Jing-Tao Sun

Mining and characterization of sequence tagged microsatellites from the brown planthopper Nilaparvata lugens.

Abstract The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an important pest of rice. To better understand the migration pattern and population structure of the Chinese populations of N. lugens, we developed and characterized 12 polymorphic microsatellites from the expressed sequence tags database of N. lugens. The occurrence of these simple sequence repeats was assessed in three populations collected from three provinces of China. The number of alleles per locus ranged from 3 to 13 with an average of 6.5 alleles per locus. The mean observed heterozygosity of the three populations ranged from 0.051 to 0.772 and the expected heterozygosity ranged from 0.074 to 0.766. The sequences of the 12 markers were highly variable. The polymorphism information content of the 12 markers was high and ranged from 0.074 to 0.807 (mean = 0.503). Sequencing of microsatellite alleles revealed that the fragment length differences were mainly due to the variation of the repeat motif. Significant genetic differentiation was detected among the three N. lugens populations as the Fst ranged from 0.034 to 0.273. Principle coordinates analysis also revealed significant genetic differentiation between populations of different years. We conclude that these microsatellite markers will be a powerful tools to study the migration routine of the N. lugens.

Development and characterization of nine polymorphic microsatellites for the small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae).

Nine microsatellite loci were isolated from the genome of Laodelphax striatellus (Homoptera: Delphacidae) by constructing (TC)(6)(AC)(5) and (AG)(6)(AC)(5) compound SSR-enriched libraries using suppression-PCR procedures. These loci were found to be highly polymorphic, with 13 to 30 alleles per locus in the three populations that we investigated (Jiangsu, Shandong and Zhejiang). The observed and expected heterozygosities ranged from 0.255 to 0.833 and 0.392 to 0.929, respectively. These microsatellite markers can be used for the study of population genetic structure and genetic diversity of L. striatellus.

Sex-dependent activity of de novo methyltransferase 3 (Tudnmt3) in the two-spotted mite, Tetranychus urticae Koch

DNA methylation is an epigenetic mechanism for regulating developmental and other important processes in eukaryotes. Several components of the DNA methylation machinery have been identified, such as DNA methyltransferases. However, little is known about DNA methyltransferases in chelicerates, which is the second largest arthropod group. Epigenetics are expected to have a crucial role in the metabolism and development of this group. Here, we investigated the role of DNA methyltransferase 3 in the development of Tetranychus urticae Koch. In silico analyses clearly showed that this enzyme possesses the necessary conserved motifs for the catalytic activity of de novo methylation of DNA. Real‐time PCR revealed that T. urticae de novo methyltransferase 3 (Tudnmt3) is expressed ubiquitously and throughout the life cycle of the two‐spotted spider mite. However, the pattern of Tudnmt3 expression was sex‐dependent during the adult stage. Whole in situ hybridization provided supportive evidence that Tudnmt3 is linked to the differentiation of the gonads in adult females and males. Methylation‐sensitive amplification polymorphism analyses of 119 loci showed that the status of DNA methylation is partially different between adult females and males, raising the possibility that this sex‐dependent DNA methylation pattern is mediated by different methylation activity of Tudnmt3.

Transcriptome and proteome analyses reveal complex mechanisms of reproductive diapause in the two-spotted spider mite, Tetranychus urticae

Although a variety of factors underlying diapause have been identified in arthropods and other organisms, the molecular mechanisms regulating diapause are still largely unknown. Here, to better understand this process, we examined diapause‐associated genes in the two‐spotted spider mite, Tetranychus urticae, by comparing the transcriptomes and proteomes of early diapausing and reproductive adult females. Amongst genes underlying diapause revealed by the transcriptomic and proteomic data sets, we described the noticeable change in Ca2+‐associated genes, including 65 Ca2+‐binding protein genes and 23 Ca2+ transporter genes, indicating that Ca2+ signalling has a substantial role in diapause regulation. Other interesting changes in diapause included up‐regulation of (1) glutamate receptors that may be involved in synaptic plasticity changes, (2) genes involved in cytoskeletal reorganization including genes encoding each of the components of thick and thin filaments, tubulin and members of integrin signalling and (3) genes involved in anaerobic energy metabolism, which reflects a shift to anaerobic energy metabolism in early diapausing mites.

