Identification of optimal reference genes in Bombyx mori (Lepidoptera) for normalization of stress-responsive genes after challenge with pesticides.

Pesticides are frequently used to control pests in agriculture due to their ease of use and effectiveness, but their use causes serious economic losses to sericulture when their production overlaps with agriculture. However, no suitable internal reference genes (RGs) have been reported in the study of silkworms in response to pesticides. In this study, a standard curve was established to detect the expression levels of seven RGs in different tissues of different silkworm strains after feeding with pesticides using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), including BmGAPDH, BmActin3, BmTBP, BmRPL3, Bm28sRNA, Bmα-tubulin, and BmUBC, and the stability of them was evaluated by using NormFinder, geNorm, Delta CT, BestKeeper, and RefFinder. The results showed that BmGAPDH and Bmα-tubulin were relatively stable in the midgut after feeding with fenvalerate, BmGAPDH and Bmactin3 were relatively stable in the fat body, and Bmα-tubulin and Bmactin3 were relatively stable in the hemolymph, indicating that Bmactin3 was the most suitable RG when evaluating fenvalerate, followed by BmGAPDH and Bmα-tubulin. Besides, BmGAPDH and Bmactin3 were relatively stable in the midgut after treatment with DDVP, BmGAPDH and Bmα-tubulin were relatively stable in the fat body, and BmGAPDH and Bmα-tubulin were relatively stable in the hemolymph, indicating that Bmα-tubulin was the most stable RG when evaluating DDVP, followed by BmGAPDH and Bmactin3. Of note, BmGAPDH was shared by the two pesticides. The results will be valuable for RG selection in studying the pesticide response mechanism of silkworms and other lepidopteran insects.

Precocious Metamorphosis of Silkworm Larvae Infected by BmNPV in the Latter Half of the Fifth Instar.

The mulberry silkworm (Bombyx mori) is a model organism, and BmNPV is a typical baculovirus. Together, these organisms form a useful model to investigate host-baculovirus interactions. Prothoracic glands (PGs) are also model organs, used to investigate the regulatory effect of synthetic ecdysone on insect growth and development. In this study, day-4 fifth instar silkworm larvae were infected with BmNPV. Wandering silkworms appeared in the infected groups 12 h earlier than in the control groups, and the ecdysone titer in infected larvae was significantly higher than that of the control larvae. We then used RNA sequencing (RNA-seq) to analyze silkworm PGs 48 h after BmNPV infection. We identified 15 differentially expressed genes (DEGs) that were classified as mainly being involved in metabolic processes and pathways. All 15 DEGs were expressed in the PGs, of which Novel01674, BmJing, and BmAryl were specifically expressed in the PGs. The transcripts of BmNGDN, BmTrypsin-1, BmACSS3, and BmJing were significantly increased, and BmPyd3, BmTitin, BmIGc2, Novel01674, and BmAryl were significantly decreased from 24 to 72 h in the PGs after BmNPV infection. The changes in the transcription of these nine genes were generally consistent with the transcriptome data. The upregulation of BmTrypsin-1 and BmACSS3 indicate that these DEGs may be involved in the maturation process in the latter half of the fifth instar of silkworm larvae. These findings further our understanding of silkworm larval development, the interaction between BmNPV infection and the host developmental response, and host-baculovirus interactions in general.

The validation of the role of several genes related to Bombyx mori nucleopolyhedrovirus infection in vivo.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of primary silkworm pathogens and causes a serious damage of cocoon losses every year. Recent years, many works have been done to clarify the silkworm anti-BmNPV mechanism, and a significant progress has been made in screening and studying of genes and proteins related to BmNPV infection, but several of them lacked the proofs in vivo. In this study, to further validate the function of seven newly reported genes in vivo, including BmAtlatin-n, Bmferritin-heavy chain (BmFerHCH), Bmthymosin (BmTHY), Bmseroin1, Bmseroin2, Bmnuclear hormone receptors 96 (BmNHR96), and BmE3 ubiquitin-protein ligase SINA-like 10 (BmSINAL10), the response of them in the midgut, fat body, and hemolymph of differentially resistant strains (resistant strain YeA and susceptible strain YeB) at 48 h following BmNPV infection were analyzed. The results showed that the relative stable or upregulated expression level of BmAtlatin-n, BmTHY, Bmseroin1, and Bmseroin2 in YeA resistant strain following BmNPV infection further indicated their antiviral role in vivo, compared with susceptible YeB strain. Moreover, the significant downregulation of BmFerHCH, BmNHR96, and BmSINAL10 in both strains following BmNPV infection revealed their role in benefiting virus infection, as well as the upregulation of BmFerHCH in YeB midgut and BmSINAL10 in YeB hemolymph. These data could be used to complementary the proofs of the function of these genes in response to BmNPV infection.

