Agronomically important thrips: development of species-specific primers in multiplex PCR and microarray assay using internal transcribed spacer 1 (ITS1) sequences for identification.

Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.

Development of Species-Specific Primers for Agronomical Thrips and Multiplex Assay for Quarantine Identification of Western Flower Thrips.

While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.

Phylogeny of Glaucosomatidae inferred from molecular evidence.

Most species of glaucosomatids (Teleostei: Glaucosomatidae) are endemic to Australia, except Glaucosoma buergeri that is widely distributed from Australia to Japan. This study elucidated phylogenetic relationships among glaucosomatids based on the morphological characters of the saccular-otolith sagitta, in addition to molecular evidence of mitochondrial 16S rDNA, cytochrome oxidase I (COI) and cytochrome b (cyt b) sequences, and nuclear rhodopsin sequences. The topologies of individuals' phylogenetic trees, based on 16S rDNA, COI and cyt b sequences, were statistically indistinguishable from one another, and were only slightly different from a tree based on rhodopsin sequences. These molecular tree topologies, however, differed from species relationships in morphology-based phylogenetic hypothesis proposed in previous studies. Specimens of G. buergeri from Australia and Taiwan showed differences in the sagitta and molecular differentiation at the four genes, suggesting a possible speciation event. Both molecular and morphological evidences indicate that Glaucosoma magnificum is the plesiomorphic sister species of other glaucosomatid species. Glaucosoma hebraicum is the sister species of a clade composed of G. buergeri and Glaucosoma scapulare. Molecular and morphological evidences also support the species status of G. hebraicum.

Molecular differentiation and diversity of Forcipomyia taiwana (Diptera: Ceratopogonidae) based on the mitochondrial cytochrome oxidase II sequence.

Forcipomyia taiwana (Shiraki), a biting midge, is one of the most annoying blood-sucking pests in Taiwan. In this study, partial DNA sequences of cytochrome c oxidase II from 113 individuals collected from 11 locations around the island were analyzed to delineate the differentiation pattern and possible dispersal processes of F. taiwana in Taiwan. The uncorrected nucleotide divergences, composed of mostly transition substitutions, were high (up to 2.7%) among the samples. Average comparable variations (approximately equal to 0.7%) were found within and between populations. Phylogenetic analysis suggested that several distinct lineages exist and some can be found simultaneously in some populations. A relationship between sequence divergences among populations and their relative geographical distances was observed. Moreover, haplotype diversity was high in all populations, and low to middle levels (Fst = 0.004-0.288) of genetic differentiation were found among populations. Linearized calibration from sequence divergences and phylogenetic analysis showed that different ancestral lineages of F. taiwana possibly emerged as early as 0.6 million years ago. Taken together, genetic exchanges among these divergently ancestral lineages, likely caused by recent artificial events, have possibly led to the similarly diversified compositions of F. taiwana populations all around Taiwan nowadays.

The thymidylate synthase gene of Hz-1 virus: a gene captured from its lepidopteran host.

The sequence analysis of a thymidylate synthase gene was identified in the Hz-1 virus HindIII-D fragment. The viral thymidylate synthase gene encodes a protein of 295 amino acids, and is closely related to that of insect, mammals and herpesvirus. The thymidylate synthase gene identified was a genuine viral gene in that it was only detected in cells infected with Hz-1 virus but not in the mock infected cells, by Southern blot analysis and by RT-PCR. Results of phylogenetic analysis based on non-synonymous and amino acid distances suggested that the TS gene of Hz-1 virus was grouped closely with that of Bombyx mori. High bootstrap values confirmed that the thymidylate synthase of Hz-1 virus was acquired by a capture event from its lepidopteran host. Results of both sequence divergences and phylogenetic analysis suggested that TS genes in insect viruses, Hz-1, CIV, and MsEPV may have a different history or originated from different capture events.