Sequence polymorphisms of the An. sinensis vgsc gene

The 267-bp DNA fragments individually amplified from a total of 89 An. sinensis mosquitoes were used for sequence polymorphism analysis. This sequence covered partial exon 19 (contains the codon 1014), intron 19, and partial exon 20 of the An. sinensis vgsc gene (KE525266.1). In general, 17 nucleotide polymorphic sites (PSs) were identified. The first to fifth PSs were located on exon 19, the sixth to 15th PSs on intron 19, and the 16th and 17th PSs on exon 20. The polymorphisms in the fourth and fifth PSs resulted in amino acid substitutions (L/F/S) at codon 1014, and the nucleotide variations in the first, second, third, 16th, and 17th PSs represented synonymous mutations (Fig. 1).

Fig. 1
figure 1

The nucleotide region of the An. sinensis vgsc gene addressed in this study. Dots indicate the polymorphic sites (PSs) in the obtained sequences. The red dots represent sites leading to nonsynonymous mutations. The positions of PSs in the 267-bp sequence are numbered below the dots. The nucleotides for each PS are given

Diversity and frequency of kdr haplotypes

Point mutations at position 1014 of the domain II of the VGSC protein have been documented to confer kdr [9, 10]. To better understand the diversity and frequency of kdr haplotypes in this area, we downloaded 70 kdr sequences of An. sinensis from NCBI, combined with our original data from 89 samples collected in Laos. Thirty-nine haplotypes were identified from 159 An. sinensis individuals (Table 1). Seven haplotypes (i.e., 1014L19, 1014L20, 1014L21, 1014F7, 1014F8, 1014F9, 1014W1) were newly identified in other research except for one haplotype, 1014L21, which was newly identified in this study. In the present study, only a wild haplotype of kdr was detected, TTG (1014L); other mutations such as TTT (1014F), TCG (1014S), and TGT (1014C) were not found. Accordingly, nine wild haplotypes, namely 1014L1, 1014L2, 1014L3, 1014L4, 1014L7, 1014L9, 1014L10, 1014L11, and 1014L21, respectively, were identified (Table 1), in the frequencies ranging from 1.12% (1/89) to 43.82% (39/89) in the three Laos populations (Fig. 2). In the LPY (Yot Ou County, Phongsaly Province) population, 1014L1 (46.99%, 39/83), 1014L4(9.64%, 8/83), 1014L7 (12.05%, 10/83), 1014L9 (7.23%, 6/83), and 1014L10 (19.28%, 16/83) had higher frequencies than other haplotypes. In the LCP (Pathoomphone County, Champasak Province) population, only 1014L3 was identified and accounted for 100% (4/4). In the LXP (Pak lay County, Xayabuli Province) population, 1014L3 and 1014L9 were both 50% (1/2). In addition, the newly identified susceptible haplotype, 1014L21, was uniquely distributed at LPY at a low frequency (1.20%, 1/83). Due to the time limit and different sample sizes between each sampling site, further evaluation is needed.

Fig. 2
figure 2

Distribution and frequency of kdr alleles in An. sinensis populations along the China–Laos border, Thailand–Laos border, and Cambodia–Laos border. Pak lay County (Xayabuli Province); Yot Ou County (Phongsaly Province); Pathoomphone County (Champasak Province). Red line = Mekong River. The shapefile map of Laos was downloaded and prepared by using Pixel Map Generator-Beta online (amCharts, Vilnius, Lithuania) (https://pixelmap.amcharts.com/), which is copyright-free

To analyze the genetic diversity indices and neutrality tests (Fu’s Fs and Tajima’s D) based on the kdr intron of An. sinensis, 89 kdr intron sequences in this study and 65 kdr intron sequences retrieved from the GenBank database (Additional file 3: Table S3a) were used in the subsequent analysis. A total of 14 haplotypes were found in nine populations. Genetic diversity varied significantly among geographical regions. The overall haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.788 and 0.02190, respectively. Compared to the three An. sinensis populations in Laos, high haplotype diversity and nucleotide diversity were found in six populations from China. Moreover, significant departures from neutrality were detected by Fu’s Fs test in all the populations (−5.17700, P < 0.05), CN-GX (Guangxi Province, Southwest China) population (−4.51700, P < 0.02), and the CN-HaN (Hainan Province, Southwest China) population (−3.66700, P < 0.02), whereas they were not detected in all the Laos populations by Fu’s Fs or Tajima’s D test. Additionally, slightly insignificant departures from neutrality were detected by Fu’s Fs test in the CN-AH (Anhui Province, Central China) population (−1.50700, P < 0.1) and CN-YN (Yunnan Province, Southwest China) population (−1.19500, P < 0.1) (Additional file 3: Table S3a). Since the Fu’s Fs statistic is particularly sensitive to demographic effects, it is difficult to conclude whether positive selection or demographic history (e.g., population expansion) accounts for the observed pattern.

