Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects

Abstract

The complete mitochondrial genome (mitogenome) of Leucoma salicis (Lepidoptera: Lymantriidae) was sequenced and annotated. It is a circular molecule of 15,334 bp, containing the 37 genes usually present in insect mitogenomes. All protein-coding genes (PCGs) are initiated by ATN codons, other than cox1, which is initiated by CGA. Three of the 13 PCGs had an incomplete termination codon, T or TA, while the others terminated with TAA. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) was consistent with those of published lepidopteran sequences. All tRNA genes had typical clover-leaf secondary structures, except for the tRNA^Ser (AGN), in which the dihydrouridine (DHU) arm could not form a stable stem-loop structure. The A + T-rich region of 325 bp had several distinctive features, including the motif ‘ATAGA’ followed by an 18 bp poly-T stretch, a microsatellite-like (AT)₇ element, and an 11-bp poly-A present immediately upstream of tRNA^Met. Relationships among 32 insect species were determined using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) phylogenetic methods. These analyses confirm that L. salicis belongs to the Lymantriidae; and that Lymantriidae is a member of Noctuoidea, and is a sister taxon to Erebidae, Nolidae and Noctuidae, most closely related to Erebidae.

Leucoma salicis is a moth that is mainly distributed in China, Korea and Japan. It is a notorious plant pest and causes considerable economic losses. It typically consumes willow and tea leaves, influencing quality and quantity of tea products¹; and damages roadside and garden trees in urban areas. Traditionally, the identification of this species was based on morphological characteristics of adult moths². However, the moth appears mainly in June to August, the rest of its life go through egg and larva stages (which has no easily identifying morphological features), requiring eggs and larvae to be reared to adult stage for identification, which is time consuming and labor intensive. Molecular methods for identification are under development, including polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP)³. Most previous work on L. salicis has focused on sex pheromone synthesis⁴, or the nuclear polyhedrosis virus that infects larvae⁵. Previous studies have not focused on the mitochondrial genome, which can provide systematically-informative information for identification, phylogenetic analysis and evolutionary studies on L. salicis.

Insect mitochondrial DNA (mtDNA) is a double-stranded, circular molecule, ranging in size from 14 to 20 kb. It usually contains a conserved set of 37 genes, including seven NADH dehydrogenase (nad1-nad6 and nad4L), three cytochrome c oxidase (cox1-cox3), two ATPase (atp6 and atp8), one cytochrome b (cob), two ribosomal RNA (rrnL and rrnS), 22 transfer RNA (tRNA) genes, and an adenine (A) + thymine (T)-rich region that contains initiation sites for transcription and replication of the genome⁶,⁷. Due to its simple genomic organization, high rate of evolution, and almost unambiguous orthology, mtDNA is typically considered to be an informative molecular marker for species identification and in studies of phylogenetic relationships and population structure⁸,⁹.

A better understanding of the lepidopteran mitochondrial genome requires expanded taxon sampling. Lepidoptera contains more than 160,000 described species, classified into 45–48 superfamilies¹⁰. Lymantriidae includes about 360 genera and over 2500 species, many of which are agriculturally important. Only eight species have completely-sequenced mitogenomes that are publically available in GenBank, despite the large species diversity in the family. In this study, we sequenced and annotated the complete mitogenome sequence of L. salicis, and compared it with those of other members of Lymantriidae. Our results provide novel methods for species identification of an important pest, as well as phylogenetically-informative sequence data that addresses the position of L. salicis within Noctuoidea.

Results

Geno me organization and composition

The mitogenome of L. salicis was a circular DNA molecule, 15,334 bp in length (Fig. 1). It contained the typical insect mitogenome set of 22 tRNAs, 13 PCGs (nad1-6, nad4L, cox1-3, cob, atp6 and atp8), two rRNAs (rrnS and rrnL), and the non-coding A + T-rich region (Table 1). Nucleotide composition was highly A + T biased (A: 42.07%, T: 38.57%, G: 7.22%, C: 12.14%; Table 2). Nucleotide BLAST (blastn) of the entire L. salicis mitogenome against GenBank returned sequence identities with closely related species of 79% (Lachana alpherakii), 78% (Euproctis pseudoconspersa), 78% (Gynaephora menyuanensis), and 77% (Lymantria dispar) (Table S1).

Figure 1. Map of the mitogenome of L. salicis.

tRNA genes are labeled according to the IUPAC-IUB three-letter amino acids; cox1, cox2 and cox3 refer to the cytochrome c oxidase subunits; cob refers to cytochrome b; nad1-nad6 refer to NADH dehydrogenase components; rrnL and rrnS refer to ribosomal RNAs.

Table 1. Summary of characteristics of the mitogenome of L. salicis.

