2（11.8）
	小计 Subtotal（49）	82（83.7）	16（16.3）	36（73.5）	10（20.4）	3（6.1）
淮阴区 Huaiyin District	抗性Resistance（19）	37（97.4）	1（2.6）	18（94.7）	1（5.3）	0（0）
	敏感Susceptible（36）	71（98.6）	1（1.4）	35（97.2）	1（2.8）	0（0）
	小计 Subtotal（55）	108（98.2）	2（1.8）	53（96.4）	2（3.6）	0（0）
宜兴市 Yixing City	抗性Resistance（21）	41（97.6）	1（2.4）	20（95.2）	1（4.8）	0（0）
	敏感Susceptible（24）	48（100）	0（0）	24（100）	0（0）	0（0）
	小计 Subtotal（45）	89（98.9）	1（1.1）	44（97.8）	1（2.2）	0（0）
合计 Total	抗性Resistance（72）	134（93.1）	10（6.9）	63（87.5）	8（11.1）	1（1.4）
	敏感Susceptible（77）	144（94.2）	10（5.8）	70（90.9）	5（6.5）	2（2.6）
	合计 Total（149）	279（93.6）	19（6.4）	133（89.3）	13（8.7）	3（2.0）