Genome-Wide Identification of Detoxification Genes in Wild Silkworm Antheraea pernyi and Transcriptional Response to Coumaphos.

For a half-century, the commercial wild silkworm, Antheraea pernyi, has been protected by coumaphos, which is an internal organophosphorus insecticide used to kill the potential parasitic fly larvae inside. Knowledge about the detoxification genes of A. pernyi as well as the detoxification mechanism for this species remains severely limited. In this study, we identified 281 detoxification genes (32 GSTs, 48 ABCs, 104 CYPs, and 97 COEs) in the genome of this insect, which are unevenly distributed over 46 chromosomes. When compared to the domesticated silkworm, Bombyx mori, a lepidopteran model species, A. pernyi has a similar number of ABCs, but a greater number of GSTs, CYPs, and COEs. By transcriptome-based expression analysis, we found that coumaphos at a safe concentration level significantly changed the pathways related to ATPase complex function and the transporter complex in A. pernyi. KEGG functional enrichment analysis indicated that protein processing in the endoplasmic reticulum was the most affected pathway after coumaphos treatment. Finally, we identified four significantly up-regulated detoxification genes (ABCB1, ABCB3, ABCG11, and ae43) and one significantly down-regulated detoxification gene (CYP6AE9) in response to coumaphos treatment, suggesting that these five genes may contribute to detoxification of coumaphos in A. pernyi. Our study provides the first set of detoxification genes for wild silkworms from Saturniidae and highlights the importance of detoxification gene repertoire in insect pesticide tolerance.

Genome-Wide Identification and Transcriptome-Based Expression Profile of Cuticular Protein Genes in Antheraea pernyi.

Antheraea pernyi is one of the most famous edible and silk-producing wild silkworms of Saturniidae. Structural cuticular proteins (CPs) are the primary component of insect cuticle. In this paper, the CPs in the genome of A. pernyi were identified and compared with those of the lepidopteran model species Bombyx mori, and expression patterns were analyzed based on the transcriptomic data from the larval epidermis/integument (epidermis in the following) and some non-epidermis tissues/organs of two silkworm species. A total of 217 CPs was identified in the A. pernyi genome, a comparable number to B. mori (236 CPs), with CPLCP and CPG families being the main contribution to the number difference between two silkworm species. We found more RR-2 genes expressed in the larval epidermis of fifth instar of A. pernyi than B. mori, but less RR-2 genes expressed in the prothoracic gland of A. pernyi than B. mori, which suggests that the hardness difference in the larval epidermis and prothoracic gland between the two species may be caused by the number of RR-2 genes expressed. We also revealed that, in B. mori, the number of CP genes expressed in the corpus allatum and prothoracic gland of fifth instar was higher than that in the larval epidermis. Our work provided an overall framework for functional research into the CP genes of Saturniidae.

Comprehensive Analysis of Codon Usage in Quercus Chloroplast Genome and Focus on psbA Gene.

Quercus (oak) is an important economic and ecological tree species in the world, and it is the necessary feed for oak silkworm feeding. Chloroplasts play an important role in green plants but the codon usage of oak chloroplast genomes is not fully studied. We examined the codon usage of the oak chloroplast genomes in detail to facilitate the understanding of their biology and evolution. We downloaded all the protein coding genes of 26 non-redundant chloroplast reference genomes, removed short ones and those containing internal stop codons, and finally retained 50 genes shared by all genomes for comparative analyses. The base composition, codon bias, and codon preference are not significantly different between genomes but are significantly different among genes within these genomes. Oak chloroplast genomes prefer T/A-ending codons and avoid C/G-ending codons, and the psbA gene has the same preference except for the codons encoding amino acid Phe. Complex factors such as context-dependent mutations are the major factors affecting codon usage in these genomes, while selection plays an important role on the psbA gene. Our study provided an important understanding of codon usage in the oak chloroplast genomes and found that the psbA gene has nearly the same codon usage preference as other genes in the oak chloroplasts.

[Optimization of synthesizing silver nanoparticles from Trichoderma strains for inhibition of Fusarium oxysporum].

