Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis.

Ameson portunus, the recently discovered causative agent of "toothpaste disease" of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

The analyses of the complete mitochondrial genomes of three crabs revealed novel gene rearrangements and phylogenetic relationships of Brachyura.

BACKGROUND: Brachyura crab is the largest branch of Decapoda crustacean. Phylogenetic relationships within Brachyura remain controversial to be investigated. The mitochondrial genome (mitogenome) is an important molecular marker for studying the phylogenetic relationships of Brachyura. METHODS AND RESULTS: To understand the phylogeny of Brachyura, the three complete mitogenomes from Charybdis annulata, Leptodius exaratus, and Spider crab were sequenced and annotated. Their full length was 15,747, 15,716, and 16,608 bp long, respectively. The first two crabs both contained 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and a control region. However, Spider crab contained 13 PCGs, two rRNA genes, 25 tRNA genes and a control region. The mitogenomes of each of the three crabs exhibited high AT content (67.8%, 69.1%, and 70.8%), with negative AT skews (-0.014, - 0.028, and - 0.017) and GC skews (-0.269, - 0.286, and - 0.341). The gene order of C. annulata was identical to the ancestor of Brachyura. Compared with the ancestor of Brachyura, L. exaratus exhibited the gene rearrangements of Val (V)-rrnS-control region, and Spider crab had the four copies of Lys (K). Phylogenetic analyses indicated that C. annulata belonged to Portunidae family, Portunoidea superfamilies, L. exaratus belonged to Xanthidae family, Xanthoidea superfamilies, and Spider crab belonged to Mithracidae family, Majoidea superfamilies. Phylogenetic analyses showed that the two species (Somanniathelphusa boyangensis and Huananpotamon lichuanense) belonging to the Potamoidea were sister groups to the Thoracotremata, thus supporting the conclusion that Heterotremata is polyphyletic. CONCLUSION: The results of this study enriched the crab mitogenome database and enabled us to better understand the phylogenetic relationships of Brachyura.

First Natural Yeast Strain Trichosporon asahii HZ10 with Robust Flavonoid Productivity and Its Potential Biosynthetic Pathway.

Flavonoids are generally thought to be essential plant natural products with diverse bioactivities and pharmacological effects. Conventional approaches for the industrial production of flavonoids through plant extraction and chemical synthesis face serious economic and environmental challenges. Searching for natural robust flavonoid-producing microorganisms satisfying green and sustainable development is one of the good alternatives. Here, a natural yeast, Trichosporon asahii HZ10, isolated from raw honeycombs, was found to accumulate 146.41 mg/L total flavonoids intracellularly. Also, T. asahii HZ10 represents a broad flavonoid metabolic profiling, covering 40 flavonoids, among which nearly half were silibinin, daidzein, and irigenin trimethyl ether, especially silibinin occupying 21.07% of the total flavonoids. This is the first flavonoid-producing natural yeast strain worldwide. Furthermore, T. asahii HZ10-derived flavonoids represent favorable antioxidant activities. Interestingly, genome mining and transcriptome analysis clearly showed that T. asahii HZ10 possibly evolves a novel flavonoid synthesis pathway for the most crucial step of flavonoid skeleton synthesis, which is different from that in plants and filamentous fungi. Therefore, our results not only enrich the diversity of the natural flavonoid biosynthesis pathway but also pave an alternative way to promote the development of a synthetic biology strategy for the microbial production of flavonoids.

Genome-Wide Analysis of Gene Families of Pattern Recognition Receptors in Fig Wasps (Hymenoptera, Chalcidoidea).

Pattern recognition receptors (PRRs) play important roles in detecting pathogens and initiating the innate immune response. Different evolutionary histories of pollinators and non-pollinators may result in different immune recognition systems. A previous study had reported that there were significant differences in peptidoglycan recognition proteins (PGRPs) between pollinators and non-pollinators in gene number and lineage of specific genes. In this study, based on the genomic data of 12 fig wasp species, with seven pollinators and five non-pollinators, we investigated the evolution patterns of PRRs, such as Gram-negative bacteria-binding proteins (GNBPs), C-type lectins (CTLs), scavenger receptors class B (SCRBs), fibrinogen-related proteins (FREPs), galectins, and thioester-containing proteins (TEPs). Our results showed that pollinators had no GNBP, but non-pollinators all had two gene members, which were clustered into two different clades in the phylogenetic tree, with each clade having specific domain and motif characteristics. The analysis of CTL and SCRB gene families also showed that there were lineage-specific genes and specific expansion in non-pollinators. Our results showed that there were significant differences in immune recognition between pollinators and non-pollinators, and we concluded that they had undergone flexible adaptive evolution in different environments. Our study can provide more molecular evidence for future functional studies on the immune system of fig wasps.