Gene	Direction	Location	Size	Anti codon	Start codon	Stop codon	Intergenic Nucleotides
tRNAMet	F	1–66	66	CAT	—	—	0
tRNAIle	F	67–134	68	GAT	—	—	−3
tRNAGln	R	132–200	69	TTG	—	—	47
ND2	F	248–1233	986	—	ATT	TAA	3
tRNATrp	F	1237–1309	73	TCA	—	—	−8
tRNACys	R	1302–1370	69	GCA	—	—	0
tRNATyr	R	1371–1442	72	GTA	—	—	1
COI	F	1453–2987	1535	—	CGA	T	2
tRNALeu(UUR)	F	2993–3059	67	TAA	—	—	0
COII	F	3060–3740	681	—	ATT	T	0
tRNALys	F	3741–3811	71	CTT	—	—	0
tRNAAsp	F	3812–3878	67	GTC	—	—	0
ATP8	F	3879–4040	162	—	ATA	TAA	−7
ATP6	F	4034–4711	678	—	ATG	TAA	14
COIII	F	4726–5514	789	—	ATG	TAA	2
tRNAGly	F	5517–5582	66	TCC	—	—	0
ND3	F	5583–59336	354	—	ATT	TAA	10
tRNAAla	F	5947–6017	71	TGC	—	—	−1
tRNAArg	F	6017–6084	68	TCG	—	—	1
tRNAAsn	F	6086–6152	67	GTT	—	—	0
tRNASer(AGN)	F	6153–6219	67	GCT	—	—	17
tRNAGlu	F	6237–6301	65	TTC	—	—	0
tRNAPhe	R	6302–6368	67	GAA	—	—	20
ND5	R	6389–8103	1715	—	—	—	8
tRNAHis	R	8112–8175	64	GTG	—	—	1
ND4	R	8177–9516	1341	—	ATG	TA	15
ND4L	R	9532–9825	294	—	—	—	5
tRNAThr	F	9831–9897	67	TGT	—	—	0
tRNAPro	R	9898–9965	68	TGG	—	—	2
ND6	F	9968–10514	547	—	ATA	TAA	8
Cytb	F	10523–11655	1133	—	ATG	TAA	1
tRNASer(UCN)	F	11657–11721	65	TGA	—	—	10
ND1	R	11732–12684	953	—	ATT	TAA	0
tRNALeu(CUN)	R	12685–12755	71	TAG	—	—	0
lrRNA	R	12756–14099	1344	—	—	—	0
tRNAVal	R	14100–14169	70	TAC	—	—	0
srRNA	R	14170–15009	840	—	—	—	0
A + T-rich region	—	15010–15334	325	—	—	—	—

Table 2. Composition and skew in different lepidopteran mitogenomes.

