Animals and Tissues

T. urticae were collected from Solanum melongena L. in 4 regions of 3 provinces, China. Locations, host plants, collection dates and abbreviations are summarized in Table 1. DNA was isolated immediately once mites were taken to the laboratory or the samples were kept at -70°C until use.

Forty female adults were randomly selected from each geographical population to conduct the PCR amplification.

TABLE 1.

Collection location, host plant and collection date of Tetranychus urticae from China.

DNA extraction

Total DNA was extracted from individual adult females using protocols described in Xie et al. (2008). In every sampled locality, three individuals were sequenced.

Amplification of wsp gene in Wolbachia and ITS2 in T. urticae A fragment in the wsp gene of about 596bp was amplified from the DNA with the specific primers (81F: 5′-TGGTCCAATAAGTGATGAAGAAAC-3′; 691R: 5′- AAAAATTAAACGCTACTCCA-3′) (Zhou et al. 1998). The amplification reactions were performed in a 50μL volume containing 4μL DNA template solution, 28.6μL ddH2O, 5μL 10×buffer, 5μL MgCl2 (25 mmol/L), 4μL dNTPs (10mmol/L each), 0.4μL Taq DNA polymerase (5U/μ, TaKaRa, Dalian), 1.5μL each primer (20 μmol/L each) according to the following schedule: 4 minutes at 94°C followed by 30 cycles at 94°C (1 minute), 53°C (1 minute) , 72°C ( 1 minute ).

A 645bp fragment of ITS2 was amplified from the DNA with the specific primers (ITS2-S:5′- ATATGCTTAAATTCAGCGGG-3′;ITS2-A: 5′-GGGTCGATGAAGAACGCAGC-3′.) The primers were defined in the highly conserved 5.8S and 28S flanking regions as previously published in Navajas et al. (1994). The amplification reactions were performed in a 50μL volume containing 2μL DNA template solution, 30.6μL ddH2O, 5μL 10×buffer, 5μL MgCl2 (25 mmol/L), 4μL dNTPs (10mmol/L each), 0.4μL Taq DNA polymerase (5U/μ, TaKaRa, Dalian), 1.5μL each primer (20 μmol/L each) according to the following schedule: 4 minutes at 94? followed by 30 cycles at 94°C (1 minute), 53°C (1 minute), 72°C (1 minute).

The PCR products of the appropriate sizes were gel-purified with the DNA Gel Extraction Kit (Tiangen Biotech, Beijing), cloned into T-easy vector (Promega USA), transformed into Escherichia coli DH5α and sequenced in the gene-sequencing company (Bioasia company).

Gene sequencing and sequence analysis

Three samples from each geographical population infected with Wolbachia were randomly chosen to sequence. DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, and the identical sequences were used as the standard. Clustal X computer program (Thompson et al. 1997) was used to align the wsp gene of T. urticae infected with Wolbachia sequenced in our research with the published wsp genes of other mites in Tetranychidae. Analysis of genetic and phylogenetic relationships was performed using MEGA2.1 (Kumar et al. 2001). Genetic relationships among every geographical population were estimated based on the distance using the Kimura-2 Parameter method. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.

According to detection results of the infection with Wolbachia, ten female adults infected with Wolbachia and ten which were not infected were chosen from each geographical population. And the DNA isolated from each individual was used as the template to amplify ITS2 fragment with the designed primers (ITS2-S and ITS2-A). Thus, eighty ITS2 sequences were obtained. Haplotype analysis was conducted using TCS1.21 software (Clement et al. 2000).

ITS2 sequence diversity and evolution analysis

Haplotype diversity (Hd), nucleotide diversity (π) and average difference of nucleic acid (k) were chosen to determine the differences of ITS2 sequences between T. urticae infected with Wolbachia and those uninfected. A neutrality test was conducted in order to learn the evolution of ITS2 sequences. All the calculations were performed in the program DnaSP5.10 (Pablo & Rozas, 2009).

Infection with Wolbachia

PCR results showed that all four geographical populations of T. urticae were infected with Wolbachia at an average intraspecific infection rate of 50.4%. Among the four populations, the HHHT and TA populations were infected with the highest rate of 60%, and the infection rate of the YT population was the lowest at 27.5% (Table 2). The four populations were infected with two strains of Wolbachia belonging to the Ori group and Con group of B supergroup, respectively, that is, wUrtOri1 (HM486515- HM486517) and wUrtCon1(HM486518) (Figure 1).

TABLE 2.

Infection rates of four geographical populations of T. urticae with Wolbachia. Forty female adults were checked for each population.

FIGURE 1.

Phylogenetic tree of wsp genes of four geographical populations of T. urticae with other reported Tetranychidae

Alignment of 80 ITS2 gene sequences showed that there were no base insertions or deletions, and that there was just one mutable site (Fig. 2). The 80 ITS2 sequences were classified into 2 haplotypes (Fig. 3).

ITS2 sequence diversity and its evolution analysis

Hd, π and k of ITS2 from T. urticae individuals infected with Wolbachia were all lower than those of ITS2 from T. urticae individuals uninfected with Wolbachia, and the differences were not significant. Neutrality tests showed that Tajima'D and Fu&Li'F values fluctuated around 0.

FIGURE 2.

Mutation sites of ITS2 in T. urticae.

FIGURE 3.

Haplotype network of ITS2 sequences in T. urticae

*Cycle and square frame represent haplotypes. Area size indicated the number of haplotypes, and the connecting line presented one mutation step.

TABLE 3.

ITS2 diversity analysis of four geographic populations of T. urticae.

Discussion