Introduction
Opisina arenosella Walker, belonging to the family Xyloryctidae in the order Lepidoptera, is a significant quarantine leaf-feeding pest on palm plants such as Cocos nucifera and Phoenix dactylifera in the Arecaceae family (Li et al., Reference Li, Yin, Cai, Li and Lu2014, Reference Li, Shao, Song, Song, Jiang, Li and Cai2015). This insect originated in Sri Lanka and India in Asia and is now primarily distributed in Southeast Asian countries and regions (Zhu et al., Reference Zhu, Sun, An, Li, Xu, He and Liang2024). It can damage 34 species of palm plants, including coconut, and can also harm important agricultural and forestry crops such as Musa nana and Saccharum officinarum. Opisina arenosella inflicts damage to its host primarily through its larval stage, causing the affected host’s leaves to curl, wither, and turn yellow, resembling a burnt appearance. In severe cases of infestation, the caterpillars consume the leaves of the coconut tree entirely, resulting in weakened tree vigour, increased fruit drop, and reduced yield (Lowe and Eddy, Reference Lowe and Eddy1997; Lv et al., Reference Lv, Yan, Jin, Wen, Fu, Li and Peng2013). This insect has a strong adaptability and spreads rapidly. Since its initial discovery in August 2013 in Hainan Province, China, it has quickly established and spread across most areas of Hainan Province, as well as in Guangdong and Guangxi Provinces (Yang et al., Reference Yang, Li, Shameer, Yan, Lv, Jiang, Zhang, Tu and Qi2021). The rapid spread of O. arenosella and the harm that it causes have aroused great concern about the potential of this species to infest trees all over the world as well as its potential impact on the economy and environment.
Mitochondria are widely used in research on population genetics, systematics, and molecular evolution due to their maternal inheritance, stable genome composition, relatively conserved gene arrangement, and rare recombination (Nelson et al., Reference Nelson, Lambkin, Batterham, Wallman, Dowton, Whiting, Yeates and Cameron2012; Driscoll et al., Reference Driscoll, Driscoll, Hazekamp, Mitton and Wehausen2015; Feng et al., Reference Feng, Zhao and Lu2015; Wang et al., Reference Wang, Lin, Lin, Du and Zhang2025). With the advancement of whole genome sequencing technology, more molecular markers may be obtained for phylogenetic analysis, leading to more robust relationships between species (Fagua et al., Reference Fagua, Condamine, Brunet, Clamens, Laroche, Levesque, Cusson and Sperling2018; Li et al., Reference Li, Shu, Li, Meng and Li2019). Arthropods’ diffusion pathways and hybridisation dynamics are usually tracked by analysing mitochondrial sequences (Rubinoff et al., Reference Rubinoff, Holland, Shibata, Messing and Wright2010; Moore et al., Reference Moore, Sylla, Goss, Burugu, Sang, Kamau, Kenya, Bosio, Munoz Mde, Sharakova, Black and Kading2013; Wang et al., Reference Wang, Yang, Lu, Zhou and Wu2017). The mitochondrial genome is a double-stranded circular molecular structure widely used for inferring both deep and recent phylogenetic relationships in insects (Li et al., Reference Li, Wei, Tang, Wu, Shi, Sharkey and Chen2016; Zheng et al., Reference Zheng, Cao, Tang, Van Achterberg, Hoffmann, Chen, Chen and Wei2018). The mitochondrial genome is a valuable resource for clarifying phylogenetic relationships at various taxonomic levels in beetles, including those at the tribe level, subfamily level, and family level (Nie et al., Reference Nie, Breeschoten, Timmermans, Nadein, Xue and Bai2018, Reference Nie, Andújar, Gómez‐Rodríguez, Bai, Xue and Tang2020, Reference Nie, Vogler, Yang and Lin2021). When mitochondrial genome sequences are unavailable, mitochondrial lineages are often studied by amplifying the universal barcode region with degenerate primers, as well as by analysing partial sequences of COI and a few other mitochondrial genes. However, these partial sequences may not be adequate for accurately identifying mitochondrial lineages unless a significant number of genetic markers are obtained. According to the National Center for Biotechnology Information (NCBI) gene database, the mitochondrial genomes of Opisina species have not yet been sequenced and characterised. This study presents, for the first time, the complete mitochondrial genome sequence of O. arenosella using next-generation sequencing technology. This sequence was compared with 19 mitochondrial genome sequences from Lepidoptera (including three families: Cosssidae, Endromidae, and Oecophoridae), with two mitochondrial genome sequences from Coleoptera (family Cerambycidae) as an outgroup, all available in GenBank. Additionally, the phylogenetic relationships of these 21 insects were analysed to enhance understanding of Opisina phylogeny. This genomic resource will contribute valuable information for future studies in systematics and taxonomy, providing a foundation for further research in this field.
