Distribution and diversity of Wolbachia in different populations of the wheat aphid Sitobion miscanthi ( Hemiptera : Aphididae ) in China

Wolbachia is a widely distributed intracellular symbiont in the reproductive tissues of arthropods. The wheat aphid Sitobion miscanthi (Takahashi) is an important agricultural pest worldwide. Wolbachia was detected in different populations of S. miscanthi in China using 16s rDNA and wsp-specific primers. Of eighteen populations eleven were infected with Wolbachia. Several strains of Wolbachia infected these S. miscanthi populations. Of the eleven infected populations, four were infected with only one Wolbachia strain and seven with double infections. This is the most systematic survey of the distribution of Wolbachia in the wheat aphid. 49 * Corresponding author; e-mail: ipmist@163.com These aphids were kept in 100% ethanol at –20°C until DNA extraction.

The wheat aphid, Sitobion miscanthi (Takahashi) (Hemiptera: Aphididae), is a major and widespread pest of cereal crops in China.It inflicts economic damage directly by sap-sucking and indirectly by transmitting the barley yellow dwarf and millet red leaf persistent luteoviruses (Blackman & Eastop, 2000).Because of the considerable reproductive potential, life cycle and behavioural plasticity of this wheat aphid, further characterization of the Wolbachia infection of this aphid is needed in order to understand the effect of this symbiont on aphid reproduction and evolution.West et al. (1998) used 16s rDNA and ftsZ genes to survey four species of aphid: Aphis jacobaeae, Capitophorus carduinus, Microlophium carnosum and Sitobium fragariae, but did not find Wolbachia in any of these species.This was the first report on Wolbachia in aphids.Two years later, Jeyaprakash & Hoy (2000) tested sixty-three arthropod species distributed in sixteen orders using the long PCR method with wsp gene, which encodes a surface protein of Wolbachia.Among these arthropods Wolbachia was detected in two species of aphids: Toxoptera citricida (Kirkaldy) and Aphis craccivora (Koch), with A-Wolbachia identified in T. citricida (Kirkaldy).This A-Wolbachia sequence is the same as the strain wSus-A1 (GB No. AF217713).Tsuchida et al. (2002) conducted an investigation of the distribution of some endosymbiotic bacteria in Japanese populations of the pea aphid, Acyrthosiphon pisum.In spite of the prevalence of secondary endosymbiotic bacteria, Wolbachia was not detected.Nirgianaki et al. (2003) analysed twenty-four DNA samples of aphids provided by Paul Baumann (University of California, Davis, USA).These aphids included Acyrthosiphon pisum, Aphis craccivora, Diuraphis noxia, Myzus persicae, Rhopalosiphum padi, Uroleucon spp.etc.None of these species was infected with Wolbachia.Then Kittayapong et al. (2003) investigated tropical rice-field community insects in Thailand.Forty-nine of 209 rice-field insect species were infected with Wolbachia.Of these insects most were Homoptera (54.2%), but the aphid Hysteroneura setariae (Thomas) was not infected with Wolbachia.In 2004, Gómez-Valero et al. amplified and sequenced the 16s rDNA and wsp genes of Wolbachia in the aphid Cinara cedri.The phylogenetic analysis based on the wsp gene indicated that this kind of Wolbachia belonged to group Con of supergroup B. In addition, their results indicate that Wolbachia coexists with two other endosymbionts: Buchnera aphidicola (the primary endosymbiont in aphids) and S symbiont (a secondary symbiont).This is the first record of Wolbachia in an aphid observed using electron microscopy.
In this paper, we characterized the pattern of Wolbachia infection in natural populations of wheat aphid in China by identifying the strains and determining their relationships with the supergroups of Wolbachia already described.Eighteen natural populations of wheat aphid, S. miscanthi, were screened for infection using Wolbachiaspecific 16s rDNA and wsp genes.

Aphid samples
All the aphid samples used in this study were collected from eighteen areas of China where wheat is grown (Fig. 1).In order to avoid collecting offspring of the same mother, only one aphid was collected from each location, which were ten meters apart.
These aphids were kept in 100% ethanol at -20°C until DNA extraction.

DNA extraction
DNA was extracted according to the protocols described by Vavre et al. (1999) with slight modifications.An aphid was washed in double distilled water and then ground in 200 µl extraction buffer (100 mmolL -1 Tris-HCl, PH7, 1.4 molL -1 NaCl, 20 mmolL -1 EDTA, 2% CTAB) and incubated at 65°C for 1 h.Then 1 µl RNase was added and the solution incubated at 37°C for 1 h.500 µl chloroform-isoamylic alcohol (24 : 1) was added before centrifugation for 15 min at 13,000 rpm.The supernatant was collected and gently mixed with double volumes of 100% ethanol and tenth of volume of Na-acetate (3 mol L -1 , PH 5.2).After precipitation over night at -20°C and centrifuged for 20 min at 13,000 rpm and the precipitate of DNA collected.The precipitate was washed with 70% ethanol and air dried.Finally 20 µl 1 × TE buffer was added to dissolve the DNA sample, which was then stored at -20°C until tested.

