Characterization of the complete mitochondrial genome of the Oriental armyworm , Mythimna separata ( Lepidoptera : Noctuidae )

The complete mitochondrial genome (mitogenome) of Mythimna separata (Lepidoptera: Noctuidae) was determined to be 15,329 bp, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A+T-rich region. The AT skew of this mitogenome was slightly negative and the nucleotide composition was also biased toward A+T nucleotides (81.00%). All PCGs were initiated by ATN codons, except for cytochrome c oxidase subunit 1 (cox1) gene, which was initiated by CGA. Five of the 13 PCGs have the incomplete termination codon, T or TA. All the tRNA genes displayed a typical clover-leaf structure of mitochondrial tRNA. The A+T-rich region of the mitogenome was 372 bp in length and consisted of several features common to the Noctuidae. Phylogenetic analysis confirmed the placement of M. separata within the Noctuidae. * These authors contributed equally to this work. ** Corresponding author.


INTRODUCTION
The insect mitochondrial genome (mitogenome) is a circular molecule, 14-19 kb in size and contains 22 transfer RNA (tRNA) genes and two ribosomal RNA genes encoding the small and large subunit rRNAs (rrnS and rrnL) that are involved in the translation of 13 protein coding genes (PCGs), ATPase subunits 6 and 8 (atp6 and atp8), cytochrome c oxidase subunits 1-3 (cox1-cox3), cytochrome B (cob), NADH dehydrogenase subunits 1-6 and 4L (nad1-6 and nad4L), and a large non-coding element termed the A+T-rich region, containing both the origin for replication and transcription (Moritz et al., 1987;Wolstenholme, 1992;Cameron, 2014).Because of their unique features, including coding content conservation, maternal inheritance, and rapid evolution, mitogenome sequences have been widely used as an informative molecular marker for diverse evolutionary studies among species in the fields of molecular evolution, phylogenetics, population genetics, and comparative and evolutionary genomics (Harrison, 1989;Boore, 1999;Whinnett et al., 2005;Lopez-Vaamonde et al., 2012;Timmermans et al., 2014).
Whilst the Lepidoptera rank as the second most numerous order of insects, comprising > 160,000 species, classified into 45-48 superfamilies, to date only a few complete or near-complete lepidopteran mitogenomes are available.These include species in the superfamilies Bombycoidea, Geometroidea, Noctuoidea, Pyraloidea, Tortricoidea, Hepialoidea and Papilionoidea.The superfamily Noctuoidea is a very large assemblage that accounts for about 40% of all described Lepidoptera (Speidel & Naumann, 2004); however, only a few mitogenomes of this superfamily have so far been sequenced (Table 1).
The Oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae) is a seasonal migrating agricultural pest moth in China that causes serious economic losses, and as such, has been used as a test insect to explore new agricultural pesticides (Chen et al., 1995;Fan et al., 2014).A better understanding of the lepidopteran mitogenome requires an expansion of taxon and genome samplings.Characterization of the M. separata mitogenome has advanced knowledge of lepidopteran mitogenomes and provided new insights intounderstanding the mechanisms underlying mitochondrial DNA (mtDNA) evolution, especially in terms of gene rearrangements.In the present study, the complete mitogenome of M. separata was sequenced and the mitogenome was then subjected to phylogenetic analyses, involving comparison with selected species belonging to the orders Lepidoptera, Orthoptera and Diptera (see below).

DNA extraction
Adult M. separata of both sexes were collected in Yancheng, Jiangsu Province.Total DNA from individuals was isolated using the Genomic DNA Extraction Kit (SangonBiotech Co., Shanghai, China), according to the manufacturer's instructions.Extracted DNA was used for PCR amplification of the complete mitogenome.

PCR amplification and sequencing
For PCR amplification of the entire M. separata mitogenome, nine primer sets designed in accordance with the known mito-were predicted using the Tandem Repeats Finder program (http:// tandem.bu.edu/trf/trf.html)(Benson, 1999).

Phylogenetic analysis
To reconstruct the phylogenetic relationship among lepidopteran insects, the complete mitogenomes of lepidopteran species were obtained from the GenBank database.These mitogenomes were divided into seven lepidopteran suborders, including, as aforementioned, Bombycoidea, Geometroidea, Hepialoidea, Noctuoidea, Pyraloidea, Tortricoidea and Papilionoidea.The mitogenomes of Locusta migratoria L. (NC_001712), Droso phila yakuba Burla (NC_001322) and Anopheles gambiae Giles (NC_002084) were used as outgroups.The amino acid sequences of each of the 13 mitochondrial PCGs were aligned using default settings and concatenated.The concatenated set of amino acid sequences from the 13 PCGs was used in phylogenetic analysis, which were performed using the Maximum Likelihood method using the MEGA version 6.06 program.This method was used to infer phylogenetic trees with 1000 bootstrap replicates.Substitution model selection was also conducted based on the lowest BIC scores (Bayesian Information Criterion) using MEGA 6.06.The mtREV24 + G + F model was the appropriate models for the amino acid sequence dataset (Tamura et al., 2013).

