Biology of Rhynchaenus maculosus provides insights and implications for integrated management of this emerging pest.

Rhynchaenus maculosus is an emerging insect pest with an increasingly serious tendency. Lack of biology information results in the bottleneck of integrated management of this pest. To facilitate an available design of integrated pest management strategy, biology of R. maculosus, including voltinism, life cycle, distribution, and damage has been investigated. Results reveal that R. maculosus is oligophagous and distributes in Heilongjiang, Jilin, and Liaoning provinces, China. This pest produces one generation per year (univoltinism) and overwinters as adults in leaf litter. From mid-April to late-April, active overwintering adults emerge from overwintering sites. The next generation of adult R. maculosus appears from mid-May to early June until mid-August to early September when the beetles move into the overwintering places. The entire time span of adult occurrence ranges from 315.6 ± 3.6 to 336.4 ± 3.2 days (Mean ± SD). Larvae undergo 3 instars with a total duration of 20 to 23 days. R. maculosus larvae feed on Q. wutaishanica and Q. mongolica without host-specific preference between the two host species, but do not harm Q. acutissim. Three species of larval parasites were collected and identified as Braconidae sp., Eulophidae sp., and Ceraphronidae sp. Biological information of R. maculosus provides essential insights for design and implementation of integrated management of this pest.

Germplasm Resources of Oaks (Quercus L.) in China: Utilization and Prospects.

Oaks exhibit unique biological characteristics and high adaptability to complex climatic and soil conditions. They are widely distributed across various regions, spanning 40 degrees latitude and 75 degrees longitude. The total area of oak forest in China is 16.72 million hm2. There are 60 lineages of Quercus in China, including 49 species, seven varieties, and four subgenera. Archaeological data indicate that oaks were already widely distributed in ancient times, and they are dominant trees in vast regions of China's forests. In addition, the acorn was an important food for ancestral humans, and it has accompanied human civilization since the early Paleolithic. Diverse oak species are widely distributed and have great functional value, such as for greening, carbon sequestration, industrial and medicinal uses, and insect rearing. Long-term deforestation, fire, diseases, and pests have led to a continuous decline in oak resources. This study discusses the Quercus species and their distribution in China, ecological adaptation, and the threats facing the propagation and growth of oaks in a changing world. This will give us a better understanding of Quercus resources, and provide guidance on how to protect and better utilize germplasm resources in China. The breeding of new varieties, pest control, and chemical and molecular research also need to be strengthened in future studies.

Nitric oxide plays a crucial role in midgut immunity under microsporidian infection in Antheraea pernyi.

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-L-nitro-arginine methyl ester, hydrochloride (L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.

Polar tube structure and three polar tube proteins identified from Nosema pernyi.

Microsporidian spores contain a single polar filament that is coiled around the interior of the spore. Upon germination the polar tube (post-germination polar filament) is ejected by inversion into a host cell. The sporoplasm flows through the polar tube, directly infecting the cytoplasm of the cell. Various species of microsporidia display differences in the number of coils in the polar filament and in the amino acid sequence of the polar tube proteins (PTPs). Nosema pernyi is a lethal pathogen that causes microsporidiosis in the Chinese oak silkworm, Antheraea pernyi. In this study, we identified three PTPs in N. pernyi using RT-PCR and LC-MS/MS. Polar tube protein 3 was localized in the polar tube using immuno-histochemical staining and an immunofluorescence assay. Co-immunoprecipitation data and LC-MS/MS analysis revealed that some potential proteins, like immune related proteins in A. pernyi may interact with PTP3.

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.

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.

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.

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.

Upregulation of a Trypsin-Like Serine Protease Gene in Antheraea pernyi (Lepidoptera: Saturniidae) Strains Exposed to Different Pathogens.

