Identification and expression profile analysis of chemosensory genes in pine needle gall midge, Thecodiplosis japonensis (Diptera: Cecidomyiidae).

Insects have highly specialized and sensitive olfactory systems involving several chemosensory genes to locate their mates and hosts or escape from predators. Pine needle gall midge, Thecodiplosis japonensis (Diptera: Cecidomyiidae), has invaded China since 2016 and caused serious damage. Till now, there is no environmentally friendly measure to control this gall midge. Screening molecules with high affinity to target odorant-binding protein to develop highly efficient attractants is a potential pest management method. However, the chemosensory genes in T. japonensis are still unclear. We identified 67 chemosensory-related genes in the transcriptomes of antennae, including 26 OBPs, 2 CSPs, 17 ORs, 3 SNMPs, 6 GRs, and 13 IRs, using high throughput sequencing. Phylogenetic analysis of these six chemosensory gene families among Dipteran was performed to classify and predict the functions. The expression profiles of OBPs, CSPs and ORs were validated by quantitative real-time PCR. 16 of the 26 OBPs were biased expressed in antennae. TjapORco and TjapOR5 were highly expressed in the antenna of unmated male and female adults. The functions of related OBPs and ORs genes were also discussed. These results provide a basis for the functional research on chemosensory genes at the molecular level.

A LAMP Assay for the Detection of Thecodiplosis japonensis, an Alien Gall Midge Species Pest of Pine Trees.

Pine needle gall midge (T. japonensis), native to Japan, has become a serious invasive pest in South Korea and, more recently in 2006, in China. It was first discovered in Qingdao, Shandong Province, and has caused serious damage to local Pinus thunbergii. The insect's small size makes morphological-based identification difficult; therefore, molecular detection techniques are urgently needed for monitoring and preventing its further spread. At present, there is no simple and accurate field molecular identification tool. To solve this problem, a LAMP-based molecular diagnosis technology of T. japonensis was developed. Four LAMP primers were designed to specifically amplify T. japonensis DNA. Positive LAMP reactions usually produce amplification in one hour. The optimal incubation conditions for LAMP detection were determined with 4 LAMP primers for 60 min at 61 °C. The LAMP detection range of gDNA concentrations is wide, with a minimum detectable gDNA concentration of 300 fg. A non-destructive DNA-releasing procedure, HotSHOT "HS6", which could extract "crude DNA" for LAMP assay in 10 min, was used for larval and adult samples. Therefore, we established a LAMP-based rapid molecular identification method that can be applied in the monitoring and management of T. japonensis.