Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects.

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.

Introduction

The insect mitochondrial DNA (mtDNA) is a closed-circular molecule ranging in size from 14,000 to 19,000 bp [1]. 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 [13], and population genetics [14].

The Lepidoptera (butterflies and moths) comprises over 160,000 described species, classified into 45–48 superfamilies and is cosmopolitan in distribution [15]. 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 [19]. However, overlapping generations and irregularity of abundance in the field from May to October make it very difficult to control [19]. 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.

Table 1

Details of the primers used to amplify the mitogenome of O. olivacea Warre.

Primer pair	Primer sequence (5’ -3’)
F1	TAAAAATAAGCTAAATTTAAGCTT
R1	TATTAAAATTGCAAATTTTAAGGA
F2	AAACTAATAATCTTCAAAATTAT
R2	AAAATAATTTGTTCTATTAAAG
F3	ATTCTATATTTCTTGAAATATTAT
R3	CATAAATTATAAATCTTAATCATA
F4	TGAAAATGATAAGTAATTTATTT
R4	AATATTAATGGAATTTAACCACTA
F5	TAAGCTGCTAACTTAATTTTTAGT
R5	CCTGTTTCAGCTTTAGTTCATTC
F6	CCTAATTGTCTTAAAGTAGATAA
R6	TGCTTATTCTTCTGTAGCTCATAT
F7	TAATGTATAATCTTCGTCTATGTAA
R7	ATCAATAATCTCCAAAATTATTAT
F8	ACTTTAAAAACTTCAAAGAAAAA
R8	TCATAATAAATTCCTCGTCCAATAT
F9	GTAAATTATGGTTGATTAATTCG
R9	TGATCTTCAAATTCTAATTATGC
F10	CCGAAACTAACTCTCTCTCACCT
R10	CTTACATGATCTGAGTTCAAACCG
F11	CGTTCTAATAAAGTTAAATAAGCA
R11	AATATGTACATATTGCCCGTCGCT
F12	TCTAGAAACACTTTCCAGTACCTC
R12	AATTTTAAATTATTAGGTGAAATT
F13	TAATAGGGTATCTAATCCTAGTT
R13	ACTTAATTTATCCTATCAGAATAA

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).

Sequence annotation

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 [22]. PCGs were initially identified by sequence identity with Pyralidae species and aligned with the other lepidopteran using ClustalX version 2.0 [23]. 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]) [24]. Relative Synonymous Codon Usage (RSCU) values were calculated in MEGA 6.0 [25]. Tandem repeats in the A+T-rich region were predicted using the Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html) [26].

Phylogenetic analysis

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) [27], Locusta migratoria (NC_001712.1) [28], and Drosophila yakuba (NC_001322) [29] 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.

Table 2

Details of the lepidopteran mitogenomes used in this study.

Superfamily	Family	Species	Size (bp)	GenBank accession no.	Reference
Bombycoidea	Bombycidae	Bombyx mandarina	15,682	AY301620	[30]
		Bombyx mori	15,643	NC_002355	Direct submission
		Rondotia menciana	15,301	KC881286.1	[31]
	Saturniidae	Antheraea pernyi	15,566	AY242996	[32]
		Antheraea yamamai	15,338	NC_012739	[33]
	Sphingidae	Manduca sexta	15,516	NC_010266	[34]
		Sphinx morio	15299	KC470083.1	[35]
Noctuoidea	Lymantriidae	Lymantria dispar	15,569	NC_012893	Unpublished
		Euproctis pseudoconspersa	15461	KJ716847.1	[36]
	Erebidae	Amata formosae	15,463	KC513737	[37]
	Notodontidae	Ochrogaster lunifer	15,593	NC_011128	[38]
	Noctuidae	Ctenoplusia agnata	15261	KC414791.1	[39]
		Agrotis ipsilon	15,377	KF163965	[40]
	Nolidae	Gabala argentata	15,337	KJ410747	[41]
Geometroidea	Geometridae	Apocheima cinerarium	15,722	KF836545	[42]
		Biston thibetaria	15,484	KJ670146.1	Unpublished
Pyraloidea	Crambidae	Chilo suppressalis	15,395	NC_015612	[43]
		Diatraea saccharalis	15,490	NC_013274	[44]
		Ostrinia furnacalis	14,536	NC_003368	[45]
		Ostrinia nubilalis	14,535	NC_003367.1	[45]
		Cnaphalocrocis medinalis	15388	NC_015985	[43]
		Paracymoriza distinctalis	15354	KF859965.1	[46]
		Tyspanodes hypsalis	15329	NC_025569	[47]
		Paracymoriza prodigalis	15,326	NC_020094.1	[48]
		Elophila interruptalis	15,351	NC_021756.1	[49]
		Pseudargyria interruptella	15.231	NC_029751.1	Direct submission
		Chilo auricilius	15,367	NC_024644.1	[50]
		Chilo sacchariphagus	15,378	NC_029716.1	Direct submission
		Evergestis junctalis	15,438	NC_030509.1	Direct submission
		Nomophila noctuella	15,309	NC_025764.1	[51]
		Tyspanodes striata	15,255	NC_030510.1	Direct submission
		Glyphodes quadrimaculalis	15,255	NC_022699.1	[52]
		Spoladea recurvalis	15,273	NC_027443.1	[53]
		Dichocrocis punctiferalis	15,355	NC_021389.1	[54]
		Glyphodes pyloalis	14,960	NC_025933.1	Unpublished
		Maruca vitrata	15,385	NC_024099.1	Unpublished
		Maruca testulalis	15,110	NC_024283.1	[55]
		Haritalodes derogat	15,253	NC_029202.1	Unpublished
		Pycnarmon lactiferalis	15,219	NC_033540.1	[56]
		Loxostege sticticalis	15,218	NC_027174.1	Unpublished
	Pyralidae	Orthaga olivacea Warre			This study
		Lista haraldusalis	15213	NC_024535	[57]
		Galleria mellonella	15320	KT750964	Unpublished
		Corcyra cephalonica	15,273	NC_016866.1	[58]
		Amyelois transitella	15,205	NC_028443.1	[59]
		Plodia interpunctella	15,264	NC_027961.1	Unpublished
		Ephestia kuehniella	15,295	NC_022476.1	Direct submission
		Meroptera pravella	15,260	NC_035242.1	[60]
		Hypsopygia regina	15,212	NC_030508.1	Direct submission
		Endotricha consocia	15,201	NC_037501.1	[61]
		Euzophera pyriella	15,184	NC_037175.1	[62]
Tortricoidea	Tortricidae	Grapholita molesta	15,717	NC_014806	[63]
		Spilonota lechriaspis	15,368	NC_014294	[64]
Papilionoidea	Papilionidae	Luehdorfia taibai	15,553	KC952673	[65]
		Teinopalpus aureus	15,242	NC_014398	Unpublished
		Apatura ilia	15,242	NC_016062	[66]
		Apatura metis	15,236	NC_015537	[67]
Yponomeutoidea	Plutellidae	Plutella xylostella	16,179	JF911819	[68]
	Lyonetiidae	Leucoptera malifoliella	15,646	NC_018547	[69]

