| The Chinese oak silkworm (Antheraea pernyi Guerin-Meneville,1855; Lepidoptera: Saturniidae) is a well-known wild silkworm used for many fields, such as insect food, textile & clothing industry, cosmetics industry, and health care industry, etc. Currently, the Chinese annual output of tussah cocoons is more than 8×104 t, which is nearly 90% of the total output of wild silk worldwide, and with a high economic value, tussah industry has become one of the main economic sources in many sericultural areas. Meanwhile, as a typical lepidopteran insect, A. pernyi larvae are fed on the leaves of oak trees in tussah-feeding oak forests until cocooning during the larval stage. There are a lot of similarities in biological rules and pathological process between A. pernyi and Iepidopterous pest. So A. pernyi can be studied as a model insect to provide some reference for pest control. A. pernyi empty-gut disease is one of the most important diseases in tussah rearing, this disease affects seriously the yield of tussah cocoon and causes great economic losses. And it was widely distributed in national sericultural areas and was greatly harmful to tussah rearing. However, there is limited information about this disease in A. pernyi, it is necessary to carry on data accumulation and system research. Therefore, for the health and sustainable development of tussah industry, it is very important to study the pathogen of A. pernyi empty-gut disease, interaction between pathogen and host, and host innate immunity to microbial infections for understanding the pathogenesis and development of targeted prevention and control of empty-gut disease.In this paper, the complete genome sequence of the pathogen of A. pernyi empty-gut disease was determined by Illumina Solexa technology, and sequences assembly was performed using SOAPdenovo-V2.04. The comparative proteomics of hemolymph from the fifth instar larvae of health A. pernyi and A. pernyi infected by the pathogen of empty-gut disease have been studied using iTRAQ. A novel spatzle gene (ApSPZ) from the Chinese oak silkworm, A. pernyi, which is a key signal transducer and a ligand for Toll receptors for activating the Toll pathway in response to different microorganisms, has been identified using RT-PCR and RACE, its prokaryotic expression vector has been constructed, and its polyclonal antibody has been prepared. The expression patterns of genes involved in the Toll pathway have been examined in A. pernyi using semi-quantitative RT-PCR, and the relative expression levels of these genes have been determined in A. pernyi infected with different microorganisms using realtime PCR. The genome of the pathogen of empty-gut disease was sequenced with a view to better understanding the taxonomic status of this bacterium and providing more genomic information for further study to mine functional genes, such as virulence factors. The differently expressed proteins were identified from the health A. pernyi and A. pernyi infected with the pathogen of empty-gut disease, the results can provide a foundation for studying the interaction between pathogen and host. The function of Toll pathway in response to different microorganisms was investigated, the results can provide a foundation for further studying the innate immunity of A. pernyi. These fingings of this paper have important reference value to revealing the pathogenesis, selecting resistant varieties and development of prevention and control of A. pernyi empty-gut disease.The results were shown as follows:1. The genome size of the pathogen of A. pernyi empty-gut disease is 3.09 Mb with C+C content 38.35%. A total of 626 Mb clean data were generated, reaching over 200-fold genome coverage depth. Sequences were assembled into 23 contigs with a total length of 3 181 210 bp (largest,603 828 bp, and smallest,654 bp) and with an N50 contig size of 370 188 bp. Finally, there were a total of 9 scaffolds with a total length of 3 188 572 bp (largest,3 086 269 bp, and smallest,654 bp) and with an N50 scaffold size of 3 086 269 bp). The genome contains 3 153 coding DNA sequences (CDSs) with an average length of 854 bp, which represent 84.48% of the whole genome. The annotation results showed that only 224 CDSs (7.1%) were not annotated into any databases, and there were 2 929 CDSs annotated into at least one database, there were 2 916,1 537,1 577,1 487,1 242,2 812,21,39 and 1 CDSs annotated into NCBInr, COG, GO, KEGG, Swiss-Prot, TrEMBL, PHI, VFDB and ARDB, respectively. Meanwhile,65 tRNAs,19 rRNAs and 13 small RNAs were identified. There were 108 interspered repeats and 223 tandem repeats indentified in the genome. Moreover, the genome" contains 9 genomic islands (GIs) with an average length of 14 058 bp, and contains 3 prophages with an average length of 37 430 bp. There was no clustered regularly interspaced short palindromic repeat (CRISPR) identified in the genome. The whole-genome sequences of the pathogen of A. pernyi empty-gut disease have been deposited at DDBJ/EMBL/GenBank under the accession LPVT00000000. Furthermore, phylogenetic trees constructed based on genome level indicated that the pathogen of A. pernyi empty-gut disease is Enterococcus pernyi.2. The comparative proteomics of hemolymph from the fifth instar larvae of health A. pernyi and A. pernyi infected by the pathogen of empty-gut disease, Enterococcus pernyi, have been studied using iTRAQ.2 206 proteins from A. pernyi and 33 proteins from E. pernyi were identified. The annotation results of proteins identified from E. pernyi showed that,129 protcins were annotated into 24 GO terms, including 10 GO biological process terms (49 proteins),9 GO cellular component terms (43 proteins) and 5 GO molecular function terms (37 proteins). In molecular function, the number of proteins annotated to