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.

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.

Peptidoglycan recognition proteins regulate immune response of Antheraea pernyi in different ways.

Peptidoglycan recognition proteins (PGRPs) are evolutionarily conserved molecules that act in innate immune responses against invading pathogens. Insect PGRPs activate the Toll and/or immune deficiency (IMD) signal transduction pathways. They induce cellular and humoral immune defense reactions that effectively protect insects from invasion of microorganisms. In this study, four cDNA clones encoding PGRPs (ApPGRP-A, ApPGRP-B, ApPGRP-C, ApPGRP-LE) were isolated from the Chinese oak silkworm, Antheraea pernyi. The deduced amino acid sequences of ApPGRP-B and -C share high identity with enzymatically active PGRP proteins and contain the amino acids required for amidase activity. The spatiotemporal expression patterns of ApPGRPs and their response to immune stimulations were determined, and suggest that PGRPs might act as a broad-spectrum pattern recognition protein and participate in the pathway activation system. Here, using an RNA-interference approach, we examined the function of PGRPs in response to immune stimulation. We observed that RNAi-mediated silencing of ApPGRP-A decreased the tested antimicrobial peptides (Attacin, Attacin 2, Ceropin, Gloverin, lysozyme and Lebocin) in response to Enterococcus pernyi challenge. However, reducing the ApPGRP-B, -C mRNA levels led to a strong increase of the AMP genes (except for Lebocin). These results suggest that ApPGRPs are necessary for pathway initiation complex formation and activate AMP generation. Our data also indicated that PGRP-B and -C down-regulate AMPs generation in the immune response.

Transcriptome sequencing to unravel the molecular mechanisms underlying the cuticle liquefaction of Antheraea pernyi following Antheraea pernyi nucleopolyhedrovirus challenge.

Baculovirus causes liquefaction of insect cuticle to enhance the dissemination of progeny virions away from the host cadavers for increasing viral transmission rates. Antheraea pernyi nucleopolyhedrovirus (ApNPV) infects A. pernyi larvae with circular pus blotches formed in cuticle in the early stage of liquefaction. To investigate the formation mechanism of those pus blotches, the transcriptome profile changes of the cuticles between ApNPV-infected and non-infected A. pernyi larvae were analyzed using RNA-Seq. The transcriptome was de novo assembled using the Trinity platform. Comparison of gene expression levels revealed that a total of 2990 and 4427 unigenes were up- and down-regulated respectively in ApNPV-infected cuticle, of which 2620 and 1903 differentially expressed genes (DEGs) could be enriched in different GO terms and KEGG pathways. In this study, we focused on chitin metabolism related DEGs, and screened 10 genes involved in chitin synthesis and degradation with down-regulated trends, indicating that the chitin metabolism pathway was inhibited by ApNPV infection, which may promote liquefaction of A. pernyi cuticle. Besides, we also identified a large number of DEGs involved in immune related pathways via KEGG analysis, indicating that intense immune responses occurred in A. pernyi cuticle. Our research findings will serve as a basis for further researching the molecular mechanisms underlying cuticle liquefaction of A. pernyi induced by ApNPV infection.

The Complete Mitochondrial Genome of the Longhorn Beetle Dorysthenes paradoxus (Coleoptera: Cerambycidae: Prionini) and the Implication for the Phylogenetic Relationships of the Cerambycidae Species.

The longhorn beetle Dorysthenes paradoxus (Faldermann, 1833) (Coleoptera: Cerambycidae) is not only a serious agricultural pest but also a traditionally edible insect in China. However, no genetic information on this species has been acquired. In the present study, we report the mitochondrial genome (mitogenome) of Do. paradoxus, as the first complete mitogenome of Prioninae. The circular mitogenome of 15,922 bp encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), and it contains an A+T-rich region. This mitogenome exhibits the lowest A+T content (71.13%) but harbors the largest AT skew (0.116) among the completely sequenced Cerambycidae species. Eleven of the 13 PCGs have a typical ATN start codon, whereas COI and ND1 are tentatively designated by AAT and TTG, respectively. Only 4 of the 13 PCGs harbor a complete termination codon, and the remaining 9 possess incomplete termination codons (T or TA). Apart from tRNASer(AGN), the other 21 tRNAs can fold into a typical clover-leaf secondary structures. The Do. paradoxus A+T-rich region contains two poly-T stretches and a tandem repeat that comprises two 47-bp-long copies. Both Bayesian inference and Maximum likelihood analyses confirmed the subfamily ranks of Cerambycidae ([Prioninae + Cerambycinae] + Lamiinae) and the close relationship between Philinae and Prioninae/Cerambycinae. However, the data did not support the monophyly of Prioninae and Cerambycinae. The mitogenome presented here provides basic genetic information for this economically important species.

Morphological characterization and HSP70-, IGS-based phylogenetic analysis of two microsporidian parasites isolated from Antheraea pernyi.

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 µm, and the other type was short-axis-oval, measuring 3.64 × 2.17 µm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 µm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.

Codon usage in Alphabaculovirus and Betabaculovirus hosted by the same insect species is weak, selection dominated and exhibits no more similar patterns than expected.