Materials and methods
Sample collection and DNA extraction
Specimens of O. arenosella were gathered on 17 June 2019, from the host plant C. nucifera L. located at Huxin Island in Haikou People’s Park, Haikou City, Hainan Province, China (20.03°N, 110.34°E, 20 m elevation). These specimens were preserved in 95% ethanol and kept at −24°C in the specimen collection room at China West Normal University for long-term storage. Genomic DNA was isolated from the thoracic muscle of each specimen using the Ezup Column Animal Genomic DNA Purification Kit (Shanghai, China) following the manufacturer’s protocol. To ensure tissue purity, specimens were dissected under a stereomicroscope, and the thoracic muscle was carefully separated from surrounding tissues, including the gut, fat body, and exoskeleton, using sterile forceps and scissors. Any non-muscle tissue was meticulously removed to minimise contamination. The purified DNA was stored at −24°C until sequencing.
DNA sequencing, mitogenome assembly, and annotation
The complete mitogenome of O. arenosella was sequenced and assembled by Beijing Aoweisen Gene Technology Co., Ltd. (Beijing, China) using next-generation sequencing. Total genomic DNA was quantified, and a whole-genome shotgun strategy was utilised for sequencing on the Illumina HiSeq platform with 150-bp paired-end reads. The assembly was carried out using methods outlined by Hahn et al. (Reference Hahn, Bachmann and Chevreux2013), and gene annotation was performed with Geneious 11.0.2 based on protocols from Greiner et al. (Reference Greiner, Lehwark and Bock2019). tRNA genes were verified using tRNAScan-SE (Lowe and Eddy, Reference Lowe and Eddy1997) and the MITOS Web Server (Bernt et al., Reference Bernt, Donath, Jühling, Externbrink, Florentz, Fritzsch, Pütz, Middendorf and Stadler2013). Base composition and relative synonymous codon usage (RSCU) values were computed using the Shengxin Cloud Online Server. Strand asymmetry was determined using the following formulas: AT-skew = (A – T)/(A + T) and GC-skew = (G – C)/(G + C) as described by Perna and Kocher (Reference Perna and Kocher1995).
Phylogenetic analyses
This study examined 19 complete mitochondrial genomes of Lepidoptera, including a newly sequenced species O. arenosella, and incorporated two outgroup species, Agapanthia amurensis and Agapanthia daurica, these two species belong to Coleoptera, Cerambycidae. The phylogenetic dataset includes all of the sequences analysed, including outgroup sequences. So, 21 species in four families (one of these families is in order Coleoptera) were analysed (table 1). Phylogenetic analysis was conducted in PhyloSuite (v1.2.3) (ChuanYu et al., Reference ChuanYu, Fangluan, Ivan, HongPeng, Ye, Hong, Hong, GuiTang and Dong2023). A phylogenetic tree was constructed using 13 protein-coding genes (PCGs) and ribosomal RNA (rRNA) sequences, analysed through maximum likelihood (ML) and Bayesian inference (BI) methods. The sequence alignment was performed with MAFFT, and the alignment was trimmed using trimAl. ModelFinder was employed to select the most appropriate model for the analysis. For ML analysis, 1,000 ultrafast bootstrap replicates and 1,000 SH-aLRT (Shimodaira-Hasegawa-like Approximate Likelihood Ratio Test) replicates were conducted to assess node reliability. Bayesian analysis involved two independent chains with one million generations, sampling every 100 generations, and 25% of the initial iterations were discarded as burn-in. The resulting phylogenetic tree was visualised and edited using the iTOL online server.