Comparative Mitochondrial Genome Analyses of Sesarmid and Other Brachyuran Crabs Reveal Gene Rearrangements and Phylogeny.

Mitochondrial genomes (mitogenomes) are important for understanding molecular evolution and phylogenetic relationships. The complete mitogenome of Perisesarma bidens was determined, which is 15,641 bp in length. The A + T content of P. bidens mitogenome was 74.81%. The AT skew was slightly negative (-0.021). The 22 tRNAs ranged from 65 to 73 bp and were highly A + T biased. All tRNA genes had typical cloverleaf structures, except for the trnS1 gene, which lacked a dihydrouridine (DHU) arm. The gene order within the P. bidens mitogenome was identical to the pancrustacean ground pattern, except for the translocation of the trnH. Additionally, the gene order of trnI-trnQ-trnM in pancrustacean ground pattern became trnQ-trnI-trnM in P. bidens. Phylogenetic analyses supported the inclusion of P. bidens in Sesarmidae and the promotion of Sesarminae to Sesarmidae. The results will help us to better understand the status and evolutionary history of Grapsoidea crabs.

Characterization of the complete mitochondrial genome of Helice latimera and its phylogenetic implications in Brachyura.

Mitochondrial genomes (mitogenomes) help advance our learning of molecular evolution and phylogenetic relationships. The mitogenome of H. latimera is 16,246 bp in length, which typically contains 37 animal mitogenome genes consisting of 13 protein-coding genes (PCGs), two rRNA genes, and 22 tRNA genes, as well as a control region. The AT content of H. latimera is 69.1%. The A + T skew of the mitogenome of H. latimera was slightly negative (-0.017). The size of Thirteen PCGs is from 162 bp to 1731 bp. Twenty-two tRNA genes ranged from 62 to 73 bp and were highly A + T biased. All tRNA genes owed a typical cloverleaf structure, not including the trnS1 gene lacking a dihydroxyuridine arm. One PCG, two rRNAs, and 12 of the tRNAs were rearranged compared to the pancrustacean gene order. Phylogenetic analysis revealed the locationt of H. latimera among the Varunidae family.

Transcriptome analysis of the male polymorphisms of fig wasp species Philotrypesis tridentata.

Intraspecific male polymorphism exhibiting extreme differences in morphology, behavior and life history presents good opportunities to explore adaptation mechanisms to different environments. In this study, we examined the transcriptomic differences between wingless and winged morphs of a fig wasp species Philotrypesis tridentata to investigate molecular basis to maintain polymorphisms. The winged male adults fly outside fig syconia to mate, while the wingless only stay and mate inside fig syconia where they have developed. We identified 2,391 differentially expressed genes (DEGs) with 1,396 highly expressed in winged morphs and 995 in wingless morphs. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses on the DEGs and differential alternative splicing genes and analyzed the top ten DEGs with the highest differential expression in each morph. The results showed that genes related to biosynthesis processes, lipid metabolism, energy production, flight and defense of the complex environments outside fig syconia were up-regulated in winged morphs. Genes involved in substance and energy metabolism and chemical reception were up-regulated in wingless morphs which might relate to their living inside fig syconia. The differences in highly expressed genes between two morphs prove adaptation of P. tridentata male polymorphism to different living environments.

Comparative mitochondrial genome analysis of Grammodes geometrica and other noctuid insects reveals conserved mitochondrial genome organization and phylogeny.

