Insect mitochondrial genomes (mitogenomes) are typically circular molecules 14–19 kb in length that contain 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes (Wolstenholme 1992, Boore 1999), and an A+T-rich region, which contains initiation sites for transcription and replication (Zhang et al. 1995, Zhang and Hewitt 1997, Taanman 1999).

Mitogenome sequences, which exhibit very low levels of recombination, are widely used in population genetics, comparative and evolutionary genomics, reconstruction of phylogenetic relationships, and evolutionary biology (Avise 1987, Ballard 2000, Ballard and Rand 2005, Cameron and Whiting 2008, Hao et al. 2012). The silk-producing insects in the lepidoptera with economic value belong to two families of moth, Bombycidae and Saturniidae (Mahendran et al. 2006). The complete mitogenomes of Bombyx mori and Bombyx mandarina of Bombycidae (Yukuhiro et al. 2002; Hu et al. 2010; Li et al. 2010a; Liu et al. 2013), and Antheraea pernyi (Liu et al. 2012b), Antheraea yamamai (Kim et al. 2009), Eriogyna pyretorum (Jiang et al. 2009), Samia cynthia ricini (Kim et al. 2012), Actias selene (Liu et al. 2012a), and Caligula boisduvalii (Hong et al. 2008) of Saturniidae have been sequenced. The origin of bombycidae insects had been studied more according to the mitogenomes (Hu et al. 2010; Li et al. 2010a).

The mulberry white caterpillar, Rondotia menciana Moore (Lepidoptera: Bombycidae) is a silk-producing insects from Bombycidae and has been exploited since the Yangshao culture period (approximately 5,500–6,000 years ago). As all the other insects from lepidoptera, R. menciana is a bivoltine insect that exhibits four molts and a dormant period after the formation of resting eggs, too (Xu et al. 1994). The number of chromosomes (22) in R. menciana differs from that of B. mori, (28) or B. mandarina (27 or 28) (Deng and Xiang 1993), and, thus, the genetic information of R. menciana is important for understanding the diversity of the Bombycidae. R. menciana larvae feed on mulberry leaves and can, in serious cases, defoliate trees. So, the natural R. menciana populations have been decreasing, due to effective control of the insect by the Chinese government to prevent destruction of mulberry trees in recent years. At the same time, the research on genetic or the other aspects about R. menciana was rare. In this study, the complete mitogenome sequence of R. menciana was obtained (GenBank accession number: KC881286), and the phylogenetic analyses based on the mitogenome of the selected insects from lepidoptera were performed using the maximum-likelihood (ML) method.

Materials and Methods

Specimen Sampling and DNA Extraction. Adult specimens of R. menciana were collected from the Tsinling Mountains (106° 55′19″ E, 34° 14′29″ N), Shaanxi Province, China, in September 2011, preserved in 100% ethanol, and stored at -80°C until DNA extraction. Total genomic DNA was extracted from heads excised from frozen insects using the MagSi Tissue DNA Kit (Omega, GA).

Polymerase Chain Reaction Amplification and Sequencing. To amplify the entire mitogenome of R. menciana, 10 primer sets (Table 1) were designed according to known mitochondrial DNA sequences from Bombycidea insects. Purified genomic DNA was amplified using the polymerase chain reaction (PCR) technique and the Taq PCR Kit (NEB, MA), under the following cycling parameters: 94°C for 3 min; 35 cycles of 30 s at 94°C, 40 s at 55–60°C, 1–3 min at 72°C; and 72°C for 10 min. The PCR products were detected by 1.0% agarose-gel electrophoresis and purified using a DNA gel extraction kit (TaKaRa, Japan). The purified PCR products were ligated into the T-vector (TaKaRa) and sequenced at least three times at Sangon.

Sequence Analysis and Gene Annotation. The BLASTN ( http://www.ncbi.nlm.nih.gov/blast) was used to determine sequence similarity. Sequence assembly was performed using DNAStar software. The location of tRNA genes and potential stem-loop secondary structures were identified using tRNAscan-SE software version 1.21 (Lowe and Eddy 1997), specifying mito and chloroplast DNA as the source and using invertebrate mitochondrial genetic code predictors. Thirteen PCGs were identified using an open reading frame finder, using the invertebrate mitochondrial genetic code. The nucleotide composition and codon usage were calculated using MEGA 5.05 (Tamura et al. 2011) and the composition skewness to the formulas: AT Skew=[A-T]/[A+T]; GC skew=[G-C]/[G+C] (Perna and Kocher 1995). The putative control region was identified by alignment with sequences from the closely related species B. mori and B. mandarina, and the tandem repeats in the control region were predicted using the Tandem Repeats Finder program (Benson 1999).

Table 1.

Primers used in this study

Table 2.

