Studies On The Eight Sibling Species Of Sclerodermus (Hymenoptera: Bethylidae) And The Biotypes Of Dastarcus Helophoroides (Coleoptera: Bothrideridae) By Using Molecular Markers

Currently the parasitoid beetle Dastarcus helophoroides(Coleoptera: Bothrideridae) is used as main insect natural enemy to control the longhorned beetles in China. Biological control has been widely used in large beetle species, such as, Anoplophora glabripennis(Coleoptera: Cerambycidae), Monochamus alternatus Hope(Coleoptera: Cerambycidae), Apriona swainsoni Hope(Coleoptera: Cerambycidae) and Massicus raddei Blessig(Coleoptera: Cerambycidae) in general. And D. helophoroides only parasitic above the 3 instar larvae and pupae of cerambycid beetles. However, Sclerodermus spp.(Hymenoptera: Bethylidae) could parasitic 1-3 instar cerambycid beetles larvae and buprestid beetles larvae. High parasitic rate and good control effect has been confirmed. But each Sclerodermus species and different biotypes of D. helophoroides are quite similar on morphology, it is still difficult to identify correctly the species of Sclerodermus, and their classification status are still unknown. It will obviously affect the control effect. In this paper, we chose to use molecular methods to identify Sclerodermus putative species that are difficult to distinguish morphologically. Also we used molecular methods to investigate the intraspecific genetic subdivision among six different biotypes of Dastarcus helophoroides in China. Furthermore, we provided reliable evidence for traditional morphological identification and conducive to biological control process releasing insect natural enemies, so as to achieve a better control effect. And the results are as fallowed:1. The SDS method and Kit method for DNAextraction was compared using individual Sclerodermus. The results of agarose gel electrophoresis showed that the Extraction Kit method extraction genomic DNA has large quantity, high extraction rate and high concentration. The species belonging to genus Sclerodermus(Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borers, mainly buprestid and cerambycid larvae, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. So we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I(COI) and 28 S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: Sclerodermus guani Xiao et Wu, Sclerodermus sichuanensis Xiao, Sclerodermus pupariae Yang et Yao, and Sclerodermus spp.(Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28 S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences(Kimura-2-parameter distances) between congeneric species averaged to 4.5%, whereas intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp.(No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining(NJ) tree. The NJ tree inferred from the 28 S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae Yang et Yao, and Sclerodermus spp.(Nos. 1–4) were clustered together and only Sclerodermus sp.(No. 5) was clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp.(No. 5) might be a new species of Sclerodermus.2. The parasitoid wasps Sclerodermus(Hymenoptera: Bethylidae) are indigenous natural enemies, of the longhorned beetle pest. It is very difficult to distinguish these Sclerodermus adult species accurately according to their morphological and ecological characteristics. Although the sequences of internal transcribed spacer of the nuclear ribosomal DNA(ITS1 and ITS2) of these parasitoids are very similar, according to the deletion of one CTTCT repeats between 1319 and 1333 bp on ITS1, we discover that the ITS sequences could separate eight Sclerodermus species into two groups, as well as one group is the known species, contained Sclerodermus guani, Sclerodermus sichuanensis, Sclerodermus pupariae, and Sclerodermus sp.(No. 4), the other group is the unnamed species which are Sclerodermus sp.(No. 1), Sclerodermus sp.(No. 2) and Sclerodermus sp.(No. 3). Thus the ITS sequence could identify the three unknown Sclerodermus(Sclerodermus sp.(No. 1), Sclerodermus sp.(No. 2) and Sclerodermus sp.(No. 3)) from the known species(S. guani, S. sichuanensis, S. puparia). So the ITS sequences could be an initial important basis for discovery new species.3. Sclerodermus spp. are parasitoids of buprestid and cerambycid larvae, are economically and ecologically important insect natural enemies of wood-boring pests in China. Some named species(S. guani, S. sichuanensis, and S. pupariae) of this genus have successfully parasitize numerous wood borers. Further, these species have demonstrated that there are variations in the range of hosts. To determine genetic variation and relationships among different species of Sclerodermus, we analyzed nucleotide sequence data for three mitochondrial DNA(mtDNA) genes(12S rDNA, cytb, 16 S rDNA) using concatenated mtDNA data and two species tree inference methods(neighbor-joining and maximum likelihood). All analyses provide strong support for Sclerodermus sp.