With the popularity of green concepts, the use of biological method to synthesize metal nanoparticles is a favored method. Silver nanoparticles (AgNPs) have attracted more and more attention in the control of agricultural diseases, because of their strong antifungal activity and not easy inducing resistance for pathogens. In this study, Trichoderma citrinoviride and Trichoderma velutinous were used to study the most suitable synthesis conditions for AgNPs, and their antifungal activity against Fusarium oxysporum. The silver nanoparticles had an absorbance peak at 400-500 nm, the most suitable synthesis conditions were at standing and light with CL method (mycelium filtrate), AgNO3 concentration of 2.0 mmol/L, pH of 7, and reaction temperature of 45 °C. AgNPs synthesized by T. citrinoviride and T. velutinous inhibited F. oxysporum, the inhibition effect was better with the increase of silver nanoparticle concentration. When the concentration of AgNPs was 200 mg/L, the antifungal rate from T. citrinoviride and T. velutinous was 33.75% and 36.08%, respectively.

Comparison of bacterial communities between midgut and midgut contents in two silkworms, Antheraea pernyi and Bombyx mori.

Bacterial communities living inside the midgut of insects have been attracting increasing interest. Previous studies have shown that both the midgut and midgut contents harbor bacterial communities. However, whether the bacterial communities of the insect midgut are similar to those of the insect midgut contents (including the peritrophic membrane, food particles, and digestive fluids secreted by the midgut in this study) remains unknown. In the present study, we analyzed two economically important silkworms, the Chinese oak silkworm Antheraea pernyi (Lepidoptera: Saturniidae) and the mulberry silkworm Bombyx mori (Lepidoptera: Bombycidae), through Illumina MiSeq technology to address this issue. In A. pernyi larvae, 17 phyla and 162 genera were found in the midgut, while 7 phyla and 36 genera were found in the midgut contents. For B. mori larvae, 30 phyla and 465 genera were found in the midgut, but 22 phyla and 344 genera were found in the midgut contents. This evidence from the two silkworms suggests that the bacterial composition and diversity in the midgut are more diverse than those in the midgut contents. Principal component analysis revealed a significant difference in the bacterial community structure between the midgut and midgut contents of B. mori. To our knowledge, this is the first study to compare the bacterial communities between the midgut and midgut contents in insects, and the results will provide useful information for probing the functional differentiation within the midgut in the future.

A high level of chloroplast genome sequence variability in the Sawtooth Oak Quercus acutissima.

The Sawtooth Oak, Quercus acutissima Carruth., is an economically and ecologically important tree species in the family Fagaceae with a wide distribution in China. Here, we examined its intraspecific chloroplast (cp) genome variability using available and a newly sequenced genome. The new cp genome comes from a Q. acutissima individual collected from Shenyang (Northeast China; "Q. acutissima Shenyang" in the following), and then is compared with two recently published cp genomes from Tongchuan (Northwest China) and Nanjing (East China). The cp genome of Q. acutissima Shenyang exhibits a slightly larger genome size than the other two individuals, although each encodes 86 protein-coding genes, 40 tRNA genes and eight rRNA genes. We also found the length difference for the IR/SC boundary region among the three cp genomes. Sequence comparison revealed a high intraspecific genetic divergence: the three cp genomes differ by 332 sequence patterns including 77 single nucleotide polymorphisms, and 255 indels (each gap considered) scattering across 67 regions. Phylogenetic analyses based on the cp genome recovered the split between the subgenus Cerris and the subgenus Quercus, but revealed that three Q. acutissima individuals did not cluster together, indicating that even complete cp genome data fail to reproduce species boundaries in Asian members of section Cerris. Our results show that more complete plastomes covering remote ranges needs to be sequenced to provide a solid backbone for future population-scale in-depth studies and phylogenetic analysis of section Cerris.

Codon Usage in the Iflaviridae Family Is Not Diverse Though the Family Members Are Isolated from Diverse Host Taxa.

All iflavirus members belong to the unique genus, Iflavirus, of the family, Iflaviridae. The host taxa and sequence identities of these viruses are diverse. A codon usage bias, maintained by a balance between selection, mutation, and genetic drift, exists in a wide variety of organisms. We characterized the codon usage patterns of 44 iflavirus genomes that were isolated from the classes, Insecta, Arachnida, Mammalia, and Malacostraca. Iflaviruses lack a strong codon usage bias when they are evaluated using an effective number of codons. The odds ratios of the majority of dinucleotides are within the normal range. However, the dinucleotides at the 1st-2nd codon positions are more biased than those at the 2nd-3rd codon positions. Plots of effective numbers of codons, relative neutrality analysis, and PR2 bias analysis all indicate that selection pressure dominates mutations in shaping codon usage patterns in the family, Iflaviridae. When these viruses were grouped into their host taxa, we found that the indices, including the nucleotide composition, effective number of codons, relative synonymous codon usage, and the influencing factors behind the codon usage patterns, all show that there are non-significant differences between the six host-taxa-groups. Our results disagree with our assumption that diverse viruses should possess diverse codon usage patterns, suggesting that the nucleotide composition and codon usage in the family, Iflaviridae, are not host taxa-specific signatures.

