Daojun Cheng

A Genome-Wide Survey for Host Response of Silkworm, Bombyx mori during Pathogen Bacillus bombyseptieus Infection

Host-pathogen interactions are complex relationships, and a central challenge is to reveal the interactions between pathogens and their hosts. Bacillus bombysepticus (Bb) which can produces spores and parasporal crystals was firstly separated from the corpses of the infected silkworms (Bombyx mori). Bb naturally infects the silkworm can cause an acute fuliginosa septicaemia and kill the silkworm larvae generally within one day in the hot and humid season. Bb pathogen of the silkworm can be used for investigating the host responses after the infection. Gene expression profiling during four time-points of silkworm whole larvae after Bb infection was performed to gain insight into the mechanism of Bb-associated host whole body effect. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism. Similar to Bacillus thuringiensis (Bt), Bb can also induce a silkworm poisoning-related response. In this process, genes encoding midgut peritrophic membrane proteins, aminopeptidase N receptors and sodium/calcium exchange protein showed modulation. For the first time, we found that Bb induced a lot of genes involved in juvenile hormone synthesis and metabolism pathway upregulated. Bb also triggered the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs), including of Attacin, Lebocin, Enbocin, Gloverin and Moricin families, were upregulated at 24 hours post the infection.

MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori)

Retraction articleBackgroundMicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm.ResultsOur results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275).ConclusionWe present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal.

Reference genes identified in the silkworm Bombyx mori during metamorphism based on oligonucleotide microarray and confirmed by qRT‐PCR

Gene expression quantification at mRNA level is very important for post‐genomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real‐time polymerase chain reaction (qRT‐PCR), require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome‐wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT‐PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 (represented by sw22934, swl4876, and swl3956) were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin (sw22671), the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.

Nuclear receptors in Bombyx mori: insights into genomic structure and developmental expression.

Nuclear receptors (NRs) function as ligand-dependent transcription factors and are involved in diverse biological processes in different animals. The updated assembly of complete genome sequence of the Bombyx mori enabled a systematic analysis of the NRs in the five holometabolous insects including B. mori, Drosophila melanogaster, Anopheles gambiae, Apis mellifera, and Tribolium castaneum. As a result, nineteen NRs were identified in the B. mori genome, each of eighteen NRs has 1:1:1:1 ortholog in the other four insects. Interestingly, the average intron number of ligand-binding domain (LBD) of each NR gene in B. mori was 2.4, much higher than that in the other four insects; the genomic position of introns in LBDs of all orthologs for each NR presents more diversity. Phylogenetic trees of all NRs from the five insects were consistent or aberrant with classical phylogeny of these insect species. The characteristics in number, genomic structure and phylogeny of all NRs revealed their evolutionary conservation and divergence during insect evolution. The expression patterns of several NR genes displayed temporal specificity similar to that in D. melanogaster and may be associated with the key biological processes during silkworm metamorphosis. The RNAi of BmbetaFTZ-F1 resulted in abnormality in larva-pupa transition, further suggesting it is also crucial for silkworm metamorphosis. In conclusion, the present study provided new insights into the structure, evolution, expression, and functions of silkworm NRs.

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects.

Identification and characterization of Sox genes in the silkworm, Bombyx mori

Sox genes encode a family of transcription factors with important roles in metazoan development, including sex-determination, embryogenesis, neurogenesis, and skeletogenesis. We identified Sox genes in the Bombyx mori genome and characterized their evolution and expression patterns. Nine Sox genes were annotated, and could be classified into five groups, B–F. Four Sox genes in the B group were tandemly clustered on one chromosome, a characteristic common to their orthologs in other insects. The intron number in the high-mobility group (HMG) box of Sox genes exhibited low diversity across surveyed insects. Based on 40 different silkworm variety genomes, we found a similar number of single nucleotide polymorphisms (SNPs) in the coding sequences of each Sox gene, for domesticated and wild groups. However, a gene-based examination showed that SoxB3 and SoxD might be evolving under positive selection during silkworm domestication. Phylogenetic analysis showed that SoxC, SoxD, and SoxF originated before the radiation of insects, and groups B and E evolved through gene duplication after the radiation of insects. Furthermore, BmSox21a, BmSoxB3,BmSoxD, and BmSoxE reveal stage, tissue, or sex-dependent expression patterns. These results provide a foundation for further surveying the functions of Sox genes during silkworm development and domestication.

