Resistance to pyrethroids and the relationship between adult resistance and knockdown resistance (kdr) mutations in Aedes albopictus in dengue surveillance areas of Guizhou Province, China

The Ae. albopictus mosquito has gained global attention due to its ability to transmit viruses, including the dengue and zika. Mosquito control is the only effective way to manage dengue fever, as no effective treatments or vaccines are available. Insecticides are highly effective in controlling mosquito densities, which reduces the chances of virus transmission. However, Ae. albopictus has developed resistance to pyrethroids in several provinces in China. Pyrethroids target the voltage-gated sodium channel gene (VGSC), and mutations in this gene may result in knockdown resistance (kdr). Correlation studies between resistance and mutations can assist viruses in managing Ae. albopictus, which has not been studied in Guizhou province. Nine field populations of Ae. albopictus at the larval stage were collected from Guizhou Province in 2022 and reared to F1 to F2 generations. Resistance bioassays were conducted against permethrin, beta-cypermethrin, and deltamethrin for both larvae and adults of Ae. albopictus. Kdr mutations were characterized by PCR and sequencing. Additionally, the correlation between the kdr allele and pyrethroid resistance was analyzed. All nine populations of Ae. albopictus larvae and adults were found to be resistant to three pyrethroid insecticides. One kdr mutant allele at codon 1016, one at 1532 and three at 1534 were identified with frequencies of 13.86% (V1016G), 0.53% (I1532T), 58.02% (F1534S), 11.69% (F1534C), 0.06% (F1534L) and 0.99% (F1534P), respectively. Both V1016G and F1534S mutation mosquitoes were found in all populations. The kdr mutation F1534S was positively correlated with three pyrethroid resistance phenotypes (OR > 1, P < 0.05), V1016G with deltamethrin and beta-cypermethrin resistance (OR > 1, P < 0.05) and F1534C only with beta-cypermethrin resistance (OR > 1, P < 0.05). Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper. Outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae. albopictus populations in Guizhou Province.


Frequency and distribution of genotype combinations at loci 1016, 1532 and 1534 in Ae. albopictus
In all populations (2626 individuals in total), we found that some mosquito individuals carried kdr mutations in two codons together, and a total of 23 genotype combinations were identified, with no individuals carrying simultaneous mutations in all three loci (Table 4).Except for the GY and XY populations, one wild type (V/V + I/I + F/F) was present in all populations, with the highest frequency observed in the CJ population (18.27%).Twelve single mutant types were observed, including V/V + I/I + S/S and V/V + I/I + F/S, which were the most widespread (found in 9 populations) with frequencies of occurrence of 41.36% and 15.73%, respectively.Additionally, there were ten double mutant phenotypes, with the highest frequency of V/G + I/I + F/S mutations (12.83%), also found in 9 populations, and the highest frequency of 33.11% in GY.

Correlation between kdr mutant genes and resistance phenotypes
The OR values and 95% CIs were calculated for the six kdr mutant alleles at loci 1016, 1532 and 1534 in Ae. albopictus exposed to deltamethrin, permethrin and beta-cypermethrin.The frequency of mutations (GGA/G) at codon 1016 was 9.29% in susceptible individuals and 14.78% in resistant individuals after exposure to deltamethrin.The OR was 1.69 (95% CI 1.06-2.71)and P < 0.05 (Table 5).The study found that in the Ae.albopictus population, the OR values were 1.33 (95% CI 0.83-2.11)(P > 0.05) and 2.21 (95% CI 1.33-3.70)(P < 0.05) against permethrin and beta-cypermethrin, respectively.Generally, the V1016G mutant allele was positively correlated with deltamethrin and beta-cypermethrin resistance genotypes.No statistical correlation (P > 0.05) was revealed between the I1532T mutant allele and the three pyrethroid resistance phenotypes at locus 1532.At codon 1534, the OR values were 1.95 (P < 0.05), 1.75 (P < 0.05), and 2.90 (P < 0.05) in the Ae.albopictus populations to deltamethrin, permethrin, and beta-cypermethrin, respectively (95% CI 1.44-2.65,1.28-2.41,and 2.17-3.88,respectively), reflecting that the F1534S mutant allele was positively associated with the three pyrethroid resistance phenotypes.The association between the F1534C mutant allele and beta-cypermethrin insecticide resistance phenotype was positive (OR 5.57, 95% CI 3.01-10.31,P < 0.05).On the other hand, no statistically significant difference was observed between the F1534C mutant allele and deltamethrin and permethrin insecticide resistance phenotypes (P > 0.05).There were no statistically significant differences (P > 0.05) between the F1534P and F1534L mutant alleles and the resistance phenotypes after exposure to deltamethrin, permethrin, and beta-cypermethrin, respectively.However, a positive correlation was found between F1534P and the total pyrethroid resistance phenotype.
To explore whether the simultaneous presence of mutations at multiple loci in the one individual was the result of insecticide pressure, we classified the combinatorial genotypes into three categories: 0 (representing no mutations at loci 1016, 1532, and 1534), 1 (mutations at only one of the three loci), and 2 (mutations at only two of the three loci), and used chi-square tests to explore whether the combinatorial genotypes varied for different resistance phenotypes.The results showed significant differences (P < 0.05) in the types of genotypic combinations of different resistance phenotypes under the pressure of the three pyrethroid insecticides.The results of the comparison between the two types are summarized in Table 6.

