Orthaga olivacea Warre (Lepidoptera: Pyralidae) is an important agricultural pest of camphor trees (Cinnamomum camphora). To further supplement the known genome-level features of related species, the complete mitochondrial genome of Orthaga olivacea is amplified, sequenced, annotated, analyzed, and compared with 58 other species of Lepidopteran. The complete sequence is 15,174 bp, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative control region. Base composition is biased toward adenine and thymine (79.02% A+T) and A+T skew are slightly negative. Twelve of the 13 PCGs use typical ATN start codons. The exception is cytochrome oxidase 1 (cox1) that utilizes a CGA initiation codon. Nine PCGs have standard termination codon (TAA); others have incomplete stop codons, a single T or TA nucleotide. All the tRNA genes have the typical clover-leaf secondary structure, except for trnS(AGN), in which dihydrouridine (DHU) arm fails to form a stable stem-loop structure. The A+T-rich region (293 bp) contains a typical Lepidopter motifs ‘ATAGA’ followed by a 17 bp poly-T stretch, and a microsatellite-like (AT)13 repeat. Codon usage analysis revealed that Asn, Ile, Leu2, Lys, Tyr and Phe were the most frequently used amino acids, while Cys was the least utilized. Phylogenetic analysis suggested that among sequenced lepidopteran mitochondrial genomes, Orthaga olivacea Warre was most closely related to Hypsopygia regina, and confirmed that Orthaga olivacea Warre belongs to the Pyralidae family.
Citation: Yang L, Dai J, Gao Q, Yuan G, Liu J, Sun Y, et al. (2020) Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects. PLoS ONE 15(3): e0227831. https://doi.org/10.1371/journal.pone.0227831
Editor: Jeffrey M. Marcus, University of Manitoba, CANADA
Received: August 31, 2019; Accepted: December 30, 2019; Published: March 6, 2020
Copyright: © 2020 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All sequences date files are available from the GenBank at NCBI database (accession number mitochondrion MN078362).
Funding: GQW was supported by the grant from the National Natural Science Foundation of China (31472147) the earmarked fund for Anhui International Joint Research and Development Center of Sericulture Resources Utilization (2017R0101). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The insect mitochondrial DNA (mtDNA) is a closed-circular molecule ranging in size from 14,000 to 19,000 bp . It generally contains 37 genes, of which seven are NADH dehydrogenase subunits (nad1-nad6 and nad4L), three cytochrome C oxidase subunits (cox1-cox3), two ATPase subunits (atp6 and atp8), one cytochrome b (cytb) subunit, two ribosomal RNAs (rrnL and rrnS), and 22 transfer RNAs (tRNA) [2, 3], and a variable length A+T-rich region, the largest noncoding sequence that modulates transcription and replication [4, 5, 6]. Whole mitochondrial genomes are a useful data source for several research areas [7, 8], such as evolutionary genomics [9, 10] and comparative molecular evolution [11, 12], phylogeography , and population genetics .
The Lepidoptera (butterflies and moths) comprises over 160,000 described species, classified into 45–48 superfamilies and is cosmopolitan in distribution . Pyralidae is one of the largest families in Lepidoptera, including over 25,000 species and some of pyralids are important agricultural pests, such as Ostrinia nubilalis and Cnaphalocrocis medinalis, whose complete mitogenomes had been sequenced [16–18]. Despite their diversity and great importance as pests of agricultural and forestry plants, they are also valuable for pollinating plants of economic importance. Most species in the family Pyralidae do not yet have sequenced mitogenomes.
Orthaga olivacea Warre (Lepidoptera: Pyralidae) is a notorious pest, widely distributed in East China. The larvae feed on Cinnamomum camphora leaves and cause considerable economic losses. Farmers apply chemical prevention and removal strategies to combat this pest species particularly during larval and pupa life stages . However, overlapping generations and irregularity of abundance in the field from May to October make it very difficult to control . Previous studies have investigated the host preference, distribution and morphological characteristics of Orthaga olivacea Warre, and the control of it by bio-pesticide has been investigated [20, 21]. However, the use of pesticides is harmful to the environment. Therefore, it is necessary to find new strategies to prevent this pest. In this study we sequenced the complete mitogenome of Orthaga olivacea Warre, and compared it with other insect species, especially with the members of Pyralidae species. Phylogenetic relationships among lepidopteran superfamilies were reconstructed using the nucleotide sequences from the 13 PCGs to test the position of Orthaga olivacea within Pyralidae. The study of mitogenomes of Orthaga olivacea can provide fundamental information for mitogenome architecture, phylogeography, future phylogenetic analyses of Pyralidae, and biological control of pests.