binding was most (17 proteins,45.95%), followed by the proteins annotated to catalytic activity (14 proteins, 37.84%). Moreover,35 proteins were annotated into 15 COG categories,28 proteins were annotated into 43 pathways. The annotation results of proteins identified from A. pernyi showed that,7 495 proteins were annotated into 51 GO terms, including 22 GO biological process terms (3 495 proteins),15 GO cellular component terms (2 438 proteins) and 14 GO molecular function terms (1 562 proteins). In molecular function, the number of proteins annotated to catalytic activity was most (692 proteins,44.30%), followed by the proteins annotated to binding (611 proteins,39.12%). Moreover,1445 proteins were annotated into 24 COG categories,1783 proteins were annotated into 295 pathways.Quantitative analysis showed that there were 305 differently expressed proteins (DEPs) identified from hemolymph of A. pernyi infected by E. pernyi compared with that of health A. pernyi, including 104 up-regulated proteins and 201 down-regulated proteins. GO significant enrichment analysis on DEPs showed that,67 DEPs were enriched to 76 GO terms in cellular component, among them 9 GO terms were significantly enriched (P-Value<0.05), including extracellular region, extracellular space, and extracellular region part, etc. In molecular function,119 DEPS were enriched to 100 GO terms, among them 25 GO terms were significantly enriched (P-Value<0.05), DEPs mainly have a variety of enzyme activity regulation function, including peptidase, endopeptidase, hydrolase, monooxygenase, etc. Moreover, DEPs have binding function, including glycosaminoglycan binding, peptidoglycan binding, cell surface binding, and bacterial cell surface binding, etc. In biological process,103 DEPs were enriched to 352 GO terms, among them 27 GO terms were significantly enriched (P-Value<0.05), DEPs were involved in defense response, immune response, innate immue response, reponse to stress, and melanin biosynthetic process, etc, meanwhile, DEPs were associated with aminoglycan catabolic process, tyrosine metabolic process, organic hydroxy compound metabolic process, secondary metabolite biosynthetic process, leukotriene metabolic process, peptidoglycan catabolic process, etc. Pathway enrichment analysis showed that 213 DEPs were enriched to 175 pathways, among them 23 pathways were significantly enriched (P-Value<0.05), including amoebiasis, arginine and proline metabolism, cell adhesion molecules (CAMs), ECM-receptor interaction, epithelial cell signaling in Helicobacter pylori infection, glycosaminoglycan degradation, leishmaniasis, leukocyte transendothelial migration, lvsosome, malaria, ohagosome, and protein digestion and absorption, etc.3. A novel spatzle gene (ApSPZ) from the Chinese oak silkworm A. 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%,33.15% and 13.58% amino acid identity with SPZs from Manduca sexta, Bombyx mori and Drosophila melanogaster, respectively. Phylogenetic analysis indicated that ApSPZ might be a new member of the Spatzle type 1 family, which belongs to the Spatzle superfamily.The prokaryotic expression vector of ApSPZ, pET30a-ApSPZ has been constructed, the molecular weight of induced fusion protein is about 29 kD, which corresponds to the predicted molecular weight, and the fusion protein is mostly in the form of inclusion bodies in the cytoplasm. Moreover, polyclonal antibody has been prepared using the recombinant ApSPZ protein as antigen. The content of the antiserum with high purity is 0.2 mg·ml-1, and antiserum titer was greater than 121 K. The antiserum has good specificity and high sensitivity,16 ng ApSPZ protein can be detected using the antiserum with dilution of 1000 times.4. The expression patterns of several genes involved in the Toll pathway were examined at different developmental stages and various tissues in fifth instar larvae. The examined targets included A. pernyi spatzle, GNBP, MyD88, Toll, Cactus and dorsalA. During four developmental stages of A. pernyi, only Spatzle and MyD88 were expressed in eggs; all genes expect Toll were expressed in larvae; all genes were expressed in pupae, and the expression levels were generally higher; Spatzle, Cactus and dorsalA were expressed in moths. The results indicated that Toll pathway plays a role in the larval and pupal stages. A. pernyi spatzle (ApSPZ) was expressed during four developmental stages, including eggs, larvae, pupae and moths, indicating that ApSPZ has an important role throughout the entire life cycle of A. pernyi. In different tissues of the fifth instar larvae, all genes involved in Toll pathway were predominantly expressed in immune-responsive fat body tissue and malpighian tubules, indicating that the genes play a crucial role in A. pernyi innate immunity. However, only MyD88 was expressed in midgut, while others not, indicating that Toll pathway is not the main immune mechanism in A. pernyi midgut.To investigate the role of the A. pernyi Toll signaling pathway in the response to different pathogens, the relative mRNA levels of genes in the Toll pathway were assessed by qRT-PCR after A. pernyi was challenged by different pathogenic microorganisms. After infection with Enterococcus pernyi and Nosema pernyi, significant elevated changes were observed in the transcriptional levels of Toll pathway genes in A. pernyi. Moreover, A. pernyi against N. pernyi were immune delayed than that in response to E. pernyi. However, no significant differences were observed in A. pernyi after infection with Escherichia coli. In conclusion, A. pernyi infection with the fungus N. pernyi and the gram-positive bacterium E. pernyi, but not the gram-negative bacterium E. 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. |
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