Genome organization and base composition
The complete mitogenome of M. separata is a closed circular molecule of size 15,329 bp.The gene content is typical of other insect mitochondrial genomes, including 22 tRNA genes (one for each amino acid, two for leucine and serine), 13 nad4L,cob,atp6 and atp8), two mitochondrial ribosomal RNAs (rrnS and rrnL), and a major non-coding region known as the A+T-chondrial sequences from other noctuids, were synthesized by Sunbiotech (Co., Ltd.Beijing, China) (Table 2).Mitochondrial DNA sequences were amplified using Aidlab 2 × PCR Master Mix (Beijing, China) according to the manufacturer's instructions.PCR was performed under the following conditions: a 4 min denaturation step at 94°C, followed by 35 cycles of 30 s at 94°C, a 1-3 min annealing step at 50-60°C, concluding with a 10 min extension step at 72°C.The products were separated by agarose gel electrophoresis (1% w/v) and purified using a DNA Gel Extraction Kit (Transgen Co., Beijing, China).The purified products were then ligated into the T-vector (SangonBiotech Co., Shanghai, China) and sequenced at least three times.

Sequence assembly and gene annotation
Sequence annotation was performed using the blast tools available at NCBI web site (http://blast.ncbi.nlm.nih.gov/Blast) and using DNAStar package (DNAStar Inc.Madison, USA).Identification of tRNA genes was verified using the tRNAscan-SE program.The potential stem-loop secondary structures within these tRNA gene sequences was calculated using the tRNAscan-SE Search Server (http://lowelab.ucsc.edu/tRNAscan-SE/)(Lowe & Eddy, 1997).The secondary structures of tRNA genes that could not be predicted using the tRNAscan-SE were analyzed by comparison with the nucleotide sequences of other insect tRNA sequences (Yin et al., 2010;Chai & Du, 2012;Gong et al., 2013;Wu et al., 2013a).The PCGs were identified by sequence similarity with that of the beet armyworm, Spodoptera exigua (Hübner) (Noctuidae) (Wu et al., 2013a).The nucleotide sequences of PCGs were translated with the invertebrate mitogenome genetic code.Alignments of PCGs from various lepidopteran mitogenomes was performed using Clustal X (Thompson et al., 1997), whilst composition skewness was calculated according to the et al., 2004).Tandem repeats in the A+T-rich region rich region (Fig. 1) (Table 3).The order of genes and the orientation of the mitogenome of M. separata are identical to the Noctuid mitogenomes.Compared with the ancestral insect gene order, the placement of the trnM tRNA gene in the M. separata mitogenome differs from that of ancestral insects.In M. separata, the order is as follows: A+T-rich region, trnM, trnI, trnQ, nad2, whereas in the ancestral order it is: A+T-rich region, trnI, trnQ, trnM, nad2 (Fig. 2) (Boore et al., 1998).The placement of trnM may therefore represent a molecular feature exclusive to lepidopteran mtDNAs (Lavrov et al., 1999).The tRNA gene rearrangements are commonly considered to be a consequence of tandem duplication of part of the mitogenome, followed by random (or nonrandom) loss of duplicated copies (Lavrov et al., 2002;Carapelli et al., 2006;Juhling et al., 2012).However, the ancestral arrangement of the trnM gene cluster was also found in the ghost moths; their ancestral gene order indicates that this gene rearrangement event likely occurred after Hepialoidea diverged from other lepidopteran lineages (Cao et al., 2012).

Protein-coding genes
All protein-coding sequences in the M. separata mitogenome were initiated by typical ATN codons (nad1 gene for ATA, nad6 gene for ATC, cob, atp6, cox3, nad4 and nad4L genes for ATG, atp8, cox2, nad2, nad3, and nad5 genes for ATT), with the exception of the cytochrome oxidase subunit 1 (cox1) (Table 3).The sequence of the 13 PCGs is 11,211 bp in length, whilst the arrangement of the PCGs is same as that in the other sequenced lepidopterans.The A+T nucleotide composition of 13 PCGs in the mitogenome of M. separata was 79.6%.Sequence alignment revealed that the open reading frame of cox1 in M. separata also starts with a CGA codon for encoding arginine.Different from the other PCGs, cox1 is initiated by a CGA codon, this noncanonical putative start codon also being found in some other insects (Yin et al., 2010;Chai & Du, 2012;Gong et al., 2013).The TTAG tetra-nucleotide is a putative start codon in C. raphaelis (Kim et al., 2006), whilst the sequence TATTAG also represents a putative start codon in the moth species, O. nubilalis and O. furnicalis (Coates et al., 2005).An unusual start codon for the cox1 gene has also been described in various arthropod mtDNAs (Negrisolo et al., 2004).The canonical termination codon (TAA) occurs in 8 PCGs in the M. separata mitogenome.The remaining five PCGs, including atp6, cox1, cox2, nad2, and nad4, were terminated with a single T or TA.The cox2 gene has incomplete stop codons, as found in all lepidopteran mtDNA sequenced to date.