GenBank accession numbers of species used in this study
Results
Mitogenome organisation and composition
The complete mitochondrial genome of O. arenosella is 15,389 base pairs (bp) long and forms a closed circular molecule (fig. 1). It contains 37 genes: 22 transfer RNAs (tRNAs), 2 rRNAs, 13 PCGs, and an A + T-rich region, which is characteristic of other typical Oecophoridae mitochondrial genomes. Out of the 13 PCGs, 9 (ATP8, ATP6, COI, COII, COIII, ND2, ND3, ND6, and CYTB) are located on the J strand, along with 14 tRNAs (Ile, Met, Trp, Leu2, Lys, Asp, Gly, Ala, Arg, Asn, Ser1, Glu, Thr, and Ser2) and the control region. The remaining four PCGs (ND5, ND4, ND4L, and ND1), eight tRNAs (Gln, Cys, Tyr, Phe, His, Pro, Leu1, and Val), and two rRNAs (12S rRNA and 16S rRNA) are situated on the N strand (fig. 1). The arrangement of genes in this mitochondrial genome follows the ancestral order proposed for all arthropods.
The mitochondrial genome map of O. arenosella is presented with specific colour coding: ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 are highlighted in yellow; COI, COII, and COIII are shown in pink; ATP6 and ATP8 are depicted in green; CYTB is marked in purple; rrnl and rns are displayed in red; all tRNAs are represented in dark blue; and the control region is shown in orange.
High-throughput sequencing using the Illumina platform generated a total of 85,070,724 paired-end reads, comprising approximately 12.76 Gb of raw data with an average read length of 150 bp. The sequencing quality was high, with 99.06% of bases achieving Q20 and 96.61% achieving Q30. The GC content of the raw reads was 38.42%. After quality filtering and assembly, 87,011 reads mapped to the mitochondrial genome, covering 11,539,094 bp in total, with a mean coverage depth of approximately 749.8×. The assembly coverage was 100%, with 15,389 positions covered at least 1×, 15,180 at 50×, 15,022 at 100×, and 14,532 at 200× or greater, indicating high assembly reliability.
The nucleotide composition of the O. arenosella mitochondrial genome is 39.24% adenine (A), 41.33% thymine (T), 12.02% cytosine (C), and 7.41% guanine (G), resulting in a highly A + T-biased content of 80.57% (table 2). The asymmetry in nucleotide content was assessed using the formulas AT-skew = (A − T)/(A + T) and GC-skew = (G − C)/(G + C). For the O. arenosella mitochondrial genome, the AT-skew and GC-skew values are −0.03 and −0.24, respectively (table 2), reflecting a negative skew in both AT and GC content. These nucleotide composition and genomic characteristics are consistent with those typically found in Lepidoptera mitochondrial genomes.
Base composition in different regions of the mitochondrial genome of O. arenosella
PCGs and codon usage
The combined length of the 13 PCGs in the O. arenosella mitochondrial genome is 11,167 bps, which represents 72.56% of the total genome. Of the 13 PCGs, four genes (ND4, ND4L, ND5, and ND1) are located on the N strand, whereas the other nine genes (COI, COII, COIII, ATP8, ATP6, ND2, ND3, ND6, and CYTB) are found on the J strand. Among these PCGs, ND5 is the longest, with an A + T content of 82.45%, while ATP8 is the shortest but has the highest A + T content at 90.74%. The start codon for COI is CGA, while COII, COIII, ATP6, ND1, ND4, ND4L, and CYTB all use ATG as their start codon. ATP8 starts with ATC, and the start codons for ND2, ND3, ND5, and ND6 are ATT (table 3). Among these 13 PCGs, ND1, ND2, ND3, ND4L, ND6, COIII, ATP6, ATP8, and CYTB terminate with the codon TAA. The other four PCGs use a single T residue as their termination codon (table 3). It is commonly accepted that incomplete codon structures indicate the end of protein translation in insects and other invertebrates (Cheng et al., Reference Cheng, Zhang, Yu, Storey and Zhang2016). Furthermore, calculating RSCU values and examining biases in base composition are crucial for understanding the replication and transcription mechanisms of mitochondrial genomes (Wei et al., Reference Wei, Shi, Chen, Sharkey, Van Achterberg, Ye and He2010). We have compiled the data on RSCU, revealing that AUG, UUA, UAA, and ACG are the four codons that are used most frequently (fig. 2).
