Mosquito strain and deltamethrin selection

Five strains of Cx. quinquefasciatus, including two field-collected parental strains (Cq_SP and Cq_NiH), two deltamethrin-selected strains (Cq_SP-R and Cq_NiH-R), and a reference susceptible Cq_Sus strain, were used in this study. All five mosquito strains have been maintained and reared at 25 ± 2 ℃ under a photoperiod of 12:12 (L: D) h in the insectary of the Department of Parasitology, Faculty of Medicine, Chiang Mai University (CMU), Thailand.

Larvae and pupae of Cx. quinquefasciatus were collected from the Sri Phum Sub-District, Muang District, Chiang Mai Province, Thailand (18๐47′35″ N, 98๐58′54′′ E) in 2019 [10] and were subsequently maintained as a colony designated as the Cq_SP strain. The Cq_NiH strain was obtained from the National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Thailand and has been maintained continuously in the CMU insectary since 2015. This strain was a long-established colony originally collected from Pom Prab Satru Phai District, Bangkok, Thailand in 1978 [20]. The susceptible Cq_Sus strain continuously had over 98% mortality in the adult bioassay tested with 0.05% deltamethrin-impregnated WHO paper for over ten generations.

Establishing a deltamethrin-resistant Cq_SP-R strain, the parental Cq_SP strain was subjected to selection pressure with deltamethrin throughout four generations in a controlled laboratory environment. The larvae were exposed to deltamethrin concentrations of 0.18 µg/L, 0.50 µg/L, 1.17 µg/L, and 1.20 µg/L for the first through fourth generations, respectively. These concentrations were determined to effectively induce mortality in 50% of the treated individuals within 24 h. Similarly, the parental Cq_NiH strain was subjected to deltamethrin selection for five generations, leading to the establishment of the deltamethrin-resistant Cq_NiH-R strain. Adults were exposed to 0.05% deltamethrin-impregnated WHO paper in the first two generations. Larvae from the third to fifth generations were then exposed to deltamethrin concentrations of 0.18 µg/L, 0.50 µg/L, and 1.17 µg/L, respectively. Survivors from each generation were retained to continue the selection process.

Insecticide susceptibility test

The larval susceptibility test was performed following the WHO standard guidelines [21] with slight modifications as previously described [4]. Briefly, the bioassays were carried out using batches of 25 early 4th instar larvae per beaker containing 250 ml of distilled water. Seven to eight different insecticide concentrations (0.05–5 µg/L) giving 0–100% mortality were tested against the larvae. The bioassays were conducted with four replicates for each concentration using a single beaker for each replication. The stock and serial dilutions of deltamethrin (Supelco, Bellefonte, PA, USA) were prepared in ethanol. In the control experiments, 0.4% ethanol was included in 250 ml of water. Larval mortality was recorded after 24 h exposure. Adult susceptibility tests were conducted following WHO standard methods [22, 23]. Due to the high levels of pyrethroid resistance observed in Cx. quinquefasciatus in Thailand [4, 10, 19], Anopheles mosquito discriminating concentrations were used instead of Culex mosquito discriminating concentrations. Four batches of 25, one–two days old, non-blood-fed females were exposed to 0.05% deltamethrin-impregnated paper and 0.75% permethrin-impregnated paper for 60 min in the standard WHO bioassay test tubes. The mortality was scored and recorded after 24 h. Dead (susceptible) and survivor (resistant) mosquitoes after the bioassays were stored at − 20 ℃ until tested.

Gene expression analysis of the Culex quinquefasciatus-cytochrome P450 genes using quantitative real-time PCR (qRT-PCR)

The expression levels of the candidate cytochrome P450 genes, CYP4C52v1, CYP4H34, CYP6AA7, CYP6P14, CYP9AL1, CYP9J34, CYP9J45, and CYP9M10, were measured using quantitative real-time PCR (qRT-PCR). Those candidate genes were selected for expression analysis based on previous evidence indicating their involvement in metabolic resistance [13,14,15]. The total RNA was isolated from the fourth instar larvae and one-day-old females of each mosquito strain. The pool of ten mosquitoes was used to extract total RNA for three replications using the illustra™ RNAspin Mini Isolation Kit (GE Healthcare, Buckinghamshire, UK), following the manufacturer’s instruction. The total RNA was reversed transcribed to single-strand cDNA using the SuperScript™ III First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA). The qRT-PCR was performed with the SensiFAST™ SYBR® Lo-ROX Kit (Bioline, Meridian Bioscience, Germany) in the 7500 Fast Real-Time PCR machine (Applied Biosystems, USA). Each qRT-PCR reaction was carried out in 25 µl final volume containing 1 × SYBR® Green master mix, 1 µl of cDNA, and the cytochrome P450 genes specific primer pairs [24] at a final concentration of 5 µmol/L. All samples and negative control were performed in triplicate. The qRT-PCR reaction cycle consists of a denaturing step of 50 ℃ for 2 min, then 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. The specificity of the PCR reactions was assessed by a melting curve analysis using Dissociation Curves software. Relative expression levels for the cytochrome P450 genes were calculated by the 2−∆∆CT method using 7500 software v2.3 (Applied Biosystems, USA). The expression of the target gene was normalized using the 18S ribosome RNA gene as an endogenous control [24].