(No. 3) merged into one haplogroup with the high bootstrap support for all three gene sequences. cytb and 12 S gene analysis also strongly supported a single haplogroup for Sclerodermus sp.(No. 2). This comparative gene analysis sheds light on the identification and evolution of mitochondrial genes in the genus Sclerodermus.4. Sequence analysis on genes region from COII to COIII in mtDNA of several Sclerodermus species for Chinese biological control in recent years, the results showed the sequence contains partial mt DNA COII, tRNA-Asp, tRNA-Lys, ATPase6, ATPase8 and part COIII, is about 1700 bp. There was 100% sequence similarity between S. guani and S. sichuanensis. And the lowest sequence similarity was 96.88% between S. pupariae and Sclerodermus sp.(No. 2). The 1700-bp region of mtDNA COII-COIII was obtained for the 106 individuals, it included 68 variable sites. And sequence divergences(K2P) averaged 0.011. NJ analysis of the mtDNA COII-COIII sequences revealed that the S. pupariae were merged into one haplogroup, and Sclerodermus sp.(No. 1) were merged into one haplogroup. The comparative analysis results of different mtDNA sequences showed that the ATPase6 gene was the most suitable for distinguishing the sibling species of Sclerodermus.5. The mtDNA- COI and mtDNA- ND5 genes were used to investigate the intraspecific genetic subdivision among six different biotype populations of Dastarcus helophoroides in China. As for the 556 bp COI sequences of 130 individuals, 16 haplotypes and 21(3.8%) polymorphic sites were detected, including 21 parsimony informative sites. The results of the analysis of molecular variance(ANOVA) showed that the Fst value was 0.24358(P<0.001), and the genetic divergence within populations was significant. And as for the 665 bp ND5 sequences of 130 individuals, 16 haplotypes and 23(3.4%) polymorphic sites were detected, including 20 parsimony informative and 3 singleton variable sites. The results of the analysis of molecular variance(ANOVA) showed that the Fst value was 0.11861(P<0.001), and the genetic divergence within populations was significant. Maximum likelihood(ML) analysis revealed disapproved the six biotypes distinction based on host species.6. The mtDNA- cytb and ND1 genes and 12 S gene sequences were used to investigate the intraspecific genetic subdivision among six different biotype populations of D. helophoroides in China. As for the 393 bp cytb sequences of 130 individuals, 11 haplotypes and 17(4.3%) polymorphic sites were detected, including 15 parsimony informative sites. The results of the analysis of molecular variance(ANOVA) showed that the Fst value was 0.19217(P<0.001), and the genetic divergence within populations was significant. And as for the 681 bp ND1 sequences of 130 individuals, 17 haplotypes and 23(3.4%) polymorphic sites were detected, including 20 parsimony informative. The results of the analysis of molecular variance(ANOVA) showed that the Fst value was 0.14549(P<0.001), and the genetic divergence within populations was significant. Phylogenetic analysis revealed disapproved the six biotypes distinction based on host species. However the 531 bp of the 12 S gene sequence of 6 six different biotype populations of D. helophoroides were completely consistent.This study we made use of molecular methods to identify Sclerodermus spp. and D. helophoroides biotype which are parasitoids of buprestid and cerambycid larvae but difficult to distinguish morphologically. ITS sequences could separate eight Sclerodermus species into named group and unnamed group. We using COI and 28 S sequences speculate that Sclerodermus sp.(No. 5) might be a new specie. cytb, 16 S and 12 S gene analysis also strongly supported two single haplogroups for Sclerodermus sp.(No. 2) and Sclerodermus sp.(No. 3). We using COI sequences speculate that S. harmandi and S. guani were two species. There was 100% sequence similarity between S. guani and S. sichuanensis. And the lowest sequence similarity was 96.88% between S. pupariae and Sclerodermus sp.(No. 2). The resultes showed that S. pupariae were merged into one haplogroup and the other five Sclerodermus species(S. guani, S. sichuanensis, Sclerodermus spp.(Nos. 1-3)) were merged into the other haplogroup. But the Sclerodermus sp.(No. 5) had the farthest genetic relationship with all the other Sclerodermus spp.. The comparative analysis results of different mtDNA sequences showed that the ATPase6 gene was the most suitable for distinguishing the sibling species of Sclerodermus. The mtDNA COI, ND5, cytb, ND1, 12 S gene sequences were used to investigate the intraspecific genetic subdivision among six different biotype populations of D. helophoroides. And the results showed that it did not match that D. helophoroides divided into six different biotypes based on different hosts. And the genetic divergence within the one biotype was significant. This study provided a valuable exploration to distinguish the species and biological species of insect natural enemies, and had important significance in biological control and screening of insect natural enemies.