Improved hybrid de novo genome assembly and annotation of African wild rice, Oryza longistaminata, from Illumina and PacBio sequencing reads.

African wild rice Oryza longistaminata, one of the eight AA- genome species in the genus Oryza, possesses highly valued traits, such as the rhizomatousness for perennial rice breeding, strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. To obtain the high-quality reference genome for O. longistaminata we employed a hybrid assembly approach through incorporating Illumina and PacBio sequencing datasets. The final genome assembly comprised only 107 scaffolds and was approximately ∼363.5 Mb, representing ∼92.7% of the estimated African wild rice genome (∼392 Mb). The N50 lengths of the assembled contigs and scaffolds were ∼46.49 Kb and ∼6.83 Mb, indicating ∼3.72-fold and ∼18.8-fold improvement in length compared to the earlier released assembly (∼12.5 Kb and 364 Kb, respectively). Aided with Hi-C data and syntenic relationship with O. sativa, these assembled scaffolds were anchored into 12 pseudo-chromosomes. Genome annotation and comparative genomic analysis reveal that lineage-specific expansion of gene families that respond to biotic- and abiotic stresses are of great potential for mining novel alleles to overcome major diseases and abiotic adaptation in rice breeding programs. This reference genome of African wild rice will greatly enlarge the existing database of rice genome resources and unquestionably form a solid base to understand genomic basis underlying highly valued phenotypic traits and search for novel gene sources in O. longistaminata for the future rice breeding programs.

Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution.

Oryza rufipogon and O. longistaminata are important wild relatives of cultivated rice, harboring a promising source of novel genes for rice breeding programs. Here, we present de novo assembled draft genomes and annotation of O. rufipogon and O. longistaminata. Our analysis reveals a considerable number of lineage-specific gene families associated with the self-incompatibility (SI) and formation of reproductive separation. We show how lineage-specific expansion or contraction of gene families with functional enrichment of the recognition of pollen, thus enlightening their reproductive diversification. We documented a large number of lineage-specific gene families enriched in salt stress, antifungal response, and disease resistance. Our comparative analysis further shows a genome-wide expansion of genes encoding NBS-LRR proteins in these two outcrossing wild species in contrast to six other selfing rice species. Conserved noncoding sequences (CNSs) in the two wild rice genomes rapidly evolve relative to selfing rice species, resulting in the reduction of genomic variation owing to shifts of mating systems. We find that numerous genes related to these rapidly evolving CNSs are enriched in reproductive structure development, flower development, and postembryonic development, which may associate with SI in O. rufipogon and O. longistaminata.

Comparative Proteomic Analysis Reveals Immune Competence in Hemolymph of Bombyx mori Pupa Parasitized by Silkworm Maggot Exorista sorbillans.

The silkworm maggot, Exorista sorbillans, is a well-known larval endoparasitoid of the silkworm Bombyx mori that causes considerable damage to the silkworm cocoon crop. To gain insights into the response mechanism of the silkworm at the protein level, we applied a comparative proteomic approach to investigate proteomic differences in the hemolymph of the female silkworm pupae parasitized by E. sorbillans. In total, 50 differentially expressed proteins (DEPs) were successfully identified, of which 36 proteins were upregulated and 14 proteins were downregulated in response to parasitoid infection. These proteins are mainly involved in disease, energy metabolism, signaling pathways, and amino acid metabolism. Eight innate immune proteins were distinctly upregulated to resist maggot parasitism. Apoptosis-related proteins of cathepsin B and 14-3-3 zeta were significantly downregulated in E. sorbillans-parasitized silkworm pupae; their downregulation induces apoptosis. Quantitative PCR was used to further verify gene transcription of five DEPs, and the results are consistent at the transcriptional and proteomic levels. This was the first report on identification of possible proteins from the E. bombycis-parasitized silkworms at the late stage of parasitism, which contributes to furthering our understanding of the response mechanism of silkworms to parasitism and dipteran parasitoid biology.

