The complete mitochondrial genome of Chlaenius bimaculatus Dejean, 1826 (Coleoptera: Carabidea) and its phylogenetic analyses

Abstract Chlaenius bimaculatus Dejean, 1826 (Coleoptera: Carabidea) is a predator of several lepidopteran pests, including Spodoptera frugiperda, S. litura and Helcystogramma triannulella. However, there has been little research into using C. bimaculatus to control crop pests. In this study, we sequenced the complete mitochondrial genome of C. bimaculatus. The results showed that the entire mitochondrial genome was 16,419 bp and contained 24% GC. 13 protein-coding, 22 transfer RNA, and two ribosomal RNA genes were identified. C. bimaculatus shares the same genetic arrangement and composition as other Coleoptera insects. In addition, phylogenetic analysis revealed that C. bimaculatus is closely related to Diplocheila zeelandica.


Introduction
Chlaenius bimaculatus Dejean, 1826 (Coleoptera: Carabidea) is distinguished by its golden cyan body, enlarged end segments of the lower lip and jaw whiskers, and a large reddishyellow spot on the sub posterior portion of the forewing (Figure 1) (Li et al. 1990;Qiu 1996).C. bimaculatus overwinters as adults in weeds, humus-rich dirt clods, and stones before laying eggs in April the following year in Fujian Province, China, where it is often found in sweet potato and rice fields (Chen and Chen 1984).
Biological control is an essential strategy for the long-term control of Spodoptera frugiperda and other lepidopteran pests, with natural enemy insect resources being the most important (Chen et al. 2019).C. bimaculatus can effectively manage crop pests and reduce economic loses from agricultural products.S. frugiperda, S. litura, Helcystogramma triannulella, Omiodes indicata, and other lepidopteran pest larvae are preyed upon by both adults and larvae of C. bimaculatus (Chen and Chen 1982;1991;Weng 1995;Huang et al. 2020).C. bimaculatus is a predatory natural predator with significant application potential, however, there has been little research on its utilization (Huang et al. 2022).Therefore, in this study, the complete mitochondrial genome sequence of C. bimaculatus was sequenced and annotated to provide data for future molecular studies on this species.

Mitochondrial genome assembly and annotation
The leg of an adult C. bimaculatus individual was used for total genomic DNA extraction, which was subsequently purified using the TruSeq DNA Sample Preparation Kit (Vazyme, Fuzhou, China) and the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).The quality and concentration of the extracted DNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).Then, DNA sequencing was performed on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA).The total sequencing data obtained for the mitotic genome was 6 Gb.After filtration steps were applied to remove low-quality reads and artifacts from the raw data set consisting of 61,332,980 reads in total; a final set of 3,835,348 clean reads remained.These clean reads were assembled using MitoZ (Meng et al. 2019) and metaSPAdes software (Nurk et al. 2017).The MITOs web server (Matthias et al. 2013) was used to annotate the assembly results based on the reference sequence of Harpalus pensylvanicus (GenBank accession number: NC_046953.1).The obtained genomic sequence data has been deposited in the NCBI database under accession number OR536810.Visualization of the mitogenome map was accomplished with Chloroplot software available at https:// irscope.shinyapps.io/Chloroplot/.Additionally, transfer RNA (tRNA) genes were predicted utilizing tRNA scan software (Lowe & Eddy 1997).

Phylogenetic analysis
To gain a more comprehensive understanding of the taxonomic status of C. bimaculatus, we constructed a phylogenetic tree utilizing the complete mitochondrial genomes of C. bimaculatus and 15 other Carabidae species selected from NCBI-BLAST (http://blast.ncbi.nlm.nih.gov), with Limonius californicus (Coleoptera: Elateroidea; GenBank accession number: KT852377.1)serving as the outgroup (Table 1).The complete mitochondrial genomes were aligned using the MUSCLE alignment software (Edgar 2022).Subsequently, an evolutionary tree employing the maximum-likelihood (ML) method was generated in MEGA 7 with bootstrap values for 1000 replications (Kumar et al. 2016).Finally, iTol v6 (https://itol.embl.de/) was employed to visualize the resulting phylogenetic tree.

Results
The mitogenome of C. bimaculatus was assembled based on the depth of the coverage (high coverage of over 5000�) (Figure S1).The complete mitochondrial genome of C. bimaculatus had a length of 16,419 bp and a GC content of 24% (A ¼ 41%, T ¼ 38.4%, C ¼ 12%, G ¼ 8.6%).It comprised 37 annotated genes, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (tRNAs), and 22 transfer RNA genes (tRNAs).This genome arrangement is consistent with that commonly observed in other insects (Cameron 2014).Fourteen genes were transcribed on the minority strand (Nstrand), consisting of eight tRNAs, four PCGs, and two rRNAs; while the remaining fourteen tRNAs and nine PCGs were transcribed on the majority strand (J-strand).The total length of the 13 PCGs was 11,085 bp, encoding a total of 3683 amino acids.The lengths of the 16S and 12S rRNA were measured as 1330 bp and 789 bp, respectively.Amongst the 22 tRNA molecules present in this genome assembly ranged from 60 bp (tRNA-Glu, tRNA-Phe) to 71 bp (tRNA-Val, tRNA-Lys) (Figure 2).Regarding start codons for protein-coding genes: COX1, ATP8, ND3, and ND6 utilized ATT as their start codon; COX2, ATP6, COX3, and CYTB employed ATG as their start codon; whereas ND5 and ND4L used TAA as their respective start codons.In contrast, the ND2 gene started with ATA, the ND1 gene began with AAC, and the ND4 gene initiated with TAC.Six genes (ND2, COX1, ATP8, ATP6, COX3, and ND6) terminated with TAA as their stop codon.ND3 and CYTB utilized TAG as a stop codon.ND4 and ND4L ended with ATT as a stop codon.The stop codons for COX2 and ND5 genes were completed by adding 3 0 A residues to the mRNA.
The phylogenetic analysis revealed that the analyzed species were classified into three major clades.The first clade, located at the root of the tree, consisted of L. californicus, which belongs to the Polyphaga of Coleoptera.The remaining 16 species belonged to the Adephaga of Coleoptera.The second clade comprised H. sinicus, H. anxius, H. pensylvanicus, and H. griseus from the Harpalinae subfamily within Caraboidea.In addition, the third branch included C. bimaculatus and other species from the same family as C. bimaculatus was found to be closely related to Diplocheila zeelandica in terms of mitochondrial genomes; both C. bimaculatus and D. zelandica belonged to Licininae subfamily within Caraboidea (Figure 3).

Discussion and conclusions
In this study, we successfully sequenced and annotated the complete mitochondrial genome of C. bimaculatus.The total length of the mitochondrial gene group is 16,419 bp, with a GC content of 24%.Phylogenetic analysis based on the maximum-likelihood method revealed a close relationship between C. bimaculatus and the mitochondrial genome of D. zeelandica.This phylogenetic analysis provides crucial insights into the evolutionary dynamics of the mitochondrial genome in this taxonomic group, while also establishing an indispensable foundation for future genetic investigations.Moreover, the availability of comprehensive mitochondrial genomic data for C. bimaculatus will greatly facilitate advancements in developing novel biological control strategies against Lepidopteran pests such as S. frugiperda.

Figure 1 .
Figure 1.Morphological photograph of a female Chlaenius bimaculatus (photographed by Jun Li at Fujian Agriculture and Forestry University, Fuzhou City, China.).