The relative synonymous codon usage (RSCU) for the obtained mitogenome is shown with the RSCU values on the y-axis and the codons corresponding to their respective amino acids for O. arenosella on the x-axis.
Characteristics of O. arenosella mitochondrial genome
tRNA genes, rRNA genes, and A + T-rich region
The 22 tRNA genes in O. arenosella have a combined length of 1,484 bp, with individual genes ranging from 63 to 72 bp each (table 3). The nucleotide composition of these tRNAs is 42.43% A, 38.90% T, 7.67% C, and 11.00% G, with AT-skew and GC-skew values of 0.04 and 0.18, respectively (table 2). Of these tRNA genes, 8 are located on the N strand, while the other 14 are found on the J strand. The secondary structure of O. arenosella tRNAs was predicted using the tRNAScan-SE server v1.21, revealing that all tRNA genes exhibit the typical cloverleaf structure, except for tRNA-Ser1, which lacks the dihydrouridine arm (fig. 3). This finding is consistent with previous studies, such as those on Batocera horsfieldi (Wu et al., Reference Wu, Xu, Wei, Liao, Li and Zhuo2023).
Secondary structure of O. arenosella tRNAs.
The 16S rRNA gene is 1,348 bp long, while the 12S rRNA gene has a length of 778 bp (table 2). The 16S rRNA gene, situated between tRNA-Leu and tRNA-Val, has a nucleotide composition of 43.27% A, 40.47% T, 5.15% C, and 11.11% G. The 12S rRNA gene, located between tRNA-Val and the A + T-rich region, is 778 bp in length, with 45.49% A, 40.03% T, 4.70% C, and 9.78% G. The AT-skew and GC-skew values for the rRNAs are 0.05 and 0.37, respectively (table 2). The positions and features of these rRNA genes are consistent with those observed in other Oecophoridae caterpillars. The A + T-rich region in O. arenosella, found between the trnI and rns genes, measures 350 bp and has an A + T content of 96.00%. The AT-skew and GC-skew for this region are −0.04 and −0.86, respectively.
Phylogenetic analysis
The goal of this study was to examine the phylogenetic relationships among 19 Lepidoptera species and two outgroup species (A. amurensis and A. daurica) by utilising mitochondrial genome data. Phylogenetic tree of O. arenosella was constructed using 13 PCGs and two rRNA sequences, applying both ML and BI methods (figs 4 and 5). The results from both BI and ML consistently indicated that Cossidae was the earliest diverging lineage. However, according to the results from BI, Oecophoridae and Endromidae formed a highly supported monophyletic clade, with high node support values, which was not supported in the ML analysis. In both BI and ML analyses, O. arenosella and Ripeacma umbellate clustered together with high node support. Interestingly, in the BI analysis, (O. arenosella + R. umbellate) clustered with ((Promalactis suzukiella + Promalactis odaiensis) + (Stathmopoda auriferella + Casmara patrona)), forming a clade with high node support values. In the ML analysis, although (O. arenosella +R. umbellate) clustered with ((P. suzukiella + P. odaiensis) + (S. auriferella + C. patrona)), this clade did not have high node support. Furthermore, both BI and ML analyses showed that Comparmustilia, Oberthueria, Pseudandraca, and Andraca formed a highly supported monophyletic clade with high node support values. However, in the ML analysis, the clade formed by Comparmustilia and Oberthueria did not have high node support.
The maximum likelihood phylogenetic relationships among 19 selected Lepidoptera and two Coleoptera outgroups (A. amurensis and A. daurica) were inferred based on 13 protein-coding genes and 2 rRNA genes. The numbers on the branches represent the bootstrap support value.
The Bayesian phylogenetic relationships among 19 selected Lepidoptera and two Coleoptera outgroups (A. amurensis and A. daurica) were inferred based on 13 protein-coding genes and 2 rRNA genes. The numbers on the branches represent posterior probabilities.