Species	Size (bp)	A%	G%	T%	C%	A + T %	AT skewness	GC skewness
Whole genome
L. salicis	15334	42.07	7.22	38.57	12.14	80.64	0.043	−0.254
C. agnata	15261	39.58	7.71	41.52	11.2	81.1	−0.023	−0.184
H. cunea	15481	40.58	7.55	39.81	12.06	80.39	0.009	−0.229
G. menyuanensis	15770	40.88	6.77	40.6	11.75	81.48	0.003	−0.268
L. dispar	15507	40.38	7.61	39.26	12.5	79.64	0.014	−0.243
E. pseudoconspersa	15461	40.42	7.61	39.51	12.46	79.93	0.011	−0.241
G. argentata	15337	39.64	7.56	42.05	10.75	81.69	−0.029	−0.174
A. formosae	15463	38.67	7.53	40.83	12.98	79.49	−0.027	−0.265
P. 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	−0.006	−0.171
B. thibetaria	15484	42.38	7.55	37.24	12.83	79.62	0.064	−0.259
R. menciana	15301	41.42	7.82	37.45	13.31	78.86	0.050	−0.259
B. mori	15666	43.09	7.31	38.26	11.34	81.35	0.059	−0.216
S. morio	15299	40.64	7.58	40.53	11.26	81.17	0.0013	−0.195
S. lechriaspis	15368	39.86	7.63	41.34	11.17	81.19	−0.018	−0.188
C. benjaminii	15272	40.08	7.52	40.7	11.7	80.78	−0.007	−0.217
PCG
L. salicis	11171	42.24	7.89	37.16	12.71	79.39	0.063	−0.233
C. agnata	11238	39.12	8.37	40.79	11.72	79.91	−0.020	−0.166
H. cunea	11205	39.99	8.35	38.6	13.06	78.59	0.017	−0.219
G. menyuanensis	11228	40.37	7.5	39.41	12.72	79.78	0.012	−0.258
L. dispar	11236	39.52	8.44	38.18	13.62	77.71	0.017	−0.234
E. pseudoconspersa	11187	3969	8.43	38.3	13.58	77.99	0.017	−0.233
G. argentata	11203	39.05	8.29	41.27	11.38	80.33	−0.027	−0.157
A. formosae	11,217	38.18	8.28	39.62	13.92	77.8	−0.018	−0.254
P. distinctalis	11189	40.54	8.12	40.53	10.81	81.07	0	−0.142
L. haraldusalis	11,193	39.88	8.47	40.16	11.49	80.04	−0.003	−0.151
B. thibetaria	11212	41.66	8.36	35.94	14.04	77.6	0.073	−0.253
R. menciana	11225	40.97	8.58	36.12	14.33	77.1	0.063	−0.251
B. mori	11177	42.93	8.16	36.64	12.28	79.57	0.079	−0.201
S. morio	11179	40.28	8.27	39.56	11.89	79.84	0.009	−0.179
S. lechriaspis	11258	39.31	8.35	40.41	11.93	79.72	−0.013	−0.176
C. benjaminii	11153	39.44	8.23	39.74	12.59	79.18	−0.003	−0.209
tRNA
L. salicis	1498	43.19	6.88	40.72	9.21	83.91	0.029	−0.145
C. agnata	1477	41.23	8.19	40.22	10.36	81.45	0.012	−0.117
H. cunea	1474	41.86	7.87	39.89	10.38	81.75	0.024	−0.138
G. menyuanensis	1504	41.29	7.38	41.76	9.57	83.05	−0.006	−0.129
L. dispar	1466	41.41	7.98	39.5	10.91	80.9	0.024	−0.155
E. pseudoconspersa	1466	41.41	8.19	40.18	10.23	81.58	0.015	−0.111
G. argentata	1469	41.32	8.24	40.23	10.21	81.55	0.013	−0.107
A. formosae	1457	40.43	7.96	40.36	11.26	80.78	0.001	−0.172
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
B. thibetaria	1478	42.08	7.85	39.24	10.83	81.33	0.035	−0.160
R. menciana	1485	41.08	8.08	39.93	10.91	81.01	0.014	−0.149
B. mori	1470	42.04	7.89	39.52	10.54	81.56	0.031	−0.144
S. morio	1463	40.6	8.2	41.01	10.18	81.61	−0.005	−0.108
S. lechriaspis	1516	41.03	7.92	41.09	9.96	82.12	−0.001	−0.114
C. benjaminii	1467	40.9	8.04	40.49	10.57	81.39	0.005	−0.136
rRNA
L. salicis	2184	41.39	5.04	40.8	12.77	82.19	0.007	−0.434
C. agnata	2112	40.01	5.07	44.65	10.27	84.66	−0.055	−0.339
H. cunea	2234	42.08	4.92	42.75	10.25	84.83	−0.008	−0.351
G. menyuanensis	2311	41.89	4.28	42.84	10.99	84.73	−0.011	−0.439
L. dispar	2140	42.52	4.81	41.82	10.42	84.35	0.008	−0.368
E. pseudoconspersa	2225	42.56	4.54	42.11	10.79	84.67	0.005	−0.408
G. argentata	2165	40.6	4.76	45.13	9.52	85.73	−0.053	−0.333
A. formosae	2163	38.93	4.72	44.85	11.51	83.77	−0.071	−0.418
P. distinctalis	2174	41.31	5.34	44.02	9.34	85.33	−0.032	−0.272
L. haraldusalis	2121	442.2	4.67	43.33	9.81	85.53	4.664	−0.355
B. thibetaria	2241	45.52	4.77	39.58	10.13	85.1	0.070	−0.360
R. menciana	2147	43.04	4.84	40.71	11.41	83.74	0.028	−0.404
B. mori	2161	43.73	4.58	41.09	10.6	84.82	0.031	−0.397
S. morio	2152	41.73	4.83	43.08	10.36	84.8	−0.016	−0.364
S. lechriaspis	2160	41.71	4.95	43.84	9.49	85.56	−0.025	−0.314
C. benjaminii	2132	41.7	4.88	43.76	9.66	85.46	−0.024	−0.329
AT RICH
L. salicis	325	34.46	2.46	57.23	5.85	91.69	−0.248	−0.408
C. agnata	334	46.71	1.5	46.71	5.09	93.41	0.000	−0.545
H. cunea	357	45.66	1.12	49.3	3.92	94.96	−0.038	−0.556
G. menyuanensis	449	43.65	2.45	49.67	4.23	93.32	−0.065	−0.266
L. dispar	371	44.74	2.43	49.6	3.23	94.34	−0.052	−0.141
E. pseudoconspersa	388	43.56	2.32	50.26	3.87	93.81	−0.071	−0.250
G. argentata	340	43.24	1.47	52.06	3.24	95.29	−0.093	−0.376
A. formosae	482	42.95	2.9	49.79	4.36	92.74	−0.074	−0.201
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	2.9	96.13	−0.047	−0.499
B. thibetaria	350	44.29	2.57	48.29	4.86	92.57	−0.043	−0.308
R. menciana	357	43.7	3.36	47.34	5.6	91.04	−0.040	−0.250
B. mori	494	44.74	1.82	50.61	2.83	95.34	−0.062	−0.217
S. morio	316	44.3	2.53	48.42	4.75	92.72	−0.044	−0.305
S. lechriaspis	441	40.36	2.49	52.38	4.76	92.74	−0.130	−0.313
C. benjaminii	293	46.42	3.07	45.73	4.78	92.15	0.007	−0.218

