Upf2-Mediated Nonsense-Mediated Degradation Pathway Involved in Genetic Compensation of TrpA1 Knockout Mutant Silkworm (Bombyx mori).

Genetic mutations leading to premature termination codons are known to have detrimental effects. Using the Lepidoptera model insect, the silkworm (Bombyx mori), we explored the genetic compensatory response triggered by mutations with premature termination codons. Additionally, we delved into the molecular mechanisms associated with the nonsense-mediated mRNA degradation pathway. CRISPR/Cas9 technology was utilized to generate a homozygous bivoltine silkworm line BmTrpA1-/- with a premature termination. Transcript levels were assessed for the BmTrpA paralogs, BmPyrexia and BmPainless as well as for the essential factors Upf1, Upf2, and Upf3a involved in the nonsense-mediated mRNA degradation (NMD) pathway. Upf2 was specifically knocked down via RNA interference at the embryonic stage. The results comfirmed that the BmTrpA1 transcripts with a 2-base deletion generating a premature termination codon in the BmTrpA1-/- line. From day 6 of embryonic development, the mRNA levels of BmPyrexia, BmPainless, Upf1, and Upf2 were significantly elevated in the gene-edited line. Embryonic knockdown of Upf2 resulted in the suppression of the genetic compensation response in the mutant. As a result, the offspring silkworm eggs were able to hatch normally after 10 days of incubation, displaying a non-diapause phenotype. It was observed that a genetic compensation response does exist in BmTrpA1-/-B. mori. This study presents a novel discovery of the NMD-mediated genetic compensation response in B. mori. The findings offer new insights into understanding the genetic compensation response and exploring the gene functions in lepidopteran insects, such as silkworms.

RNA N6-methyladenosine of DHAPAT and PAP involves in regulation of diapause of Bombyx mori via the lipid metabolism pathway.

Environment-induced epigenetics are involved in diapause regulation, but the molecular mechanism that epigenetically couples nutrient metabolism to diapause regulation remains unclear. In this study, we paid special attention to the significant differences in the level of N6-adenosine methylation (m6A) of dihydroxyacetone phosphate acyltransferase (DHAPAT) and phosphatidate phosphatase (PAP) genes in the lipid metabolism pathway of the bivoltine silkworm (Bombyx mori) strain Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, non-diapause egg producer). We knocked down DHAPAT in the pupal stage of the QFLT group, resulting in the non-diapause destined eggs becoming diapausing eggs. In the PAP knockdown group, the colour of the non-diapause destined eggs changed from light yellow to pink 3 days after oviposition, but they hatched as normal. Moreover, we validated that YTHDF3 binds to m6A-modified DHAPAT and PAP mRNAs to promote their stability and translation. These results suggest that RNA m6A methylation participates in the diapause regulation of silkworm by changing the expression levels of DHAPAT and PAP and reveal that m6A epigenetic modification can be combined with a lipid metabolism signal pathway to participate in the regulation of insect diapause traits, which provides a clearer image for exploring the physiological basis of insect diapause.

BmINR and BmAC6 genes involve in diapause regulation via the insulin/IGF signaling pathway in the silkworm (Bombyx mori).

Diapause of the silkworm (Bombyx mori) is an important ecological adaptation strategy regulated by multiple signaling pathways. As an evolutionarily conserved signaling pathway, the insulin/IGF signaling (IIS) pathway is essential in regulating lifespan, energy accumulation, and stress resistance in diapause insects. However, the regulatory mechanism of IIS on diapause in B. mori is still not fully understood. To investigate the role of the IIS pathway in regulating diapause, we first analyzed the transcription levels of the insulin receptor (BmINR) and its downstream gene adenylate cyclase 6 (BmAC6). The diapause-terminated eggs of a bivoltine strain QiuFeng (V2-QF) were incubated at 25 °C in natural room light for preparing diapause egg producers (DEPs) and at 17 °C in total darkness for preparing non-diapause egg producers (NDEPs), respectively. Then we investigated the effects of BmINR and BmAC6 on diapause phenotype and expression of diapause-related genes by RNA interference (RNAi) and overexpression techniques. The results showed that the mRNA expression levels of BmINR and BmAC6 in the head and ovary of NDEPs were higher than those in DEPs during the early and middle pupal stages. Furthermore, when BmINR was knocked down in the NDEPs, approximately 14.43% of eggs were in light red color and subsequently changed into gray-purple color after 48 h post-oviposition, then stayed in a diapause state. On the other hand, overexpression of BmINR or BmAC6 via recombinant baculoviruses did not cause any obvious phenotypic alterations in NDEPs, but it upregulated the expression of genes related to carbohydrate metabolism, which provides energy for embryonic growth and development. Therefore, it can be concluded that BmINR and BmAC6 genes regulate embryonic diapause in bivoltine B. mori.

Exorista sorbillans (Diptera: Tachinidae) parasitism shortens host larvae growth duration by regulating ecdysone and juvenile hormone titers in Bombyx mori (Lepidoptera: Bombycidae).

The tachinid fly, Exorista sorbillans, is a notorious ovolarviparous endoparasitoid of the silkworm, Bombyx mori, causing severe damage to silkworm cocoon industry. Silkworm larvae show typically precocious wandering behavior after being parasitized by E. sorbillans; however, the underlying molecular mechanism remains unexplored. Herein, we investigated the changes in the levels of 20-hydroxyecdysone (20E) and juvenile hormone (JH) titer, and they both increased in the hemolymph of parasitized silkworms. Furthermore, we verified the expression patterns of related genes, which showed an upregulation of 20E signaling and biosynthesis genes but a significant downregulation of ecdysone oxidase (EO), a 20E inactivation enzyme, in parasitized silkworms. In addition, related genes of the JH signaling were activated in parasitized silkworms, while related genes of the JH degradation pathway were suppressed, resulting in an increase in JH titer. Notably, the precocious wandering behavior of parasitized silkworms was partly recoverable by silencing the transcriptions of BmCYP302A1 or BmCYP307A1 genes. Our findings suggest that the developmental duration of silkworm post parasitism could be shortened by regulation of 20E and JH titers, which may help silkworm to resist the E. sorbillans infestation. These findings provide a basis for deeper insight into the interplay between silkworms and E. sorbillans and may serve as a reference for the development of a novel approach to control silkworm myiasis.

m6A-dependent mevalonate kinase in juvenile hormone synthesis pathway regulates the diapause process of bivoltine silkworm (Bombyx mori).