Antheraea pernyi Guérin-Méneville is used for silk production and as a food resource. Its infection by exogenous pathogens, including microsporidia, fungi, bacteria, and virus, can lead to silkworm diseases, causing major economic losses. A trypsin-like serine protease gene (TLS) was found in A. pernyi transcriptome data resulting from two different infection experiments. The cDNA sequence of ApTLS was 1,020 bp in length and contained an open reading frame of 774 bp encoding a 257-amino acid protein (GenBank KF779933). The present study investigated the expression patterns of ApTLS after exposure to different pathogens, and in four different A. pernyi strains. Semiquantitative RT-PCR indicated that ApTLS was expressed in all developmental stages and was most expressed in the midgut. Quantitative real-time PCR indicated ApTLS was upregulated in the midgut of A. pernyi exposed to nucleopolyhedrovirus (ApNPV), Nosema pernyi, Enterococcus pernyi, and Beauveria bassiana infections, and the highest gene expression level was found under ApNPV infection. The strain Shenhuang No. 2 presented the lowest infection rate and the highest ApTLS gene expression level when exposed to ApNPV. Thus, ApTLS seems to be involved in innate defense reactions in A. pernyi.

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.

Transcriptome Analysis of the Midgut of the Chinese Oak Silkworm Antheraea pernyi Infected with Antheraea pernyi Nucleopolyhedrovirus.

The Antheraea pernyi nucleopolyhedrovirus (ApNPV) is an exclusive pathogen of A. pernyi. The intense interactions between ApNPV and A. pernyi cause a series of physiological and pathological changes to A. pernyi. However, no detailed report exists regarding the molecular mechanisms underlying the interactions between ApNPV and A. pernyi. In this study, four cDNA libraries of the A. pernyi midgut, including two ApNPV-infected groups and two control groups, were constructed for transcriptomic analysis to provide new clues regarding the molecular mechanisms that underlie these interactions. The transcriptome of the A. pernyi midgut was de novo assembled using the Trinity platform because of the lack of a genome resource for A. pernyi. Compared with the controls, a total of 5,172 differentially expressed genes (DEGs) were identified, including 2,183 up-regulated and 2,989 down-regulated candidates, of which 2,965 and 911 DEGs were classified into different GO categories and KEGG pathways, respectively. The DEGs involved in A. pernyi innate immunity were classified into several categories, including heat-shock proteins, apoptosis-related proteins, serpins, serine proteases and cytochrome P450s. Our results suggested that these genes were related to the immune response of the A. pernyi midgut to ApNPV infection via their essential roles in regulating a variety of physiological processes. Our results may serve as a basis for future research not only on the molecular mechanisms of ApNPV invasion but also on the anti-ApNPV mechanism of A. pernyi.

The Toll Signaling Pathway in the Chinese Oak Silkworm, Antheraea pernyi: Innate Immune Responses to Different Microorganisms.

The Toll pathway is one of the most important signaling pathways regulating insect innate immunity. Spatzle is a key protein that functions as a Toll receptor ligand to trigger Toll-dependent expression of immunity-related genes. In this study, a novel spatzle gene (ApSPZ) from the Chinese oak silkworm Antheraea pernyi was identified. The ApSPZ cDNA is 1065 nucleotides with an open reading frame (ORF) of 777 bp encoding a protein of 258 amino acids. The protein has an estimated molecular weight of 29.71 kDa and an isoelectric point (PI) of 8.53. ApSPZ is a nuclear and secretory protein with no conserved domains or membrane helices and shares 40% amino acid identity with SPZ from Manduca sexta. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in 5th instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Tolloid, cactus and dorsalA. The RT-PCR results showed that these genes were predominantly expressed in immune-responsive fat body tissue, indicating that the genes play a crucial role in A. pernyi innate immunity. Moreover, A. pernyi infection with the fungus Nosema pernyi and the gram-positive bacterium Enterococcus pernyi, but not the gram-negative bacterium Escherichia coli, activated the Toll signaling pathway. These results represent the first study of the Toll pathway in A. pernyi, which provides insight into the A. pernyi innate immune system.

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.

Genome Sequence of Enterococcus pernyi, a Pathogenic Bacterium for the Chinese Oak Silkworm, Antheraea pernyi.

We report the draft genome assembly of Enterococcus pernyi The genome sequence is 3.09 Mb in length with a G+C content of 38.35%. It covers 3,153 genes with an average length of 854 bp, and contains 65 tRNAs, 13 small RNAs, and 18 rRNAs. Moreover, it contains 9 genomic islands with an average length of 14,058 bp and 3 prophages with an average length of 37,430 bp.

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.