The mitochondrial genome (mitogenome) plays an important role in revealing molecular evolution. In this study, the complete mitogenome of Grammodes geometrica (G. geometrica) (Lepidoptera: Erebidae) was sequenced and characterized. The nucleotide composition of the genome is highly A + T biased, accounting for 80.49%. Most protein-coding genes (PCGs) are initiated by ATN codons except for the cytochrome oxidase subunit 1 (cox1) gene, which was initiated by CGA. The order and orientation of genes with the order trnM-trnI-trnQ-nad2 is a typical rearrangement compared with those ancestral insects in which trnM is located between trnQ and nad2. Most tRNA genes were folded into the typical cloverleaf structure except for trnS1 (AGN). The A + T-rich region contains the conserved motif "ATAGA" followed by a 19 bp poly-T stretch, which was also observed in other Noctuoidea species. In addition, we reconstructed phylogenetic trees among the nucleotide alignments of five families of Noctuoidea species except the Oenosandridae. Finally, we achieved a well-supported tree, which showed that G. geometrica belongs to the Erebidae family. Moreover, the relationships at the family-level can be displayed as follows: (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))).

Transcriptomic analysis of immune-related genes in the lipopolysaccharide-stimulated hepatopancreas of the mudflat crab Helice tientsinensis.

The mudflat crab Helice tientsinensis is one of the most economically important aquaculture species in China. Nevertheless, it is susceptible to various diseases caused by viruses, bacteria and rickettsia-like organisms. A better understanding of the immune system and genes related to the responses to bacterial and viral infection is required. Herein, the hepatopancreas transcriptome of H. tientsinensis was analyzed by comparing control and lipopolysaccharide (LPS)-stimulated RNA-Seq data, yielding 91,885,038 bp and 13.78 Gb of clean reads. Following assembly and annotation, 93,207 unigenes with an average length of 883 bp were identified, of which 31,674 and 13,700 were annotated in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. Following LPS, 4845 differentially expressed genes (DEGs) were identified, of which 2491 and 2354 were up- and down-regulated, respectively. To further investigate immune-related DEGs, KEGG enrichment analysis identified immune response pathways, most notably the peroxisome and Toll-like receptor signaling pathways. Quantitative real time-PCR (qRT-PCR) confirmed the up-regulation of a random selection of DEGs. This systematic transcriptomic analysis of the innate immune pathway in H. tientsinensis expands our understanding of the immune system in crabs.

The complete mitochondrial genome of Clostera anastomosis (Lepidoptera: Notodontidae) and implication for the phylogenetic relationships of Noctuoidea species.

In the present study, the complete mitochondrial genome (mitogenome) of Clostera anastomosis (C. anastomosis) has been determined for the first time. The mitogenome is 15,390 base pairs (bp) in length, comprised of 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and one non-coding control region (CR). The gene order shows a typical trnM rearrangement (trnM-trnI-trnQ) compared to ancestral insects (trnI-trnQ-trnM). Almost all the PCGs have the same start codon (ATN) except for cox1 (CGA), and almost all tRNAs have a typical cloverleaf secondary structure except for trnS1. At the beginning of the CR, we found a conserved motif "ATAGA + poly-T" as found in other lepidopteran insects. There are 20 intergenic regions and 11 overlapping regions, ranging from 1 to 53 bp and 1 to 9 bp, respectively. The A + T content is relatively high across the whole mitogenome. The optimal tree topologies of Noctuoidea were given by the dataset consisting of all 13 PCGs from five families (exclude Oenosandridae). Our trees suggested a topology of (Notodontidae + (Erebidae + (Nolidae + (Euteliidae + Noctuidae)))) and identified that C. anastomosis belongs to Notodontidae.

Transcriptome Analysis Reveals Potential Antioxidant Defense Mechanisms in Antheraea pernyi in Response to Zinc Stress.

The growth and development of the Chinese oak silkworm, Antheraea pernyi, are strongly influenced by environmental conditions, including heavy metal pollution. An excess of heavy metals causes cellular damage through the production of free radical reactive oxygen species. In this study, transcriptome analysis was performed to investigate global gene expression when A. pernyi was exposed to zinc infection. With RNA sequencing (RNA-Seq), a total of 25 795 510 and 38 158 855 clean reads were obtained from zinc-treated and control fat body libraries, respectively. We identified 2399 differential expression genes (DEGs) (1845 upregulated and 544 downregulated genes) in the zinc-treated library. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were related to the peroxisome pathway that was associated with antioxidant defense. Our results suggest that fat bodies of A. pernyi constitute a strong antioxidant defense against heavy metal contamination.