Characterization of a highly conserved Antheraea pernyi spermidine synthase gene.

In the present study, we isolated a spermidine synthase gene from Antheraea pernyi (ApSpds) using expressed sequence tag method. The obtained cDNA sequence of 1483 bp contains an open-reading frame of 864 bp encoding a polypeptide of 287 amino acids. Sequence analysis revealed that ApSpds belonged to class I of AdoMet-MTase family, and exhibited 30% identity to those from bacteria, 45-48% identity to fungi, 36-47% identity to plants, 52-54% identity to vertebrates and 53-80% identity to invertebrates. Phylogenetic analysis found that the used Spds protein sequences were well divided into five groups corresponding to bacteria, fungi, plants, invertebrates and vertebrates, respectively. These results further confirmed that Spds is highly conserved through evolution of life organisms. The ApSpds mRNA is expressed during all four developmental stages and is present in all examined tissues with the highest abundance in the muscle, in which the relative mRNA expression level was 1.6 times higher than in the fat body. Although not significant, the mRNA level decreased after high-temperature exposure suggesting that the Spds gene may not be involved in temperature stress tolerance in A. pernyi. Taken together, our results suggested that ApSpds play an important role in development of silkworm.

Transcriptomic analysis of the prothoracic gland from two lepidopteran insects, domesticated silkmoth Bombyx mori and wild silkmoth Antheraea pernyi.

The prothoracic gland (PG) is an important endocrine organ of synthesis and secretion of ecdysteroids that play critical roles in insects. Here, we used a comparative transcriptomic approach to characterize some common features of PGs from two lepidopteran species Bombyx mori and Antheraea pernyi. Functional and pathway annotations revealed an overall similarity in gene profile between the two PG transcriptomes. As expected, almost all steroid hormone biosynthesis genes and the prothoracicitropic hormone receptor gene (Torso) were well represented in the two PGs. Impressively, two ecdysone receptor genes, eleven juvenile hormone related genes, more than 10 chemosensory protein genes, and a set of genes involved in circadian clock were also presented in the two PGs. Quantitative real time -PCR (qRT-PCR) validated the expression of 8 juvenile hormone and 12 clock related genes in B. mori PG, and revealed a different expression pattern during development in whole fifth larval instar. This contribution to insect PG transcriptome data will extend our understanding of the function and regulation of this important organ.

Comparative mitochondrial genomes provide new insights into the true wild progenitor and origin of domestic silkworm Bombyx mori.

The domestication of domestic silkworm Bombyx mori, the only truly domesticated insect, is a distinctive event in agricultural history. The domestication and origin of domestic silkworm remains unclear, although it has connected with human for ~5500 years. In the present study, we would like to highlight our evidence from whole mitochondrial genome for the presence of two genetically distinctive subtypes in Chinese B. mandarina populations, corresponding to northern Chinese B. mandarina and southern Chinese B. mandarina, respectively. The mitochondrial genomes and mitochondrial phylogenetic tree provide a solid molecular evidence that the true wild ancestor of domestic silkworm is northern Chinese B. mandarina, rather than southern Chinese B. mandarina, thus implying that the early domestication event may have occurred in northern China. Our finding provides new insights into the origin and evolution of domestic silkworm.

Characterization of the Adult Head Transcriptome and Identification of Migration and Olfaction Genes in the Oriental Armyworm Mythimna separate.