Discussion
In the study, nine field populations of Ae. albopictus were obtained from dengue surveillance areas in Guizhou Province, including surrounding and central areas, with 2,626 mosquitoes, the largest number in the same study to date.The results showed that Ae. albopictus (larvae and adult mosquitoes) of Guizhou had already accumulated serious resistance to pyrethroid insecticides (deltamethrin, permethrin, and beta-cypermethrin), approaching or exceeding the results of southern and coastal provinces and cities such as Zhejiang and Hainan in China 9,25,34 .In  resistance phenotypes revealed that the V1016G mutation was significantly associated with Ae. albopictus resistance phenotypes against deltamethrin (OR > 1, P < 0. 05) and beta-cypermethrin (OR > 1, P < 0.05), while the F1534C mutation was positively correlated with beta-cypermethrin (OR > 1, P < 0.05) and the F1534S mutation was positively correlated for resistance to deltamethrin, permethrin and beta-cypermethrin (OR > 1, P < 0.05).However, no correlation was identified between the I1532T mutation and the three pyrethroid resistance phenotypes, which may be due to the low frequency of the mutation.The difference between the appearance of the kdr mutation and the absence of the mutation was significant (P < 0.0167) for all three insecticides, whereas the occurrence of one locus mutation and the occurrence of two showed a significant difference only in the case of beta-cypermethrin, suggesting that the difference between the resistant phenotype and the susceptible phenotype is mainly the production of the kdr mutation, and that simultaneous mutation of multiple loci may be the result of enhanced resistance.
In the absence of reports on the existence of Aedes aegypti in Guizhou currently, Ae. albopictus is the dominant vector for the transmission of dengue virus.The WHO recommended the global promotion of the use of pyrethroid insecticides for mosquito control in the late 1980s 35 .Since 1992, pyrethroid insecticides have been widely available because of their affordability, rapid knockdown, low toxicity to mammals and relative safety to humans 36 .We have reported pyrethroid resistance in wild populations of Ae. albopictus in 2018 and 2021, having observed susceptibility to pyrethroids in GY, CS and XY populations.In this study, bioassays showed increased resistance to pyrethroids in Ae. albopictus compared to the GY population in 2018 37 , and little more or slightly increased resistance in the CS and XY populations in 2021 38 .During our interventions, we noticed that householders in residential areas habitually spray commercially purchased insecticides around flowers and plants, which are complex insecticides containing several pyrethroids.The literature reports that 23 types of pyrethroid active ingredients are approved by the government of the People's Republic of China as public health insecticides 11 .Second, several cities, including ZY, CS and XY, had contracted with PCOs for regular insecticide spraying to reduce mosquito densities and breeding as part of their hygiene city campaigns.In these habitats, mosquitoes may have developed high levels of resistance at the larval and adult stages under the selective pressure of continuous exposure to pyrethroid insecticides.In addition, the XY and JS populations of Ae. albopictus showed lower mortality rates after exposure to the three pyrethroids than other populations, which may explain the difference in geographical location.In recent years, resistance of Ae. albopictus to pyrethroid insecticides has also been reported in other provinces and cities in China 2,39,40 , but the differences should be carefully analyzed as more factors affect the results of the bioassay, including the rearing sites, the samples tested and the assessment of mortality standards 9 .The results of this study enriched the information on the resistance of Ae. albopictus to pyrethroids in the dengue surveillance areas of Guizhou Province, providing more direct supporting data to guide the utilization of insecticides.
In the current study, the individuals we used for kdr gene testing were mosquitoes after bioassay, facilitating the accurate determination of the association between the mutation and pyrethroid resistance.We identified seven mutant alleles at codons 1016 (13.86%), 1532 (0.53%) and 1534 (70.75%) and further confirmed the correlation of F1534S with pyrethroids, which is consistent with the results of population studies in several geographical regions of China 9,25,34 .Second, in the face of the emergence of outbreaks in surrounding provinces and the existence of imported Dengue instances [41][42][43][44] , the higher mutation frequency may represent a serious threat to the control of potential Dengue outbreaks, even if there has been no indigenous Dengue outbreak at present in Guizhou Province.Moreover, the present study identified the V1016G mutation as positively associated with the resistance phenotype of Ae. albopictus to deltamethrin and permethrin, which has been demonstrated to be responsible for the insensitivity against these insecticides in reports of Aedes Aegypti but has hardly been reported in studies of Ae. albopictus.Some studies have documented a positive correlation between the I1532T mutation and the deltamethrin resistance phenotype 45 , but there are also conflicting views 9,34 .The I1532T mutation has a low mutation frequency and was not found to be associated with its insecticide resistance in the present results, which needs to be further investigated.Our results showed that F1534C was only insensitive to betacypermethrin.Although a resistance relationship between F1534C and pyrethroids has been detected in Aedes www.nature.com/scientificreports/aegypti 46 , few correlations have been reported in Ae. albopictus in China 9,13,33 .F1534C could be a weaker indicator of resistance.Significantly, a new mutation, F1534P, was identified in the current study, but it was only found in one population, and its association with resistance needs to be further investigated.A total of 29 different genotypic combinations were observed at codons 1016, 1532 and 1534.Among these, the F1534S single mutation at three loci was the most common combination, distributed across all populations, corresponding to the findings of Zhao et al. on field populations of Ae. albopictus in 11 dengue-endemic provinces 12 .The interactions between mutations at different loci remain to be further investigated.Differences in mutation rates at different loci in different geographical regions may result from uneven distribution of resistance 39 and the presence of multiple resistance mechanisms, resulting in the kdr mutation not playing a dominant role.Further investigation of other resistance mechanisms, such as metabolic resistance 1,47 , should also be conducted.In addition, Chen and Zhao et al. found that the mutation rates at loci 1016, 1532, and 1534 were correlated with average annual temperature, rainfall, and dengue-endemic areas 12,48 .Therefore, monitoring the kdr mutation frequency is not only conducive to the monitoring of Ae. albopictus resistance to pyrethroids and controlling dengue vector density, but also to preventing dengue outbreaks in advance.

