DNA samples extraction

Corcyra eggs were collected from Guangdong Province of China and raised in the laboratory in Beijing. The hatched adults were collected, preserved in 100% ethanol, and stored at —20 °C. Total DNA was extracted and isolated from single specimens using the DNeasy Tissue kit (QIAGEN,  www.qiagen.com) according to manufacturer instructions.

Primer design, PCR, and sequencing

The short fragment amplifications were performed using the universal PCR primers from Simon et al. (1994). The degenerate and specific primer pairs were designed based on the known mitochondrial sequences in Lepidoptera, or designed by Primer 5.0 software on the fragments that we previously sequenced (Table 1). All the primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd,  www.sangon.com. For fragments of length less than 2 kb, PCR conditions were as follows: 95 °C for five min, 34 cycles of 94 °C for 30 sec, 50–55 °C (depending on primer combinations), 1–3 min (depending on putative length of the fragments) at 68 °C, and a final extension step of 72 °C for 10 min. For fragments of length longer than 2 kb, PCR conditions were as follows: 92 °C for two min, 40 cycles of 92 °C for 30 sec, 50–55 °C for 30 sec (depending on primer combinations), 60 °C for 12 min, and a final extension step of 60 °C for 20 min.

The entire mitogenome of the Corcyra was amplified in 17 fragments. For most fragments, 2× Taq PCR MasterMix (Tiangen Biotech Co., Ltd.,  www.tiangen.com) was used in the amplification; fragments longer than 2 kb (e.g., rrnL-rrnS and nad4-cob) and with higher AT contents (e.g., rrnS-nad2 and cox3-nad5) were amplified using Takara LA Taq (Takara Co.  www.takara-bio.com). All amplifications were performed on an Eppendorf Mastercycler and Mastercycler gradient in 50 µL reaction volumes. The reaction volume of 2 × Taq PCR MasterMix contained 22 µL sterilized distilled water, 25 µL 2× Master Mix, 1 µL of each primer (10 uM), and 1 µL of DNA template; the reaction volume of Takara LA Taq consisted of 26.5 µL sterilized distilled water, 5 µL 10×LA PCR Buffer II (Takara), 5 µL 25 mM MgCl₂, 8 µL of dNTPs Mixture, 2 µL of each primer (10 µM), 1 µL of DNA template, and 0.5 µL (1.25 U) of Takara LA Taq polymerase (Takara).

The PCR products were detected via electrophoresis in 1% agarose gel, purified using the 3 S Spin PCR Product Purification Kit, and sequenced directly with ABI-377 automatic DNA sequencer. All fragments were sequenced from both strands. Short amplified products were sequenced directly by internal primers, and long amplified products were sequenced completely by primer walking. The rrnS-nad2 region was sequenced after cloning. The purified PCR products were ligated to the pEASY-T3 Cloning Vector (Beijing TransGen Biotech Co., Ltd., transgen.com.cn) and then sequenced by M13F and M13-R primers and walking. Sequencing was performed using ABI BigDye ver 3.1 dye terminator sequencing technology and run on ABI PRISM 3730×1 capillary sequencers.

Analysis and annotation

Sequence annotation was performed using the DNAStar package (DNAStar Inc.,  www.dnastar.com). The sequence was checked manually for consistency by alignment, and tRNA genes were found using tRNAscan-SE software v. 1.21 (Lowe and Eddy 1997) with manual editing. The undermined putative tRNAs were identified by sequence alignment with other insects of Pyralidae (Diatraea, O. furnacalis, and O. nubilalis) using Bioedit (Hall 1999). Secondary structure was inferred using DNASIS v.2.5. The trnS1(AGN) secondarystructure was developed as proposed by Steinberg and Cedergren (1994). PCGs and rRNAs were identified by similarity to other lepidopteran sequences. The nucleotide sequences of the PCGs were translated based on the invertebrate mtDNA genetic code. Since the Corcyra does not utilize the AGG codon, use of the variant arthropod genetic code (Abascal et al. 2006) was unnecessary. Nucleotide composition and codon usage were calculated using MEGA4.0 (Tamura et al. 2007).

Genome structure and organization

The Corcyra mitogenome is a circular molecule 15,273 bp in length; data were uploaded to Genbank (HQ897685). The Corcyra mitogenome showed the standard gene complement containing 13 PCGs, 2 rRNAs, 22 tRNAs, and non-coding regions typical for lepidopterans. The trnM is coded between the A+T rich region and tRNA-Ile (order is A+T region-trnM-trnl-trnQ), which was different from the ancestral gene order of insects (A+T region-trnl-trnQ-trnM). Since the trnS2(UCN) was not found by tRNAScan-SE, it was later determined by sequence comparison with other lepidopteran insects.

The Corcyra mitogenome was biased toward A+T content (80.43%) with the value falling into the lepidopteran range of 77.84% in Ochrogaster lunifer (Salvato et al. 2008) to 82.66% in Coreana raphaelis (Kim et al. 2006). Additionally, the A+T content was 78.96% in PCGs, 82.95%, in rrnL genes, and 85.86% in rrns genes. These values were also well within the range reported for other lepidopterans. The A+T content (96.58%) of A+T rich region was the highest value among the known lepidoteran MtDNA sequences (Table 3).

Protein-coding genes

The initial and termination codons of thirteen PCGs are shown in Table 2. Twelve PCGs started with a typical ATN codon (ATT for nad2, cox2, atp8, nad3, nad6; ATA for nad5, cob, nad1; ATG for atp6,cox3, nad4, nad4l). One exception is cox1gene, which used CGA as a start codon.

