Sequence variation and haplotype network
PCR amplification and sequencing of the mitochondrial cox1 gene resulted in a 796 bp fragment for each individual study subject, with no insertions or deletions. All of the 120 sequences were identical or possessed > 99% similarity with Ae. albopictus (GenBank: KR068634). 18 variable sites were observed and 14 of them were parsimony informative (Table 1). Among eight populations, the Zhanjiang (ZJ) population had the highest values of polymorphism sites (S = 7), haplotype diversity (Hd = 0.886), nucleotide diversity (π = 0.261) and the highest average number of nucleotide differences (k = 2.076). However, the Beijing (BJ) population had the lowest values of these genetic indices for it had only one haplotype. Tajima’s D tests for all populations were not statistically significant (Table 1), indicating that the populations are in genetic equilibrium, consistent with the neutral mutation hypothesis. Likewise, Fu’s Fs tests were not statistically significant and rejected the population expansion/bottleneck model, with the exception of one Hanzhou population (HZ, Fs = -2.233, P = 0.015) (Table 1).
A total of 20 haplotypes of mtDNA cox1 were detected in the 120 specimens (GenBank: MT188111-MT188130, Additional file 1: Table S1). To determine the relationships among the samples or haplotypes, we constructed a median-joining network using haplotypes based on sequence variation (Fig. 1). The most samples were identified as H01 (49.2%) in Ae. albopictus populations, but no haplotype H01 in Lingshui population (LS) (Fig. 1). Other haplotypes were either unique to a specific population (such as H03 in HZ; H06, H07, H08, H10 in MS; H11 in GZ; H12, H14, H15 in ZJ; H17,H18,H19,H20 in LS), or had a limited geographical distribution (such as H13 and H16 in southern China (ZJ and LS), H09 in southern-western China (MS, GZ and ZJ)) (Fig. 1).
Genetic clustering and differentiation
Based on Bayesian clustering analysis and the Delta K method, the optimal partitioning of all samples was obtained for K = 4 with structure analysis by the Delta K method (Fig. 2). The proportional membership coefficient of individuals was showed in the pie charts from the eight Ae. albopictus populations studied (Fig. 3, Additional file 2: Table S2). The largest membership coefficient value (Q), or the proportion of individuals assigned to a cluster, were high for Lingshui (Q = 0.630), suggesting a strong affinity to be included in a single cluster (cluster 3). This cluster was shared with some of the individuals from Zhanjiang and Guangzhou, consistent with the results obtained in the network analysis (Fig. 1). Individuals from the rest populations mainly constituted three genetic clusters (clusters 1, 2, and 4; Fig. 3), suggesting a strong gene flow among these populations. Based on these results and previous study [25], we divided the eight populations into two groups: southern (GZ, ZJ and LS) and other populations (BJ, SJZ, HZ, WH, MS). AMOVA results (Table 2) indicate that the majority of the variation in Ae. albopictus was within populations, accounting for 54.19% of the variation, while variations among groups and populations within groups accounted for 35.10% and 10.72% of the total variation, respectively. Fisher’s exact test showed that there was significant genetic variation at these three levels.
Table 2
Analysis of molecular variance (AMOVA) of two groups of populations of Ae. albopictus in China
Source of variation | df | SS | Variance components | Percentage of variation | P-value | Fixation index |
Among groups | 1 | 21.018 | 0.337 | 35.10 | P = 0.012 | FCT = 0.351 |
Among populations within groups | 6 | 12.382 | 0.103 | 10.72 | P < 0.001 | FSC = 0.165 |
Within populations | 112 | 58.267 | 0.520 | 54.19 | P < 0.001 | FST = 0.458 |
Total | 119 | 91.667 | 0.960 | | | |
Abbreviations: df, degrees of freedom; SS, sum of squares |
Twenty-one of 28 pairwise tests were significant at P < 0.05 after Bonferroni correction, and pairwise FST values ranged from 0.117 (between HZ and GZ) to 0.620 (between BJ and LS), with an average of 0.333 (Table 3). The Mantel test showed statistically significant correlation (y = 0.52x – 3.18, R2 = 0.364, P = 0.003) between the genetic distance (y, estimated as FST /(1 − FST)) and geographical distance (x, estimated as Ln (km)) between populations.