Study on the Role of Cytc in Response to BmNPV Infection in Silkworm, Bombyx mori (Lepidoptera).

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens of the silkworm. Cytochrome c (cytc) showed a significant response to BmNPV infection in our previous transcriptome study. However, little is known about the role of Bombyx mori cytc (Bmcytc) in resistance to BmNPV infection. In this study, the expression levels analysis of Bmcytc showed stable expression levels in selected tissues of the resistant strain AN following BmNPV infection, while there was downregulation in the susceptible strain p50, except in the malpighian tubule. To further study the role of Bmcytc in viral infection, Bmcytc was knocked down with siRNA in vitro, resulting in significant downregulation of selected downstream genes of the mitochondrial pathway, including Bmapaf, Bmcaspase-Nc, and Bmcaspase-1; this was also confirmed by overexpression of Bmcytc using the pIZT/V5-His-mCherry insect vector, except Bmcaspase-1. Moreover, knockdown of Bmcytc significantly promoted the infection process of BmNPV in vitro, while the infection was inhibited by overexpression of Bmcytc at the early stage and subsequently increased rapidly. Based on these results, we concluded that Bmcytc plays a vital role in BmNPV infection by regulating the mitochondrial apoptosis pathway. Our work provides valuable data for the clarification of the mechanism of silkworm resistance to BmNPV infection.

The regulation of crecropin-A and gloverin 2 by the silkworm Toll-like gene 18 wheeler in immune response.

The innate immune system is conserved among different insect species in its response to microorganism infection. The transmembrane receptors of the Toll superfamily play an important role in activating immune response, however, the function of silkworm Toll family member 18 Wheeler (18 W) remained unclear. Here, the 18w gene in silkworm was characterized. A relatively high transcription level of Bm18w mRNA was found in Malpighian tubules, and in eggs, larvae pre-molt to fourth instar, pupae and adults. When silkworm larvae were infected with E. coli or S. aureus, Bm18w showed a significant response, especially to E. coli, but did not have antibacterial activity. To further identify the downstream antimicrobial peptide genes of Bm18w, expression of Bm18w was knocked down with siRNA in vitro, resulting in significant decreases of cecropin-A, gloverin 2, and moricin B3. The overexpression of Bm18w was carried out using pIZT/V5-His-mCherry insect vector in BmN cells and significant upregulation of cecropin-A and gloverin 2 was detected, as well as upregulation of attacin and defensin. Based on the results, we concluded that Bm18w is involved in response to bacterial infection by selectively inducing the expression of antimicrobial peptide genes, especially cecropin-A and gloverin 2. This study provides valuable data to supplement understanding of the immune pathway of the silkworm.

Rapid diversification of five Oryza AA genomes associated with rice adaptation.

Comparative genomic analyses among closely related species can greatly enhance our understanding of plant gene and genome evolution. We report de novo-assembled AA-genome sequences for Oryza nivara, Oryza glaberrima, Oryza barthii, Oryza glumaepatula, and Oryza meridionalis. Our analyses reveal massive levels of genomic structural variation, including segmental duplication and rapid gene family turnover, with particularly high instability in defense-related genes. We show, on a genomic scale, how lineage-specific expansion or contraction of gene families has led to their morphological and reproductive diversification, thus enlightening the evolutionary process of speciation and adaptation. Despite strong purifying selective pressures on most Oryza genes, we documented a large number of positively selected genes, especially those genes involved in flower development, reproduction, and resistance-related processes. These diversifying genes are expected to have played key roles in adaptations to their ecological niches in Asia, South America, Africa and Australia. Extensive variation in noncoding RNA gene numbers, function enrichment, and rates of sequence divergence might also help account for the different genetic adaptations of these rice species. Collectively, these resources provide new opportunities for evolutionary genomics, numerous insights into recent speciation, a valuable database of functional variation for crop improvement, and tools for efficient conservation of wild rice germplasm.

Phylogenetic relationships and genome divergence among the AA- genome species of the genus Oryza as revealed by 53 nuclear genes and 16 intergenic regions.

