Infection Rate Assay by Nested PCR and the Phylogenetic Analysis of Himetobi P Virus in the Main Pests of Rice-Wheat Cropping Systems.

Himetobi P virus (HiPV) is an ssRNA in the family Dicistroviridae that infects rice pests belonging to Hemiptera. To determine its host range, a nested PCR method was designed to detect HiPV in some of the main rice pests (Hemiptera) in eastern China. The incidence of infection in the grain aphid Sitobion avenae Fabricius (Hemiptera: Aphididae) was low (3%), while high incidences of infection occurred in the planthoppers Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae) (100%) and Nilaparvata lugens (Hemiptera: Delphacidae) (51%) and in the leafhoppers Cicadella viridis (Hemiptera: Cicadellidae) (90%) and Nephotettix cincticeps (Hemiptera: Cicadellidae) (57%). Phylogenetic analysis by maximum likelihood tree and median-joining networks implied the HiPVs from the same hosts were genetically close. Neutral equilibrium evolution for the polymorphism data was tested by the Tajima's D test and by Fu and Li's D and F tests. Test values were negative, which indicates a selection on the HiPV haplotypes. We sequenced the complete genome sequence of HiPV to look for evidence of recombination. We identified a recombination event in which two genomes recombined in the region of ORF2. The two open reading frames of the HiPV had been selected with low Ka/Ks ratios compared with two previous genome sequences.

The complete mitochondrial genomes of two rice planthoppers, Nilaparvata lugens and Laodelphax striatellus: conserved genome rearrangement in Delphacidae and discovery of new characteristics of atp8 and tRNA genes.

BACKGROUND: Nilaparvata lugens (the brown planthopper, BPH) and Laodelphax striatellus (the small brown planthopper, SBPH) are two of the most important pests of rice. Up to now, there was only one mitochondrial genome of rice planthopper has been sequenced and very few dependable information of mitochondria could be used for research on population genetics, phylogeographics and phylogenetic evolution of these pests. To get more valuable information from the mitochondria, we sequenced the complete mitochondrial genomes of BPH and SBPH. These two planthoppers were infected with two different functional Wolbachia (intracellular endosymbiont) strains (wLug and wStri). Since both mitochondria and Wolbachia are transmitted by cytoplasmic inheritance and it was difficult to separate them when purified the Wolbachia particles, concomitantly sequencing the genome of Wolbachia using next generation sequencing method, we also got nearly complete mitochondrial genome sequences of these two rice planthoppers. After gap closing, we present high quality and reliable complete mitochondrial genomes of these two planthoppers. RESULTS: The mitogenomes of N. lugens (BPH) and L. striatellus (SBPH) are 17, 619 bp and 16, 431 bp long with A + T contents of 76.95% and 77.17%, respectively. Both species have typical circular mitochondrial genomes that encode the complete set of 37 genes which are usually found in metazoans. However, the BPH mitogenome also possesses two additional copies of the trnC gene. In both mitochondrial genomes, the lengths of the atp8 gene were conspicuously shorter than that of all other known insect mitochondrial genomes (99 bp for BPH, 102 bp for SBPH). That two rearrangement regions (trnC-trnW and nad6-trnP-trnT) of mitochondrial genomes differing from other known insect were found in these two distantly related planthoppers revealed that the gene order of mitochondria might be conservative in Delphacidae. The large non-coding fragment (the A+T-rich region) putatively corresponding responsible for the control of replication and transcription of mitochondria contained a variable number of tandem repeats (VNTRs) block in different natural individuals of these two planthoppers. Comparison with a previously sequenced individual of SBPH revealed that the mitochondrial genetic variation within a species exists not only in the sequence and secondary structure of genes, but also in the gene order (the different location of trnH gene). CONCLUSION: The mitochondrial genome arrangement pattern found in planthoppers was involved in rearrangements of both tRNA genes and protein-coding genes (PCGs). Different species from different genera of Delphacidae possessing the same mitochondrial gene rearrangement suggests that gene rearrangements of mitochondrial genome probably occurred before the differentiation of this family. After comparatively analyzing the gene order of different species of Hemiptera, we propose that except for some specific taxonomical group (e.g. the whiteflies) the gene order might have diversified in family level of this order. The VNTRs detected in the control region might provide additional genetic markers for studying population genetics, individual difference and phylogeographics of planthoppers.

