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

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.

Keywords: Halyomorpha halys; PCR identification; nondestructive DNA extraction; samurai wasp; species-specific primers.

Figure 1

Alignment of TJ234F and TJ460R primers to various Hymenoptera CO1 sequences, and location of primers within CO1 locus. Detail of the CO1 sequence alignment at the TJ234F/TJ460R primer binding sites of T. japonicus specimens used for primer design and other wasp species related to or coexisting in the same habitat as T. japonicus. Identical nucleotides in primers and the target sequences are marked with dots. Colored letters and dashes highlight nucleotide dissimilarities between primers and target sequences. The number of nucleotide mismatches is summarized in the column at the right side of each alignment. Asterisks above the alignments show the position of conserved nucleotides within primer sequences. The cartoon at the bottom of the figure represents an approximate location at the 5′ end of the analyzed partial CO1 sequences (dark blue rectangle) and primer binding sites (arrows) within the complete CO1 gene sequence (pale blue rectangle), as well as the theoretical amplicon size.

Figure 2

Alignment of TJ234F/TJ460R primers with sequences of eight BMSB parasitoids other than T. japonicus. Mismatch patterns of T. basalis, T. belenus, T. cultratus, T. edessae, T. hullensis, T. plautiae, T. mitsukurii and T. utehensis to our primers are shown. For each of the wasp species included in the figure, we analyzed a variety of sequences and identified diverse haplotypes. Frequently, two or more haplotypes showed the same pattern of mismatches across our two primer binding sites. Accordingly, a mismatch pattern in the figure may be found in one or more CO1 haplotypes of the corresponding species. Dots denote identical nucleotides in primers and the target sequences. Colored letters or dashes indicate nucleotide dissimilarities, and the total number of mismatches for each species’ mismatch pattern is listed in columns to the right of the alignment. Asterisks indicate the position of conserved nucleotides within primer sequences. TJ234F/TJ460R are shown.

Figure 3

Sensitivity of the novel T. japonicus specific PCR using TJ234F and TJ460R primers. Total DNA amounts used in PCR were 50, 10, 5, 1, 0.5, 0.1, 0.05 and 0.01 ng, and a negative control (Neg) with 0 ng in a 25 μL reaction. The rightmost well contains 1 μL of 1 Kb Plus DNA Ladder (Invitrogen, Carlsbad, CA, USA).