Materials and methods
Sample collection and DNA isolation
Orthaga olivacea Warre, larvae (the larvae are about 22–30 mm long, brown, reddish-brown on the head and anterior thoracic plate, and have a brown wide band on the back of the body, with two yellow-brown lines on each side.) were collected from the camphor trees on the campus of Anhui Agricultural University (Hefei, China). Specimens were preserved with 100% ethanol and stored at -80°C. This insect is not an endangered or protected species. Total genomic DNA was extracted from the larvae using the Aidlab Genomic DNA Extraction Kit (Aidlab Co., Beijing, China) according to the manufacturer’s instructions. Extracted DNA quality was assessed by 1% agarose (w/v) gel electrophoresis.
Amplification and sequencing
Thirteen pairs of conserved primers were designed from the mitogenomes of previously sequenced Pyralidae species (synthesized by BGI Tech Co., Shenzhen, China) (Table 1). All PCRs were performed in 50 μL reaction volumes; 34.75 μL sterilized distilled water, 5 μL 5 × Taq buffer (Mg2+ plus), 4 μL dNTPs (2.5 mM), 2 μL genomic DNA, 2 μL of each primer (10 μM) and 0.25 μL (1.25 unit) Taq polymerase (TaKaRa Co., Dalian, China). A two-step PCR was performed under the following conditions: initial denaturation at 94°C for 5 min followed by 35 cycles of 30s at 94°C, annealing 2–3 min (depending on putative length of the fragments) at 51–58°C (depending on primer combination) and a final extension step of 72°C for 10 min.
PCR amplicons were analyzed on 1.0% agarose gel electrophoresis, and purified using a gel extraction kit (CWBIO Co., Beijing, China). Purified fragments were ligated into the T-vector (TaKaRa Co., Dalian, China) and transformed into Escherichia coli DH5α. Positive recombinant colonies with insert DNA were sequenced in both directions and at least three times by Invitrogen Co. Ltd. (Shanghai, China).
The complete mtDNA sequence was assembly using the DNAStar package (DNAStar Inc. Madison, USA) and sequence annotation was performed using the blast tools from NCBI (http://blast.ncbi.nlm.nih.gov/Blast). The sequences were submitted to GenBank at NCBI under the accession number MN078362. The tRNA genes were identified using the tRNAscan-Se program software available online at http://lowelab.ucsc.edu/tRNAscan-SE/, and visually identify sequences using the alignment with the appropriate anticodons capable of folding into the typical clover-leaf structure . PCGs were initially identified by sequence identity with Pyralidae species and aligned with the other lepidopteran using ClustalX version 2.0 . Nucleotide sequences of the PCGs were translated into their putative amino acids based on the invertebrate mtDNA genetic code. Composition skew was performed according to the formulas AT skew = [A−T]/[A+T], GC skew = [G−C]/[G+C]) . Relative Synonymous Codon Usage (RSCU) values were calculated in MEGA 6.0 . Tandem repeats in the A+T-rich region were predicted using the Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html) .
To reconstruct the phylogenetic relationships of Lepidoptera, 58 lepidopteran mitogenomes (Table 2) representing seven lepidopteran superfamilies (Bombycoidea, Noctuoidea, Geometroidea, Pyraloidea, Tortricoidea, Papilionoidea and Yponomeutoidea) were used. The mitogenomes of Limnephilus hyalinus (NC_044710.1) , Locusta migratoria (NC_001712.1) , and Drosophila yakuba (NC_001322)  were used as outgroups. The 13 PCGs concatenated nucleotide sequences of these lepidopterans were initially aligned using ClustalX version 2.0. Phylogenetic analysis was performed using Maximum Likelihood (ML) method with the MEGA 6.0 program. This method was used to infer phylogenetic trees with 1000 bootstrap replicates.
Results and discussion
Genomic structure, organization and composition
The complete mitogenome of Orthaga olivacea Warre is a circular molecule with 15,174 base pairs (bp) in size (Fig 1). This is comparable to the mitogenome sizes documented for other sequenced lepidopterans which range from 14,535 bp in Ostrinia nubilalis to 16,179 bp in Plutella xylostella, and it is similar to Lista haraldusalis (15213) (Table 2). The Orthaga olivacea Warre mitogenome is identical to that of other lepidopterans in terms of gene organization, including all 13 PCGs (cox1–3, nad1–6, nad4L, cytb, atp6 and atp8), 22 tRNA genes, two ribosomal RNAs (rrnS and rrnL), and the important non-coding region also known as “A+T-rich region” [70, 71] (Fig 1; Table 3). Variety in non-coding regions is the primarily reason for size differences across Lepidoptera mitochondrial genomes. Nucleotide composition revealed that the most common base is T = 6249 (41.18%) and the least common base is G = 1249 (8.23%) and AT skew  (As to Ts) is slightly negative (−0.042). This trend has also been reported from Manduca sexta (−0.005) , Ctenoplusia agnata (−0.023) , Paracymoriza distinctalis (−0.002) , and Lista haraldusalis (−0.007) . In addition, the GC skew (Gs to Cs) is also negative (−0.215). Base composition of the Orthaga olivacea Warre mitogenome is A+T rich (79.02% A+T content and 20.98% G+C content). Highly A+T biased mitogenomes have been previously sequenced from lepidopterans (ranging from 77.8% in Rondotia menciana to 81.94% in Cnaphalocrocis medinalis) [17, 31], (Table 4). Nucleotide skew is negative, similar to the mitogenome of other lepidopterans, such as M. sexta (-0.005 and -0.181)  and C.medinalis (-0.030 and -0.175)  (Table 4).