Wolbachia detection
Three diagnostic PCRs were performed to amplify a fragment of the 28s rDNA gene of the aphid and of the 16s rDNA and wsp genes of Wolbachia.
The 28s rDNA gene is universally present in eukaryotes and highly conserved.The primers based on the 28s rDNA gene were used to check for the quality of DNA extraction.The primers were forward (5'TAC CGT GAG GGA AAG TTG AAA) and reverse (5'AGA CTC CTT GGT CCG TGT TT).PCR cycling conditions were a 2 min pre-dwell at 94°C followed by 38 cycles of 30 s at 94°C, 50 s at 58°C, 90 s at 72°C and a post-dwell period of 10 min at 72°C.Samples negative for 28s rDNA gene were discarded.The 16s rDNA primers, which were forward (5'CAT ACC TAT TCG AAG GGA TAG) and reverse (5'AGC TTC GAG TGA AAC CAA TTA), were used to screen for Wolbachia infection.PCR cycling conditions were a 2 min pre-dwell at 94°C followed by 38 cycles of 30 s at 94°C, 45 s at 55°C, 90 s at 72°C and a post-dwell period of 10 min at 72°C.The aphid samples that were positive were reamplified using 16s rDNA and wsp primers (81F/522R; 136F/691R) using the PCR conditions described above Zhou et al. (1998).

Cloning and sequencing
PCR products of the 16S rDNA and wsp gene segment were purified using a DNA Fragment Purification kit (Sangon).Purified PCR products were cloned in the plasmid vector pMD19-T (TaKaRa) and transformed into Escherichia coli DH5competent cells.The nucleotide sequences of selected clones were sequenced on an ABI automated sequencer (ABI Prism 377, USA).Both strands of plasmids were sequenced using universal primers (M13+, M13-) with forward and reverse reads.At least three independent clones were sequenced from each Wolbachia strain in order to identify polymerase errors.

Alignments and genetic analyses
Similar sequences to these of the 16S rDNA and wsp genes obtained from the wheat aphid were searched for in GenBank, using BLAST.The 16S rDNA sequences were aligned with the representative dataset of sequences from all supergroups described except supergroup G. Sequences of wsp were aligned with sequences from 33 A-and B-Wolbachia strains downloaded from GenBank (Table 2) followed by manual adjustments based on the amino acid translation of the different genes.Phylogenetic analyses were conducted with neighbour-joining (NJ) and maximum parsimony (MP) methods using MEGA 4.0 (Tamura et al., 2007).For maximum parsimony analysis, the close-neighbour-interchange (CNI) search method was used with the initial tree using random addition trees (10 repetitions).Alignment gaps were excluded and bootstrap analysis carried out with 1,000 replications.For NJ analysis, distances were calculated using the Kimura 2-Parameter model and bootstrap tests performed with 1,000 replications.The phylogenetic tree was constructed using the NJ method.

Recombination analyses of wsp gene
Analysis of recombination was done using the part of the wsp gene.Four wsp sequences, wMisBJA1, wMisBJA1, wMisBJA2, wMisBJA3 and wMisBJB, from infected wheat aphids were aligned with 33 published sequences (Table 2) for recombination analyse.Automated RDP tool implemented in the program RDP2 was used.Default parameters were used and the highest acceptable P value cutoff was 0.01.

Prevalence of Wolbachia in S. miscanthi
This extensive targeted survey for Wolbachia infection in S. miscanthi using PCR amplification of the 16S rDNA gene revealed two different 16S rDNA genes in this wheat aphid.The phylogenetic analysis indicated that the two 16S rDNA sequences belonged to the separate supergroups A and B (Fig. 2).
Of the eighteen geographical populations of S. miscanthi sampled in China, eleven (61%) were infected with Wolbachia, with all the individuals in the population infected.The same phenomenon is also found in whiteflies and leafhoppers (Nirgianak et al., 2003).The reason for this is not clear.Four populations (HBXT, SDJN, AHAQ and GZDY) were infected with only one strain of Wolbachia.There were double infections of all individuals in seven populations: BJ, HBCZ, HBHS, HNXX, HNLY, HNLH and YNKM, and seven populations (SDWF, SDTA, SXLF, AHMC, HBSY, HBXG and SCCD) were not infected by any type of Wolbachia (Table 1).