List of taxa used in this study

Phylogenetic Analysis. The complete mitogenomes of 29 lepidopteran species (Table 2) were used to reconstruct the phylogenetic relationship. The mitogenomes of Drosophila melanogaster (Diptera: Drosophilidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) were used as outgroups (Clary et al. 1982, Friedrich and Muqim 2003). The amino acid sequences of each of the 13 mitochondrial PCGs were aligned by Clustal X 1.83 using default settings (Thompson et al. 1997) and then backtranslated into nucleotide sequences after alignment. The concatenated set of nucleotide sequences were performed in phylogenetic analysis, using ML method with the MEGA version 5.05 program.

Results

Genome Organization and Base Composition. In this study, the organization of R. menciana mitogenome was shown in Fig. 1. The complete mitogenome is a closed circular molecule of 15,301 bp in length, containing 13 PCGs (coxI–III, nad1–6, nad4L, cyt B, atp6, atp8), 22 tRNA genes, 2 rRNAs (rrnL and rrnS), and an A+T-rich region (Table 3). The order and orientation of R. menciana mitochondrial genes are identical to those found in available lepidopteran mitogenomes (Cameron and Whiting 2008, Liu et al. 2013, Yang et al. 2013). The gene order trnM, trnI, and trnQ of the lepidopteran mitogenomes differs from the most common type trnI, trnQ, and trnM, which was found in a variety of insect orders and inferred to be ancestral for insects (Boore et al. 1998). The nucleotide composition of the R. menciana mitogenome was as follows: A (41.42%), T (37.45%), G (7.82%), and C (13.31%), and the A+T content (78.87%) was the lowest in the Bombycidae (Table 4). The ATskewness and GC skewness of R. menciana mitogenome was 0.050 and -0.26, respectively, as observed in other Bombycidae, more biased toward A (the value of AT skewness is above zero) and C (Table 4).

Fig. 1.

Map of the mitogenome of R. menciana. The direction of all PCGs is designed by the underlined arrows. The transfer RNA genes are designated by single-letter amino acids codes. L1, L2, S1, and S2 denote trnL(UUR), trnL(CUN), trnS(AGN), and trnS(UCN), respectively.

PCGs and Codon Usage. The total length of 13 PCGs is 11,194 bp, and the A+T content of them is between 69.9% (coxI) and 89.51% (atp8) (Table 3). All PCGs in the R. menciana mitogenome were initiated by typical ATN codons: ATT for nad2, coxI, atp8, and nad3 genes, ATA for nad5, nad6, and cytb genes, and ATG for the other six genes. Twelve of the PCGs were terminated with the canonical stop codons TAA or TAG, and coxII gene was terminated with a single T (Table 3). The presence of an incomplete stop codon seems a common phenomenon and had been found in several invertebrate mitochondrial genes (Jiang et al. 2009, Liu et al. 2013, Yang et al. 2013).

Codon usage of the PCGs exhibited a notable AT bias with an A+T composition of 77.05% (Table 4), which plays a major role in the A+T bias of the entire mitogenome. The six most frequently used codons in the R. menciana mitogenome (TTA for Leu, ATT for Ile, TTT for Phe, ATA for Met, AAT for Asn, and TAT for Tyr) are composed of T or a combination of A and T, and the least frequent codons (CCG for Pro, TCG, AGG, and AGC for Ser, CGC for Arg, CTG for Leu, and CGG for Arg) have a high CG content (Table 5).

The analysis of the base composition at each codon position of the 13 PCGs of R. menciana mitogenome shows that the third codon position (87.4%) is considerably higher in A+T content than the first (72.8%) and the second (70.3%) ones (Table 6). Further analysis of the base composition at third codon position among Bombycidae insects shows that the highest and lowest A+T content appeared in the R. menciana (Kim et al. 2014) and this article, respectively, whereas the C+Tcontent of each codon position was similar.

Overlapping and Intergenic Spacer Regions. Eleven overlaps, a total of 34 bp, were observed in the R. menciana mitogenome (Table 3). The largest overlap, 8 bp, occurred between the trnW and trnC genes, as reported in other Bombycidae species, such as B. mandarina (Li et al. 2010a) and B. mori Dazao (Liu et al. 2013). As in other lepidopteran species, a 7 bp overlap occurred between the atp8 and atp6 genes, as well. The R. menciana mitogenome harbors 17 short non-coding regions of 1–52 bp, for a total of 149 bp, as in the other insects from Bombycidae, the longest spacer is located between the trnQ and nad2 genes (Table 3).