Mutations shape synonymous codon usage bias in certain organism genomes, while selection shapes it in others. Lepidopteran-specific Alphabaculovirus and Betabaculovirus are two large genera in the family of Baculoviridae. In this study, we analyzed the codon usage patterns in 17 baculoviruses, including 10 alphabaculoviruses and 7 betabaculoviruses, which were isolated from seven insect species, and we characterized the codon usage patterns between Alphabaculovirus and Betabaculovirus. Our results show that all the baculoviruses possessed a general weak trend of codon bias. The differences of ENc (effective number of codons) values, nucleotide contents and the impacts of nucleotide content on ENc value within alpha-/betabaculovirus pairs were independent of whether the host species are the same or different. Furthermore, the majority of amino acid sequences adopted codons unequally in all viruses, but the numbers of common preferred codons between alpha- and betabaculoviruses hosted by the same insect species were not significantly different from the differences observed between alpha- and betabaculoviruses hosted by different insect species. In addition, the amino acids that adopt the same synonymous codon composition between alpha- and betabaculoviruses hosted by the same insect species were statistically as few as those between alpha- and betabaculoviruses hosted by different insect species. Correspondence analysis revealed that no major factors resulted in the codon bias in these baculoviruses, implying multiple minor influential factors exist. Neutrality plot analysis indicated that selection pressure dominated mutations in shaping the codon usage. However, the levels of selection pressure were not significantly different among viruses hosted by the same insect species. We expect that evolution would cause the alpha- and betabaculoviruses hosted by the same insect species to share more patterns, but this effect was not observed.

Identification and Characterization of a Novel Microvitellogenin from the Chinese Oak Silkworm Antheraea pernyi.

Microvitellogenin (mVg) is a relatively small vitellogenic protein only characterized in the eggs of the lepidopteran insects Manduca sexta and Bombyx mori. In the present study, we report a novel mVg (ApmVg) isolated from the Chinese oak silkworm Antheraea pernyi. The obtained ApmVg cDNA sequence contains an open reading frame of 783 bp encoding a protein of 260 amino acids with a predicted molecular weight of 29.96 kDa. This gene does not contain introns. Structural analysis revealed that this protein shares putative conserved domains with the lepidopteran low-molecular weight lipoprotein, which belongs to the lipoprotein_11 superfamily. The protein sequence of ApmVg exhibits 48% sequence identity with mVg from M. sexta and 40-47% sequence identity with the 30K lipoproteins from B. mori. Phylogenetic analysis suggests that ApmVg is a novel member of the lepidopteran low-molecular weight lipoproteins. Transcriptional analysis indicated that ApmVg mRNA is mainly expressed in the fat body (both female and male) during post-diapause development of the pupal stage, and it was also detected in ovaries and spermaries in smaller amounts. RT-PCR and Western blot analyses revealed that ApmVg is synthesized by the fat body and secreted into hemolymph and ultimately accumulates in eggs. The ApmVg transcript can be detected in the fat bodies of female pupae four days after treatment with 20-hydroxyecdysone and shows an expression pattern distinct from that of vitellogenin (Vg), which is detectable throughout diapausing and in post-diapause development. ApmVg decreased dramatically during embryonic development. These results represent the first study of mVg outside M. sexta and B. mori and provide insight into the physiological role and evolution of mVgs.

Morphological and molecular characterization of Nosema pernyi, a microsporidian parasite in Antheraea pernyi.

Nosema pernyi is a lethal pathogen that causes microsporidiosis in the Chinese oak silkworm, Antheraea pernyi. In this study, we presented its morphological and some molecular characteristics. The mature spores were measured to be 4.36 × 1.49 µm. The spore wall consisted of an electron-dense exospore (EX) and electron-lucent endospore (EN) layer. The polar filament (PF) was isofilar with 10-12 coils that were frequently arranged in a single row. Investigation results indicated that N. pernyi can infect the gut wall, silk glands, and other tissues. A full-length SMART cDNA library of N. pernyi was constructed, and then 824 expressed sequence tags (ESTs) were sequenced. Ninety unigenes, out of 197 assembled unigenes, showed significant homology to known genes of Nosema ceranae, Nosema bombycis, Encephalitozoon cuniculi, and other microsporidian species. Based on the nucleotide sequence of the α- and ß-tubulin genes and amino acid sequence of actin gene, phylogenetic trees analysis showed that N. pernyi was closely related to Nosema philosamiae and Nosema antheraeae. It was correctly assigned to the Nosema group.

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.

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina.

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.

Characterization of Antheraea pernyi nucleopolyhedrovirus p11 gene, a homologue of Autographa californica nucleopolyhedrovirus orf108.

Antheraea pernyi nucleopolyhedrovirus (ApNPV) p11 gene is 309 bp long, potentially encoding 102 amino acids with a predicted molecular weight of 11.2 kDa. ApNPV p11 gene was cloned into the prokaryotic expression vector pQE-30 and P11 was expressed in E. coli M15. Polyclonal antiserum was made against 6xHis tagged P11 protein expressed in E. coli M15. P11 gene transcription was detected as early as 36 h post-infection (p.i.) in tussah pupa and remain at high level up to 96 h p.i. Structural localization revealed that P11 protein was present in polyhedral inclusion bodies (PIB) dilute alkaline saline (DAS) pellet (P) fractions and occlusion-derived virus (ODV), but not in PIB DAS supernatant (S) fractions and budded virus (BV). These results indicated that P11 was associated with ApNPV structure.