Labeling tRNA genes according to the IUPAC-IUB single-letter amino acids: cox1, cox2 and cox3 present the three subunits of cytochrome c oxidase; cob present cytochrome b; nad1-nad6 constitutes NADH dehydrogenase; rrnL and rrnS refer to ribosomal RNAs. Genes named above the bar are located on major strand, while the others are located on minor strand. Anti-clockwise rRNA or PCGs genes are located on L strand and others are located on H strand.
The concatenated protein-coding genes are 11,147 bp in length, accounting for approximately 73.46% of the mitogenome. All PCGs are initiated by typical ATN start codons, except cox1, which is initiated by CGA (Table 3). The use of a non-canonical start codon for this gene is common across lepidopterans [17, 37, 73, 74], and cox1 transcripts do not overlap with the upstream tRNA, as has been proposed for several insect species . Annotation of cox1 start codon can be justifiably conducted on the basis of comparative amino acid alignments, aiming to identify conserved sites downstream of the flanking tRNA, and there is thus no justification for continued speculation about polynucleotide start codon .
Nine PCGs have canonical termination codons TAA or TAG, while four have incomplete termination codons single T (cox3 and cytb) or TA (nad4 and nad1) (Table 3). Incomplete stop codons have been observed in most other lepidopteran mitogenomes and are common across mitogenomes . It has been proposed that polycistronic pre-mRNA transcripts are processed by endonucleases, cleaving between tRNAs, and that polyadenylation of adjacent PCGs produces functional stop-codons from the partial termination codons such as a single T .
Complete mitogenome sequences of several lepidopterans were evaluated for codon usage. These species belonged to seven superfamilies (three species belonging to Pyraloidea, two species belonging to Bombycoidea, and one from each Noctuoidea, Geometroidea, Tortricoidea, Papilionoidea and Yponomeutoidea) (Fig 2). The analysis of codon usage showed that Asn, Ile, Leu2, Lys, Tyr and Phe were the amino acids with high relative usage frequency, while Arg was the least used amino acid. Three species of Geometroidea have consistent codon distributions in and each amino acid has equal content in them (Fig 3). The least used codons are those with high G and C, possibly due to high AT skew in lepidoptera PCGs [37, 79], for instance, L. haraldusalis, G. mellonella, B. mori, B. thibetaria, and L. malifoliella species all lack GCT codons, while G. molesta lacks CGT codons. However, in the present study all of these codons were observed in the mitogenome of Orthaga olivacea Warre (Fig 4) like that of A. yamamai, L. dispar and A. metis species [33, 67].
The lowercase letters above species name (a, b, c, d, e, f and g) indicate the superfamily which the species belong to (a: Pyraloidea, b: Bombycoidea, c: Noctuoidea, d: Geometroidea, e: Tortricoidea, f: Papilionoidea, g: Yponomeutoidea).
Transfer and ribosomal RNA genes
Orthaga olivacea Warre mitogenome has 22 tRNA genes, ranging in size from 53 bp (tRNASer1) to 70 bp (tRNACys and tRNALeu). TRNAs show high A+T content (80.17%) and negative AT-skew (−0.015). All the tRNAs display typical cloverleaf secondary structures, except trnSAGN which is missing a stable dihydrouridine (DHU) arm (Fig 5); this phenomenon is common across insects [17, 80, 81].
The rRNAs showed higher A+T content (84.00%) in comparison to the PCGs and tRNAs; this value falls within the range of sequenced insects (Table 4).
Overlapping and intergenic spacer regions
Six overlapping sequences with a total length of 23 bp were identified in the Orthaga olivacea Warre mitogenome. These sequences varied in length from 1 to 8 bp, and between tRNATrp and tRNACys with the biggest overlapping region (8 bp). The overlapping region located between atp8 and atp6 was 7 bp, 3 bp between tRNAIle and tRNAGln, while the remainders were shorter than 3 bp (Table 3). The 7 bp overlapping region “ATGATAA” (Fig 6B) has also been documented in several lepidopterans sequenced to date [82, 83].