Global expression profile of silkworm genes from larval to pupal stages： Toward a comprehensive understanding of sexual differences

Abstract  Sexual dimorphism is a widespread phenomenon in many higher animals. The genes and gene networks that underlie sex differences are poorly understood. Using microarray data we analyzed sex‐related differences in the global expression profiles of silkworm genes from larval to pupal stages. Sex‐biased genes could be divided into three clusters. Cluster 1 contained 932 genes that showed a female‐biased expression trend at first and a male‐biased trend afterward. Cluster 2 included 283 male‐biased genes. Cluster 3 was comprised of 497 female‐biased genes that were expressed during the late pupal stage. Cluster 1 genes were found to be related closely to cuticle proteins, hormones, binding proteins, enzyme regulators, structural proteins, transcription regulators and so on. Several genes in clusters 2 and 3 were associated with spermatogenesis and oogenesis, respectively. The chromosomal distribution of sex‐biased genes showed evidence of chromosomal enrichment. In particular a large number of the silkworms’ male‐biased genes are located on the Z chromosome. These results provide new insights into the molecular differences that dictate sexual dimorphism in the silkworm.

Species-specific expansion of C2H2 zinc-finger genes and their expression profiles in silkworm, Bombyx mori

Most C2H2 zinc-finger proteins (ZFPs) function as sequence-specific DNA-binding transcription factors, and play important roles in a variety of biology processes, such as development, differentiation, and tumor suppression. By searching the silkworm genome with a HMM model of C2H2 zinc-fingers, we have identified a total of 338 C2H2 ZFPs. Most of the ZFP genes were clustered on chromosomes and showed uneven distribution in the genome. Over one third of genes were concentrated on chromosome 11, 15 and 24. Phylogenetic analysis classified all silkworm C2H2 ZFPs into 75 families; 63 of which belong to evolutionarily conserved families. In addition, 188 C2H2 ZFP genes (55.6%) are species-specific to the silkworm. A species-specific expansion of a family with 39 members in a tandem array on chromosome 24 may explain the higher number of species-specific ZFPs in silkworm compared to other organisms. The expression patterns of C2H2 ZFP genes were also examined by microarray analysis. Most of these genes were actively expressed among different tissues on day 3 of the fifth instar. The results provide insight into the biological functions of the silkworm C2H2 ZFP genes in metamorphism and development.

The Homeodomain Transcription Factors Antennapedia and POU-M2 Regulate the Transcription of the Steroidogenic Enzyme Gene Phantom in the Silkworm

Background: The transcriptional regulation of steroidogenic enzymes in the silkworm remains poorly understood. Results: Antp and POU-M2 are expressed in the PG and regulate the transcription of Phantom. Conclusion: Antp and POU-M2 coordinate the transcription of Phantom via a protein interaction. Significance: Our study indicates new roles for homeodomain proteins in regulating insect ecdysteroidogenesis. The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis.

The silkworm homolog of Methoprene‐tolerant (Met) gene reveals sequence conservation but function divergence

Abstract  Methoprene‐tolerant (Met) gene has been found to be involved in juvenile hormone (JH) action in insects. Herein, we isolated a silkworm (Bombyx mori) homolog of Met gene from Drosophila melanogaster using bio‐informatics analysis and rapid amplification of cDNA ends – polymerase chain reaction method, and defined it as BmMet. The full‐length cDNA of BmMet gene consists of 1 917 nucleotides and includes a 1 368 bp of open reading frame for a deduced protein of 455 amino acids. All deduced protein sequences from Met genes in B. mori and other surveyed insects contain four typical domains of bHLH, PAS‐A, PAS‐B and PAC, highlighting a high sequence conservation of Met genes during insect evolution. Also, genomic structure and phylogenic analysis suggested that Met in both B. mori and Drosophila species may originate from an ancestor gene with gce, another member of bHLH‐PAS family, via gene duplication. In addition, BmMet was detected in all surveyed tissues and throughout the whole life of silkworm at transcriptional levels. Furthermore, silkworm individuals with RNAi silencing of BmMet gene in the early stage of the fourth instar larvae could molt normally and pupate successfully. This result was different from the observation in T. castaneum but similar to that in D. melanogaster after Met knockdown, revealing that the action mode of Met in B. mori and D. melanogaster should be divergent with that in other insect species.