Abstract: Despite extensive use of insect mitochondrial genomes in phylogenetic and evolutionary studies, large-scale and systematic comparative analyses of protein-coding genes (PCGs) in Orthoptera remain limited. In this study, 13 mitochondrial PCGs from 404 Orthoptera species are analyzed, including representatives from both Caelifera and Ensifera, to investigate sequence length variation, nucleotide composition, codon usage patterns, and nucleotide substitution rates. The results revealed a clear AT bias across all PCGs, with ATP8 and ND6 exhibiting the highest levels of sequence variability, while COX1 was the most conserved. Start codons were predominantly ATN types, and TAA was the most frequently used stop codon, with incomplete stop codons (T or TA) widely observed. Codon usage bias analysis showed a strong preference for synonymous codons ending in A or U, with relative synonymous codon usage (RSCU) and effective number of codons (ENC) indicating substantial codon usage bias driven primarily by natural selection rather than mutation pressure. Furthermore, Ka/Ks analysis demonstrated that all PCGs were subject to strong purifying selection, with COX1 evolving at the slowest rate and ND4L at the fastest. These findings provide new insights into the molecular evolution of Orthoptera mitochondrial genomes and offer a valuable foundation for further research on insect phylogeny, molecular adaptation, and evolutionary genetics.

Key words: Orthoptera, mitochondrial genome, protein-coding gene, codon usage bias, comparative genomics