Amplification and DNA sequencing of the fragments of the Culex quinquefasciatus-vgsc gene

The total RNA was isolated from a single and pooled ten mosquitoes of the one-day-old females of each strain for three replications. The single-strand cDNA was synthesized and used as a template for PCR amplification. Four fragments of the Cx. quinquefasciatus-vgsc gene were amplified for encompassing four domains of VGSC protein (Fig. 1) using four pairs of primer, newly designed in this study (Table 1). All PCR reactions were carried out in a volume of 20 µl that contained a final concentration of 1.25 units of Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA, USA), 1 × High Fidelity PCR buffer, 0.5 mol/L dNTPs, 2.5 mol/L MgSO4 and 0.5 µmol/L each of the forward and reverse primers. The amplifications consisted of an initial heat activation step at 94 ℃ for 90 s, followed by 35 cycles of 94 ℃ for 45 s, 62 ℃ for 45 s, and 68 ℃ for 60 s with a final extension step at 68 ℃ for 10 min. PCR products were analyzed by electrophoresis on 2% agarose gel (Invitrogen, Carlsbad, CA, USA) using a voltage of 120 v for 25 min and visualized under UV light by RedSafe™ Nucleic Acid Staining (iNtRON biotechnology, Korea). The PCR products were purified using illustra™ ExoStar™ 1-Step kit (GE Healthcare, Buckinghamshire, UK). The PCR product was purified and then sent to Macrogen (Seoul, Korea) for DNA sequencing in both directions.

Fig. 1

The VGSC pore-forming α-subunit consists of four homologous repeat domains (I–IV), each with six transmembrane segments (S1–S6) connected by intracellular and extracellular loops. The four fragments of the vgsc gene, including the IS2–IS6 region of domain I (green fragment; F1), the IIS1–IIS6 region of domain II (dark green fragment; F2), the IIIS1–IIIS6 region of domain III (tan fragment; F3) and the IVS1–IVS6 region of domain IV (dark tan fragment; F4) were amplified in this study. Three non-synonymous mutations in the vgsc gene were identified in the resistant strains of Cx. quinquefasciatus. These vgsc mutations, including V240M, novel L925F and kdr L1014F, were indicated by the red and orange circles. The red circle denotes the kdr L1014F mutation observed in both the Cq_SP and Cq_SP-R strains. The orange circle represents two vgsc mutations, V240M and L925F, exclusively found in the Cq_SP-R strain

Schematic of the voltage-gated sodium channel.

Table 1 Sequences of primers for amplifying the Culex quinquefasciatus voltage-gated sodium channel geneGenotyping of the CYP9M10 gene and the kdr L1014F mutation in vgsc gene

After exposure to 0.05% deltamethrin WHO paper, the survivor and dead mosquitoes were randomly selected for genotyping. Genomic DNA was extracted from a single mosquito using the method previously described [27]. Genotyping of the CYP9M10 gene was performed using PCR I and II, previously described [28], with some modifications. In brief, the PCR reaction was carried out in a 25 μl PCR master mix that contained 1.5 mol/L MgCl2, 1 × PCR buffer, 0.2 µmol/L of each primer, 200 µmol/L dNTP, 0.2 U/µl Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). Each PCR reaction was performed in a 25 μl reaction volume. The PCR master mix contains 1 × PCR buffer, 1.5 mol/L MgCl2, 200 µmol/L dNTP, 0.2 U/μl of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 0.2 µmol/L each primer of Gen1F and Gen1R for genotyping PCR I, and Gen2Fa and Gen2Fb for genotyping PCR II, and 0.4 µmol/L of Gen2R for genotyping PCR II. The PCR reaction began with a 2-min denaturation step at 94 ℃, followed by 35 cycles of 30 s at 50 ℃, 30 s at 72 ℃, and 30 s at 72 ℃, with a 2-min extension step at 72 ℃. PCR products were analyzed by electrophoresis on 2% agarose gel (Invitrogen, Carlsbad, CA, USA) using a voltage of 100 v for 30 min and visualized under UV light by RedSafe™ Nucleic Acid Staining (iNtRON biotechnology, Korea). The kdr L1014F mutations in the Cx. quinquefasciatus-vgsc gene was detected using the tetra-primer AS-PCR and real-time PCR with melt curve analysis methods as described previously [19, 29].

Data analysis

The concentration-mortality responses for the larvae susceptibility test were determined by probit analysis [30] using the LdP Line software (www.Ehabsoft.com/LDPline). The statistical significance of the gene expressions was calculated using a Student’s t-test for all 2-sample comparisons and one-way analysis of variance (ANOVA) for multiple sample comparisons using SPSS version 17.0 (SPSS Inc., Chicago, USA); a value of P ≤ 0.05 was considered statistically significant. Significant overexpression was determined using a cut-off value of a ≥ twofold change in expression [31]. Nucleotide sequences of the vgsc gene were aligned using Geneious Prime software version 2023.1.2 (Biomatters, Auckland, New Zealand). The genotypes and allele frequencies of the CYP9M10 gene and the kdr L1014F mutation in the vgsc gene of Cx. quinquefasciatus mosquitoes were calculated, and statistical differences between the survivor and dead mosquito groups were examined by Fisher’s exaction test using Genepop [32].