Complete mitochondrial genome of Appasus japonicus Vuillefroy, 1864 (Hemiptera: Belostomatidae)

Abstract We describe the initial sequencing and assembly of the complete mitochondrial genome of Appasus japonicus Vuillefroy, 1864 (Hemiptera; Belostomatidae; Appasus). The mitochondrial genome of A. japonicus was found to be 18,608 bp. It contains thirteen protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and an AT-rich region. The overall base composition of A. japonicus is A-41.9%, C-17.5%, G-11.9%, and T-28.7%. A phylogenetic analysis of 21 species within the order Hemiptera suggests that Diplonychus rusticus is most closely related to A. japonicus.


Introduction
Some hemiptera prefer slow-flowing environments and habitats rich in aquatic plants. These preferences also play a role in population regulation in freshwater ecosystems in terms of predator positions in the food chain (Mukai et al. 2005). Appasus japonicus Vullefroy 1864 (Hemiptera, Belostomatidae) had been classified as a genus of Appasus (Figure 1). In Korea, the family Belostomatidae includes Diplonychus esakii, Lethocerus deyrollei, Appasus japonicus and Appasus major. However, genetic research on Belostomatidae is lacking in Korea. For this reason, this study analyzed the complete mitochondrial genome of Appasus japonicus, which shows the widest distribution range in Korea among the species in Belostomatidae.

Materials and methods
A. japonicus was collected from Nakwaamcheon, Buseokmyeon, Yeongju-si, 128 39'34.76"E). The specimens were identified by Hwang-Goo Lee (morningdew@sangji.ac.kr) and then stored in the Department of Biological Science of Sangji University in Korea under voucher number SJAEAJ001. A. japonicus is an invertebrates and is neither an endangered or protected species, thus meeting the requirements for ethical approval.
We also compared each gene to the previously published mitochondrial genome of D. rusticus (GenBank FJ456940), which was suggested to be the most closely related species (Choi et al. 2021), for correct gene annotation. To reveal the phylogenetic position of A. japonica, the species used in the mitochondrial genome paper of D. rusticus and Hemiptera species registered in Korea were downloaded. As outgroups, Bemisia tabaci was downloaded and a total of 20 species were used. Their GenBank registration numbers are shown in Figure 2. Phylogenetic analyses were done using thirteen PCGs and two rRNA genes from the 21 species. Total genomic DNA was extracted from the specimen using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Genome sequencing was performed on the MiSeq (Illumina Inc., San Diego, CA) platform. The treatments of the raw data, such as trimming, were performed with Geneious prime 2021.1.1 (Biomatters Ltd, Auckland, New Zealand). The sequences were aligned using MAFFT (Katoh et al. 2002) and the maximum-likelihood (ML) and neighbor-joining (NJ) trees were created with MEGA X (Kumar et al. 2018).

Results
The assembled mitogenome of A. japonicus (GenBank accession No: OK012605) showed a length of 18,608 bp and an overall GC content of 29.4%, along with a total nucleotide composition of A À 41.9%, C À 17.5%, G À 11.9%, and T À 28.7%. The mitogenome consists of 13 protein-coding genes (PCGs), 22 tRNA genes, and two ribosomal RNA (rRNA) genes ( Figure 3). The genome structure, gene order, and total gene number of the A. japonicus mt genome are identical to those in D. rusticus (FJ456940), which is a closely related genus. The phylogenetic tree revealed that A. japonicus distributed in the clade containing the species D. rusticus and the relationships was supported by a high bootstrap value (BS ¼ 100) (Figure 2).

Conclusion
The mitochondrial genome of A. japonicus was 18,608 bp and contained thirteen protein-coding genes, 22 transfer RNAs, two ribosomal RNAs and an AT-rich region. A phylogenetic analysis of 21 complete mitochondrial genomes of the registered Order Hemiptera suggested that D. rusticus was most closely related to A. japonicus. Also, A. japonicus and the genus Lethocerus formed a monophyletic group. We expect that the present results will facilitate further investigations into the phylogenetic relationships in the Belostomatidae.

Author contributions
Kyeong-Sik Cheon was involved in the conception and design; Jung-Soo Han, Jun-Kil Choi and Hwang-Goo Lee was involved in the analysis and interpretation of the data; Jung-Soo Han and Ji-Eun Jang was involved in the DNA extraction experiment; Jung-Soo Han, Jun-Kil Choi and Hwang-Goo Lee drafted the paper; all authors revised it critically for intellectual content and gave the final approval of the version to be published; and that all authors agree to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was performed with the support of the Graduate School of Sangji University.