Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by Japanese encephalitis virus (JEV) (Dong et al., 2023). JE is mainly harmful to children and adolescents, causing central nervous system lesions (Kumar, 2020). The mortality rate caused by JE is about 10% - 30%, and up to 50% of survivors are left neurological defects (Campbell et al., 2011; Tolsa-Garcia et al., 2023). According to WHO statistics (https://www.who.int/data/collections), there are about 68,000 clinical cases of JE each year, with more than 3 billion people at risk of infection. Mainland China used to be a high-epidemic area for JE, but the incidence of JE has declined rapidly since the end of the 20th century (Tong et al., 2023; Yan et al., 2024). However, in recent year, JE outbreaks have been reported in central and western regions of China including Sichuan Province (Shi et al., 2022).

Currently, the main measures to control the epidemic of JE include vaccination and mosquito control. Although vaccination is the most economical and reliable measure for JE control in China, mosquito control remains an important means to prevent and control JE due to the change of the dominant JEV genotype and incomplete vaccination coverage in China (Cao et al., 2016; Dong et al., 2023; Yan et al., 2024), especially when the outbreak of JE.

While more than 20 species of mosquitoes are possible vector of JEV in China, Culex tritaeniorhynchus is the most important one (Yan et al., 2024). The breeding areas of Cx. tritaeniorhynchus are mostly rice fields, which are heavily exposed to agricultural insecticides. Due to the continuous use of insecticides in agriculture and the short generation and high fertility of Cx. tritaeniorhynchus, field populations of this mosquito species have developed resistance to organophosphate (OP), carbamate (CB) and pyrethroid insecticides in different regions of China (Wu et al., 2016; Shi et al., 2019; Wu et al., 2021). For example, Cx. tritaeniorhynchus collected from four different provinces of China were resistant to deltamethrin, beta-cypermethrin, permethrin, dichlorvos, and propoxur (Wu et al., 2016).

Two major mechanisms of insecticide resistance have been documented in mosquitoes, i.e. metabolic resistance and target resistance (Bandibabone et al., 2021; Li et al., 2021; Wang et al., 2023). Metabolic resistance is caused by enhanced insecticide detoxification, and target resistance is resulted from reduced sensitivity of insecticidal targets including acetylcholinesterase, nerve axon sodium ion channel, and γ-aminobutyric acid receptor chloride ion channel. As the target of organochlorine and pyrethroid insecticides, the voltage-gated sodium channel (VGSC) can regulate the sodium ions of cardiomyocytes, neurons and other cells, depolarization and repolarization process, and determine the state of excitable cells (Orjuela et al., 2019). Insensitivity to dichloro-diphenyl-trichloroethane (DDT) and pyrethroids caused by sodium channel point mutations is called knockdown resistance (kdr) (Roca-Acevedo et al., 2023). The first reported kdr mutation is a change of Leu to Phe at site 1014 of the Musca domestica sodium channel gene (VGSC) (Rinkevich et al., 2012). Acetylcholinesterase (AChE), as a key enzyme in biological nerve conduction, is the target site of OP and CB insecticides (Vale and Lotti, 2015). Several mutations of AChE related to insecticide resistance have been reported in mosquitoes (reviewed in Liu, 2015), including the F455W substitution identified in Cx. tritaeniorhynchus (Nabeshima et al., 2004). Cyclopentadiene insecticides (e.g. lindane and dieldrin) and other chemicals (e.g. fipronil) act on Gamma-aminobutyric acid (GABA) receptor in insects (Chebib et al., 2001; Chen et al., 2006; Nakao, 2017). A point mutation leading to a substitution of Ala 296 to Ser/Gly (A296S/G) in the GABA receptor Rdl subuint (Rdl) was reported to be responsible for dieldrin resistance in several species of mosquitoes (Liu, 2015).

Mianyang City is located in the northwest of Sichuan Basin of China within the north subtropical mountainous humid monsoon climate zone. The climate in Mianyang is mild throughout the year and rainfall is abundant. These natural and social factors are suitable for the spread of mosquito-borne infectious diseases. As the global climate warms and population mobility increases, Mianyang City is facing an increasing threat of these mosquito-borne diseases.