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 [72] (As to Ts) is slightly negative (−0.042). This trend has also been reported from Manduca sexta (−0.005) [34], Ctenoplusia agnata (−0.023) [39], Paracymoriza distinctalis (−0.002) [46], and Lista haraldusalis (−0.007) [57]. 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) [33] and C.medinalis (-0.030 and -0.175) [17] (Table 4).

Fig 1

Map of the mitogenome of O. olivacea Warre.

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.

Table 3

Summary results for characteristics of the mitogenome of Orthaga olivacea Warre.

Gene	Location	Direction	Size	Intergenic Nucleotides	Start codon	Stop codon
tRNA-Met	1–67	F	67	1	—	—
tRNA-Ile	69–132	F	64	-3	—	—
tRNA-Gln	130–198	R	69	52	—	—
ND2	251–1264	F	1014	0	ATT	TAA
tRNA-Trp	1265–1332	F	68	-8	—	—
tRNA-Cys	1325–1394	R	70	4	—	—
tRNA-Tyr	1399–1464	R	66	3	—	—
COX1	1468–2973	F	1506	0	CGA	TAA
tRNA-Leu1	2974–3040	F	67	0	—
COX2	3041–3712	F	672	0	ATT	TAA
tRNA-Sup	3713–3781	F	69	4	—	—
tRNA-Asp	3786–3853	F	68	0	—	—
ATP8	3854–4015	F	162	-7	ATC	TAA
ATP6	4009–4689	F	681	-1	ATG	TAA
COX3	4689–5478	F	790	2	ATG	T
tRNA-Gly	5481–5548	F	68	0	—	—
ND3	5549–5902	F	354	12	ATT	TAA
tRNA-Ala	5915–5980	F	66	0	—	—
tRNA-Arg	5981–6044	F	64	2	—	—
tRNA-Asn	6047–6112	F	66	3	—	—
tRNA-Ser1	6116–6168	F	53	19	—	—
tRNA-Glu	6188–6253	F	66	-2	—	—
tRNA-Phe	6252–6318	R	67	0	—	—
ND5	6319–8052	R	1734	0	ATT	TAA
tRNA-His	8053–8118	R	66	0	—	—
ND4	8119–9455	R	1337	0	ATA	TA
ND4L	9456–9746	R	291	2	ATG	TAA
tRNA-Thr	9749–9812	F	64	0	—	—
tRNA-Pro	9813–9877	R	65	0	—	—
ND6	9878–10398	F	521	9	ATA	TAA
CYTB	10408–11566	F	1159	-2	ATG	T
tRNA-Ser2	11565–11631	F	67	20	—	—
ND1	11652–12577	R	926	1	ATG	TA
tRNA-Leu2	12579–12648	R	70	0	—	—
rRNA-16s	12649–14032	R	1384	0	—	—
tRNA-Val	14033–14096	R	64	0	—	—
rRNA-12s	14097–14881	R	785	0	—	—
A-T-rich region	14882–15174	F	293		—	—

Table 4

Composition and skewness in different Lepidopteran mitogenomes.