Phylogeny and recombination of wsp gene
The twenty wsp sequences obtained belong to group Kue, Eva, Mis and Pip, respectively, of which Kue, Eva and Mis belong to supergroup A, Pip to supergroup B (Fig. 3).Group Kue, Eva and Pip are described but Mis is a new group.Intragenic recombination within the wsp gene was shown using the Siscan and RDP methods in RDP2.One recombination fragment was detected in wMisBJA3.The major and minor parents were identified as wMors and wHa.The size of the recombination frag-51   ment is 43 bp (beginning breakpoint 421/ending breakpoint 464).

Distribution of Wolbachia in S. miscanthi
This survey of Wolbachia infections in the wheat aphid, S. miscanthi, in the main wheat growing areas of China detected Wolbachia in eleven of eighteen populations.Several kinds of Wolbachia infected the S. miscanthi populations.Of the eighteen populations examined eleven were infected, four with only one and seven with two Wolbachia strains.Double infections (at least two Wolbachia strains found in one host individual) are only recorded in some homopteran species, such as whitefly (Nirgianaki et al., 2003) and the zig-zag leafhopper (Kittayapong et al., 2003).This is the first report of a double infection in aphids.
The nature of the Wolbachia infections in the different populations differed.The reason for this is unknown, but several factors might have contributed.Migration in wheat aphids is universal in China, and may have affected the distribution of Wolbachia.The direction of migration of S. miscanthi in China is uncertain, so a large-scale investigation of the distribution of Wolbachia in this wheat aphid is needed.
The A-Wolbachia is rarely detected in Hemiptera but the wSus-A1 strain belonging to group Mel is recorded from Toxoptera citricida (Kirkaldy) (Jeyaprakash & Hoy, 2000).However, in Chinese populations of Trichogramma and fruit flies, A-Wolbachia strains are common (Fig. 3).Most of A-Wolbachia strains detected in Chinese insect populations belong to group Kue or Eva.The wDroHN1 [Bactrocera dorsalis (Hendel)] strain belongs to group Mel along with the wSus-A1 strain.A greater diversity of B-Wolbachia are recorded for Hemiptera (Fig. 3).
The Wolbachia recorded in S. miscanthi are very similar to those in other insects, such as trichogramma (Trichogramma ostriniae, Trichogramma evanescens), fruit fly [Bactrocera dorsalis (Hendel)], drosophila [Drosophila simulans (mauritiana)], mosquito (Culex pipiens), leafhopper (Nephotettix nigropictus) and rice moth (Corcyra cephalonica) (Fig. 3).These insects have no direct relationship to S. miscanthi, and Wolbachia is not recorded from plants, however, these insects should be linked with the complex food chain, so the most likely route is horizontal transmission.Another hypothesis is that Wolbachia was present in a distant ancestor of S. miscanthi and that some populations may have lost it.While this may certainly be the case for the B-Wolbachia, the A-Wolbachia appears in different groups in the phylogenetic tree indicating at least some degree of horizontal transfer.

Origins and evolution of Wolbachia in S. miscanthi
In recent years, several studies have revealed that high rates of recombination have occurred in the wsp gene (Baldo et al., 2005a, b;Roy & Harry, 2007;Verne et al., 2007), so using this gene for phylogenetic reconstruction could be misleading.Moreover, a Multilocus Sequence Typing (MLST) scheme exists for genotyping Wolbachia (Baldo et al., 2006;Baldo & Werren, 2007).MLST is an effective means of detecting diversity among strains within a single host, as well as for identifying closely related strains found in different hosts.In this study, the recombination test performed on wsp sequences of Wolbachia infecting S. miscanthi revealed slight intragenic recombination.However, the sequences of the wsp gene used in the analysis of recombination consisted of only part of the whole wsp gene sequence.Analysis using the whole wsp gene would provide more information about recombination.As recently reported, the complete genome of wMel of A-supergroup encodes the necessary machinery for recombination and has experienced both extensive intragenomic homologous recombination and introduction of foreign DNA (Wu et al., 2004).The implications of recombination are clearly of great interest.It may provide a potential motor for evolutionary change and the acquisition of new mechanisms by bacteria.Intracellular symbiosis in aphids is common.The coexistence of Wolbachia with other symbionts in aphids (Gómez-Valero et al., 2004) is recorded.So the patterns of recombination in Wolbachia genomes could clarify important aspects of the evolution of this host-symbiont system (Baldo et al., 2005a).
The prevalence and distribution of the Wolbachia in the wheat aphid S. miscanthi suggest that the effect of Wolbachia on aphid populations merits further study.

Fig. 3 .
Fig. 3. Phylogenetic tree of Wolbachia based on wsp gene constructed using NJ method.S. miscanthi indicated by " ", other aphids by " ", other insects from China are underlined.

TABLE 1 .
Distribution of Wolbachia in different populations of S. miscanthi in China based on 16S rDNA gene.