The complete mitochondrial genome sequence of Sogatella furcifera (Horváth) and a comparative mitogenomic analysis of three predominant rice planthoppers.

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the most destructive pests of rice crops in many Asian countries. Using long-PCR and shotgun library methods, we sequenced the entire mitochondrial genomes (mt-genomes) of two WBPH individuals. Total lengths of the mt-genome of the two WBPH individuals were 16,612 bp and 16,654 bp with an identical AT content of 76.19%. Among the 13 protein coding genes (PCGs), only nad5 used an atypical initiation codon GTG. Most of the tRNA genes had the typical cloverleaf secondary structure except that the dihydrouridine (DHU) arms in two trnS genes and the TΨC arm of trnG gene did not form a stable stem-loop structure. Similar to the brown planthopper (BPH), Nilaparvata lugens (Stål), and the small brown planthopper (SBPH), Laodelphax striatellus (Fallén), some extraordinary features were observed in the WBPH mt-genome. These include similar gene rearrangement pattern, unusually short length of the atp8 gene and variable numbers of tandem repeat (VNTR) structure in control region. Interestingly, the same tandem repeat unit with stable secondary structure appeared in two different planthoppers, WBPH and SBPH, which belong to two different genera of the Delphacidae. This peculiar feature provides a direct evidence for the close relationship between the two planthoppers and updates our understanding of the evolutionary characteristics of mitochondrial control region. Comparison with two other predominant rice planthoppers (BPH and SBPH) revealed that different PCGs of mitochondria exhibit different evolutionary patterns.

The complete mitochondrial genomes of six species of Tetranychus provide insights into the phylogeny and evolution of spider mites.

Many spider mites belonging to the genus Tetranychus are of agronomical importance. With limited morphological characters, Tetranychus mites are usually identified by a combination of morphological characteristics and molecular diagnostics. To clarify their molecular evolution and phylogeny, the mitochondrial genomes of the green and red forms of Tetranychus urticae as well as T. kanzawai, T. ludeni, T. malaysiensis, T. phaselus, T. pueraricola were sequenced and compared. The seven mitochondrial genomes are typical circular molecules of about 13,000 bp encoding and they are composed of the complete set of 37 genes that are usually found in metazoans. The order of the mitochondrial (mt) genes is the same as that in the mt genomes of Panonychus citri and P. ulmi, but very different from that in other Acari. The J-strands of the mitochondrial genomes have high (∼ 84%) A+T contents, negative GC-skews and positive AT-skews. The nucleotide sequence of the cox1 gene, which is commonly used as a taxon barcode and molecular marker, is more highly conserved than the nucleotide sequences of other mitochondrial genes in these seven species. Most tRNA genes in the seven genomes lose the D-arm and/or the T-arm. The functions of these tRNAs need to be evaluated. The mitochondrial genome of T. malaysiensis differs from the other six genomes in having a slightly smaller genome size, a slight difference in codon usage, and a variable loop in place of the T-arm of some tRNAs by a variable loop. A phylogenic analysis shows that T. malaysiensis first split from other Tetranychus species and that the clade of the family Tetranychoidea occupies a basal position in the Trombidiformes. The mt genomes of the green and red forms of T. urticae have limited divergence and short evolutionary distance.

Wolbachia play an important role in affecting mtDNA variation of Tetranychus truncatus (Trombidiformes: Tetranychidae).

The prevalence of the endosymbiont Wolbachia and its effects on mitochondria variation were analyzed in seven natural populations of Tetranychus truncatus Ehara (Trombidiformes: Tetranychidae) in current study. Five Wolbachia strains (wtru1, wtru5, wtru7, wtru8, and wtru12) were detected based on the surface protein of Wolbachia (wsp) sequence data and the multiple locus sequences typing data, suggesting that multiple separate invasions have occurred. Part of mitochondrial cytochrome oxidase subunit I gene was sequenced from infected individuals revealing 10 different haplotypes. As predicted, the haplotype and nucleotide diversity were lower in infected individuals than that in uninfected individuals. Furthermore, phylogenetic and analysis of molecular variance analyses revealed that the distribution of mtDNA haplotypes is not associated with geography. Rather, it is strongly concordant with infection status. These data support the hypothesis that Wolbachia infection can affect the genetic structure and diversity of the host mites.