Species	Size (bp)	A%	G%	T%	C%	A+T %	ATskewness	GCskewness
Whole genome
O. olivacea Warre	15174	37.83	8.23	41.18	12.75	79.02	−0.042	−0.215
B. mori	15643	43.05	7.32	38.27	11.36	81.32	0.051	−0.216
R. menciana	15301	41.42	7.82	37.45	13.31	78.86	0.050	−0.259
M. sexta	15516	40.67	7.46	41.11	10.76	81.79	−0.005	−0.181
E. pseudoconspersa	15461	40.42	7.61	39.51	12.46	79.93	0.011	−0.241
C. agnata	15261	39.58	7.71	41.52	11.2	81.1	−0.023	−0.184
A. cinerarium	15722	41.51	7.80	39.32	11.37	80.83	0.027	−0.186
D. saccharalis	15490	40.87	7.42	39.15	12.56	80.02	0.021	−0.258
C. medinalis	15388	40.36	7.45	41.58	10.61	81.94	−0.030	−0.175
1P. distinctalis	15354	41.04	7.49	41.22	10.24	82.27	−0.002	−0.155
L. haraldusalis	15213	40.47	7.66	41.04	10.83	81.52	−0.007	−0.172
G. mellonella	15320	38.62	7.47	41.80	12.11	80.42	−0.039	−0.237
S. lechriaspis	15368	39.86	7.63	41.34	11.17	81.19	−0.018	−0.188
A. ilia	15,242	39.77	7.75	40.68	11.80	80.45	−0.011	−0.207
P. xylostella	16179	40.66	7.68	40.22	10.82	80.89	0.005	−0.170
PCG
O. olivacea Warre	11147	37.12	9.11	40.24	13.53	77.36	−0.040	−0.195
B. mori	11177	42.92	8.17	36.66	12.26	79.57	0.079	−0.200
R. menciana	11225	40.97	8.58	36.12	14.33	77.1	0.063	−0.251
M. sexta	11185	40.41	8.23	39.88	11.48	80.30	0.007	-0.165
E. pseudoconspersa	11187	3969	8.43	38.3	13.58	77.99	0.017	−0.233
C. agnata	11238	39.12	8.37	40.79	11.72	79.91	−0.020	−0.166
A. cinerarium	11227	40.63	8.78	38.19	12.39	78.83	0.031	−0.171
D. saccharalis	11206	40.34	8.27	37.55	13.83	77.90	0.036	−0.252
C. medinalis	11210	39.88	8.15	40.69	11.28	80.56	−0.010	−0.161
P. distinctalis	11189	40.54	8.12	40.53	10.81	81.07	0	−0.142
L. haraldusalis	11193	39.88	8.47	40.16	11.49	80.04	−0.003	−0.151
G. mellonella	11196	38.03	8.20	40.84	12.92	78.88	−0.036	−0.224
S. lechriaspis	11256	39.30	8.35	40.41	11.93	79.72	−0.014	−0.177
A. ilia	11,148	39.41	8.41	39.49	12.69	78.89	−0.001	−0.203
P. xylostella	11049	40.47	8.82	38.85	11.86	79.32	0.020	−0.147
tRNA
O. olivacea Warre	1452	39.461	8.26	40.70	11.57	80.17	−0.015	−0.167
B. mori	1468	42.10	7.90	39.31	10.69	81.40	0.034	−0.150
R. menciana	1485	41.08	8.08	39.93	10.91	81.01	0.014	−0.149
M. sexta	1554	40.99	7.92	41.06	10.04	82.05	−0.001	−0.118
E. pseudoconspersa	1466	41.41	8.19	40.18	10.23	81.58	0.015	−0.111
C. agnata	1477	41.23	8.19	40.22	10.36	81.45	0.012	−0.117
A. cinerarium	1483	42.01	8.02	39.45	10.52	81.46	0.031	−0.135
D. saccharalis	1478	41.81	7.713	40.32	10.15	82.14	0.018	−0.136
C. medinalis	1475	41.29	8.00	40.81	9.90	82.10	0.006	−0.106
P. distinctalis	1536	42.19	8.14	39.78	9.9	81.97	0.029	−0.098
L. haraldusalis	1451	41.08	7.86	41.42	9.65	82.49	−0.004	−0.102
G. mellonella	1489	40.09	8.06	40.90	10.95	80.51	−0.010	−0.152
S. lechriaspis	1450	40.97	8.00	40.90	10.14	81.86	0.001	−0.118
A. ilia	1433	40.61	8.30	40.96	10.12	81.58	−0.004	−0.099
P. xylostella	1468	42.51	8.17	38.83	10.49	81.34	0.045	−0.124
rRNA
O. olivacea Warre	2169	39.65	4.84	44.35	11.16	84.00	−0.056	−0.389
B. mori	2158	43.74	4.59	41.06	10.61	84.80	0.032	−0.396
R. menciana	2147	43.04	4.84	40.71	11.41	83.74	0.028	−0.404
M. sexta	2168	41.37	4.84	44.05	9.73	85.42	−0.031	−0.335
E. pseudoconspersa	2225	42.56	4.54	42.11	10.79	84.67	0.005	−0.408
C. agnata	2112	40.01	5.07	44.65	10.27	84.66	−0.055	−0.339
A.cinerarium	2179	43.97	4.77	41.17	10.10	85.13	0.033	−0.358
D. saccharalis	2193	41.45	6.84	43.59	10.17	85.04	−0.025	−0.360
C. medinalis	2170	41.47	5.02	43.87	9.63	85.35	−0.028	−0.314
P. distinctalis	2174	41.31	5.34	44.02	9.34	85.33	−0.032	−0.272
L. haraldusalis	2121	42.20	4.67	43.33	9.81	85.53	−0.013	−0.355
G. mellonella	2143	40.18	4.95	44.19	10.69	84.37	−0.048	−0.367
S. lechriaspis	2160	41.71	4.95	43.84	9.49	85.56	−0.025	−0.314
A. ilia	2109	40.11	4.98	44.86	10.05	84.97	−0.056	−0.337
P. xylostella	2162	41.44	4.90	43.94	9.71	85.38	−0.029	−0.329
A+T-rich region
O. olivacea Warre	293	44.03	2.73	49.83	3.41	93.86	−0.062	−0.111
B. mori	449	44.69	1.60	50.70	3.00	95.39	−0.063	−0.304
R. menciana	357	43.7	3.36	47.34	5.6	91.04	−0.040	−0.250
M. sexta	324	45.06	1.54	50.31	3.09	95.37	−0.005	−0.335
E. pseudoconspersa	388	43.56	2.32	50.26	3.87	93.81	−0.071	−0.250
C. agnata	334	46.71	1.5	46.71	5.09	93.41	0.000	−0.545
A. cinerarium	625	47.20	1.92	48.64	2.24	95.84	−0.015	−0.077
D. saccharalis	335	43.28	0.60	51.64	4.48	94.93	−0.088	−0.765
C. medinalis	339	42.48	0.88	53.39	3.24	95.87	−0.114	−0.571
P. distinctalis	349	46.13	1.15	49	3.72	95.13	−0.030	−0.528
L. haraldusalis	310	45.81	0.97	50.32	2.90	96.13	−0.047	−0.499
G. mellonella	350	44.29	0.29	52.86	2.57	97.14	−0.088	−0.8
S. lechriaspis	441	40.36	2.49	52.38	4.76	92.74	−0.130	−0.313
A. ilia	403	42.93	3.23	49.63	4.22	92.56	−0.072	−0.133
P. xylostella	1081	37.74	2.50	45.42	5.09	83.16	−0.092	−0.341