Discussion
The complete mitochondrial genome sequence of O. arenosella obtained in this study provides crucial new information for the Opisina genus within the Oecophoridae family. Comparative and phylogenetic analyses were performed using 13 PCGs and 2 rRNA genes from 19 Lepidoptera species available in public databases. This research establishes a basis for population genetics studies on O. arenosella and enhances the phylogenetic understanding of the Oecophoridae family.
In Oecophoridae mitochondrial genomes, the secondary structures of tRNAs generally exhibit a typical cloverleaf structure, except for tRNA-Ser1 (AGN), which lacks the D-arm, as seen in other insect orders such as Coleoptera, Diptera, and Hemiptera (Liao et al., Reference Liao, Wang, Wu, Li, Zhao, Huang, Niu, Liu and Li2010; Wang et al., Reference Wang, Lin, Lin, Du and Zhang2025). Most of the mitochondrial PCGs in O. arenosella start with typical ATN (ATT, ATG) start codons, except the COI gene, which starts with CGA. The stop codons are generally TAA, except for COI, COII, ND4, and ND5 genes, which have incomplete stop codons (T or TA). These incomplete stop codons can be completed by the addition of an A at the 3′ end of the transcript (Boore, Reference Boore1999; Chu et al., Reference Chu, Su, Zan, Lin, Yao, Xu and Liu2024; Dong et al., Reference Dong, Cai, Dong, Wang and An2025; Wen et al., Reference Wen, Shang, Chen, Guo, Rong, Jiang and Sun2025). The use of the CGA start codon in the COI gene is consistent with other Lepidoptera species such as Hyphantria cunea (Liao et al., Reference Liao, Wang, Wu, Li, Zhao, Huang, Niu, Liu and Li2010; Wei et al., Reference Wei, Shi, Chen, Sharkey, Van Achterberg, Ye and He2010). The A + T-rich region is highly variable in length even among closely related species and is characterised by a very high A + T content (Zhang et al., Reference Zhang, Szymura and Hewitt1995; Yang et al., Reference Yang, Cao and Geng2017; Zhu et al., Reference Zhu, Sun, An, Li, Xu, He and Liang2024; Liu et al., Reference Liu, Ni, Chen, Xu, Pei, Yang, Xu, Wang, Fu and Yu2025).
Traditional morphological features are still predominantly used for classifying Lepidoptera and Coleoptera insects. However, due to the complexity and variability of these features, there are ongoing classification challenges. In this study, a phylogenetic tree was constructed using 13 PCGs and 2 rRNAs with BI and ML methods, yielding results consistent with previous research. For instance, our analyses suggested that Oberthueria falcigera is the earliest branching species within the genus Oberthueria, and Oberthueria yandu shares a close relationship with Oberthueria caeca, aligning with Zolotuhin and Wang (Zhang et al., Reference Zhang, Szymura and Hewitt1995). Additionally, P. odaiensis and P. suzukiella were clustered together with high support values, consistent with findings by Kim and Lee (Zhang et al., Reference Zhang, Szymura and Hewitt1995; Kim and Lee, Reference Kim and Lee2019; ChuanYu et al., Reference ChuanYu, Fangluan, Ivan, HongPeng, Ye, Hong, Hong, GuiTang and Dong2023; Qiao et al., Reference Qiao, Zhuang, Wang, Xie, Zhang, Shen and Zhou2025). The primary aim of this work was to analyse phylogenetic relationships among 19 Lepidoptera species and two outgroups (A. amurensis and A. daurica) using mitochondrial genome data. Although the topologies of the ML and BI trees were not identical, they were quite similar (Chen et al., Reference Chen, Chen, Chen, Yu and Lin2019; Hassan et al., Reference Hassan, Liu, Mehboob, Bilal, Arain, Siddique, Chen, Li, Zhang, Shi, Lv and Lin2023). Notably, the BI trees showed higher node support values than the ML trees, a trend observed in other studies (Li et al., Reference Li, Wei, Tang, Wu, Shi, Sharkey and Chen2016; Yuan et al., Reference Yuan, Zhang, Zhang, Guo, Liu, Shen and Shao2016; Chen et al., Reference Chen, Zhao, Xu and Du2018; Lin et al., Reference Lin, Sun, Hu, Yang, Li, Wang, Zhang, Lu, Li, Li, Zhang and Li2025). Our results underscore the utility of mitochondrial genome sequences for inferring species relationships within Lepidoptera.
