Molecular Identification of Trissolcus japonicus, Parasitoid of the Brown Marmorated Stink Bug, by Species-Specific PCR

SIMPLE SUMMARY: The brown marmorated stinkbug Halyomorpha halys, is an invasive pest that causes millions of dollars of crop damage each year in the US. A promising biocontrol agent for this pest is the samurai wasp, Trissolcus japonicus, which is a potential long term control method with few negative ecological impacts. However, the wasps’ small size of only 1 mm in length can make it difficult to identify morphologically. We developed a DNA-based approach to determine whether a wasp specimen is T. japonicus using conventional methods of DNA extraction, PCR, and gel electrophoresis. When tested against eight families of Hymenoptera, including three Trissolcus species, our method identified samurai wasp samples with 100% accuracy. Additionally, in silico analyses of various T. japonicus sequences provide evidence that the method will work throughout the US, Europe, and parts of Asia. We expect that this method will be a valuable tool for reliably identifying T. japonicus during distribution and recapture efforts to assess its survival, establishment, and dispersal for biocontrol purposes. ABSTRACT: The samurai wasp, Trissolcus japonicus (Ashmead), has been proposed as a biocontrol agent against brown marmorated stink bugs (BMSB), due to its ability to parasitize and kill BMSB eggs. However, the wasps’ small size makes it challenging for those untrained in morphological identification to determine the wasps’ species. To circumvent this problem, a molecular method was created to identify T. japonicus. The method uses species-specific primers, designed in this study, which target the variable region of the mitochondrial Cytochrome Oxidase 1 (CO1) locus. After confirming successful DNA extraction from samples, the PCR amplification using our primers produced 227-bp PCR products for all T. japonicus specimens and no amplification in other microhymenoptera candidates. Additionally, DNA from BMSB-parasitized eggs gave positive PCR amplification, while the control BMSB samples showed no amplification. This indicates that PCR with our primers specifically and sensitively differentiates T. japonicus specimens from other similar wasp species and discriminates between T. japonicus-parasitized and non-parasitized BMSB eggs. Finally, an in silico analysis of CO1 sequences demonstrated that our primers match the sequences of four different haplotypes of T. japonicus, indicating that our diagnostic method could potentially be applied to analyze T. japonicus populations throughout North America, Europe, and parts of Asia.