TaqMan Real-time PCR Quantification Of Frankliniella Occidentalis (thysanoptera: Thripidae) Remains In Domestic Predator Guts

Frankliniella occidentalis is a worldwide invasion pest, which has caused tremendous threat to agricultural production in China. Currently it has only harmed in few areas of mainland China. Due to its high insecticide resistance, the indigenous natural enemies can be used as a necessary and effective measure for biological control of this thrips. Invasive pests can be easily outbreak when escaped from natural enemies in their indigenous districts, while introduction of natural enemies from the original country may form a new issue of alien invasive species. Two molecular markers, COI and SCAR, were developed to qualitatively detect the predation on Frankliniella occidentalis by local natural enemies. According to the COI sequences of F. occidentalis in Genbank, a pair of F.occidentalis-specific primers, FO340 (SS-COI primers), was designed. The fragment amplified by the primers was 340 bp. a pair of F. occidentalis species-specific primers FOMF/FOMR (SCAR primer) was designed based on RAPD-SCAR PCR.The fragment amplified by the primers was 320 bp. Specificity test indicated that the above two pairs of primers only have positive reaction to the DNA from F. occidentalis, and Propylaea japonica, Harmonia axyridis and Chrysopa septempunctata, feeding a female adult of F. occidentalis, and no cross reactions with other 28 species (SS-COI marker) or 41 thrips species (SCAR markers), as well as 18 other pest species and predators. The results showed that these two molecular marker systems are available for screening predators of F. occidentalis in a new invaded area. In addition, SS-COI and SCAR systems were not only well, used for testing different development stages of the thrips, but also well testing 14 different populations from five different countries and areas of China (SS-COI maker). Meanwhile, the SCAR maker can detect the eggs in the host plants from infested areas. These results demonstrated that the above two molecular marker systems can be used in quarantine at ports and in monitoring of flowers and seedlings.TaqMan real time quantitative PCR detection primers and probe, FO-77F/R and FO-77 prober, were designed according to the 340 bp F. occidentalis-specific fragment. A standard curve was developed by using plasmid DNA. The numbers of DNA copies of different development stages of F. occidentalis, were egg 1.32×105, first instar larva 6.43×105, second instar larva 3.42×106, pre-pupa 4.24×106, pupa 1.02×107, male adult 1.96×106, and female adult 7.95×106. Furthermore, the relationships between quantitative detection ability of predation and environmental factors were established, the correlation fomulas were digestion time:Y=-0.2169x +6.259 (R2=0.851), environment temperature Y=-0.0368x+7.2289 (R2=0.9761),predator number:Y=0.0705x+6.2467, (R2=0.9674), and other prey in the same field (bean aphid):Y=-0.2589x+5.479 (R2=0.955), respectively. These are crucial for evaluating the predation on F. occidentalis by domestic predators in field.The natural enemies of F. occidentalis were collected regularly in pepper fields in Dounan village, Chenggong County, Kunming, Yunnan. The numbers of spiders were significantly greater than those of ladybird beetles and predatory bugs (Pentatomidae);while there was no significant defferences between ladybird beetles and predatory bugs. The results of TaqMan real time quantitative PCR showed that Orius sauteri was an important predator of F. occidentalis, then followed by Coccinella septempunctata, Propylaea japonica, Heteropodidae, Oirus niger, Orius similis, Erigonidium graminicolum and Pirata subpiraticus. Harmonia axyridis was not as important as above mentined ones, the numbers of F. occidentalis female adults preyed were 0.0785,0.0412,0.0393,0.0335,0.0321,0.0229,0.0172,0.0151 and 0.0027, respectively.The establishment of qualitative and quantitative evaluation system on the predatory ablity to F. occidentalis provided useful tools for screening predators, quantitative evaluation predatory ability on F. occidentalis by domestic natural enemies. The techniques were also important in protection and use natural enemies, establishment sustainable management measues on alien species. The establishment of SS-COI and SCAR technology systems can be used at ports and in transportation of flowers, vegetable and seedlings, consequently, preventing further spreading of F. occidentalis in China, as well as explain the mechanisms of expansion.