Genealogical analysis of kdr mutations

Network analysis showed that haplotypes H6-1014L7 and H8-1014L11 were derived from single mutational steps through T231C and G202T, respectively, in the intron 19 from ancestor H1-1014L1, while H1-1014L21 reported in this study derived from H1-1014L1 with a single mutation at C245T in the exon 20. Haplotype H3-1014L3, which was only detected in LXP and LCP populations, and H6-1014L10, were derived from single mutational steps through G202T and T215C, respectively, in the intron 19 from ancestor H2-1014L2. Additionally, H4-1014L4 was derived from H3-1014L3 with a single mutation at T215C in intron 19, while H4-1014L9 exhibited one more single mutation at C245T in exon 20 from the ancestor H3-1014L3 (Fig. 3a).

Fig. 3
figure 3

The network of kdr haplotypes identified in An. sinensis populations. a Networks showing the genealogical relationship in Laos. Yellow, green, and blue circles represent 1014L haplotypes from LPY (Phongsaly Province: Yot Ou County), LCP (Champasak Province: Pathoomphone County), and LXP (Xayabuli Province: Pak lay County) populations, respectively. The size of each circle is proportional to its corresponding frequencies. H represents the type of intron haplotypes (numbers in brackets). The straight line indicates the possible mutational step. The note above the line referred to the mutation position and base. b Networks showing the genealogical relationship among different kdr haplotypes from the present study and NCBI data. Yellow, blue, green, red, and pink circles represent 1014L, 1014S, 1014C, 1014F, and 1014 W haplotypes, respectively. The size of each circle is proportional to its corresponding frequencies

To estimate the evolutionary relationship of kdr mutations, nine haplotypes identified in this study and 30 haplotypes retrieved from the GenBank database (Table 1) were used to construct a network using Network 4.0. The network analysis revealed complex reticulate patterns and multiple independent mutation events leading to kdr haplotypes. The genealogical analysis revealed that a single mutation might result in the resistant phenotype from the susceptible one. For example, 1014S2 shared an identical intron sequence with 1014L2, L5, and S1, and a single mutation (T165C) might change the susceptible 1014L2 to the resistant 1014S2. Haplotypes 1014F1, F2, F3, F5, F7, and F9 were possibly derived from 1014L1, 1014L3, 1014L3, 1014L7, 1014L2, and 1014L11, respectively, while 1014F6 and F8 derived from the ancestor 1014L1 with two mutational steps, i.e., the additional mutations in exon 19 (T140A) or intron 19 (T229A) of 1014F1. Likewise, 1014F4 was derived from the ancestor 1014L11 with two mutational steps, i.e., the additional mutations in exon 19 (T140A) of 1014F9. Haplotypes 1014S1, S2, S3, S4, S5, and S6 were perhaps the results of an independent mutational step from six different wild haplotypes 1014L5, 1014L2, 1014L3, 1014L4, 1014L1, and 1014L6, respectively. Additionally, 1014W1 was derived from the ancestor 1014L1, while 1014C1 and 1014C2 were derived from 1014F1 with two or more mutational steps (Fig. 3b, Table 1).

Mitochondrial DNA sequence variation

Eighty-nine sequences for COII were generated for the three populations (Additional file 3: Table S3b). The COII sequence alignment revealed 26 variable sites. The overall haplotype diversity (Hd), number of haplotypes, and nucleotide diversity (Pi) were 0.799, 22, and 0.00351, respectively. Significant departures from neutrality were both detected by Fu’s Fs test (−11.48900, P < 0.001) and Tajima’s D test (−1.79501, P < 0.02) in all the populations. In contrary to the LXP and LCP populations, high haplotype diversity (0.783), number of haplotypes (20), and nucleotide diversity (0.00282) were found in the LPY population. Additionally, significant departures from neutrality were both detected by Fu’s Fs test (−11.80300, P < 0.001) and Tajima’s D test (−1.61923, P < 0.05) in the LPY population, whereas not detected in the LXP and LCP population.

