International Journal of Aquaculture, 2025, Vol.15, No.3, 99-115 http://www.aquapublisher.com/index.php/ija 101 The 13 protein-coding genes include 7 subunits related to the NADH dehydrogenase complex (ND1-ND6, ND4L), 1 subunit related to cytochrome c reductase (cyt b), 3 subunits related to cytochrome oxidase (COX1–COX3), and 2 ATP synthase subunits (ATP6 and ATP8). The arrangement of these coding genes in the mitochondrial genome of Snakehead follows a typical order: for example, tRNAPhe-12S rRNA-tRNAVal-16S rRNA-ND1-ND6-cyt b-ND4L-ND5-D-loop. The length of each coding gene and the start and stop codons are basically the same among different species of snakehead. For example, the COX1 gene starts with the standard start codon ATG and ends with the complete stop codon TAA. There are a small amount of intervals or overlaps between genes, and some gene intervals have only 0~4 non-coding bases, and the genome utilization rate is close to the extreme. Overall, there is no abnormality or novelty in the composition and arrangement of the mitochondrial genome of snakehead, indicating that its genome structure is relatively conservative in evolution. 2.2 Characteristics of the control region and non-coding segments The mitochondrial control region (D-loop) is the longest non-coding region in the genome and is also a key region for regulating mitochondrial replication and transcription. The mitochondrial control region of the snakehead genus is located between the tRNAProgene and the tRNAPhe gene, with a length ranging from 800 bp to 1 000 bp, and is one of the regions with the highest sequence variability in the entire genome. The control region of the snakehead genus has a typical three-segment structure: the terminal termination fragment (EVT), the middle conserved region (CSBs) and the terminal conserved sequence. It contains multiple highly conserved short sequence elements (such as CSB-1, CSB-2, CSB-3) and AT-rich regions. These elements are relatively conserved in various species of the genus Snakehead, which helps to maintain the functional integrity of the control region. Studies have shown that the control region sequence of Snakehead presents a significant base composition bias: rich in A+T bases, and the total content of A and T accounts for more than 60% of the control region sequence. This strong AT bias is a common feature of the mitochondrial control region of fish, which may be related to the need for easy melting of replication initiation (Alam et al., 2019). In addition to the control region, there are very few other non-coding sequences in the mitochondrial genome of snakehead, and there are only a few base spacing regions between a few genes. For example, there is usually a very short non-coding interval between the ND5 and ND6 genes, with a length of 2 bp~5 bp. There may be a small amount of overlap or gaps between some genes, such as tRNAle and tRNAGln, tRNAThr and tRNAPro. In addition to the control region, there are almost no large insertions or repetitive sequences in the mitochondrial genome of the Snakehead, and no pseudogenes or extra genes have been found. This compact genome structure is consistent with the characteristics of the mitochondrial genome of most bony fish. It is worth mentioning that the mitochondrial genome occasionally undergoes gene rearrangement or duplication during evolution, but no similar abnormalities have been found in the currently sequenced Snakehead species. Therefore, it can be inferred that the structure of the mitochondrial genome of Snakehead is relatively stable in evolution, and its variation is mainly reflected in base substitutions and small fragment insertions and deletions, rather than changes in the macroscopic structure. 2.3 Arrangement of tRNA, rRNA, and protein-coding genes The gene arrangement order of the mitochondrial genome of Snakehead is consistent with that of most bony fish. The gene organization presents a highly conservative pattern: the genes are staggered on the two chains, the H chain contains 12 protein genes, 14 tRNAgenes and 2 rRNAgenes, and the L chain contains the ND6 gene and the remaining 8 tRNA genes. Specifically, the genome starts with tRNAPhe, followed by 12S rRNA and tRNAVal, 16S rRNA. The structure and length of these two rRNAgenes are almost completely consistent among Snakehead species (12S rRNA is about 950 bp, 16S rRNA is about 1 650 bp). It is followed by tRNALeu (UUR) and protein-coding gene ND1 (Li et al., 2018). tRNA genes are interspersed between most protein-coding genes, such as tRNAIle and tRNAGln inserted between ND1 and ND2, and tRNATrp inserted after ND2. This typical "coding gene-tRNA" alternating arrangement pattern reflects the compactness and functional intensiveness of the mitochondrial genome. It is worth noting that the mitochondrial genome has asymmetric replication of the two chains during replication, resulting in differences in the base composition of the H chain and the L chain, generally manifested as H chain
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