Advancements in mitochondrial genome sequencing and assembly techniques will facilitate future mitochondrial genome sequencing projects (Crampton-Platt et al., Reference Crampton-Platt, Timmermans, Gimmel, Kutty, Cockerill, Vun Khen and Vogler2015; Yan et al., Reference Yan, Liu, Li, Lv, Tan, Peng and Li2015; Bourguignon et al., Reference Bourguignon, Dahlsjö and Jacquemin2017; Song et al., Reference Song, Li, Gao, Yan, Zhang, Liu and Zhou2024; Qiao et al., Reference Qiao, Zhuang, Wang, Xie, Zhang, Shen and Zhou2025). Expanding species sampling and genome sequencing will help resolve classification challenges in Lepidoptera. Although our data may not fully resolve intrafamilial phylogenetic relationships within Lepidoptera, it provides a significant reference for further research. The complete mitochondrial genome of O. arenosella obtained in this study offers valuable information for future phylogenetic and taxonomic investigations, laying a foundation for related research.
Conclusions
In this study, we first successfully sequenced the complete mitochondrial genome of O. arenosella, which spans 15,389 bp. Aligning with typical Lepidoptera molecular patterns, the genome comprises 37 gene segments, including 22 tRNAs, 2 rRNAs, 13 PCGs, and a characteristic A + T-rich region. Notably, four PCGs are on the N strand, while nine are on the J strand. Various start and stop codons were identified across the PCGs, adding nuance to O. arenosella genetic make-up. Phylogenetic analyses consistently position Cossidae as the earliest branching clade. BI highlights a well-supported monophyletic group of Oecophoridae and Endromidae, while this result lacks support in ML analysis. Opisina arenosella and Ripeacma umbellata form a robust clade in both BI and ML analyses. Intriguingly, the BI analysis reveals a clade including (O. arenosella + R. umbellate) grouping with ((P. suzukiella + P. odaiensis) + (S. auriferella + C. patrona)), displaying high node support. However, in the ML analysis, this clade lacks high node support. Both BI and ML analyses demonstrate a monophyletic group of Comparmustilia, Oberthueria, Pseudandraca, and Andraca, though the Comparmustilia and Oberthueria clade lacks high node support in ML analysis. These comprehensive mitochondrial genome data enhance our understanding of O. arenosella genetic landscape and contribute to broader insights into Lepidoptera phylogenetics.
Acknowledgements
We thank Dr. Hongjun Yang for sample collection (College of Life Sciences, Sichuan University). We also wish to express our deep appreciation to the anonymous reviewers for their comments that helped to improve the quality of this manuscript.
Author contributions
Z.Z. participated in the data interpretation, conceptualisation of the project, and scientific paper writing. D.X. participated in the formal data analysis, data interpretation, and conceptualisation of the project. X.T. participated in the formal data analysis, funding acquisition, and data management. H.A. participated in the investigation, data analysis, and interpretation. Z.Z. participated in the formal data analysis, funding acquisition, data management, supervision, scientific paper writing, conceptualisation, and investigation methodology. All authors read and approved the final manuscript.
Financial support
This work was supported by the Sichuan Provincial Department of Science and Technology (2026JDKP0121).
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Not applicable because this article does not contain any studies with human or animal subjects.
Data availability statement
The complete mitochondrial genome assembly generated in this study has been deposited in GenBank under accession number MK467611.1. The raw sequencing reads (SRA data) are available in the NCBI Sequence Read Archive under accession number SRR34143417, which is part of BioProject (PRJNA1281560).
Data archiving statement
The genome sequence of O. arenosella was sequenced and assembled by Beijing Aoweisen Gene Technology Co., Ltd. (Beijing, China) using next-generation sequencing.