Transfer RNA genes
The structure of tRNA genes was predicted using the tRNAscan-SE Search Server.The M. separata mitogenome contains 22 tRNA genes, similar to the mitogenomes of most animals (Boore, 1999).A total of 15 unmatched base pairs were found to occur in the M. separata mitochondrial tRNA genes; ten of them are G-U pairs, which form a weak bond.The trnL1, L2, trnV and trnA genes contain an U-U mismatch.All tRNAs displayed the typical clover-leaf secondary structure observed in mitochondrial tRNA genes (Fig. 3); trnS1 (AGN) lacking a stable dihydrouridine (DHU) arm has been observed in several insects and metazoan mitogenomes (Wolstenholme, 1992;Sima et al., 2013;Liu et al., 2014b).Among the 22 tRNA genes, 14 were encoded by the H-strand,, eight by the L-strand (Table 3).The A+T content of all tRNAs was 81.8%.

Ribosomal RNA genes
As typically observed in other insect mitogenomes, two rRNA genes (rrnS and rrnL) were present in the M. separata mitogenome, which were located between trnL1 (CUN) and trnV, and between trnV and the A+T-rich region, respectively.The lengths of the rrnL and rrnS were 1359 bp and 783 bp, respectively.The A+T content of the two rRNA genes as analyzed were 84.73%, which is within the range reported for other lepidopterans (Table 4).

The A+T-rich region
The A+T-rich region of M. separata extends over 372 bp (14,958-15,329 nt) and is located between the rrnS and trnM genes.Comparison with other lepidopteran species revealed that this region is shorter than that of C. boisdu valii (330 bp), A. yamamai (334 bp), C. raphaelis (375 bp), P. atrilineata (457 bp), A. melete (351 bp), A. honmai (489 bp), B. mori (494 bp), A. pernyi (552 bp) and B. mandarina (747 bp), and longer than that of M. sexta (324 bp) and O. lunifer (319 bp).The A+T-rich region contained the highest A+T content (94.35%) in the mitogenome, which is within the range reported for other lepidopteran insects (Table 4).Several conserved structures of other lepidopteran mitogenomes were also observed in the A+T-rich region of the M. separata mitogenome, including the motif "ATAGA" and a 20-bp poly-T stretch at the downstream of the rrnS gene, which is widely conserved in lepidopteran mitogenomes (Timmermans et al., 2014), and may represent the origin of minority or light strand replication (Taanman, 1999).A poly-A commonly observed in other lepidopteran mitogenomes was also found immediately upstream of the trnM gene.We also identified microsatellite (TA) 6 and (TA) 8 elements in the A+T-rich region of the M. separata mitogenome.The presence of multiple tandem repeat elements is a characteristic of the insect A+T-rich region; however, the repeat element is variable, even within the same species (Pan et al., 2008;Hu et al., 2010).The A. melete and Artogeia napi L. mitogenomes contain a duplicate 36 bp repeat element comprising a 26 bp core sequence flanked by 10 bp perfectly inverted repeats (Hong et al., 2009).In A. pernyi, the A+T-rich region harbours a repeat element of 38 bp, occurring six times in tandem, while in the wild type of A. pernyi, this element is present in five repeats (Liu et al., 2012b).In M. separata, a tandem repeat was also found in the A+T-rich region of the mitogenome containing a duplicate 51 bp repeat element and occurring twice (Fig. 4).

Phylogenetic analyses
According to the most recent consensus view of lepidopteran relationships, Bombycoidea, Noctuoidea and Geometroidea are designated as the Macrolepidoptera, and Pyraloidea with Macrolepidoptera as Obtectomera.In the present study, the concatenated amino acid sequences of the 13 PCGs of the mitochondrial genome were used to reconstruct the phylogenetic relationships among the seven superfamilies of Lepidoptera (Fig. 5).Phylogenetic  analyses showed that the Noctuoidea were clustered in one branch in the phylogenetic tree.Bombycoidea was the closest to Noctuoidea..More mitogenomes from lepidopteran insects are required to resolve uncertainties concerning the position of the Noctuoidea and the relationships among these superfamilies.Our phylogenetic analyses of these superfamilies supports the traditional morphology-based classification (Kristensen & Skalski, 1999;Timmermans et al., 2014).

Fig. 2 .
Fig. 2. The trnM gene rearrangement in the M. separata comparison with an ancestral insect.

Fig. 4 .
Fig. 4. Features present in the A+T-rich region of Mythimna separata.The sequence is shown in the reverse strand.The coloured nucleotides sequentially indicate the ATATG motif (orange), poly-T stretch (blue), two microsatellite T/A repeat sequences (red), and a poly-A stretch (green).51 bp of two tandem repeats are shown underlined in red and black.

TaBLe 1 .
List of the complete mitogenome of the superfamily Noctuoidea.

TaBLe 3 .
Summary of the mitogenome of Mythimna separata.
Fig. 3. Putative secondary structures for the tRNA genes of the Mythimna separata mitogenome.