Given that resistance will undermine the effectiveness of current insecticide based JE vector control interventions, it is of great significance to carry out regular monitoring of vector resistance to commonly used chemical insecticides. Unfortunately, except a recent study on genetic mutations in populations of Cx.tritaeniorhynchus in southeast of Sichuan Basin of China (Liu et al., 2023), there is no other report on the status of pesticide resistance in Cx. tritaeniorhynchu field populations in Sichuan Province of China to our knowledge. In this study, as the first effort in this regard, we investigated the possible presence of the resistance conferring mutations in three genes encoding insecticide targets in six representative Cx. tritaeniorhynchoides populations in Mianyang City of Sichuan Province.

Although other measures help, chemical insecticides have long been used to prevent and control mosquito biting and mosquito-borne diseases. Consequently, many mosquito populations have developed resistance to commonly used insecticides (Pai et al., 2023; Tungu et al., 2023). Considering the facts that Cx. tritaeniorhynchus is the dominant mosquito species, and little is known about the status and involved mechanism of insecticide resistance in Cx. tritaeniorhynchus populations in Sichuan Province including Mianyang City, we investigated the occurrence of insecticide resistance-related genetic mutations.

Previous studies have well demonstrated that the L1014F mutation in VGSC is commonly present in several species of mosquitoes including Cx. tritaeniorhynchus (Liu, 2015; Wu et al., 2016; Liu et al., 2023). As expected, this mutation was also detected in Cx. tritaeniorhynchus samples collected from Mianyang City. The frequency of resistant allele (1014F) ranged from 16.98% (PW) to 27.78% (ST), which is similar to that reported in Cx. tritaeniorhynchus samples in Neijiang City of Sichuan Province (Liu et al., 2023), and in 12 sites in other four provinces of China (Wu et al., 2016).

The point mutation resulting in a substitution of Ala 296 to Ser (A296S) in the channel-lining region within the Rdl molecule has been documented for dieldrin resistance in several species of mosquitoes (Liu, 2015). In Sichuan, the A296S mutation is present at high frequencies in many populations of Anopheles sinensis (Qian et al., 2021). In this study, we found that the conserved A296S mutation was distributed in Cx. tritaeniorhynchus populations in Mianyang. To our knowledge, this is the first record of Rdl A296S mutation in this mosquito species. Notably, The A296S mutation was present at high frequencies (62.04% to 94.00%). In addition to the V327I mutation which has been reported to coexist with A296S in dieldrin-resistant An. funestus (Wondji et al., 2011) and in An. sinensis (Qian et al., 2021), a low frequency (0.93% to 1.80%) of the same V327I mutation was detected for the first time in Cx. tritaeniorhynchus in this study. Interestingly, the V327I was found only in individuals that harbored the A296S mutation. Until now, the impact of the V327I mutation on Rdl sensitivity remains unknown and is worthy of further investigation.

The Gly-119-Ser (G119S) substitution in AChE is commonly detected in mosquitoes (Liu, 2015), but not reported in Cx. tritaeniorhynchus. Instead, the F455W mutation leading to insensitivity of AChE to OP and CB has been identified in Cx. tritaeniorhynchus from Japan (Nabeshima et al., 2004), India (Misra and Gore, 2015), and China (Wu et al., 2016; Liu et al., 2023). The distribution scenario of the AChE F455W mutation observed in this study further supports the notion that the resistance Ace-455W allele is widely distributed at a very high frequency in China (Wu et al., 2016; Liu et al., 2023).

The results revealed the prevalent existence of resistance-conferring mutations in multiple insecticide target proteins of Cx. tritaeniorhynchus in Mianyang City. The co-occurrence of Ace- F455W and Rdl-296S at high levels warns a risk of failure in the control of Cx. tritaeniorhynchus using chemical insecticides targeting the AChE and Rdl in this region. It is highly recommended to monitor phenotypic resistance in Cx. tritaeniorhynchus on a regular basis.