Mitochondrial genome of Argopecten irradians reveals higher-level phylogenetic relationships in Anisomyaria.

The complete mitochondrial genome of Argopecten irradians strain Zhongkehong was sequenced and annotated: it is 16,212 bp in length and contains twelve protein-coding genes (atp8 is absent, as in most species in Anisomyaria), two ribosomal RNA genes, and 21 transfer RNA genes (trnS is absent and there are two copies of trnF). The heavy strand has an overall A + T content of 57.3%; GC and AT skews are 0.249 and -0.262, respectively, indicating more Gs and more Ts than Cs and As. Phylogenetic analysis based on Bayesian Inference and Maximum Likelihood of the twelve protein-coding genes shows that A. irradians has close relationships with A. purpuratus and A. ventricosus; this indicated that A. irradians belongs to the Pectinidae family. The Pectinidae was sister to (Ostreidae + Mytilidae). This work provides general information on the evolution of cultured scallops.

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level.

A small heat shock protein 21 (sHSP21) mediates immune responses in Chinese oak silkworm Antheraea pernyi.

Small heat shock proteins (sHSPs) are conserved among insects and play an important role in the regulation of many biological processes, including temperature stress, abiotic stress, immune responses, metamorphosis, and embryo development. Antheraea pernyi is an economically valuable silk-producing moth and source of insect food containing high-quality protein. The aim of this study was to quantify expression of the ApsHSP21 gene in response to pathogen-associated molecular patterns (PAMPs) and nucleopolyhedrovirus (NPV) challenge. The deduced ApsHSP21 protein sequence consists of 186 residues with a calculated molecular mass of 21.0 kDa and an isoelectronic point (pI) of 6.63. The protein contains a conserved α-crystallin domain (ACD), and includes two casein kinase II phosphorylation sites, a protein kinase C phosphorylation site, two tyrosine kinase phosphorylation sites, and various polypeptide binding sites. Phylogenetic analysis revealed that ApsHSP21 is closely related to homologs from other insects. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis revealed that expression of ApsHSP21 was significantly up-regulated at different timepoints following simulated pathogen challenge with lipopolysaccharide (LPS), peptidoglycan (PGN), glucan, and NPV. The results suggest sHSP21 is involved in innate immune responses in A. pernyi.

Comparative mitochondrial genome analysis of Spilarctia subcarnea and other noctuid insects.

This study was performed to better understand the phylogenetic relationships within the lepidopteran superfamily Noctuoidea. The mitochondrial genome (mitogenome) has been extensively used for studying phylogenetic relationships at different taxonomic levels. In this study, the complete mitogenome of Spilarctia subcarnea (Noctuoidea: Erebidae) was sequenced and annotated. The mitogenome is 15,441bp in length, containing 13 typical protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a noncoding control region (CR). The order and orientation of genes of S. subcarnea mitogenome with the order trnM-trnI-trnQ-nad2 is different from the ancestral insects in which trnM is located between trnQ and nad2 (trnI-trnQ-trnM-nad2). The phylogenetic relationships based on mitochondrial sequences using Bayesian inference and Maximum likelihood methods showed that S. subcarnea was closely related to Lemyra melli, supporting that S. subcarnea belongs to Erebidae. These analyses confirm that Lymantriidae should be included as subfamilies within Erebidae. The Erebidae was sister to (Nolidae+(Euteliidae+Noctuidae)); Notodontidae is sister to the other families of Noctuoidea in our study.

De novo transcriptome assembly and analysis of differential gene expression following lipopolysaccharide challenge in Pelteobagrus fulvidraco.