Protein-coding genes and codon usage

The PCG region formed 72.9% of the L. salicis mitogenome, and was 11,172 bp long. Nine of 13 PCGs (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6 and cob) were encoded on the H-strand, while the remaining four (nad5, nad4, nad4L and nad1) were encoded on the L-strand. Each PCG was initiated by a canonical ATN codon, except for cox1 (Table 1), which was initiated by a CGA codon. Ten of 13 PCGs used a typical TAA termination codon; but cox1 and cox2 terminated with a single T and nad4 terminated with TA (Table 1).

Relative synonymous codon usage (RSCU) analysis of PCGs in L. salicis revealed that the codons encoding Asn, Ile, Leu (UUA, UUG), Lys, Tyr and Phe were the most frequently present, while those encoding Cys and Arg were rare (Fig. 2). In the PCGs of the eight moth species examined, codon distributions and amino acid content were largely consistent among species (Fig. 3). Codons with A or T in the third position were overused in comparison to other synonymous codons: for example, the codons for valine GTC and GTG were rare, while the synonymous codons GTT and GTA were prevalent (Fig. 4). All used codons were present in the PCGs of the L. salicis mitogenome, except for CGC and GGC. This is similar to codon usage in Hyphantria cunea, Spilonota lechriaspis, and Gabala argentata, which respectively lack CGG and CGC, GCG and CGG, and CGG and CGC.

Figure 2. Comparison of codon usage within the mitochondrial genome of members of the Lepidoptera.

Lowercase letters (a,b,c,d and e) above species names represent the superfamily to which the species belongs (a: Noctuoidea, b: Geometroidea, c: Bombycoidea, d: Pyraloidea, e: Tortricoidea).

Figure 3. Codon distribution in members of the Lepidoptera.

CDspT = codons per thousand codons.

Figure 4. Relative Synonymous Codon Usage (RSCU) of the mitochondrial genome of five superfamilies in the Lepidoptera.

Codon families are plotted on the x-axis. Codons indicated above the bar are not present in the mitogenome.

Ribosomal RNA and transfer RNA genes

The large (rrnL) and small (rrnS) ribosomal RNA subunit genes of L. salicis were located between the tRNA^Leu1(CUN)/tRNA^Val and the tRNA^Val/A + Trich regions, respectively (Fig. 1, Table 1). The rrnL gene was 1,344 bp long, while rrnS was 840 bp long. A + T content of the rRNA genes was 83.91%. AT and GC skews were positive (0.029) and negative (−0.144), respectively.

The L. salicis mitogenome included 22 tRNA genes, ranging from 64 bp (tRNA^His) to 73 bp (tRNA^Trp) long. Of these, 14 genes were encoded on the H-strand and eight on the L-strand (Table 1). The tRNA genes were highly A + T biased (82.19%) with a positive AT-skew (0.007) (Table 2). All the tRNAs possessed a typical clover-leaf secondary structure, except tRNA^Ser(AGN), which lacks the dihydrouridine (DHU) arm and forms a simple loop (Fig. 5). Ten of the tRNA genes were each found to have 11 G-U mismatches in their respective secondary structures, which form a weak bond. Ten U-U mismatches were present in the respective amino acid acceptor stems of tRNA^Gln, tRNA^Trp, tRNA^Leu(UUR), tRNA^Ala, tRNA^Thr, tRNA^Leu(CUN), and tRNA^Val (Fig. 5). All tRNA secondary structures of the tRNA genes were calculated using the tRNAscan-SE program.

Figure 5. Predicted secondary structures of the 22 tRNA genes of the L. salicis mitogenome.

Overlapping and intergenic spacer regions

We identified four overlapping gene sequences, varying from 1 bp to 8 bp, making up 19 bp in total. The longest overlapping region was 8 bp between tRNA^Trp and tRNA^Cys; there was a 7 bp overlap between atp8 and atp6; 3 bp overlap between tRNA^Ile and tRNA^Gln, and 1 bp between tRNA^Ala and tRNA^Arg (Table 1).

Intergenic spacers were spread over 18 regions, and ranged in length from 1 bp to 47 bp. The longest (47 bp) contained an A + T-rich region and occurred between tRNA^Gln and nad2. The 10 bp spacer region between tRNA^Ser (UCN) and nad1 included an ‘ATACTAA’ motif (Fig. 6A).

