Background

Malaria, caused by infection with Plasmodium spp., is one of the most significant life-threatening infectious diseases in humans worldwide. According to the WHO Malaria Report 2022, the total number of deaths due to malaria worldwide reached 619,000 in 2021, with approximately 247 million reported malaria cases [1]. Anopheles sinensis is the predominant Anopheles species in Henan Province, China and the main vector of the malaria parasite Plasmodium vivax [2]. In 2006, there was an outbreak of malaria caused by P. vivax in Yongcheng county, Henan Province [3]. Malaria vectors are known to still exist in the original malaria-endemic areas and if there are also imported malaria cases, the risk of a malaria outbreak is extremely high. It has become an important measure to control the population density of An. sinensis and other malaria vectors and prevent secondary transmission caused by imported cases [4, 5]. Indigenous malaria transmission has been effectively controlled, and China officially obtained the WHO certification for malaria elimination in 2021. With the development of global trade and the transnational economy, infections imported from overseas have become the main source of malaria cases in Henan Province. Imported malaria has introduced new challenges to achieving the overall elimination of malaria [6,11].

COI gene amplification of deltamethrin-resistant and -sensitive mosquitoes

To clarify the genetic evolutionary relationship between resistant and sensitive mosquitoes of An. sinensis, the mtDNA-COI gene was amplified in deltamethrin-resistant and deltamethrin-sensitive An. sinensis using the forward primer GGTCAACAAATCATAAAGATATTGG and the reverse primer TAAACTTCAGGGTGACCAAAAAATCA, following the methods previously described by Liang et al. [27]. All PCR products were analyzed by 2% agarose gel electrophoresis and the purified products were sequenced using an ABI 3730XL automated sequencer (Applied Biosystems®, Thermo Fisher Scientific, Waltham, MA, USA.

Statistical analysis

The mortality rates of the test and control samples were calculated after the bioassay for each insecticide group. If the control mortality was > 20%, the tests were discarded. When control mortality was 5% and 20%, then the observed mortality was corrected using Abbott’s formula [28]. Mosquito resistance status was interpreted in accordance with WHO guidelines as: (i) confirmed resistance (CR), mortality rate < 90%; (ii) possible resistance (PR), mortality rate between 90% and 97%; and (iii) susceptibility (S), mortality rate ≥ 98% [24]. Statistical analyses were performed using SPSS version 21.0 software (IBM Corp., Armonk, NY, USA). The mortality rates of An. sinensis at the survey sites, ace-1 and kdr mutation rates and gene mutation rates of the different populations were calculated using the Chi-square (χ2) test. Nucleotide polymorphism, natural selection and population differentiation were described according to He et al. [29]. In addition, we constructed a haplotype network diagram among the different geographical populations based on the median connection method, with the aim to analyze the genetic relationship among different geographical populations using PopArt software. A P value of < 0.05 was considered to indicate significance.

Results

Molecular identification of Anopheles species and insecticide susceptibility test

The molecular identification of all samples used for insecticide resistance determination was carried out based on the length of the electrophoresis fragments of the PCR product. A total of 1409 Anopheles mosquitoes were identified by species-specific PCR, of which 1334 (94.68%) were An. sinensis, 28 (1.99%) were An. yatsushiroensis, 43 (3.05%) were An. anthropophagus and four (0.28%) were An. belenrae (Table 1). Analysis of the insecticide resistance of the 1334 An. sinensis mosquitoes showed that the 24-h mortality rates in mosquitoes exposed to deltamethrin, beta-cyfluthrin and propoxur ranged from 3.28% to approximately 85.85%, which reached the CR level. The 24-h mortality rate of An. sinensis to malathion in **qiao and Tanghe was 97.43% and 97.67%, respectively, which reached the PR level. The 24-h mortality rates for malathion in ** resistance to organophosphorus and carbamate insecticides. Other researchers found that the G285A and F350Y mutations on the ace gene are related to resistance to organic phosphorus, and that the F105S, G285A and F305Y mutations are related to resistance to carbamate [37]. Higher G119S allele frequencies were observed in both propoxur- and malathion-resistant mosquitoes than in propoxur- and malathion-sensitive mosquitoes in the Tanghe population, but such a development was not observed in the other three sites, suggesting that the production of resistance might be the result of the joint action of multiple factors.