Transfer and rRNA Genes. As in other lepidopteran insects, all 22 tRNA genes with characteristic cloverleaf secondary structure in the R. menciana mitogenome were predicted using the tRNAscan-SE Search Server. The length of tRNA genes ranged from 64 bp (trnI) to 71 bp (trnK) and the A+T content ranged from 73.53% (trnL(UUR)) to 92.54% (trnE) (Table 3). A total of six mismatches were found in five tRNA genes, two in amino acid acceptor stems, three in anticodon stems, and one in pseudouridine (TΨC) stems (Fig. 2). The mismatched bases show significant nucleotide bias, four U-U, one A-G, and one U-G.

Table 3.

Summary of the mitogenome of R. menciana

Table 4.

Comparison of the nucleotides composition and skewness of Bombycoidea insects

Two rRNA genes were identified on the N-strand in the R. menciana mitogenome: the rrnL gene, located between trnL(CUN) and trnV genes, and the rrnS gene, between the trnV gene and the A+T-rich region (Table 3). The length of rrnL and rrnS genes was 1,365 bp and 782 bp, and their A+Tcontent was 83.37% and 84.4%, respectively.

A+T-Rich Region. The A+T-rich region of R. menciana mitogenome was exactly same as that of Kim et al. (2014). The A+T-rich region was 360-bp long and located between the rrnS and trnM genes. The A+T content of the region was 91.11%, lower than the other Bombycidae insects (94.42–95.55%) (Yukuhiro et al. 2002; Hu et al. 2010; Li et al. 2010b; Liu et al. 2013). Several structures conserved in other Bombycidae mitogenomes were also observed in the R. menciana A+T-rich region (Fig. 3). The conserved “ATAGA+poly T” motif with 17 consecutive Ts was located 24 bp downstream of the rrnS gene. A microsatellite (ATAT)n element and a 12-bp poly-A region, commonly observed in other lepidopteran mitogenomes, were also found immediately upstream of the trnM gene. We also identified 2.6 tandem repeats elements of 37 bp in the R. menciana A+T-rich region (Fig. 3).

Table 5.

Codon usage of PCGs in R. menciana mitogenome

Table 6.

Summary of base composition at each codon position of PCGs in the Bombycidae mitogenome

Fig. 2.

Putative secondary cloverleaf structures for the tRNA genes of the R. menciana mitogenome with mismatch bases. The blue point and red point indicate Watson—Crick base pairing A-U and G-C, respectively, and the blank indicate the mismatch bases. Six mismatches (four U-U, one A-G, and one U-G) lies in five tRNA genes (two in amino acid acceptor stems, three in anticodon stems and one in pseudouridine) (TΨC).

Phylogenetic Analysis. In this study, the concatenated nucleotides sequences of 13 PCGs of 29 mitogenomes were used to reconstruct the phylogenetic relationships by ML method (Fig. 4). These 29 mitogenomes represent five superfamilies within the lepidopteran suborder: Bombycoidea, Geometroidea, Noctuoidea, Pyraloidea, and Tortricidea. The results show that the phylogenetic relationships among these five superfamilies are Tortricidea+(Pyraloidea+(Noctuoidea+(Geome troidea+Bombycoidea))), the relationship between Geometridae and Bombycoidea was close. The phylogenetic relationships inside Bombycoidea and Bombycidae were Bombycidae+(Sphingidae +Saturniidae)) and R. menciana+(B. mori+B. mandarina), respectively.

Discussions

The complete mitogenome of R. menciana with a circular molecule of 15,301 bp was determined using the PCR method, which is the shortest in known complete mitogenomes of Bombycidae. The gene organization and order of R. menciana mitogenome are identical to the studied lepidopteran mitogenomes (Cameron and Whiting 2008, Liu et al. 2013, Yang et al. 2013). The order of the trnM tRNA gene in the lepidopteran mitogenomes is A+T-rich region, trnM, trnI, trnQ, and nad2, whereas the deduced ancestral gene order is A+T-rich region, trnI, trnQ, trnM, nad2 (Boore et al. 1998). This observation suggests that lepidopteran insects may have acquired the typical gene orientation and order independently after diverging from the ancestral insect.

Fig. 3.

Alignments of the A+T-rich region in Bombycidae. The thread underlined and thick overlined indicate the tandom repeat in R. menciana, and B. mori and B. mandarina, respectively.

The A+Tcontent (78.87%) of the R. menciana mitogenome is lower than the other Bombycidea insects (Yukuhiro et al. 2002; Cameron and Whiting 2008; Hong et al. 2008; Jiang et al. 2009; Kim et al. 2009; Hu et al. 2010; Li et al. 2010a; Liu et al. 2012b, 2013; Kim et al. 2014) (Table 4). The AT skewness of the R. menciana mitogenome was 0.050 (Table 4), higher than the R. menciana mitogenome (0.021) of Kim et al. (2014), and lower than that (0.054∼0.059) of the B. mandarina and B. mori, indicating a higher occurrence of A comparedwith T nucleotides in Bombycidae. Different from the Bombycidae, there were higher occurrence of T compared with A nucleotides in the two families of Sphingidae and Saturniidae (Table 4). The GC skewness of the R. mencianamitogenome was -0.260, indicating a heavy bias toward C versus G nucleotides and much more negative than observed for other Bombycoidea mitogenome (<0.236).