Figure 6.

(A) Alignment of the intergenic spacer region between tRNA^Ser (UCN) and nad1 of several Lepidopteran insects. (B) Features present in the A + T-rich region of L. salicis. The ‘ATATG’ motif is shaded. The poly-A stretch is double underlined, and the poly-T stretch is underlined. The single microsatellite T/A repeat sequence is indicated by dotted underlining.

The A + T-rich region

The 325 bp long A + T-rich region of L. salicis was located between the rrnS and tRNA^Met genes (Table 1). A + T content in the A + T-rich region was 91.69%, and both AT (−0.248) and GC (−0.408) skews were negative (Table 2). The A + T-rich region did not contain long repeats, though some short repeating sequences scattered over the entire region were present: an ‘ATAGA’ motif followed by an 18 bp poly-T stretch, a microsatellite-like (AT)₇ and a poly-A element upstream of the tRNA^Met gene (Fig. 6B).

Phylogenetic relationships

We established phylogenetic relationships among 32 insects (Table 3), based on nucleotide sequences of 13 PCGs, using Maximum Likelihood (ML), Neighbor Joining (NJ) and Bayesian Inference (BI) methods. Species clustered by family (Fig. 7A, B and C). Within Lymantriidae, L. salicis was most closely related to G. menyuanensis. Lymantriidae clustered with Erebidae, while Noctuidae clustered with Nolidae. Noctuoidea was most closely related to Bombycoidea in ML and NJ trees, while in the BI tree Bombycoidea was most closely related to Geometroidea. Papilionoidea and Tortricoidea branched together in ML and NJ methods, but were separated from each other in the BI tree.

Table 3. Details of the lepidopteran mitogenomes used in this study.

Superfamily	Family	Species	Size (bp)	GenBank No.
Bombycoidea	Bombycidae	Bombyx mori	15666	KM875545.1
		Rondotia menciana	15301	KC881286.1
	Saturniidae	Actias selene	15,236	NC_018133
		Antheraea pernyi	15,566	AY242996
		Antheraea yamamai	15,338	NC_012739
	Sphingidae	Sphinx morio	15299	KC470083.1
		Manduca sexta	15,516	NC_010266
Noctuoidea	Lymantriidae	Lymantria dispar	15507	GU994783.1
		Gynaephora menyuanensis	15770	KC185412.1
		Euproctis pseudoconspersa	15461	KJ716847.1
		Leucoma salicis	15334	This study
	Noctuidae	Ctenoplusia agnata	15261	KC414791.1
		Agrotis ipsilon	15,377	KF163965
	Nolidae	Eutelia adulatricoides	15,360	KJ185131
		Gabala argentata	15,337	KJ410747
	Erebidae	Amata formosae	15463	KC513737
		Hyphantria cunea	15481	GU592049.1
	Notodontidae	Phalera flavescens	15,659	NC_016067
		Ochrogaster lunifer	15,593	NC_011128
Geometroidea	Geometridae	Apocheima cinerarium	15,722	KF836545
		Biston thibetaria	15,484	KJ670146.1
Pyraloidea	Crambidae	Chilo suppressalis	15,395	NC_015612
		Diatraea saccharalis	15,490	NC_013274
		Paracymoriza distinctalis	15354	KF859965.1
	Pyralidae	Lista haraldusalis	15213	NC_024535
Tortricoidea	Tortricidae	Spilonota lechriaspis	15368	HM204705.1
		Grapholita molesta	15,717	NC_014806
Papilionoidea	Papilionidae	Papilio maraho	16,094	NC_014055
		Teinopalpus aureus	15,242	NC_014398
	Nymphalidae	Apatura ilia	15,242	NC_016062
		Apatura metis	15,236	NC_015537
		Fabriciana nerippe	15,140	NC_016419
		Argynnis hyperbius	15,156	NC_015988
Hepialoidea	Hepialidae	Thitarodes renzhiensis	16,173	NC_018094
		Ahamus yunnanensis	15,816	NC_018095
Hesperioidea	Hesperiidae	Choaspes benjaminii	15272	JX101620.1

Figure 7.

(A) Tree showing the phylogenetic relationships among 32 species, constructed using Maximum Likelihood with 1000 bootstrap replicates. (B) Neighbor Joining (NJ) tree, with 1000 bootstrap replicates. (C) Tree constructed using Bayesian Inference (BI) MCMC consensus tree, with posterior probabilities shown at nodes. Drosophila melanogaster (NC_025936) and Locusta migratoria (NC_002084) were used as outgroups.