Protein-coding genes

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 [75]. 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 [76].

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 [77]. 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 [78].

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].

Fig 2

Codon usage patterns of O. olivacea Warre mitochondrial genome compared with other species of the Lepidoptera.

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).

Fig 3

Codon distribution of O. olivacea Warre compared with other species of the Lepidoptera.

CDspT = codons per thousand codons.

Fig 4

The Relative Synonymous Codon Usage (RSCU) of the eight superfamilies mitochondrial genome of Lepidoptera.

Codon family is displayed on the X axis. Codons which are not present in mitochondrial genomes are indicated above.

Transfer and ribosomal RNA genes

Orthaga olivacea Warre mitogenome has 22 tRNA genes, ranging in size from 53 bp (tRNA) to 70 bp (tRNA and tRNA). TRNAs show high A+T content (80.17%) and negative AT-skew (−0.015). All the tRNAs display typical cloverleaf secondary structures, except trnS which is missing a stable dihydrouridine (DHU) arm (Fig 5); this phenomenon is common across insects [17, 80, 81].

Fig 5

Putative secondary structures of the 22 tRNA genes of the Orthaga olivacea Warre mitogenome.

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 tRNA and tRNA with the biggest overlapping region (8 bp). The overlapping region located between atp8 and atp6 was 7 bp, 3 bp between tRNA and tRNA, 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].

Fig 6

Conserved sequence across the Lepidoptera order.

(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 tRNA and nad2. The 20 bp intergenic spacer region located between tRNA 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.

Fig 7

Phylogenetic relationships tree among Lepidopteran insects.

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.

Conclusion

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.

25 Oct 2019

PONE-D-19-24590

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralididae) and comparison with other Lepidopteran insects

PLOS ONE

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Reviewer #1: In this manuscript, the authors sequence the complete mitochondrial genome of the moth, Orthaga olivacea. They then describe the annotation of this moth and use phylogenetic methods to compare it with the mitochondrial genomes of 34 other Lepidoptera. The writing is somewhat uneven—I have a number of suggestions for improvement, especially for the Abstract and Introduction in the Minor comments section at the end of this reviewer report. The annotation of the mitochondrial genome is acceptable, but I have a number of concerns especially about the phylogenetic analysis that will need to be addressed before this manuscript will be suitable for publication.

