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C-type lectins (CTLs) are widely distributed in mammals, insects, and plants, which act as pattern recognition receptors (PRRs) to recognize pathogens and initiate immune responses. In this study, we identified a C-type lectin gene called BmIML-2 from the silkworm Bombyx mori. Its open reading frame (ORF) encodes 314 amino acids, which contain dual tandem C-type lectin-like domain (CTLD). BmIML-2 is highly expressed in the fat body and is significantly induced at 24 h after BmNPV infection. Moreover, overexpression of BmIML-2 dramatically inhibited the proliferation of BmNPV, and knockdown assay via siRNA further validated the inhibition of BmIML-2 on viral proliferation. In addition, transcript level detection of apoptosis-related genes and observation of apoptosis bodies implied that overexpression of BmIML-2 promoted BmNPV-induced apoptosis. Immunofluorescence analysis indicated that BmIML-2 distributed throughout the cytoplasm and was slightly concentrated in the cell membrane. Taken together, our results suggest that BmIML-2 could inhibit in the proliferation of BmNPV by facilitating cell apoptosis. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have emerged as key players in host–pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have shown that the 3′ UTR of BmVMP23 was destroyed by an inserted fragment, and the BmVMP23 expression was downregulated in the lethal egg of “Ming” (l-em ). In this study, we found a miRNA (bmo-miR-1a-3p) that matches the 3′ UTR sequence of BmVMP23 perfectly. The relationship between bmo-miR-1a-3p and the BmVMP23 expression was examined by the co-transfection in vitro. The luciferase assay result showed that luc expression was strongly repressed. This result inferred that bmo-miR-1a-3p may downregulate BmVMP23 expression via complementary interaction with the target sites at the 3′ UTR. [Copyright &y& Elsevier]

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-l p ) and brown quail-like (q-l b ) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-l p mutant also presents a developmental abnormality. In this study, genes controlling q-l p and q-l b mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA_type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation. The purple quail-like (q-l p ) and brown quail-like (q-l b ) mutants originated from plain silkworm breeds 932VR and 0223JH respectively. By positional cloning, the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants, one base deletion in q-l p mutant and a 4.3 kb fragment insertion in q-l b mutant. The quail-like pigmentation appeared after BommoOK gene knockout by CRISPR/cas9, and on the contrast the mutant characteristics was rescued by injecting peptides of BommoOK, which completely indicated that orcokinin gene was the key factor of two quail-like mutants. Image 1 • We confirmed that BommoOK involves in the regulation of pigmentation by its mature peptide OKA_type2. • A new method, peptide injection was established for exploration of peptide function. • The results are of theoretical importance for the study of mechanism of insect pigmentation and function of BommoOK gene. [ABSTRACT FROM AUTHOR]

Abstract Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most damaging virus for the production of silkworm cocoons. Antivirus research continues to be an important aspect of the silkworm industry. Two-dimensional electrophoresis and mass spectrometry have been applied for analyzing the midgut proteome of BmNPV-infected silkworms. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has frequently been used when studying interaction between BmNPV and Bombyx mori , and useful information has been obtained. In this study, midgut proteins of BmNPV-infected silkworms were extracted from silkworm variety NIL·LVR with anti-BmNPV activity at 48 h, and proteome analysis was carried out using the label-free method. 2196 proteins were identified. Among them, there were 85 differentially expressed proteins, 45 upregulated proteins (immune-activated proteins), 28 downregulated proteins, and six proteins were specific for the BmNPV group and another six specific for control group. Many of the immune-activated proteins have been reported to have innate immune functions, and the downregulated proteins are involved in apoptosis or abnormal cell viability. In conclusion, this study provides evidence for host defense against BmNPV infection by both innate immunity and apoptosis, revealing the potential function of the midgut after oral infection of BmNPV in Bombyx mori. Significance Bombyx mori nuclear polyhedrosis virus (BmNPV) has a great impact on the sericulture industry. However, the mechanism of resistance to BmNPV has not been fully elucidated. The silkworm midgut is not only the major organ for food digestion and nutrient absorption but also an immune organ serving as the first line of defense against microbial invasion and proliferation. Here we combined label-free quantitative proteomic, bioinformatics, quantitative real-time PCR and SDS-PAGE analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut of NIL·LVR with anti-BmNPV activity. The results showed that many upregulated differentially expressed proteins have been reported to have innate immune functions and the downregulation proteins are involved in apoptosis or abnormal cell viability. These findings provide evidence for host defense against BmNPV infection by both innate immunity and apoptosis, and reveals the potential function of the midgut after infection of BmNPV in Bombyx mori. Graphical abstract Unlabelled Image Highlights • Quantitative proteomics of the silkworm midgut after BmNPV infected by label-free method • The research material is antiviral NIL•LVR strain. • Among the 85 differentially expressed proteins identified, six were in the BmNPV group and six were in the control group. • Some key differentially expressed proteins were validated by quantitative real-time PCR. • These data reveals the potential function of the midgut after oral infection of BmNPV in Bombyx mori. [ABSTRACT FROM AUTHOR]

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant ( q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR ( BmAR ) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications. [ABSTRACT FROM AUTHOR]

Abstract: The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-em ) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-em mutant to 360kb on chromosome 10 using 2287 F2 individuals. Using expression analysis and RNA interference, the best l-em candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100kb DNA fragment in the l-em mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-em mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-em mutant. These results indicate that BmEP80 is responsible for the l-em mutation. [Copyright &y& Elsevier]

Abstract: Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977bp in length with an open reading frame of 885bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5–87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon–5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis. [Copyright &y& Elsevier]