Expression level and immunolocalization of de novo methyltransferase 3 protein (TuDNMT3) in adult females and males of the two-spotted spider mite, Tetranychus urticae

DNA methylation is an epigenetic mechanism for regulating developmental and other important processes in eukaryotes. Several essential components of the DNA methylation machinery have been identified, such as DNA methyltransferases. In the two-spotted spider mite, Tetranychus urticae Koch, we have identified one DNA methyltransferase 3 gene (Tudnmt3) and tentatively investigated its potential role in adult females and males. Here, to better elucidate the functional role of Tudnmt3, its protein structure, expression and localization were subjected to more detailed analyses. Bioinformatic analyses clearly showed that the structure of TuDNMT3 was highly conserved, with several vital amino acid residues for the activation and stabilization of its confirmation. Western blot analyses revealed that this protein was expressed in both genders, with higher expression in adult females, which was inconsistent with the gene expression, suggesting translational regulation of Tudnmt3. Subsequent immunodetection provided supportive evidence for higher expression of the TuDNMT3 protein in adult females and indicated that this protein was generally localized in the cytoplasm and that its expression was predominantly confined to the genital region of spider mites, strengthening the hypothesis that de novo methylation mediated by Tudnmt3 in gonad development or gametogenesis has a different mechanism from maintenance methyltransferase.

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae): DNA methylation in two-spotted spider mite

The two‐spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well‐known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome‐wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation‐sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms.

Evolutionary divergence of mitochondrial genomes in two Tetranychus species distributed across different climates.

There is increasing evidence that mitochondrial genomes (mitogenomes) can be under selection, whereas the selective regimes shaping mitogenome evolution remain largely unclear. To test for mitogenome evolution in relation to the climate adaptation, we explored mtDNA variation in two spider mite (Tetranychus) species that distribute across different climates. We sequenced 26 complete mitogenomes of Tetranychus truncates, which occurs in both warm and cold regions, and nine complete mitogenomes of Tetranychus pueraricola, which is restricted to warm regions. Patterns of evolution in the two species’ mitogenomes were compared through a series of dN/dS methods and physicochemical profiles of amino acid replacements. We found that: (1) the mitogenomes of both species were under widespread purifying selection; (2) elevated directional adaptive selection was observed in the T. truncatus mitogenome, perhaps linked to the cold climates adaptation of T. truncatus; and (3) the strength of selection varied across genes, and diversifying positive selection detected on ND4 and ATP6 pointed to their crucial roles during adaptation to different climatic conditions. This study gained insight into the mitogenome evolution in relation to the climate adaptation.

Development of microsatellite markers for six Tetranychus species by transfer from Tetranychus urticae genome.

Microsatellite markers are frequently used to explore the population genetic structure of organisms. Spider mites (genus Tetranychus) are important agricultural pests. Several markers have been developed for T. urticae, but for other spider mites, few such markers are available, hampering studies of their population genetics. In this study, we developed and characterized microsatellite markers for six non-model spider mite species (T. truncatus, T. kanzawai, T. ludeni, T. piercei, T.phaselus and T. pueraricola) by cross-species amplification of markers in the T. urticae genome, in order to better understand the population structure of Tetranychus species. Among 228 screened loci, many were polymorphic, including 13 loci in T. urticae, 11 loci in T. truncatus, 15 loci in T. pueraricola, 23 loci in T. kanzawai, 19 loci in T. piercei, 11 loci in T. phaselus and 9 loci in T. ludeni. Sequence analysis determined that the fragment length variations of the transferred microsatellites were mainly due to the variations of the numbers of repeats. These new microsatellite markers should be useful for studying the population genetics of the seven Tetranychus species.