Major issues:

1. Pyraloidea taxon sampling. Line 62. “Considering the limited information of the mitochondrial sequences in Pyralidae, we sequenced the complete mitogenome of Orthaga olivacea, and compared it with other insect species, especially with the members of Pyralidae species.” This is a reasonable justification for sequencing the mitogenome of O. olivacea, but it is very curious that only 3 mitogenomes were included in the phylogenetic analysis and many of the other pyralid mitochondrial genomes that are available from Genbank were not included in these analyses including Corcyra cephalonica, Amyelois transitella, Plodia interpunctella (3 mitogenomes), Ephestia kuehniella (3 mitogenomes), Meroptera pravella, and Hypsopygia regina. Similarly, the sister-family to the Pyralidae includes an even larger number of species with sequenced mitochondrial genomes that were not included in the presented analyses including Paracymoriza prodigalis, Elophila interruptalis, Pseudargyria interruptella, Chilo auriculius, Chilo sacchariphagus, Evergestis junctalis, Nomophila noctuella, , Tryspandoes striata, Glyphodes quadrimaculalis, Spoladea recurvalis, Dichocrocis punctiferalis, Glyphodes pyloalis, Maruca vitrata, Maruca testulalis, Haritalodes derogate, Pycnarmon lactiferalis, Loxostege stricticalis, Endotricha consoci, Euzophera pyriella, Dichocrocis punctiferalis, and Cnaphalocrocis medinalis (3 mitogenomes). If one of the goals of the authors is to demonstrate that Orthaga olivacea belongs within the Pyralidae and to determine its closest relatives with sequences mitochondrial genomes, then they need to repeat their phylogenetic analysis after supplementing their current data set with all of these additional species. (Also note that Lista haraldusalis is misspelled in Fig. 7 and in other locations in the manuscript. Also, Family Pyralidae (and probably also Family Crambidae) should be indicated in Fig. 7.)

2. The authors employ 2 non-Lepidoptera outgroup species: Drosophila yakuba, a fly (Order Diptera) and Locusta migratoria, a grasshopper (Order Orthoptera), but the authors do not include any representatives of the insect Order most closely related to the Lepidoptera, the caddisflies (Order Trichoptera). There are at least 17 complete mitochondrial genomes representing several caddisfly Families available through Genbank (Al-Baeity et al. 2019). To root the Lepidopteran tree properly caddisflies sequences MUST also be included in the phylogenetic analyses.

Minor issues:

Line 2. Title: In modern usage, the lepidopteran family is usually called Pyralidae, not “Pyralididae”. Change here and throughout manuscript.

Line 18. Abstract: Suggested reword with greater specificity “Orthaga olivacea Ware (Lepidoptera Pyralidae) is an important agricultural pest of camphor trees (Cinnamomum camphora).”

Line 19. Suggested reword “To further supplement the known genome-level…”

Line 20. Suggested reword “…other species of Lepidoptera.”

Lines 31-31. Suggested reword “Phylogenetic analysis suggested that among sequenced lepidopteran mitochondrial genomes, Orthaga olivacea Warre was most closely related…”

Line 38. Suggested reword “…(mtDNA) is a circular molecule range in size from 14 to 19 kb…”

Line 42. Suggested reword “…A+T-rich region, the largest noncoding…”

Line 43-47. Suggested reword to remove repetition “Whole mitochondrial genomes are a useful data source for several research areas, such as evolutionary genomics (9, 10), comparative molecular evolution (11, 12), phylogeography (13), and population genetics (14).”

Line 50-51. Suggested reword to remove extraneous information “…over 25,000 species and some pyralids are important agricultural pests…”

Line 53-53. Suggested reword “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.”

Line 58. “remove” should be “removal”

Line 59-60. “However, overlapping generations and irregularity of abundance in the field from May to October make it very difficult to control.”

Line 72. Suggested reword “…the camphor trees on the campus of…”

Line 94. “…insert DNA were sequenced at least three times…” Query: was sequencing of the inserts done in both directions? If yes, please specify in the text.

Lines 99-100. Suggested reword “…under the accession number MN078362.”

Line 125-126. “…mitogenome sizes documented for other sequenced lepidopterans which range from 14,534 bp in Ostrinia nublilalis (incomplete)…” Since the sequencing of the mitochondrial genome of O. nublilalis is incomplete, it is inappropriate and incorrect to use this sequence to estimate the minimum mitochondrial genome size in the Lepidoptera. This data point should be replaced with the smallest completely sequenced mitochondrial genome from the Lepidoptera.

Lines 136-137. Suggested reword “In addition, the GC skew…”

Line 146. Table 4. I’m not sure that this table is necessary and perhaps should be removed.

Line 166. Suggested reword “…observed in most other lepidopteran mitogenomes and are…”

Line 177-179. Suggested reword “…for instance, L. haraldusalis, G. mellonella, B. mori, B. thibetaria, and L. malifoliella species all lack GCT codons, while G. mollesta lacks CGT codons.”

Line 246-248. “The species Orthaga olivacea…” This sentence should be revised based on the updated phylogenetic analysis after adding the taxa I suggested in the major revisions section above.

Lines 252-253. Suggested reword “…constituent with prior studies of lepidopteran phylogeny.”

References:

Al-Baeity, H., Allard, L.S., Arreza, L., et al. (2019) The complete mitochondrial genome of the North American pale summer sedge caddisfly Limnephilus hyalinus (Insecta: Trichoptera: Limnephilidae). Mitochondrial DNA Part B 4: 413-415.

Reviewer #2: The manuscript mainly determined the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralididae) and compare the mtDNA with other Lepidopteran insects. The English is acceptable. The literature cited is appropriate and draws on numerous comparative examples of similar research. Overall structure is of good quality and the raw data complete. The paper touches on the pertinent theoretical ideas proposed by earlier researchers. Overall, this manuscript is interesting, the description of the methods is complete and sound, and worthy to be published in “PLoS ONE” after minor modified.

