3.2 Protein-Coding Genes(PCGs)
The 13 PCGs of this moth mt genome include 7 NADH dehydrogenase subunits(nad1—6，nad4L)，3 cytochrome c oxidase subunits(cox1—3)，2 ATPase subunits(atp6，atp8)，and one cytochrome b gene(cob).The total length of these 13 PCGs is 11189bp，accounting for 68.73% of the entire mitochondrial genome，encoding 3，720 amino acids(Table 2).Excluding stop codons，the A+T content of the PCGs is 78.96%，lower than that of the mt genome as a whole(Table 2).The codon frequency analysis shows that a total of 62 codons are used for transcription，with the absence of UAG and UGC(Table 3).The most frequently used codon is UUA for Leu，followed by AUU for Ile(Table 3).The fraction of codons encoding the hydrophobic amino acids(Met，Trp，Phe，Val，Leu，Ile，Pro，Ala)is 55.29%(Table 3)，reflecting the biased usage of A/T nucleotides and the hydrophobic nature of respiratory membrane complexes.The codon distribution patterns of 8 compared Hepialidae species are consistent with the finding that Leu，Ile，Phe，Ser，Met and Asn are the six amino acids most frequently used，whereas Cys is rare(Fig.2).The six most common amino acids in Hepialidae differed from those of the other Lepidoptera，in which the six most common amino acids are Asn，Ile，Leu，Lys，Tyr and Phe(Dai et al.，2015).
Fig.2 Comparison of codon usage among Hepialidae mitochondrial genomes
Table 3 Codon usage of Thitarodes sp.mitochondrial PCGs
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Continued table
The base composition at each PCG codon position indicates that the third codon position(90.58%)，though somewhat lower than in the other sequenced Hepialidae，harbors a higher A/T content than the first(76.53%)and second(70.75%)codon positions(Table 2).A similar codon usage pattern was also detected in other sequenced Hepialidae，with an average of 74.80%in the first position，70.84%in the second position，and 93.33%in the third position，respectively(Table 2).
Except for cox1，all PCGs began with a canonical start codon(ATN or NTG).More specifically，six PCGs(cox2，cox3，atp6，nad4，nad4L and cob)started with ATG，three PCGs(nad2，nad3 and nad5)with ATT，two PCGs(nad6 and atp8)with ATA，and nad1 with TTG，whereas the start codon for cox1 was CGA(Table 1).When annotating the start codon of cox1，ambiguities may arise due to irregular initiation codons，such as the trinucleotides CGA in T.renzhiensis(Cao et al.，2012)and TCG in Anopheles funestus(Krzywinski et al.，2006)，the tetranucleotide TTAG in C.raphaelis(Kim et al.，2006)and the hexanucleotide ATTACGin Papilio xuthus(Feng et al.，2010).In this study，we tentatively considered that the codon CGA(coding for arginine)was involved in the initiation signaling of cox1 because this codon is found to be conserved as an initial amino acid in most Lepidoptera(Kim et al.，2009).
For the stop codon，eight PCGs(atp6，atp8，nad1，nad4，nad4L，nad6，cox3 and cob)are terminated with the typical stop codon TAA，while five PCGs(nad2，nad3，cox1，cox2 and nad5)located immediately upstream of tRNAs ended with only T.The incomplete stop codon is common in insect mt genomes，and their corresponding transcripts could be processed into mature RNA by precise endonucleolytic cleavages using the recognition signals of tRNA secondary structures.The single T stop codon would be a complete and functional stop codon(TAA)after posttranslational modification that occurs during the mRNA maturation process(Ojala et al.，1980；Ojala et al.，1981).From the perspective of evolutionary frugality，the existence of a partial stop codon is proposed to minimize the intergenic spacers and gene overlaps(Hao et al.，2012).