Table 3
Pairwise genetic differentiation and geographical distance between Ae. albopictus populations from China
| BJ | SJZ | HZ | WH | MS | GZ | ZJ | LS |
BJ | - | 5.573 | 7.021 | 6.959 | 7.347 | 7.536 | 7.685 | 7.803 |
SJZ | 0.000 | - | 6.900 | 6.714 | 7.169 | 7.403 | 7.563 | 7.696 |
HZ | 0.048 | 0.003 | - | 6.312 | 7.352 | 6.947 | 7.286 | 7.415 |
WH | 0.071 | 0.004 | -0.025 | - | 6.916 | 6.710 | 7.044 | 7.247 |
MS | 0.170* | 0.160* | 0.136* | 0.150* | - | 7.107 | 7.064 | 7.273 |
GZ | 0.208* | 0.181* | 0.117* | 0.141* | 0.160* | - | 6.084 | 6.434 |
ZJ | 0.513* | 0.498* | 0.432* | 0.448* | 0.346* | 0.243* | - | 5.668 |
LS | 0.620* | 0.602* | 0.526* | 0.542* | 0.441* | 0.354* | 0.022 | - |
Pairwise genetic differentiation (FST) between all populations displayed below the diagonal; geographical distance [ln (km)] displayed above the diagonal |
*Asterisks indicate significant values after Bonferroni correction (P < 0.05) |
Vector competence of Ae. albopictus for DENV-2
At 14 days post-infection (dpi), The IR, DR, TR and PTR were all 100.00% (30/30) in BJ and SJZ populations ; 96.67% (29/30), 96.55% (28/29), 93.10% (27/29) and 90.00% (27/30), respectively, in HZ population; 96.67% (29/30), 96.55% (28/29), 89.66% (26/29) and 86.67% (26/30), respectively, in WH population; 93.33% (28/30), 96.43% (27/28), 92.86% (26/28) and 86.67% (26/30), respectively, in MS population; 93.33% (28/30), 96.43% (27/28), 89.29% (25/28) and 83.33% (25/30), respectively, in GZ population; 93.33% (28/30), 89.29% (25/28), 85.71% (24/28) and 80.00% (24/30), respectively, in ZJ population; 96.67% (29/30), 93.10% (27/29), 89.66% (26/29) and 86.67% (26/30), respectively, in LS population (Table 4). The IR, DR and TR of DENV-2 in mosquitoes were not significantly different among the eight population of Ae. albopictus (IR: χ2 = 4.52, df = 7, P = 0.838; DR: χ2 = 5.72, df = 7, P = 0.456; TR: χ2 = 8.56, df = 7, P = 0.221). However, there was significant difference in the PTR among them (χ2 = 13.18, df = 7, P = 0.043). The PTR between populations was further compared by using ANOVA post hoc Tukey’s HSD tests. The PTR in ZJ population showed significantly different with that in BJ population (χ2 = 6.67, df = 1, P = 0.024) and SJZ population (χ2 = 6.67, df = 1, P = 0.024), respectively.
Table 4
Rates of dengue virus infection, dissemination, potential transmission and population potential transmission by Ae. albopictus females from eight different populations
Populations | IR | DR | TR | PTR |
BJ | 100.00% (30/30) | 100.00% (30/30) | 100.00% (30/30) | 100.00% (30/30) |
SJZ | 100.00% (30/30) | 100.00% (30/30) | 100.00% (30/30) | 100.00% (30/30) |
HZ | 96.67% (29/30) | 96.55% (28/29) | 93.10% (27/29) | 90.00% (27/30) |
WH | 96.67% (29/30) | 96.55% (28/29) | 89.66% (26/29) | 86.67% (26/30) |
MS | 93.33% (28/30) | 96.43% (27/28) | 92.86% (26/28) | 86.67% (26/30) |
GZ | 93.33% (28/30) | 96.43% (27/28) | 89.29% (25/28) | 83.33% (25/30) |
ZJ | 93.33% (28/30) | 89.29% (25/28) | 85.71% (24/28) | 80.00% (24/30) |
LS | 96.67% (29/30) | 93.10% (27/29) | 89.66% (26/29) | 86.67% (26/30) |
Abbreviations: IR: infection rate = no. infected midguts/no. tested midguts (%); DR: dissemination rate = no. infected heads/no. infected midguts (%); TR: potential transmission rate = no. infected salivary glands/no. infected midguts (%); PTR: potential population transmission rate = no. infected salivary glands/no. infected mosquitoes (%). |
The amount of dengue virus in the mosquito midguts, heads and salivary glands was measured by qRT-PCR (Fig. 4). At 14 days post-infection, the average DENV RNA copies (log10) in midguts for each population were 4.82 (BJ), 4.55 (SJZ), 4.38 (HZ), 5.11 (WH), 5.21 (MS), 3.24 (GZ), 4.18 (ZJ), 4.51 (LS), respectively. There was significant difference (P < 0.05) in midgut between GZ and each one of BJ, SJZ, HZ, WH, MS, LS; and between MS and ZJ. The average DENV RNA copies (log10) in heads for each population were 4.25 (BJ), 3.93 (SJZ), 3.79 (HZ), 4.69 (WH), 3.99 (MS), 3.23 (GZ), 3.51 (ZJ), 3.68 (LS), respectively. There was significant difference (P < 0.05) in heads between WH and each one of SJZ, HZ, MS, GZ, ZJ, LS; between GZ and each one of BJ, SJZ, MS; and between BJ and JZ. The average DENV RNA copies (log10) in salivary glands for each population were 3.72 (BJ), 3.63 (SJZ), 3.35 (HZ), 3.85 (WH), 3.45 (MS), 3.13 (GZ), 2.94 (ZJ), 3.13 (LS), respectively. There was significant difference (P < 0.05) in salivary glands between GZ and each one of BJ, SJZ, WH; between ZJ and each one of BJ, SJZ, WH, MS; between LS and each one of BJ, SJZ, WH; and between WH and HZ.