The complete mitochondrial genome of Orthaga achatina (Lepidoptera: Pyralidae)

Pyralidae is the largest family in Lepidoptera, with more than 25,000 species in the world, some of which are pests of agricultural and forestry plants, such as Orthaga (Yang et al. 2020). Orthaga achatina Butler (Lepidoptera: Pyralidae) is the most serious pest of camphor trees (Cinnamomum camphora) in China, Korea, Japan, and Malaysia (Wu 2006). O. achatina can also feed on other Lauraceae plants, such as Lindera glauca and Cinnamomum cassia, causing serious defoliates (Long et al. 2017). The mitochondrial genomes have the potential to be “molecular clock” due to its high mutation rate and low DNA recombination rate (Gai et al. 2020; Yang et al. 2020). However, the mitochondrial genome of O. achatina has not been publicly reported. Therefore, we determined to sequence the complete mitochondrial genome of O. achatina using the de novo sequencing techniques strategy to understand the mitogenomic background and genetic evolution relationship of O. achatina.

In the present study, samples of O. achatina were collected from camphor trees in July 2020 in Suzhou, Jiangsu Province, China (N31.16568o, E120.62638o). Some of these samples were immediately frozen at -80°C for sequencing analysis and others were preserved in the Entomological Lab of Nanjing Forestry University, and their specimen code is 2020NJEM1855-1860. The genomic DNA was extracted from O. achatina using CTAB (cetyltrimethylammonium Ammonium Bromide) method (Huanca-Mamani et al. 2015). Raw data generated by the Illumina HiSeq platform (Illumina Inc.; San Diego, CA, USA) were subject to de novo assembly by SPAdes version 3.14 (Bankevich et al. 2012). The complete mitochondrial genomes were annotated by MITOS WebServer (http://mitos.bioinf.uni-leipzig.de/index.py) (Bernt et al. 2013) and submitted to NCBI GenBank (GenBank accession number: MT916176).

The mitochondrial genome of O. achatina was 15,150 bp in size, with a nucleotide composition of 38. 9% A, 41.8% T, 11.4% C and 7.9% G. The mitochondrial genome of O. achatina comprised the entire set of 37 typical invertebrate mitochondrial genes consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a control region (D-loop). The majority-coding strand encoded 23 genes (9 PCGs and 14 tRNAs), whereas the minority-coding strand encoded 14 genes (4 PCGs, 8 tRNAs, and 2 rRNAs). The sequence and arrangement of genes were highly conserved, suggesting the similarity with typical characteristics of the genome in Lepidoptera (Liu et al. 2018; Wu et al. 2016, 2020). A total of 44 overlapping nucleotides between genes in 6 locations with a length of 2 to 25 bp were found, whereas there were 857 bp intergenic nucleotides in 22 locations, ranging from 4 to 297 bp in length.

All protein-coding genes (PCGs) were initiated with ATN as the start codon except the cox1, which is no justification for continued speculation about polynucleotide start codon similar to other Lepidoptera insects (Liu et al. 2018; Singh et al. 2017; Yang et al. 2020). Ten PCGs had canonical stop codons TAA or TAG, while three had incomplete termination codons single T (cox3 and atp6) or TA (nad4L). There were 22 tRNA genes with a length between 63 and 70 bp. All tRNA genes exhibited a typical clover-leaf secondary structure, except for tRNA-Ser(AGN) lacking the dihydrouridine (DHU) arm, which is common in Lepidoptera insects (Garey and Wolstenholme 1989). The lengths of lrRNA and srRNA were 1,362 bp and 780 bp, respectively. The control region was located between srRNA and tRNA-Met with a total length of 298 bp.

In addition, the BLAST-based ortholog detector OrthoFinder v2.2.7 (Emms and Steven 2019) with default parameter values were used to identify ortholog among all the protein sequences of the 24 mitochondrial genomes. The phylogenetic relationship of O. achatina and 23 Lepidoptera species was inferred from phylogenetic analysis of the 13 protein-coding genes using MEGA7.0 software with maximum likelihood method and 1000 replicate sets on bootstrap analysis. The amino acid identity (AAI) of the 13 protein-coding genes of each Lepidoptera species and O. achatina were calculated by NCBI BLASTP. The phylogenetic tree and AAI heatmap of each protein was visualized using EVOLVIEW version 2 (https://evolgenius.info//evolview-v2) (He et al. 2016). Phylogenetic analyses showed similar relationships among sampled families as shown in Yang et al. (2020). Each clade showed a monophyletic cluster and the following clades were highly supported (Fig 1): (1) Pyralidae + Crambidae; and (2) (Pyralidae + Crambidae) + (Noctuidae + (Bombycidae + Geometridae)). We also found that O. achatina strains had the closest relationship with the genus Hypsopygia and Endotricha, which were located in a clade in the clade of Pyralidae. This study can provide a useful resource for the genetic evolution of O. achatina and underline the potential importance of mitochondrial genomes in comparative genomic analyses of Lepidoptera species.