DNA based markers to characterize insect pest damage: diagnostic trials on Leptoglossus occidentalis (Hemiptera: Coreidae)

The exotic Western Conifer-Seed Bug (WCSB) Leptoglossus occidentalis Heidemann is considered one of the most harmful pests to stone pine nut production in the Mediterranean region. Nevertheless, the role of WCSB in stone pine conelet mortality and seed loss is still to be ascertained. An antibody-based assay was already developed in North America to detect WCSB residual saliva on fed-on conifer seeds. However, several other insects are known to feed upon stone pine fructifications. The goal of this study was to develop a PCR-based diagnostic method, to detect WCSB DNA, supposedly present inthe residual insect saliva, on attacked pine conelets and seeds . A pair of primers targeting cytochrome c oxidase I (COI) WCSB sequences were designed, and initially tested on WCSB genomic DNA as template. As negative control, DNA from stone pine tissues was used. Only when WCSB DNA was used amplicons of the expected length (658 bp) were specifically obtained. WCSB DNA was also detected into the insect saliva, with a sensitivity as low as 0.1 pg/µl. The specificity of this assay was further confirmed using DNA from 5 other insect species associated to pine fructification, and from WCSB specimens with different geographical origins. Unfortunately, this protocol failed to detect WCSB DNA when tested on WCSB-attacked seed samples. Thus, a new primer pair was designed, still targeting WCSB COI but giving a shorter amplicon (171 bp), to be used in  High Resolution Melting analysis (HRMA). Although a higher sensitivity was obtained on WCSB genomic DNA (0.01 pg/µl), HRMA analysis was also unable to detect WCSB DNA into attacked seeds, possibly due to negligible amounts of residual saliva into seed inner tissues. Nevertheless, both these PCR-based assays were sensitive enough to detect WCSB DNA in plant tissues contaminated by WCSB’s feces, implying their potential use in surveying WCSB populations.