1. the tables would be “three line”.

2. the literature 34 (line 353) was not complete.

3. “Warre” (ects.) in the figures would not be italic.

4. correct others, for examples, line 194 “TRNAs” (tRNAs ?); line 201 “The rNAs” (The rRNAs ?), ect.

Reviewer #3: In the manuscript, the mitogenome of Orthaga olivacea was determined and comparison with other lepidopteran sequences were also analyzed. The results of the study are valuable for the readers interested in the comparative mitogenome and phylogeny of Pyralididae. These results are informative and useful. I suggest this article can be published in this journal. However, the manuscript needs to be improved before acceptance for publication.

1. Introduction: the authors should provide clearly the study aim and scientific questions. It includes a description of the importance of the research and the study and reviews most of the previous literature. However, the authors have omitted a few studies of relevance and these should be included,

2. It is not clear from the manuscript that the collected Orthaga olivacea samples were verified astruly belong to the said species. It is suggested for the author to delimit the detailed morphological characters of the species to confirm.

3. Based on the dataset of 13 concatenated protein sequences, the authors reconstructed the phylogeny of Lepidoptera using MEGA with the Maximum Likelihood method. It is more persuasive and popular to carry out such analysis with RAxML method.

4. There are some errors in grammar and syntax throughout the text of the manuscript, the English writing should be further improved.

**********

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Reviewer #1: No

Reviewer #2: Yes: Ping You

Reviewer #3: No

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7 Dec 2019

Responds to the reviewer’s comments:

Reviewer #1:

Major issues:

1. Response to comment: (Pyraloidea taxon sampling. Line 62. “Considering the limited information of the mitochondrial sequences in Pyralidae, we sequenced the complete mitogenome of Orthaga olivacea, and compared it with other insect species, especially with the members of Pyralidae species.” This is a reasonable justification for sequencing the mitogenome of O. olivacea, but it is very curious that only 3 mitogenomes were included in the phylogenetic analysis and many of the other pyralid mitochondrial genomes that are available from Genbank were not included in these analyses including Corcyra cephalonica, Amyelois transitella, Plodia interpunctella (3 mitogenomes), Ephestia kuehniella (3 mitogenomes), Meroptera pravella, and Hypsopygia regina. Similarly, the sister-family to the Pyralidae includes an even larger number of species with sequenced mitochondrial genomes that were not included in the presented analyses including Paracymoriza prodigalis, Elophila interruptalis, Pseudargyria interruptella, Chilo auriculius, Chilo sacchariphagus, Evergestis junctalis, Nomophila noctuella, , Tryspandoes striata, Glyphodes quadrimaculalis, Spoladea recurvalis, Dichocrocis punctiferalis, Glyphodes pyloalis, Maruca vitrata, Maruca testulalis, Haritalodes derogate, Pycnarmon lactiferalis, Loxostege stricticalis, Endotricha consoci, Euzophera pyriella, Dichocrocis punctiferalis, and Cnaphalocrocis medinalis (3 mitogenomes). If one of the goals of the authors is to demonstrate that Orthaga olivacea belongs within the Pyralidae and to determine its closest relatives with sequences mitochondrial genomes, then they need to repeat their phylogenetic analysis after supplementing their current data set with all of these additional species. (Also note that Lista haraldusalis is misspelled in Fig. 7 and in other locations in the manuscript. Also, Family Pyralidae (and probably also Family Crambidae) should be indicated in Fig. 7.))

Response: We are very sorry for our omission that it is inadequate to the goals to demonstrate that Orthaga olivacea belongs within the Pyralidae and to determine its closest relatives with sequences mitochondrial genomes with only 3 pyralid mitochondrial genomes were included in the phylogenetic analysis. According reviewer’s suggestion, we have repeated our phylogenetic analysis after supplementing our current data set with all of these additional species. And we have corrected the misspelled of Lista haraldusalis in Fig. 7 and in other locations in the manuscript. Also, Family Pyralidae (and probably also Family Crambidae) was indicated in Fig. 7.

2. Response to comment: (The authors employ 2 non-Lepidoptera outgroup species: Drosophila yakuba, a fly (Order Diptera) and Locusta migratoria, a grasshopper (Order Orthoptera), but the authors do not include any representatives of the insect Order most closely related to the Lepidoptera, the caddisflies (Order Trichoptera). There are at least 17 complete mitochondrial genomes representing several caddisfly Families available through Genbank (Al-Baeity et al. 2019). To root the Lepidopteran tree properly caddisflies sequences MUST also be included in the phylogenetic analyses.)

Response: Considering the Reviewer’s suggestion, we have included the caddisflies sequences of Limnephilus hyalinus in the phylogenetic analyses as outgroup.

Minor issues:

1. Response to comment: (Line 2. Title: In modern usage, the lepidopteran family is usually called Pyralidae, not “Pyralididae”. Change here and throughout manuscript.)

Response: We are very sorry for our Negligence of the use of “Pyralididae”, and we have corrected it to “Pyralidae” throughout manuscript.

2. Response to comment: (Line 18. Abstract: Suggested reword with greater specificity “Orthaga olivacea Ware (Lepidoptera Pyralidae) is an important agricultural pest of camphor trees (Cinnamomum camphora).”)

