Nysius plebeius Distant, 1883 (Heteroptera: Lygaeidae) is an emerging pest of Perilla frutescens (L) Britton (Lamiaceae) that damages its seeds, foliage, and flowers (Maharjan et al., 2022). P. frutescens is one of the main upland crops used in traditional Korean cooking (KOSIS, 2022;Lee et al., 1998). The genus Nysius causes significant damage and economic loss in P. frutescens (Kim et al., 1994). In particular, genetic research on N. plebeius has been minimal compared to the persistent and clear economic damage caused by these pests (Maharjan et al., 2020). Furthermore, the phylogenetic relationships between Lygaeoidea remain disputable (Carapelli et al., 2021;Nakatani, 2015), and the relationships and placement of several taxon levels remain unresolved. Therefore, we sequenced the complete mitochondrial genome (mitogenome) of N. plebeius (GenBank accession number: MN599979), and assessed its phylogenetic relationships to nine other species of Lygaeoidea using mitogenomic data.

Materials and Methods

A population of N. plebeius was originally collected from the perilla experimental field in Miryang-si, Gyeongsangnamdo, South Korea (geographic location: 35°29'29"N, 128°44' 31"E) and mass-reared for five generations to fix the mitochondrial genome in the population. A specimen was deposited at Museum of Arthropod biodiversity, Kangwon National University, Korea (contact person; Hyeonmi Lee, email; lhl427@kangwon.ac.kr) under the voucher number KNUAM- 220929 (Fig. 1).

Following a previously reported method (Kim et al., 2021), ten adult females were used to extract genomic DNA using DNAzol (Molecular Research Center, Cincinnati, Ohio, USA). For mitochondrial genome sequencing, libraries were prepared using the TruSeq DNA Nano kit (Illumina, San Diego, CA, USA) and the Miseq platform (Illumina), and more than 5.2 Gb of the genome was sequenced (over 600 coverage). The CLC Assembly Cell package version 4.2.1 (CLCBio, Arhaus, Denmark) was used to assemble the data. After trimming the raw data using the CLC quality trim assembly, the CLC de novo assembler with low-coverage whole-genome shotgun sequencing (dnaLCW) was used. The assembled sequences were confirmed using BLASTZ (Schwartz et al., 2003). The N. plebeius mitochondrial genome was annotated using the GeSeq program (Tillich et al., 2017).

For the phylogenetic analysis of N. plebeius, a multiple sequence alignment was prepared based on 13 mitochondrial protein-coding sequences from 10 species in the NCBI GenBank database. A maximum-likelihood phylogenetic tree was constructed using MEGA11 (Tamura et al., 2021) with the GTR + G substitution model. Bootstrap values were set at 1000 repetitions.

Results

In total, a 17,367 bp complete mitochondrial genome of N. plebeius was verified, which includes 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNAs (Fig. 2). For the 13 protein-coding genes, the most common shared start codon was ATG (in ND2, ATP6, COX3, CYTB), followed by ATA (COX2, ATP8, ND3). The most common termination codon was TAA (ND1, ND2, ND4, ND4L, ND5, COX3, ATP6, ATP8), followed by the incomplete termination codon T— (COX1, COX2, ND3). The composition of the mitochondrial base is as follows: A, 46.6%; T, 33.4%; G, 9.5%; and C, 13.5%. The G+C content was found to be 23%.

The phylogenetic relationship with three congeneric species is similar to that previously reported for two species, N. cymoides and N. fuscovittatus (Cao et al., 2020). Phylogenetic analysis revealed that Nysius (and N. plebeius) and the subfamily Orsillinae is positioned in a clade together with Lygaeinae, supporting the inclusion of Orsillinae in Lygaeidae (Fig. 3).

The tree is drawn to scale, with branch lengths measured as the number of substitutions per site. Genbank accession numbers: Nysius plebeius (MN599979), Nysius sp. (MW465654), N. fuscovittatus (NC050167) (Cao et al., 2020), N. cymoides (MW291653) (Carapelli et al., 2021), Lygaeus sp. (MF497725) (Liu et al., 2019), Arocatus melanocephalus (MW619723) (Ye et al., 2022), Tropidothorax sinensis (MW547017) (Huang et al., 2021), Malcus setosus (NC063138) (Ye et al., 2022), Metatropis longirostris (NC037373) (Zhang et al., 2019), Geocoris pallidipennis (NC012424) (Hua et al., 2008), and Cimex lectularius (NC030043) (Kolokotronis et al., 2016).

Discussion

In Korea, Nysius seed bugs damage various cereal crops (Maharjan et al., 2022). Among them, the occurrence rate of N. plebeius in oil crops such as perilla is very high, but research on its control and ecology is insufficient (Kim et al., 1994;Lee et al., 1998). Additionally, the closely related species N. hidakai and N. inconspicuus occur sympatrically in the field, so accurate identification of the specific pest causing damage is required (Cao et al., 2020;Carapelli et al., 2021). Therefore, this study can be used to develop a species diagnosis method using molecular techniques together with morphological identifications to establish a comprehensive Lygaeidae management program.