Population structure and genetic differentiation

The median-joining network based on 89 COII sequences denoted the distribution pattern exhibited by 22 haplotypes in An. sinensis populations. The An. sinensis populations fell into two main clusters. Cluster 1 consisted of all the haplotypes except for one haplotype (H22) from LCP; cluster 2 consisted of only one haplotype (H22). Within cluster 1, two sub-clusters were also found; sub-cluster 1 consisted of all the haplotypes in LPY and LXP, while sub-cluster 2 consisted of only one haplotype (H21) in LCP (Fig. 4a). The most common haplotypes referred to H1 (n = 13), H4 (n = 37), and H6 (n = 7), as only identified in 67.47% (56/83) of LPY and 50% (1/2) of LXP. H21 (n = 3) and H22 (n = 1) were only identified in LCP (Fig. 4a). The unweighted pair group method with arithmetic mean (UPGMA) dendrogram based on Nei’s unbiased genetic distances between haplotypes indicated that H22 constituted one cluster, while the other haplotypes constituted the second (Fig. 4b). Additionally, two sub-clusters were also found in the dendrogram, which was consistent with the results of the median-joining network.

Fig. 4
figure 4

Phylogenetic analysis based on the COII sequences in An. sinensis populations in Laos. a Phylogenetic network of 22 mitochondrial haplotypes of the COII gene in An. sinensis. Localities are indicated by different colors (bottom right). The size of each circle is proportional to its corresponding frequencies. b UPGMA dendrogram based on Nei’s unbiased genetic distance between the 22 haplotypes of An. sinensis. Yellow, green, and blue circles/rectangles represent haplotypes from LPY (Phongsaly Province: Yot Ou County), LCP (Champasak Province: Pathoomphone County), and LXP (Xayabuli Province: Pak lay County) populations, respectively

To draw a broader comparison in haplotype from Laos and other geographical regions, we downloaded and analyzed available data in GenBank from neighboring nations (Additional file 2: Table S2). In general, a total of 148 An. sinensis COII sequences were generated for seven populations, including LPY (n = 83), LCP (n = 4), LXP (n = 2), JP (Japan, n = 3), KR (South Korea, n = 19), TH (Thailand, n = 3), and CN (China, n = 34), and 46 haplotypes were found in the median-joining network. The An. sinensis populations fell into two main clusters. Cluster 1 consisted of all the haplotypes except for one haplotype (H27) from LCP; cluster 2 consisted of only one haplotype (H27). Within cluster 1, three sub-clusters were also found: sub-cluster 1 consisted of haplotype (H26) from LCP; sub-cluster 2 consisted of five haplotypes (H29, H35, H37, H42, and H43) from China; sub-cluster 3 consisted of other haplotypes (Additional file 4: Fig. S1a). The UPGMA dendrogram based on Nei’s unbiased genetic distances between haplotypes indicated that H27 constituted one cluster, while the other haplotypes constituted the second (Additional file 4: Fig. S1b). Additionally, three sub-clusters were also found in the dendrogram, which was consistent with the results of the median-joining network.

AMOVA analysis based on COII sequences demonstrated that most of the variances were found among group variation (58.43%) rather than within populations (38.93%) and among populations within groups (2.64%), suggesting that these populations could fall into several groups. However, no statistical significance was found in evaluating the fixation index among groups (FCT), among populations within groups (FSC). In contrast, the fixation index within populations (FST) showed statistical significance (P < 0.05) (Additional file 5: Table S4). Due to the time limit and different sample sizes between each sampling site, further evaluation is needed.

The maximal level of genetic differentiation by the fixation index FST based on sequences analysis of COII was between LCP and LPY (FST = 0.61657, ˂ 0.05). while no significant genetic differentiation was found between LPY and LXP populations (0.08915, P > 0.05) or between LXP and LCP populations (0.09565, P > 0.05). Furthermore, the minimal estimate of gene flow (Nm) was between LCP and LPY (Nm = 0.31093). In contrast, high gene flows were found between LPY and LXP populations (Nm = 5.10835), as well as between LXP and LCP populations (Nm = 4.72727) (Additional file 6: Table S5).

Spatial genetic structure analysis, demographic history, and neutrality test

A Mantel test revealed a significant correlation between geographical and genetic distances in all populations (Z = 655.5437, r = 0.7995, P ≤ 0.0010), suggesting the genetic structure observed in An. sinensis populations could be partially explained by distance isolation based on COII sequence analysis (LCP and LPY populations) (Additional file 7: Fig. S2a). As indicated from Tajima’s D and Fu’s Fs tests based on COII, the LPY population exhibited significant negativity (P < 0.05, P < 0.001), suggesting a recent population expansion or selection (Table S3b). Furthermore, the observed smooth and unimodal mismatch distribution in the LPY population suggested a sudden population expansion, conforming to the mismatch distribution derived under the model of sudden expansion (Additional file 7: Fig. S2 b–d).

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