The yellow catfish, Pelteobagrus fulvidraco, has been recognized as an important freshwater aquaculture species in Eastern and Southeast Asia. To gain a better understanding of the immune response in P. fulvidraco, we analyzed its transcriptome following stimulation with lipopolysaccharide (LPS). Phosphate buffer saline (PBS) was used as control. Following assembly and annotation, 72,152 unigenes with an average length of 1090 bp were identified. A total of 370 differentially expressed genes (DEGs) in the P. fulvidraco were observed at 12 h post LPS treatment, including 197 up-regulated genes and 173 down-regulated genes. Clusters of Orthologous Groups of proteins (KOG/COG) annotation demonstrated that a total of 18,819 unigenes classified into 26 categories. Gene ontology (GO) analysis revealed 20 biological process subcategories, 7 cellular component subcategories and 20 molecular function subcategories. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified immune responses pathways. Quantitative reverse transcription polymerase chain reaction measured the expression of 18 genes involved in the immune response. CXCL2-like chemokine (CXCL2), goose-type lysozyme (LYZ G), and cathepsin K (CTSK) were significantly up-regulated. This study enriches the P. fulvidraco transcriptome database and provides insight into the immune response of P. fulvidraco against infection.

Characterisation of the complete mitochondrial genome of Helice wuana (Grapsoidea: Varunidae) and comparison with other Brachyuran crabs.

The mitochondrial genome (mitogenome) provides important information for phylogenetic analysis and understanding evolutionary origins. Herein, we sequenced, annotated, and characterised the mitogenome of the crab Helice wuana to better understand its molecular evolution and phylogeny. The 16,359bp mitogenome includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region. The genome composition is highly A+T biased 68.42%, and exhibits a negative AT-skew (-0.036) and GC-skew (-0.269) among Brachyura crabs. Gene rearrangements were detected, as was tandem duplication followed by random loss, which explains the translocation of mitochondrial genes. Phylogenetic analysis showed that H. wuana and H. tientsinensis clustered on one branch with high nodal support values. These results confirm that the placement of H. wuana within the Varunidae family of Thoracotrematan crabs. This study will provided a better understanding for gene rearrangements and crab evolution in the further.

Transcriptome-Wide Identification of Differentially Expressed Genes in Chinese Oak Silkworm Antheraea pernyi in Response to Lead Challenge.

Antheraea pernyi is a commercially cultivated silk moth and a source of insect food with high-quality protein. Insects suffer oxidative stress on exposure to heavy metals, and reactive oxygen species are cleared by antioxidant enzymes. To gain better understanding of the antioxidant defense system of A. pernyi, we analyzed transcriptomes of pupae after stimulation with lead and phosphate-buffered saline (control). In total, 72 367 unigenes were identified. Gene ontology analysis revealed that these DEGs were in 20 biological process subcategories, 19 cellular component subcategories, and 18 molecular function subcategories. Clusters of orthologous groups of protein annotation placed a total of 528 DEGs into 25 categories. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified antioxidant defense pathways, including "Peroxisome" and "Glutathione metabolism", which are reported for the first time in A. pernyi. Our study enriches A. pernyi transcriptome databases and provides insight into the heavy metal responses of antioxidant systems of this insect fat bodies.

Mitochondrial Genomes of Two Bombycoidea Insects and Implications for Their Phylogeny.

The mitochondrial genome (mt genome) provides important information for understanding molecular evolution and phylogenetics. As such, the two complete mt genomes of Ampelophaga rubiginosa and Rondotia menciana were sequenced and annotated. The two circular genomes of A. rubiginosa and R. menciana are 15,282 and 15,636 bp long, respectively, including 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes and an A + T-rich region. The nucleotide composition of the A. rubiginosa mt genome is A + T rich (81.5%) but is lower than that of R. menciana (82.2%). The AT skew is slightly positive and the GC skew is negative in these two mt genomes. Except for cox1, which started with CGA, all other 12PCGs started with ATN codons. The A + T-rich regions of A. rubiginosa and R. menciana were 399 bp and 604 bp long and consist of several features common to Bombycoidea insects. The order and orientation of A. rubiginosa and R. menciana mitogenomes with the order trnM-trnI-trnQ-nad2 is different from the ancestral insects in which trnM is located between trnQ and nad2 (trnI-trnQ-trnM-nad2). Phylogenetic analyses indicate that A. rubiginosa belongs in the Sphingidae family, and R. menciana belongs in the Bombycidae family.