(A) Intergenic spacer region alignment between trnS2 (UCN) and ND1 of several Lepidopterans. The framework ‘ATACTAA’ motif is conserved across the Lepidoptera order. (B) Intergenic overlap region alignment between ATP8 and ATP6 of several Lepidopterans. The bold ‘ATGATAA’ motif is the overlap region and it’s conserved across the Lepidoptera order. (C) Features present in the A+T-rich region of Orthaga olivacea Warre. The sequence is shown in the reverse strand. The ATAGA motif is bolded. The poly-T stretch is underlined. The single microsatellite T/A repeat sequence are double underlined.
The intergenic spacers of Orthaga olivacea Warre mitogenomes spread over fourteen regions and ranged in size from 1 to 52 bp with a total length of 134 bp. The longest intergenic spacer (52 bp) resided between tRNAGln and nad2. The 20 bp intergenic spacer region located between tRNASer2 and nad1 contained the ‘ATACTAA’ motif. The 7 bp motif is considered to be a conserved structure found in most of the insect mtDNAs (Fig 6A).
The A+T-rich region
The mitogenome of Orthaga olivacea Warre includes an A+T-rich region of 293 bp. This region showed the highest A+T content (93.86%), within the range reported of other lepidopterans (Table 4). Variation in intergenic length of noncoding regions particularly repeat sequences is responsible for most size variation in mitogenome. The control region is usually the largest noncoding part in the mitogenome [84, 85]. Several conserved structures found in other lepidopteran mitogenomes were also observed in the AT-rich region of Orthaga olivacea Warre, including the ‘ATAGA’ motif followed by a 17 bp poly-T stretch, and a microsatellite-like (AT)13 reapeat [86, 87] (Fig 6C).
Above all, there are many remarkable characteristics in nucleotide composition. Compared with reported lepidopteran species, these characteristics include the structure of tRNAs and PCGs, A+T rich region and intergenic spacer region share similarities but also some differences. And these differences and similarities between them can be used as potential markers in phylogenetic analysis.
We reconstructed the phylogenetic relationships among seven lepidopteran superfamilies using Maximum Likelihood (ML) method based on concatenated nucleotide sequences of the 13 PCGs. Phylogenetic analysis revealed that different species from the same family clustered together (Fig 7). The complete nucleotide sequences of 59 species of Lepidoptera, represent 16 families (Bombycidae, Saturniidae, Sphingidae, Lymantriidae, Erebidae, Notodontidae, Noctuidae, Nolidae, Geometridae, Crambidae, Pyralidae, Tortricidae, Papilionidae, Nymphalidae, Plutellidae, and Lyonetiidae) were downloaded from GenBank to reconstruct phylogenetic relationships among them. The species Orthaga olivacea Warre belonging to the superfamily Pyralidae, and the relationship were closer with Hypsopygia regina than that with Galleria mellonella and Corcyra cephalonica. Phylogenetic analyses showed that Pyraloidea is clustered with other superfamilies including Bombycoidea, Geometroidea, Noctuoidea, Papilionoidea, Tortricoidea, and Yponomeutoidea. Of these Bombycoidea and Geometroidea were sister groups, and the relationgship of them were closer than Noctuoidea in ML analysis (Fig 7). In the present study, the relationships at superfamily level are consistent with prior studies of lepidopteran phylogeny [88–90]. Previous classifications of Pyralidae species were mostly based on morphology, of which numerous studies are regionally limited; therefore, the precise position of Pyralidae within the Pyraloidea remained unclear, more studies are needed on the complete mitochondrial genome of the diverse Pyraloidea species in order to understand the complexity of phylogenetic relationships.
The Maximum Likelihood method was used in the tree constructing. Bootstrap values (1000 repetitions) of the branches are indicated. Limnephilus hyalinus (NC_044710.1), Drosophila incompta (NC_025936) and Locusta migratoria (JN858212) were used as outgroups.
The newly accessible mitogenome of Orthaga olivacea Warre (Lepidoptera: Pyralidae) is 15,174 bp long, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A+T-rich region. The arrangement of 13 PCGs is same to that of other sequenced lepidopterans. All PCGs of the mitogenome start with typical ATN codons, except for cytochrome c oxidase 1 (cox1) with the start codon CGA. The canonical termination codon (TAA or TAG) occurs in nine PCGs (TAA for nad2, cox1, cox2, atp8, atp6, nad3, nad5, nad4L and nad6 genes), and the remainders PCGs were terminated with a single T or TA (a single T for cox3 and cytb genes, TA for nad4 and nad1 genes). Phylogenetic analysis suggested that Orthaga olivacea Warre is more closely related to the Lista haraldusalis, and confirms that Orthaga olivacea Warre belongs to the family Pyralidae.
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