The complete mitochondrial genome sequence of Oryctes rhinoceros (Coleoptera: Scarabaeidae) based on long-read nanopore sequencing

Background
The coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is a severe and invasive pest of coconut and other palms throughout Asia and the Pacific. The biocontrol agent, Oryctes rhinoceros nudivirus (OrNV), has successfully suppressed O. rhinoceros populations for decades but new CRB invasions started appearing after 2007. A single-SNP variant within the mitochondrial cox1 gene is used to distinguish the recently-invading CRB-G lineage from other haplotypes, but the lack of mitogenome sequence for this species hinders further development of a molecular toolset for biosecurity and management programmes against CRB.

Here we report the complete circular sequence and annotation for CRB mitogenome, generated to support such efforts.

Methods
Sequencing data were generated using long-read Nanopore technology from genomic DNA isolated from a CRB-G female. The mitogenome was assembled with Flye v.2.5, using the short-read Illumina sequences to remove homopolymers with Pilon, and annotated with MITOS. Independently-generated transcriptome data were used to assess the O. rhinoceros mitogenome annotation and transcription. The aligned sequences of 13 protein-coding genes (PCGs) (with degenerate third codon position) from O. rhinoceros, 13 other Scarabaeidae taxa and two outgroup taxa were used for the phylogenetic reconstruction with the Maximum likelihood (ML) approach in IQ-TREE and Bayesian (BI) approach in MrBayes.

Results
The complete circular mitogenome of O. rhinoceros is 20,898 bp in length, with a gene content canonical for insects (13 PCGs, two rRNA genes, and 22 tRNA genes), as well as one structural variation (rearrangement of trnQ and trnI) and a long control region (6,204 bp). Transcription was detected across all 37 genes, and interestingly, within three domains in the control region. ML and BI phylogenies had the same topology, correctly grouping O. rhinoceros with one other Dynastinae taxon, and recovering the previously reported relationship among lineages in the Scarabaeidae. In silico PCR-RFLP analysis recovered the correct fragment set that is diagnostic for the CRB-G haplogroup. These results validate the high-quality of the O. rhinoceros mitogenome sequence and annotation.