Response: According reviewer’s suggestion, we have reworded with greater specificity.

3. Response to comment: (Line 19. Suggested reword “To further supplement the known genome-level…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

4. Response to comment: (Line 20. Suggested reword “…other species of Lepidoptera.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

5. Response to comment: (Lines 31-31. Suggested reword “Phylogenetic analysis suggested that among sequenced lepidopteran mitochondrial genomes, Orthaga olivacea Warre was most closely related…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

6. Response to comment: (Line 38. Suggested reword “…(mtDNA) is a circular molecule range in size from 14 to 19 kb…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

7. Response to comment: (Line 42. Suggested reword “…A+T-rich region, the largest noncoding…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

8. Response to comment: (Line 43-47. Suggested reword to remove repetition “Whole mitochondrial genomes are a useful data source for several research areas, such as evolutionary genomics (9, 10), comparative molecular evolution (11, 12), phylogeography (13), and population genetics (14).)

Response: According reviewer’s suggestion, we have removed repetition in the target location.

9. Response to comment: (Line 50-51. Suggested reword to remove extraneous information “…over 25,000 species and some pyralids are important agricultural pests…”)

Response: According reviewer’s suggestion, we have removed extraneous information in the target location.

10. Response to comment: (Line 53-53. Suggested reword “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.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

11. Response to comment: (Line 58. “remove” should be “removal”)

Response: According reviewer’s suggestion, we have corrected “remove” to “removal”.

12. Response to comment: (Line 59-60. “However, overlapping generations and irregularity of abundance in the field from May to October make it very difficult to control.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

13. Response to comment: (Line 72. Suggested reword “…the camphor trees on the campus of…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

14. Response to comment: (Line 94. “…insert DNA were sequenced at least three times…” Query: was sequencing of the inserts done in both directions? If yes, please specify in the text.)

Response: Yes, the sequencing of the inserts was done in both directins. According reviewer’s suggestion, we have specified in the text.

15. Response to comment: (Lines 99-100. Suggested reword “…under the accession number MN078362.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

16. Response to comment: (Line 125-126. “…mitogenome sizes documented for other sequenced lepidopterans which range from 14,534 bp in Ostrinia nublilalis (incomplete)…” Since the sequencing of the mitochondrial genome of O. nublilalis is incomplete, it is inappropriate and incorrect to use this sequence to estimate the minimum mitochondrial genome size in the Lepidoptera. This data point should be replaced with the smallest completely sequenced mitochondrial genome from the Lepidoptera.)

Response: Thank you for pointing out the error. We have re-searched NCBI, and found that the sequencing of the mitochondrial genome of Ostrinia nublilalis is complete with 14,535 bp. And maybe Ostrinia nublilalis is the smallest completely sequenced mitochondrial genome from the Lepidoptera. we have corrected it in the target location.

17. Response to comment: (Lines 136-137. Suggested reword “In addition, the GC skew…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

18. Response to comment: (Line 146. Table 4. I’m not sure that this table is necessary and perhaps should be removed.)

Response: Thank you for your suggestion, but we think by base preference and compared it with other species in table 4, can better understand this mitochondrial genome. Therefore, we chose to keep table 4 in the manuscript.

19. Response to comment: (Line 166. Suggested reword “…observed in most other lepidopteran mitogenomes and are…”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

20. Response to comment: (Line 177-179. Suggested reword “…for instance, L. haraldusalis, G. mellonella, B. mori, B. thibetaria, and L. malifoliella species all lack GCT codons, while G. mollesta lacks CGT codons.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

21. Response to comment: (Line 246-248. “The species Orthaga olivacea…” This sentence should be revised based on the updated phylogenetic analysis after adding the taxa I suggested in the major revisions section above.)

Response: According reviewer’s suggestion, we have revised this sentence based on the updated phylogenetic analysis.

22. Response to comment: (Lines 252-253. Suggested reword “…constituent with prior studies of lepidopteran phylogeny.”)

Response: According reviewer’s suggestion, we have reworded it in the target location.

Reviewer #2:

1. Response to comment: (the tables would be “three line”.)

Response: According reviewer’s suggestion, we have changed all tables into “three line” forms.

2. Response to comment: (the literature 34 (line 353) was not complete.)

Response: According reviewer’s suggestion, we have reworded it in the target location.

3. Response to comment: (“Warre” (ects.) in the figures would not be italic.)

Response: We are very sorry for our error application of the italic of “Warre” (ects.) in the figures, we have corrected it in all figures.

4. Response to comment: (“correct others, for examples, line 194 “TRNAs” (tRNAs ?); line 201 “The rNAs” (The rRNAs ?), ect.)

Response: We are very sorry for our incorrect in spelling, and we have already corrected them in the text and marked in color.

Reviewer #3:

1. Response to comment: (Introduction: the authors should provide clearly the study aim and scientific questions. It includes a description of the importance of the research and the study and reviews most of the previous literature. However, the authors have omitted a few studies of relevance and these should be included,)

Response: According reviewer’s suggestion, we have reworded the Introduction section to provide a clearer outline of the study aims and research question and marked in color.

2. Response to comment: (It is not clear from the manuscript that the collected Orthaga olivacea samples were verified as truly belongs to the said species. It is suggested for the author to delimit the detailed morphological characters of the species to confirm.)