Rapid radiations have long been regarded as the most challenging issue for elucidating poorly resolved phylogenies in evolutionary biology. The eight diploid AA- genome species in the genus Oryza represent a typical example of a closely spaced series of recent speciation events in plants. However, questions regarding when and how they diversified have long been an issue of extensive interest but remain a mystery. Here, a data set comprising >60 kb of 53 singleton fragments and 16 intergenic regions is used to perform phylogenomic analyses of all eight AA- genome species plus four diploid Oryza species with BB-, CC-, EE- and GG- genomes. We fully reconstruct phylogenetic relationships of AA- genome species with confidence. Oryza meridionalis, native to Australia, is found to be the earliest divergent lineage around 2.93 mya, whereas O. punctata, a BB- genome species, serves as the best outgroup to distinguish their phylogenetic relationships. They separated from O. punctata approximately 9.11 mya during the Miocene epoch, and subsequently radiated to generate the entire AA- genome lineage diversity. The success in resolving the phylogeny of AA- genome species highlights the potential of phylogenomics to determine their divergence and evolutionary histories.

Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori.

BACKGROUND: Serine proteases (SPs) and serine proteases homologs (SPHs) are a large group of proteolytic enzymes, with important roles in a variety of physiological processes, such as cell signalling, defense and development. Genome-wide identification and expression analysis of serine proteases and their homologs in the silkworm might provide valuable information about their biological functions. RESULTS: In this study, 51 SP genes and 92 SPH genes were systematically identified in the genome of the silkworm Bombyx mori. Phylogenetic analysis indicated that six gene families have been amplified species-specifically in the silkworm, and the members of them showed chromosomal distribution of tandem repeats. Microarray analysis suggests that many silkworm-specific genes, such as members of SP_fam12, 13, 14 and 15, show expression patterns that are specific to tissues or developmental stages. The roles of SPs and SPHs in resisting pathogens were investigated in silkworms when they were infected by Escherichia coli, Bacillus bombysepticus, Batrytis bassiana and B. mori nucleopolyhedrovirus, respectively. Microarray experiment and real-time quantitative RT-PCR showed that 18 SP or SPH genes were significantly up-regulated after pathogen induction, suggesting that SP and SPH genes might participate in pathogenic microorganism resistance in B. mori. CONCLUSION: Silkworm SP and SPH genes were identified. Comparative genomics showed that SP and SPH genes belong to a large family, whose members are generated mainly by tandem repeat evolution. We found that silkworm has species-specific SP and SPH genes. Phylogenetic and microarray analyses provide an overview of the silkworm SP and SPHs, and facilitate future functional studies on these enzymes.

Identification and analysis of Toll-related genes in the domesticated silkworm, Bombyx mori.

Silkworm (Bombyx mori), a model system for Lepidoptera, has contributed enormously to the study of insect immunology especially in humoral immunity. But little is known about the molecular mechanism of immune response in the silkworm. Toll receptors are a group of evolutionarily ancient proteins, which play a crucial role in the innate immunity of both insects and vertebrates. In human, Toll-like receptors (TLRs) are the typical pattern recognition receptors for different kinds of pathogen molecules. Toll-related receptors in Drosophila, however, were thought to function as cytokine receptors in immune response and embryogenesis. We have identified 11 putative Toll-related receptors and two Toll analogs in the silkworm genome. Phylogenetic analysis of insect Toll family and human TLRs showed that BmTolls is grouped with Drosophila Tolls and Anopheles Tolls. These putative proteins are typical transmembrane receptors flanked by the extracellular leucine-rich repeat (LRR) domain and the cytoplasmic TIR domain. Structural prediction of the TIR domain alignment found five stranded sheets and five helices, which are alternatingly joined. Microarray data indicated that BmToll and BmToll-2 were expressed with remarkable enrichment in the ovary, suggesting that they might play a role in the embryogenesis. However, the enriched expression of BmToll-2 and -4 in the midgut suggested that the proteins they encode may be involved in immune defense. Testis-specific expression of BmToll-10 and -11 and BmToLK-2 implies that these may be involved in sex-specific biological functions. The RT-PCR results indicated that 10 genes were induced or suppressed with different degrees after their immune system was challenged by different invaders. Expression profiles of BmTolls and BmToLKs reported here provide insight into their role in innate immunity and development.