The oriental armyworm Mythimna separate is an economically important insect with a wide distribution and strong migratory activity. However, knowledge about the molecular mechanisms regulating the physiological and behavioural responses of the oriental armyworm is scarce. In the present study, we took a transcriptomic approach to characterize the gene network in the adult head of M. separate. The sequencing and de novo assembly yielded 63,499 transcripts, which were further assembled into 46,459 unigenes with an N50 of 1,153 bp. In the head transcriptome data, unigenes involved in the 'signal transduction mechanism' are the most abundant. In total, 937 signal transduction unigenes were assigned to 22 signalling pathways. The circadian clock, melanin synthesis, and non-receptor protein of olfactory gene families were then identified, and phylogenetic analyses were performed with these M. separate genes, the model insect Bombyx mori and other insects. Furthermore, 1,372 simple sequence repeats of 2-6 bp in unit length were identified. The transcriptome data represent a comprehensive molecular resource for the adult head of M. separate, and these identified genes can be valid targets for further gene function research to address the molecular mechanisms regulating the migratory and olfaction genes of the oriental armyworm.

Supercooling Capacity and Cold Tolerance of the Wild Silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

While wild silkworms have served humans for several thousand years, little attention on cold hardiness has been paid to these economically important species. In the present study, supercooling capacity and low temperature tolerance of Chinese oak silkworm, Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae), an economic insect reared both for silk production as well as human food, were examined under laboratory conditions. The supercooling points (SCPs) of pupae dropped significantly from a mean of -15.6°C in prediapause to -20.1°C in diapause, and then increased to -17.5°C during postdiapause development. Sex and voltinism influenced body mass but had no significant effect on the SCP. Our data demonstrated that cold tolerance of A. pernyi is tightly linked to life stage. Exposure of eggs to -5°C for up to 8 h had no effect on the hatching rate, whereas silkworm larvae failed to break through the chorion and hatch following a 4-8-h exposure to -10°C. Mean SCPs of intact eggs and naked larvae one day before hatching were similar, -23.3°C and -22.3°C, respectively, indicating that chorion does not significantly affect SCP. Comparison of lower lethal temperature (LLT50) and SCP means suggested that both pupae and eggs of A. pernyi are chill intolerant. These data will improve our understanding of low temperature tolerance in this commercially important species.

Molecular characterization and phylogenetic analysis of the eukaryotic translation initiation factor 4A gene in Antheraea pernyi (Lepdoptera: Saturniidae).

Eukaryotic initiation factor 4A (eIF-4A) is an essential component for protein translation in eukaryotes. The eIF-4A gene (ApeIF-4A) was isolated and characterized from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae). The obtained cDNA sequence was 1,435-bp long with an open reading frame of 1,266 bp encoding 421 amino acids. The predicted amino acid sequence shared several conserved features as found in known eIF-4As and revealed 74 and 78% identities with eIF-4As of Homo sapiens L. and Drosophila melanogaster (Meigen), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that ApeIF-4A was transcribed at four developmental stages and in all tissues tested, suggesting that it plays an important role in development of A. pernyi. Homologous alignment suggested that eIF-4As are highly conserved throughout evolution of eukaryote organisms. Phylogenetic trees based on the amino acid and nucleotide sequences of eIF-4A demonstrated a similar topology with the classical systematics, suggesting that it has the potential value in phylogenetic inference of eukaryotes.

Complete mitochondrial genome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae) and the phylogenetic relationship of Saturniidae species.

Mitochondrial genome (mitogenome) can provide information for genomic structure as well as for phylogenetic analysis and evolutionary biology. In this study, we present the complete mitogenome of the atlas moth, Attacus atlas (Lepidoptera: Saturniidae), a well-known silk-producing and ornamental insect with the largest wing surface area of all moths. The mitogenome of A. atlas is a circular molecule of 15,282 bp long, and its nucleotide composition shows heavily biased towards As and Ts, accounting for 79.30%. This genome comprises 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an A+T-rich region. It is of note that this genome exhibits a slightly positive AT skew, which is different from the other known Saturniidae species. All PCGs are initiated by ATN codons, except for COI with CGA instead. Only six PCGs use a common stop codon of TAA or TAG, whereas the remaining seven use an incomplete termination codon T or TA. All tRNAs have the typical clover-leaf structure, with an exception for tRNA(Ser)(AGN). The A. atlas A+T-rich region contains non-repetitive sequences, but harbors several features common to the Bombycoidea insects. The phylogenetic relationships based on Maximum Likelihood method provide a well-supported outline of Saturniidae, which is in accordance with the traditional morphological classification and recent molecular works.

Selective pressure dominates the synonymous codon usage in parvoviridae.