Discussion

At the family level, the length of the L. salicis mitogenome (15,334 bp) is marginally smaller than that of Euproctis pseudoconspersa (15,461 bp), but it falls within the range (15,140–16,173 bp) of other known lepidopteran mitogenomes. Gene order and orientation are the same as in previously-sequenced Lymantriidae. Nucleotide BLAST (blastn) result of the entire mitogenome against closely related species revealed that L. salicis has a high similarity with the Lymantriidae species (77% in L. dispar–79% in L. alpherakii). The conserved regions lie in 22 tRNAs and 13 PCGs, while A + T-rich region varies in these species. These remarkable characteristics have been reported in other lepidopteran species⁷ and could be used as potential markers for identification at genus and species level in recent molarcular techniques. The highly A + T biased nucleotide composition is within the range of previously sequenced lepidopterans (79.64% in L. dispar–81.48% in G. menyuanensis). The positive AT skew (0.043) observed here, indicating the presence of more As than Ts, is similar to that seen in many lepidopterans, including L. dispar (0.014), Rondotia menciana (0.050), and Biston thibetaria (0.064) (Table 2). It is slightly higher than that of other sequenced mitogenomes in Noctuoidea, including Ctenoplusia agnata (−0.023), G. menyuanensis (0.003) and E. pseudoconspersa (0.011). A similar trend has been observed in other lepidopteran superfamilies such as Bombycoidea, where AT skew varies from 0.001 (Sphinx morio) to 0.059 (Bombyx mori)¹¹. In all sequenced lepidopteran mitogenomes, GC skew ranges from −0.268 in G. menyuanensis to −0.155 in Paracymoriza distinctalis (Table 2). The L. salicis mitogenome is moderately skewed (−0.254), showing the presence of more Cs than Gs.

The AT skew value (0.063) of the protein-coding gene region in the L. salicis mitogenome is higher than that of several previously sequenced mitogenomes. Its negative GC skew (−0.234) is similar to that seen in other animals. Cox1 is thought to initiate with CGA, as found in other lepidopteran insects¹²,¹³. Cox1 and cox2 terminate with a single T, while nad4 terminates with TA. Similar results have been documented in several sequenced lepidopteran mitogenomes, including Artogeia melete¹⁴, Phthonandria atrilineata¹⁵, Ochrogaster lunifer¹⁶, H. cunea¹⁷ and Amata emma¹⁸. The common termination codon TAA is usually created via post-transcriptional polyadenylation¹⁹. The relative synonymous codon usage of the 13 protein-coding genes (PCGs) in L. salicis is consistent with those of published lepidopteran sequences. Similarly, codons with A or T in the third codon position being overrepresented relative to other synonymous codons, is consistent with previous observations of lepidopterans⁹; likewise the absence or underrepresentation of high-GC codons¹⁸,²⁰.

The A + T content (83.91%) of rRNA genes is similar to that seen in Lymantriidae (83.05% in G. menyuanensis). The positive AT (0.029) and negative GC (−0.144) skew seen in the L. salicis mitogenome has also been reported in several sequenced lepidopterans (Table 2). For example, H. cunea has a positive AT (0.024) and negative GC (−0.137) skew¹⁷; and L. dispar also has positive AT (0.023) and negative GC (−0.155) skew.

The secondary structure of L. salicis tRNA^Ser(AGN) lacks the dihydrouridine (DHU) arm and forms a simple loop. This has also been observed in several other animal mitogenomes²¹, including those of insects¹⁵,²²,²³. Ten tRNA genes have 11 mismatches in their secondary structures; most of these are located in the acceptor, DHU and anticodon stems. In addition, tRNA^Cys and tRNA^Ser (UCN) contain an A-A mismatch in the anticodon stem. Unmatched base pairs observed in tRNA sequences can be corrected by RNA-editing mechanisms that are well known for arthropod mtDNA²⁴.

Four overlapping sequences occur in the mitogenome of L. Salicis. The 7 bp overlap between atp8 and atp6 has been documented in several other lepidopteran mitogenomes²⁵,²⁶. The 10 bp intergenic spacer region containing an ‘ATACTAA’ motif, between tRNA^Ser (UCN) and nad1, has also been documented in at least nine other species, suggesting that this region is highly conserved among most of the lepidopteran mtDNAs sequenced to date²⁷.

The length of the A + T-rich region of L. salicis (325 bp) is shorter than those of G. menyuanensis (449), L. dispar (371), H. cunea (357) and B. thibetaria (350), and longer than those of Lista haraldusalis (310) and Choaspes benjaminii (293). Extra tRNA-like structures are often found in the A + T-rich region of lepidopteran mitogenomes. For example Antheraea yamamai has tRNA^Ser(UCN)-like and tRNA^Phe-like sequences, each with correct anticodon structure and forming a clover-leaf structure, which suggests that they may be functional, though each has several mismatches in both aminoacyl and anticodon stem regions²⁸. Extra tRNA-like structures have not been seen in L. salicis. The presence of multiple tandem-repeat elements is described as being characteristic of insect A + T-rich regions²⁹. Antheraea pernyi has a repeat element of 38 bp tandemly repeated six times²⁵; and Cnaphalocrocis medinalis has a duplicated 25 bp repeat element²⁵,³⁰. Long conspicuous repeats were not observed in the A + T-rich region of L. salicis, though shorter repeating sequences, an ‘ATAGA’ motif and other features were. These characteristic features have each been found in previously sequenced lepidopteran species²⁷,³¹,³².