Kdr was first identified in the house-fly Musca domestica L. [38]. The L1014F substitution has been reported in pyrethroid-resistant pest species, including An. gambiae and Cx. p. pallens [39, 40]. The L1014C mutation, with TTG (Leu) being replaced with TGT (Cys), is a newly reported mutation [41]. The L1014F and L1014C mutations were detected in the Republic of Korea, where the TTC L1014F mutation was observed for the first time [42]. Previous studies revealed that the frequencies of the L1014F allele were significantly associated with deltamethrin-resistant and DDT-resistant phenotypes, but that this was not the case for the L1014C allele. In Kaifeng (Henan, China), the difference in kdr mutation frequency between surviving and dead mosquitoes was statistically significant [36, 43]. However, Sun et al. reported that the difference in kdr mutation frequency between surviving and dead mosquitoes was not statistically significant in Yingjiang (Yunnan, China) and Suining (Jiangsu, China) [43].

Three types of kdr mutant alleles were found in the present study, including L1014F, L1014C and L1014W. In contrast to the situation with An. sinensis in Sichuan, China, where the frequency of L1014F was observed to be higher than that of L1014L. L1014L was found to be the predominant resistance allele in Henan, but L1014C had lower frequencies than that L1014F and L1014L, which is similar to the results of the present study [33]. L1014F and L1014C mutations were found in Henan Province in a previous study, and the L1014W mutation was first found in the present study [11]. In the present study, the proportion of the L1014W mutation was very small (0.12%) and only distributed in the **qiao population. Tan et al. reported that the presence of the L1014W mutation in 52 An. sinensis in Guangxi, based on their kdr gene mutation study, accounting for a small proportion (1%) [44]. This result is similar to that of the current study. Higher L1014F and lower L1014C allele frequencies were observed in beta-cyfluthrin-resistant mosquitoes than in beta-cyfluthrin-sensitive mosquitoes, suggesting that the relationship between higher L1014F and lower L1014C allele frequencies and insecticide resistance requires further in-depth analysis.

The evolution of insect resistance is based on the genetics of insecticide-resistant populations. The Hd was > 0.95, and nucleotide diversity was > 0.008 from 2020–2021, indicating that the overall genetic diversity of An. sinensis in Henan Province is high. The values of Tajima's D and of Fu and Li's D and F suggest that the populations conform to the neutral selection hypothesis and are not subject to obvious selection pressure during the evolution process. Yang et al. studied the population genetic variation and population structure characteristics of five species groups of An. sinensis in nine sampling sites in Yunnan Province [45]. These authors reported gene exchange between the four species groups of An. sinensis in Yunnan Province, except for the YU population group, and that genetic differentiation was not apparent [45]. The values of the Fst and the haplotype network diagram indicated that there was no genetic differentiation between deltamethrin-resistant and deltamethrin-sensitive mosquito populations, possibly because the mosquitoes in this study were all adult mosquitoes collected in the field, and they lived in the same ecological environment and faced the same pressure of insecticide selection. External environmental factors might neutralize the degree of genetic differentiation, so genetic differentiation between the two populations was not observed. The results of this study show that the emergence of mosquito resistance might be the result of multiple mechanisms.

The advantages of adult female mosquitoes directly captured in the field for the study of insecticide resistance are that fewer facilities are required, and the age distribution of the vectors is representative of the wild vector population at a given time and location. The limitations of this study include failing to accurately assess adult female mosquitoes’ blood-sucking history, age and survival status, any of which might affect the test results. No further study on mutations at other sites besides the kdr and ace-1 genes and no comprehensive study of resistance mechanisms have been carried out from the perspective of enzymology and proteomics.

Conclusions

Four Anopheles species were identified in the present study: An. sinensis, An. yatsushiroensis, An. anthropophagus and An. belenrae. Anopheles belenrae was discovered for the first time in Henan Province. A high resistance to pyrethroids and propoxur was noted in the mosquitoes collected in Henan Province, but resistance to malathion varied according to the sampling location. The L1014W (TGG) mutation was detected for the first time in this study. Only one mutation, G119S, was found in the ace-1 gene. Mutations in the kdr and ace-1 genes play a role in the generation of insecticide resistance in An. sinensis. The genetic diversity of deltamethrin-resistant and deltamethrin-sensitive mosquitoes showed no genetic differentiation. The generation of resistance might be the result of the combination of multiple factors. Further experiments are needed to collect comprehensive research data on resistance mechanisms from the perspectives of enzymology and proteomics. These findings are a step towards providing missing data on the insecticide resistance of An. sinensis in Henan Province and provide the scientific basis for guiding the control of An. sinensis.