Response: According reviewer’s suggestion, we have added detailed description of the morphological characteristics of the species' larvae in the materials and methods section to better delimit the species.

3. Response to comment: (Based on the dataset of 13 concatenated protein sequences, the authors reconstructed the phylogeny of Lepidoptera using MEGA with the Maximum Likelihood method. It is more persuasive and popular to carry out such analysis with RAxML method.)

Response: We are very sorry that we didn’t reconstruct the phylogeny of Lepidoptera using the RAxML method as you suggested. Because we think the RAxML method is an alternative solution in phylogeny, we found that using MEGA method to analyze the phylogeny of Lepidoptera is also popular. For example, in the study of Cerura menciana (Dai et. al., 2015), Biston marginata (Zheng et al., 2018) and Ctenoptilum vasava (Hao et. al., 2012) in Lepidoptera, they used MEGA to reconstruct the evolutionary relationship of Lepidoptera with the Maximum Likelihood method, and also got a better evolutionary relationship tree. In this study, based on the analysis of the original evolutionary relationship, we added another 25 species of Pyralidae and finally got a better evolutionary relationship of Lepidoptera. So we think that the MEGA method can also be used to construct the evolutionary tree based on Lepidoptera mitochondria.

4. Response to comment: (There are some errors in grammar and syntax throughout the text of the manuscript, the English writing should be further improved.)

Response: We are very sorry for our incorrect in grammar and syntax, and we have already corrected them in the text and marked in color.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised manuscript. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

Yours Sincerely

Guoqing Wei

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Dec 2019

PONE-D-19-24590R1

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects

PLOS ONE

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Greetings. After receiving the first round of reviewer comments responding to your initial submission, PLOS ONE has asked me to change my role from Reviewer (I was Reviewer #1) to Guest Academic Editor to guide you through the remainder of the peer review process.

I have read your revision and overall, I am very pleased with how you have responded to the reviewer comments. However, there are a few remaining items that you will need to address before your manuscript can be considered acceptable for publication in PLOS ONE. They are listed below. Please make theses additional necessary changes and resubmit your work for final consideration by the journal.

1. Line 62. Delete entire sentence beginning with "What'more considering the limited..." It is unnecessary.

2. Table 2 includes a column of references. These table citations are not in the same format as the in-text citations in the rest of the manuscript and some of these references do not appear in the reference section at the end of the manuscript. Please correct the formatting, and ensure that all of the references listed in Table 2 also appear in the reference section.

3. The reference for the Meroptera pravella mitochondrial genome in Table 2 is listed as "Consortium et al. (2017)". This is properly referenced as "Living Prairie Consortium (2017)".

4. Fig. 7. The vertical line associated with the label "Pyraloidea" should extend from Glyphodes quadrimaculalis to Ephestia kuehniella in this figure.

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27 Dec 2019

Dear Editor:

Thank you very much for your letter and the comments concerning our manuscript entitled “Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects” (ID: PONE-D-19-24590). Those comments are all valuable and very helpful for revising our paper to meet the acceptable criterion for publication in PLOS ONE. We have studied comments carefully and have made corrections which we hope to meet with approval. Revised portions are marked in color in the manuscript. The main corrections in the paper and the responds to the comments are as flowing:

Responds to the reviewer’s comments:

1. Response to comment: (Line 62. Delete entire sentence beginning with "What'more considering the limited..." It is unnecessary.)

Response: According to the reviewer’s suggestion, we have deleted it in the target location.

2. Response to comment: (Table 2 includes a column of references. These table citations are not in the same format as the in-text citations in the rest of the manuscript and some of these references do not appear in the reference section at the end of the manuscript. Please correct the formatting, and ensure that all of the references listed in Table 2 also appear in the reference section.)

Response: We are very sorry for our negligence of the citations format and omission of some references in Table 2, and we have corrected the formatting in Table 2 and increased the omissive references in the reference section.

3. Response to comment: (The reference for the Meroptera pravella mitochondrial genome in Table 2 is listed as "Consortium et al. (2017)". This is properly referenced as "Living Prairie Consortium (2017)".)

Response: Considering the second suggestion, we have modified the reference formats in table 2.

4. Response to comment: (Fig. 7. The vertical line associated with the label "Pyraloidea" should extend from Glyphodes quadrimaculalis to Ephestia kuehniella in this figure.)

Response: According the suggestion, we modified the vertical line associated with the label "Pyraloidea" and make sure it is extended from Glyphodes quadrimaculalis to Ephestia kuehniella in fig. 7.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper. And here we did not list the changes but marked in revised manuscript. We appreciate for Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Once again, thank you very much for your comments and suggestions.

Yours Sincerely

Guoqing Wei

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

31 Dec 2019

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects

PONE-D-19-24590R2

Dear Dr. Wei,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for responding to my recommendations for revision. I am now prepared to recommend acceptance of this manuscript at PLOS ONE.

Reviewers' comments:

24 Feb 2020

PONE-D-19-24590R2

Characterization of the complete mitochondrial genome of Orthaga olivacea Warre (Lepidoptera Pyralidae) and comparison with other Lepidopteran insects

Dear Dr. Wei:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE