Identification of Nilaparvata lugens and Its Two Sibling Species (N. bakeri and N. muiri) by Direct Multiplex PCR

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is a destructive rice pest of Asia. Currently, one important monitoring method of BPH is through black light trapping. However, two sibling species of BPH, Nilaparvata bakeri (Muri) and Nilaparvata muiri China, can also be trapped by black light, and these species feed only on gramineous weeds rather than on rice. Therefore, the accurate identification of Nilaparvata species is crucial for N. lugens forecasting and management. The traditional morphological identification method is not feasible for subadults and damaged specimens. Furthermore, this error-prone morphological identification method is time and labor intensive, with the need for expertise and experience. Here, we established a direct multiplex polymerase chain reaction (dmPCR) assay using crude tissue fluid as a template, omitting purified DNA extraction. The crude tissue fluid can be obtained by grinding specimens without any biological reagent but only using distilled water. This dmPCR assay, using three pairs of diagnostic primers, is based on internal transcribed spacers (ITS). Each primer pair amplifies a species-specific fragment of a different size, which were easily and reliably separated in a 2% agarose gel. Furthermore, the dmPCR was verified to be applicable to damaged tissue specimens, such as head, thorax, or abdomen. In conclusion, this dmPCR assay is a novel, time-saving, cost-effective, and easy-to-apply molecular diagnostic method for the identification of the above three sibling species, N. lugens, N. bakeri, and N. muiri.