There was an incomplete stop codon of a single T in R. menciana coxII gene. The incomplete stop codon had been found in several invertebrate mitochondrial genes, which seems a common phenomenon of mitochondrion genes (Jiang et al. 2009, Liu et al. 2013, Yang et al. 2013). The relative synonymous codon usage exhibits extensive similarity with other lepidopteran mitogenomes in previous study (Salvato et al. 2008). The most frequent codons in R. menciana are composed of T or a combination of A and T, especially the third codon position. The observed differences in nucleotide composition may caused by the constraints on A+T content in the third codon position, which is more relaxed than those in the first and second codon positions due to degenerated genetic code (Taanman 1999). The C+T content in each of the codon positions were similar, which is agree with the point that the high A+T content in insect mitogenome were caused by the mutation of C to T (The Honeybee Genome Sequencing Consortium 2006).

The length of the intergenic spacer regions (149 bp in 17 regions) is longer than that of Manduca sexta (115 bp in 13 regions) (Cameron and Whiting 2008) and Ac. selene (137 bp in 13 regions) (Liu et al. 2012a) and is shorter than that of C. boisduvalii (194 bp in 16 regions) (Hong et al. 2008) and Bombycidae insects (about 338 bp) (Yukuhiro et al. 2002; Hu et al. 2010; Li et al. 2010a; Liu et al. 2013), which has been suggested to be constitutively synapomorphic and restricted to Ditrysia mitogenomes (Cameron and Whiting 2008, Hao et al. 2012). Similar to the mitogenome of the other insects, the tRNA genes have typical clover-leaf structure. However, the anticodon stem of trnS(UCN) could not form a stable stem-loop structure for the two U-U mismatches. The phenomenon of two U-G mismatches occurred also in the anticodon stem of trnL(CUN) of E. pyretorum (Jiang et al. 2009), B. mori Dazao (Liu et al. 2013), and Ac. selene (Liu et al. 2012a).

The exactly same A+T-rich region occurred between the R. menciana mitogenome of this article and Kim et al. (2014). The length of 360 bp was shorter than that of Bombycidae insects (494 bp∼747 bp) (Yukuhiro et al. 2002; Hu et al. 2010; Li et al. 2010a; Liu et al. 2013) and longer slightly than that of A. yamamai (334 bp) (Kim et al. 2009) and C. boisduvalii (330 bp) (Hong et al. 2008). The A+Tcontent of the region was 91.11%, higher than that of Ac. selene (87.91%) (Liu et al. 2012a), A. pernyi (90.4%) (Li et al. 2011), A. yamamai (90.40%) (Kim et al. 2009), and lower than those from Bombycidae insects (94.42–95.55%). There are some common structures in the A+T-rich region of R. menciana mitogenome, such as ATAGA+poly-T, and tandem repeats elements. The conserved motif of “ATAGA+poly T” stretch at the 24 bp downstream of the rrnS gene is thought to be the origin of the DNA replication (Taanman 1999, Jiang et al. 2009). There are 2.6 tandem repeats elements of 37 bp in the R. menciana A+T-rich region, whereas three 32-bp and three 37-bp tandem repeats elements in B. mori C108 and B. mandarina Qingzhou. In B. mori Dazao, the A+T-rich region harbors two 31-bp repeat elements and three 36-bp repeat elements (Liu et al. 2013). The sequence and location of tandem repeats elements among Bombycidae mitogenomes are nonconserved. The existence of tandem repeats elements maybe caused mainly by the replication slippage.

Fig.4. Phylogenetic

analysis inferred from the concatenated nucleotides sequences of 13 PCGs of mitogenome using Mega 5.05 software and ML method. D. melanogaster and T. castaneum were used as outgroups. The numbers above the branches specify bootstrap percentages (1,000 replicates). *This study.

Mitogenomes are effective markers for deep-level phylogenetic studies in the Lepidoptera. In our analysis, Bombycidae (B. mori, B. mandarina, and R. menciana), Sphingidae (M. sexta), and Saturniidae (Ac. selene, A. pernyi, A. yamamai, S. cynthia ricini, E. pyretorum, and C. boisduvalii) were clustered in one branch of the phylogenetic tree, and the relationship of the three family was Bombycidae+(Sphingidae+Saturniidae), which is consistent with the morphological data and some previous studies (Zwick 2008, Zwick et al. 2011, Liu et al. 2013). The relationship of Geometridae is closer with Bombycoidea in our analyses, which is similar to the study by Yang et al. (2013).