The putative start codon CGA is common across insects (Fenn et al. 2007) such as Bombyx mori (Yukuhiro et al. 2002), O. nubilalis and O. furnacalis (Coates et al. 2005), Adoxophyes honmai (Lee et al. 2006), Coreana (Kim et al. 2006), Antheraeapernyi (Liu et al. 2008), B. mandarina (Pan et al. 2008), Ochrogaster (Salvato et al. 2008), Artogeia melete (Hong et al. 2009), Eriogyna pyretorum (Jiang et al. 2009), and Hyphantria cunea (Liao et al. 2010).

Nine PCGs had the common stop codon TAA, while the nad2, cox1, cox2, nad4 have single T as an incomplete stop codon, also found in other animal mitochondrial genes (Clary and Wolstenholme 1985). The common interpretation of this phenomenon is that the TAA terminator is created via post– transcriptional polyadenylation (Ojala et al. 1981).

Transfer and ribosomal RNA genes

The 22 tRNA genes ranging from 64 to 73 nucleotides were spread over the mitogenome. Fourteen tRNAs were coded on the J-strand and eight on the N-strand, which is the same organization observed in other lepidopteran mitogenomes. Complete cloverleaf secondary structures could be inferred for 21 of the 22 tRNAs with the exception of trnS1(AGN), which lacks the DHU arm (Figure 1). A total of 43 unmatched base pairs were scattered in 20 tRNA genes, including 20 pairs in the DHU stems, eight pairs in the amino acid acceptor stems, nine pairs in the T^ψ;C stems, and six pairs in the anticodon stems. 24 of them are G-U pairs, which form a weak bond. The remaining were A-A, C-A, C-U, G-A, G-G, and U-U mismatches.

As in the other insect mitogenome sequences, two rRNA genes were present in Corcyra. The rrnL were found between trnL(CUN) and trnV, and the rrnS between trnV and the A+T rich region, respectively.

Codon usage

Relative synonymous codon usage values of Corcyra mitogenome are summarized in Table 4. The codons CTG, CCG, and AGG were not represented in the coding sequences. Leucine (14.42%), isoleucine (12.14%), phenylalanine (9.74%), and serine (9.23%) were the most common amino acids in Corcyra mitochondrial proteins (45.53%). These amino acids are abundant in other insects, averaging 45.08% (Lessinger et al. 2000).

Non-coding and overlapping region

The Corcyra mitogenome harbored 15 non-coding regions, from 1 to 351 bp to 512 bp. Intergenic spacer sequences covered four major regions of length more than 10 bp. The remaining intergenic spacer were less than 5 bp.

Spacer 1 (61 bp), located between trnQ gene and nad2 gene, is a common intergenic spacer rich in AT nucleotides (96.72%). The location of this spacer is fixed in lepidopterans, but varied in length from 40 bp (Parnassius bremeri) (Kim MI 2009) to 88 bp (Sasakia charonda) (Unpublished, AP011824). This spacer can be taken as a lepidopteran mitogenome marker not found in other insect mitogenomes. Kim MI (2009) found that the intergenic spacer sequences and the nad2 gene had higher sequence identity than other fragments of the mitogenome. There were 29 species with more than 60% identity of 32 total lepidopteran species sequenced (Table 5), suggesting that this spacer sequence originated from a partial duplication of the nad2 gene.

Spacer 2 (49 bp) was found between trnE and trnF genes, including two microsatellite-like regions, (TA) 18 and (TTAT)3, similar to other lepidopterans. The spacer in Adoxophyes (Lee et al. 2006) is 222 bp and contains a different motif (TATTA)31. The spacer in Ochrogaster (Salvato et al. 2008) is 70 bp, contains a microsatellite (TA)23, and shows triplication of a 10-nucleotide motif with some changes. In other lepidoptera insects it is shorter than 10 bp.

Spacer 3 (16 bp) was between the trnS2(UCN) and nad1 genes, commonly detectable in lepidopteran insects, and measured 16–38 bp. This intergenic spacer sequence of most lepidopterans harbored the motif (ATACTAA), except for ATACTAT in Corcyra and ATCATAT in Sesamia (Figure 2). Similarly, in Hymenoptera there is a 6 bp conserved motif (THACWW) (Wei et al. 2010), and in Coleoptera the motif is 5 bp (TACTA). Such conservation suggests that the motif is functional in Lepidoptera. This motif is possibly fundamental to site recognition by the transcription termination peptide (Taanman 1999).

Spacer 4 (10 bp) was between nad1 and trnL(CUN). Ostrinia furnacalis and O. nubilalis also showed 10 bp spacers, while other lepidopteran spacers measured 1–6 bp.

Figure 1.

Putative secondary structures for the tRNA genes of the Corcyra cephalonica mitogenome. High quality figures are available online.

Figure 2.

Alignment result of Spacer 4 tRNA-Ser(UCR)-NADI in 28 lepidopteran species. High quality figures are available online.

Spacer 5 (351 bp) was A+T rich and found between rrnS and trnM with AT nucleotides (96.58%). There was a motif ATAGA followed by a 20 bp poly-T stretch downstream of rrnS, and two microsatellitelike regions (TA)9 and (TA)8. Finally, a 10 bp poly-A was present upstream of trnM. The feature was found to be common for other lepidopterans sequenced to date.

Overlapping sequences had a total of 35 bp spread over eight regions. Like other insect species (Adoxophyes) (35), atp8 and atp6 had a seven-nucleotide overlap (ATGATAA), known to be translated from the same cistronic mRNAs. The longest overlapping sequence (8 bp) was between trnW gene and trnC genes. The remaining overlapping sequences were all less than 6 bp.