In general, the L. salicis mitogenome contains several features in nucleotide composition, structure of tRNAs and PCGs as well as in the A + T rich region. Particularly in advanced technologies like PCR–RFLP methods³ and DNA barcodes³³, these similarities and differences between L. salicis and other insects could be used as potential markers in species identification, especially the differences.

Phylogenetic relationships were established using Maximum Likelihood (ML) Neighbor Joining (NJ) and Bayesian Inference (BI) methods. Species clustered in families, and results were broadly consistent with previous work, e.g. Dong et al.²⁶ and Dai et al.³⁴. Results obtained from our analyses also supported the classification proposed by Fibiger and Lafontaine³⁵, including within Lymantriidae a clade comprised of E. pseudoconspersa, L. salicis, L. dispar and G. menyuanensis. The present analysis showed that within Lymantriidae, L. salicis was most closely related to G. menyuanensis, which is consistent with a recent study on E. pseudoconspersa²⁶. Interestingly, L. dispar is more closely related to G. menyuanensis than E. pseudoconspersa in ML and NJ trees (Fig. 7A and B), whereas in the BI consensus tree L. dispar and E. pseudoconspersa branch together with 0.6406 posterior probabilities (Fig. 7C). We conclude from the above results that differences between BI, ML and NJ methods generate different results on the relationship among different Noctuoidea species.

Because most previous classifications of Lymantriidae species have been based on morphological features, the precise position of Lymantriidae within the Noctuoidea is still unclear. Kitching has suggested that the Lymantriidae are the sister group to a paraphyletic Pantheidae, sharing apomorphies such as the presence of secondary setae in first instar larvae³⁶. Zahiri et al. reclassified the Noctuoidea on the basis of molecular analyses, making the group currently named Lymantriinae a subfamily of Erebidae³⁷. Our results suggest that Lymantriidae can be regarded as a sister group to other families (Erebidae, Nolidae and Noctuidae) in the Noctuoidea, being most closely related to Erebidae that is consistent with previous study of Fibiger and Lafontaine (2005) on higher Noctuoidea classification. They placed the Lymantriidae from a position in front of the Nolidae to a position after Arctiidae to reflect the close association of the arctiids and lymantriids, and moved the Nolidae, Arctiidae and Lymantriidae in front of the upgraded family Erebidae so that their close relationship with the “quadrifids’ is better reflected³⁵. It is concluded that further studies are needed on sequencing and characterization of mitogenomes of the family Lymantriidae that will provide insight to classification of Noctuoidea.

At the level of superfamilies, Noctuoidea was closely related to Bombycoidea in our ML and NJ analyses, while in the BI tree, Bombycoidea was closely related to Geometroidea. Papilionoidea and Tortricoidea branched together in ML and NJ trees, but in the BI tree they formed separate branches, more in line with previous studies. Hepialoidea was the sister group to all other superfamilies, as found previously by Salvato et al.¹⁶ and Chai et al.³⁸. While several previous studies have been undertaken on mitogenomes of Noctuoidea, relatively little is known about Lymantriidae specifically. Further taxon sampling within Lymantriidae and related families is required to resolve the placement of Lymantriidae in Noctuoidea.

Materials and Methods

Sample collection and mitochondrial DNA extraction

L. salicis larvae were collected from willow trees within the campus of Anhui Agricultural University, Hefei, China. Total genomic DNA was extracted using the Aidlab Genomic DNA Extraction Kit (Aidlab Co., Beijing, China) according to the manufacturer’s instructions. Quality of extracted DNA was assessed by electrophoresis on a 1% agarose gel stained with ethidium bromide.

Primer design, PCR amplification and sequencing

The full mitochondrial genome of L. salicis was PCR amplified in thirteen overlapping fragments, based on primers that were designed from known mitogenomes of Lymantriidae, and synthesized by Invitrogen Co. Ltd. Shanghai, China (Table 4). All PCRs were performed in a 50 μL reaction volume, including 35 μL sterilized distilled water, 5 μL 10 × Taq buffer (Mg^2 +), 4 μL dNTP (25 mM), 1.5 μL DNA, 2 μL of each primer (10 μM) and 0.5 μL (1 unit) Taq (TaKaRa Co., Dalian, China). PCR conditions were as follows: 4 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 40 s at 46–58 °C (Table 4), and 1–3 min (depending on putative length of the fragments) at 72 °C; and then a final extension step of 72 °C for 10 min.

Table 4. Details of the primers used to amplify the mitogenome of L. salicis.