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence's composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.

Molecular cloning, expression pattern and phylogenetic analysis of the will die slowly gene from the Chinese oak silkworm, Antheraea pernyi.

The will die slowly (wds) gene coding for a WD-repeat protein with seven repeats has been characterized in Drosophila melanogaster. In this paper, the wds gene was isolated and characterized from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae). The obtained 1733 bp cDNA sequence contains an open reading frame of 1041 bp encoding a polypeptide of 346 amino acids, with 85% sequence identity to that from D. melanogaster. RT-PCR analysis showed that the wds gene was transcribed during four developmental stages and in all the tissues tested, consistent with the result observed in Bombyx mori based on EST resources and genome-wide microarray information. The mRNA expression level of the A. pernyi wds gene was not significantly down- or up- regulated under temperature stress compared to the control, indicating that it may be not involved in temperature stress tolerance. In search of database, the wds protein homologues were found in various kinds of eukaryotes, including fungi, plants, invertebrates and vertebrates, with 50-93% amino acid sequence identities between them, suggesting that they are highly conserved during the evolution of eukaryotes. Phylogenetic analysis based on the wds protein homologue sequences clearly separated the known fungi, plants, invertebrates and vertebrates, consistent with the topology tree on the classical systematics, suggesting the potential value of wds protein in eukaryotic phylogenetic inference. In vertebrates, two apparent types of the wds proteins were also defined by sequence alignment and phylogenetic analysis.

cDNA cloning and expression pattern of homolog of alpha subunit of platelet-activating factor acetylhydrolase Ib from the Chinese oak silkworm, Antheraea pernyi.

Platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme that catalyzes the hydrolysis of platelet-activating factor (PAF). A homolog of alpha subunit of PAF-AH(Ib) from Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) (ApPAFAHIbα) was isolated and characterized. The obtained cDNA sequence was 1843 base pairs (bp) long with an open reading frame (ORE) of 678 bp encoding 225 amino acids. The predicted amino acid sequence shared several conserved features of PAF-AHs of other organisms, and revealed 88, 60, and 46% identity with the homologues of Bombyx mori, Drosophila melanogaster, and Homo sapiens, respectively. Phylogenetic analysis indicated that lepidopteran PAFAHIbαs including ApPAFAHIbα might be a new member of the PAF-AHs family of insects. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the ApPAFAHIbα gene was transcribed at four developmental stages and expressed in all tissues tested.

Comparative genetic diversity and genetic structure of three chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini donovan (Lepidoptera: Saturniidae)

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60 percent and 72 percent of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16 percent of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.

cDNA cloning and expression pattern of two enolase genes from the Chinese oak silkworm, Antheraea pernyi.

In this study, two enolase genes were isolated and characterized from the Chinese oak silkworm, Antheraea perny, which were designated as enolase I and II, respectively. The enolase I cDNA sequence was 1712 bp with an open reading frame (ORF) of 1302 bp encoding 433 amino acids. The enolase II cDNA sequence was 1549 bp with an ORF of 1296 bp encoding 431 amino acids. The amino acid sequences of the two genes share several conserved features/sites of enolase. Antheraea pernyi enolase I shows 93%-97% sequence identity to enolases of lepidopterans available to date, 75%-82% identity to enolases of other invertebrates, 60%-72% identity to enolases of other organisms including vertebrates, plants, and fungi. Antheraea pernyi enolase II shows 84% identity to Bombyx mori enolase II, but 60% identity to A. pernyi enolase I. In the phylogenetic tree, enolase II sequences from A. pernyi and B. mori were clearly separated from the majority of enolase sequences of higher organisms including A. pernyi and B. mori enolase I sequences. By sequence comparisons and phylogenetic analysis, we suggest that enolase II from A. pernyi and B. mori may be a new member of the enolase superfamily. Antheraea pernyi enolase I mRNA was found in all tested tissues whereas enolase II mRNA was expressed specifically in the spermaries and ovaries, suggesting that the product of enolase II gene may be related to reproduction. The transcript abundance of A. pernyi enolase I gene was significantly down-regulated after cold shock and significantly up-regulated after heat shock, suggesting that A. pernyi enolase I gene may be inducible by temperature stress.

Comparative genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan (Lepidoptera: Saturniidae).

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60% and 72% of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16% of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.