Conclusion
Current susceptibility status of wild populations of Ae. albopictus to insecticides and a higher frequency of kdr mutations from dengue-monitored areas in Guizhou Province are reported in this paper.The pyrethroid resistance phenotype was clearly associated with the kdr mutation F1534S, which can be considered a molecular marker for resistance.In addition, outcomes of this study can serve as data support for further research and development of effective insecticidal interventions against Ae.albopictus populations in Guizhou Province.

Collection of wild Ae. albopictus samples
Nine field populations of Ae. albopictus were collected using Pasteur pipettes from breeding sites, such as flowerpots, waste tires, metal containers, waste buckets, water tanks and other water containers from Guiyang (GY), Ziyun (ZY), Jinsha (JS), Libo (LB), Xingyi (XY), Bijiang (BJ), Congjiang (CJ), Panzhou (PZ) and Chishui (CS) in Guizhou Province during July-August 2022.The larvae were identified as Ae.albopictus species by using the taxonomic keys 49 .All the samples collected in the field in each region were brought back to the laboratory of the Guizhou Provincial Centre for Disease Control and Prevention in buckets and identified again, and finally the selected Ae. albopictus mosquitoes were reared in tubs filled with dechlorinated water.The larvae were reared to adulthood under standard conditions of 26 ± 1 °C, 75 ± 5% relative humidity and 14/10 h light/dark photoperiod.Larvae were fed a special powdered diet (pig liver: steamed bread = 1:1) and adults were fed a 10% sucrose solution.All Ae. albopictus directly collected and identified from the field (F0 generation) were reproduced (F1-F2 generations) before being tested for susceptibility.
The susceptible strains of Ae. albopictus which served as a reference, was kindly provided by National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.The strain has been maintained in the insectary over a decade, without exposure to any insecticide.

Larval resistance bioassays
The susceptibility of larvae was determined using three insecticides (permethrin 90% pure, deltamethrin 98%, beta-cypermethrin 91.16%) from the Chinese Center for Disease Control and Prevention, complying with WHO guidelines 50 .Firstly, all insecticides were diluted to 5-7 concentrations with acetone to obtain mortality from 10 to 90%.Then, 1 ml diluted insecticide solution and 20-30 late third and fourth instar larvae were added to an experimental beaker that held 199 ml water separately.Furthermore, insecticide solution was replaced by acetone in the control group.Larval of Ae. albopictus mortalities were recorded after 24 h exposure.Larvae that did not move or twitched when strongly stimulated were considered dead.The assay was repeated three times.Larval mortality was calculated by dividing the number of dead larvae by the total number tested.The extent of resistance is defined by the resistance ratio (RR 50 ), which was derived by comparing the median lethal concentration (LC 50 ) value of the field population with the LC 50 value of the sensitive laboratory population to the insecticide.

Adult resistance bioassays
The susceptibility of adults was tested using three insecticide-impregnated papers (0.4% permethrin, 0.03% deltamethrin, 0.08% beta-cypermethrin) from the Chinese Center for Disease Control and Prevention complying with WHO guidelines56 51 .In the assay, 25 female mosquitoes, non-blood-fed, aged 3-5 days were selected to the resting tube without insecticide to adaptation for 30 min.After that they were gently blown into experimental tubes containing insecticide impregnated paper for up for 1 h.Silicone oil-treated papers without insecticides were applied to the control groups.Following insecticide exposure, mosquitoes were returned to resting tubes with 10% sugar water and mortality assessed after 24 h.Approximately 100 female mosquitoes were tested for each insecticide.Mosquitoes that do not move or only tremble in their limbs and wings and cannot survive are considered dead.They are also considered susceptible phenotypes.In contrast, mosquitoes that can survive are considered resistant phenotypes.Dead and survival mosquitoes were collected individually in test tubes filled with 95% alcohol at − 80 °C for subsequent DNA analysis.The bioassay should be repeated if the mortality in the control group was ≥ 20%.The test group mortality rate would be adjusted using Abbott's formula if the control group mortality rate was between 5 and 20%: Corrected mortality (%) = (test group mortality − control group mortality)/(1 − control group mortality) × 100 51

Statistical analysis
The resistance status of adult mosquitoes was also classified according to WHO criteria 51 : a mortality < 90% was recognized as resistant, a mortality of 90-98% as probable resistance, and a mortality > 98% was susceptible.For larval bioassays, susceptible if RR 50 < 5, moderately resistant if 5 < RR 50 ≤ 10, and highly resistant if RR 50 > 10.
A chi-squared test and hazard analysis were used to analyze the correlation between the frequency of mutant alleles and their resistance phenotypes, and the dominance ratio (OR) values were calculated.Differences were determined to be statistically significant at P < 0.05; the relationship between the kdr allele and the resistance phenotype was considered positive when the OR > 1 and negative when the OR < 1, and not yet statistically significant or statistically insignificant if the 95% CI of the OR value ranged between 1 or a P > 0.05.All data were analysed using excel2013 and spss24.0software.

Figure 1 .
Figure 1.Mortality of Ae. albopictus field populations after 24 h exposure to three pyrethroids.Solid line represents mortality at 98%, dashed line as 90%.

Table 1 .
Sensitivity of Ae. albopictus larvae to deltamethrin in nine different populations in Guizhou Province, China.RR 50 (resistance ratio): LC 50 test population/LC 50 laboratory-susceptible strain.

Table 2 .
Kdr Kdr allele frequencies at loci 1016, 1532 and 1534 in field populations of Ae. albopictus in Guizhou Province, China.
allele mutation frequencies at loci 1016, 1532 and 1534 in the field population of Ae. albopictus in Guizhou Province, China.N is the sample number.Data outside parentheses are the number of individuals; data within parentheses are their frequency (%).

Table 3 .
Kdr genotype frequencies of mutations at loci 1016, 1532 and 1534 in field populations of Ae.

Table 4 .
Genotypes and frequencies of three locus combinations of the kdr gene in the Ae.albopictus field population.Data outside parentheses are the number of individuals; data within parentheses are their frequency (%); no data are unlabeled to avoid clutter.

Table 5 .
Relationship between kdr gene mutation and insecticide resistance in the field population of Ae.Albopictus.N represents the sample number.S means susceptible phenotype: R stands for resistant phenotype.Data outside brackets indicate a number of individuals; data within brackets indicate the frequency (%); no data are unlabeled to avoid clutter.*P < 0.05.