Primer pair	Primer sequence (5′–3′)	Annealing temperature
F1	TAAAAATAAGCTAAATTTAAGCTT	52 °C
R1	TATTAAAATTGCAAATTTTAAGGA
F2	AAACTAATAATCTTCAAAATTAT	46 °C
R2	AAAATAATTTGTTCTATTAAAG
F3	ATTCTATATTTCTTGAAATATTAT	46 °C
R3	CATAAATTATAAATCTTAATCATA
F4	TGAAAATGATAAGTAATTTATTT	48 °C
R4	AATATTAATGGAATTTAACCACTA
F5	TAAGCTGCTAACTTAATTTTTAGT	53 °C
R5	CCTGTTTCAGCTTTAGTTCATTC
F6	CCTAATTGTCTTAAAGTAGATAA	48 °C
R6	TGCTTATTCTTCTGTAGCTCATAT
F7	TAATGTATAATCTTCGTCTATGTAA	50 °C
R7	ATCAATAATCTCCAAAATTATTAT
F8	ACTTTAAAAACTTCAAAGAAAAA	53 °C
R8	TCATAATAAATTCCTCGTCCAATAT
F9	GTAAATTATGGTTGATTAATTCG	53 °C
R9	TGATCTTCAAATTCTAATTATGC
F10	CCGAAACTAACTCTCTCTCACCT	58 °C
R10	CTTACATGATCTGAGTTCAAACCG
F11	CGTTCTAATAAAGTTAAATAAGCA	55 °C
R11	AATATGTACATATTGCCCGTCGCT
F12	TCTAGAAACACTTTCCAGTACCTC	52 °C
R12	AATTTTAAATTATTAGGTGAAATT
F13	TAATAGGGTATCTAATCCTAGTT	48 °C
R13	ACTTAATTTATCCTATCAGAATAA

All PCR products were visualized by electrophoresis on a 1.0% TAE agarose gel, and purified using a DNA gel extraction kit (Transgen Co., Beijing, China). The purified PCR fragments were ligated into the T-vector (TaKaRa Co., Dalian, China) and transformed into Escherichia coli DH5α, using the manufacturer’s protocol. Recombinants were cultured overnight at 37 °C on Luria-Bertani (LB) solid medium containing Ampicillin (AMP), isopropylthiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal). White colonies carrying insert DNA were selected, cultured overnight in liquid media, and vector inserts were directly sequenced by Sangon Biotech Co., (Shanghai, China).

Sequence assembly and gene annotation

The complete mtDNA sequence was assembled using the SeqManII program from the Lasergene software package (DNAStar Inc., Madison, USA). Sequence annotation was performed using the NCBI’s web interface for BLAST (http://blast.ncbi.nlm.nih.gov/Blast).

Nucleotide sequences of the PCGs were translated into putative proteins based on insect sequences available in GenBank. Initiation and termination codons were identified using an alignment created in ClustalX version 2.0, with other lepidopteran sequences as references. To describe base composition, we analyzed skew as described by Junqueira³⁹: AT skew = [A − T]/[A + T], GC skew = [G − C]/[G + C]. The relative synonymous codon usage (RSCU) was obtained using MEGA 5⁴⁰.

The tRNA genes were verified using the program tRNAscan-SE with default settings⁴¹, in addition to using the alignment to visually identify sequences with the appropriate anticodons capable of folding into the typical clover-leaf secondary structure. In the A + T-rich region, tandem repeats were found with the Tandem Repeats Finder program (http://tandem.bu.edu/trf/trf.html)⁴².

Phylogenetic analysis

A total of 29 sets of 13 PCG sequences were used to perform phylogenetic analysis, including those of L. salicis. Those from other taxa were downloaded from GenBank, with Drosophila melanogaster (U37541.1)⁴³ and Locusta migratoria (JN858212)⁴⁴ sequences used as an outgroup. Alignments of the 13 concatenated PCGs were conducted using ClustalX version 2.0. Maximum likelihood (ML) phylogenetic analysis was performed using MEGA 5.0 with Tamura-Nei model⁴⁰. Neighbor Joining (NJ) distance analysis was performed using PAUP4b10⁴⁵, and Bayesian Inference (BI) MCMC phylogenetic analysis was performed using MrBayes 3.2⁴⁶. The ML analysis was pseudosampled with 1000 bootstrapped datasets. The NJ analysis was done with 1000 bootstrap replicates. The BI analysis used four chains MCMC, running for 1,000,000 generations, with trees being sampled every 1000 generations. The consensus tree was visualized using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).

Additional Information

How to cite this article: Sun, Y.-X. et al. Characterization of the Complete Mitochondrial Genome of Leucoma salicis (Lepidoptera: Lymantriidae) and Comparison with Other Lepidopteran Insects. Sci. Rep. 6, 39153; doi: 10.1038/srep39153 (2016).

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