BACKGROUND: Research has shown that epigenetic modification are involved the regulation of diapause in bivoltine silkworms (Bombyx mori), but it remains unclear how epigenetic modification in response to environmental signals precisely to regulate the diapause processing of bivoltine B. mori. METHODS AND RESULTS: In this study, the diapause terminated eggs of bivoltine B. mori, Qiufeng (QF) were divided into two groups: a QFHT group incubated at 25 °C with a natural day/night cycle to produce diapause eggs, and a QFLT group incubated at 16.5 °C in darkness to produce non-diapause eggs. On the 3rd day of the pupal stage, the total RNAs of the eggs were extracted and their N6-adenosine methylation (m6A) abundances were analyzed to explore the effects of m6A methylation on diapause in the silkworm. The results showed that 1984 m6A peaks are shared, 1563 in QFLT and 659 in QFHT. The m6A methylation level of the QFLT group was higher than that of the QFHT one in various signaling pathways. The m6A methylation rate of mevalonate kinase (MK) in the insect hormone synthesis pathway was significantly different between the two groups. The knockdown of MK by RNA interference in the pupae of QFLT resulted in females laying diapause eggs rather than non-diapause eggs after mating. CONCLUSIONS: m6A methylation involves in the diapause regulation of bivoltine B. mori by changing the expression levels of MK. This result provides a clearer image of the environmental signals on the regulation of diapause in bivoltine silkworms.

Metabolomics analysis of dietary restriction results in a longer lifespan due to alters of amino acid levels in larval hemolymph of Bombyx mori.

Dietary restriction (DR) has been a very important discovery in modern aging biology research. Its remarkable anti-aging effect has been proved in a variety of organisms, including members of Lepidoptera, but mechanisms by which DR increases longevity are not fully understood. By using the silkworm (Bombyx mori), a model of lepidopteran insect, we established a DR model, isolated hemolymph from fifth instar larvae and employed LC-MS/MS metabolomics to analyze the effect of DR on the endogenous metabolites of silkworm, and tried to clarify the mechanism of DR to prolong lifespan. We identified the potential biomarkers by analyzing the metabolites of the DR and control groups. Then, we constructed relevant metabolic pathways and networks with MetaboAnalyst. DR significantly prolonged the lifespan of silkworm. The differential metabolites between the DR and control groups were mainly organic acids (including amino acid), and amines. These metabolites are involved in metabolic pathways such as amino acid metabolism. Further analysis showed that, the levels of 17 amino acids were significantly changed in the DR group, indicating that the prolonged lifespan was mainly due to changes in amino acid metabolism. Furthermore, we identified 41 and 28 unique differential metabolites in males and females, respectively, demonstrating sex differences in biological responses to DR. The DR group showed higher antioxidant capacity and lower lipid peroxidation and inflammatory precursors, with differences between the sexes. These results provide evidence for various DR anti-aging mechanisms at the metabolic level and novel reference for the future development of DR-simulating drugs or foods.

Determination of Key Components in the Bombyx mori p53 Apoptosis Regulation Network Using Y2H-Seq.

The apoptosis pathway is highly conserved between invertebrates and mammals. Although genes encoding the classical apoptosis pathway can be found in the silkworm genome, the regulatory pathway and other apoptotic network genes have yet to be confirmed. Consequently, characterizing these genes and their underlying mechanisms could provide critical insights into the molecular basis of organ apoptosis and remodeling. A homolog of p53, a key apoptosis regulator in vertebrates, has been identified and cloned from Bombyx mori (Bmp53). This study confirmed via gene knockdown and overexpression that Bmp53 directly induces cell apoptosis and regulates the morphology and development of individuals during the metamorphosis stage. Furthermore, yeast two-hybrid sequencing (Y2H-Seq) identified several potential apoptotic regulatory interacting proteins, including the MDM2-like ubiquitination regulatory protein, which may represent an apoptosis factor unique to Bmp53 and which differs from that in other Lepidoptera. These results provide a theoretical basis for analyzing the various biological processes regulated by Bmp53 interaction groups and thus provide insight into the regulation of apoptosis in silkworms. The global interaction set identified in this study also provides a basic framework for future studies on apoptosis-dependent pupation in Lepidoptera.

The Pseudogene BMEA_B0173 Deficiency in Brucella melitensis Contributes to M-epitope Formation and Potentiates Virulence in a Mice Infection Model.

It is widely acknowledged that pseudogenes play important roles in bacterial diversification and evolution and participate in gene regulation and RNA interference (RNAi). However, the function of most pseudogenes in Brucella spp remains poorly understood, warranting further studies.To comprehensively analyze the function of the pseudogenes BMEA_B0173 in Brucella melitensis strain 63/9, a BMEA_B0173 in-frame deleted mutant strain was constructed. Then, the phenotypes of the mutant strain, such as growth characteristics and bacterial virulence, were assessed in mice infection models. Finally, iTRAQ analysis was performed to investigate the gene expression profile affected by the pseudogenes BMEA_B0173. In this study, we found that BMEA_B0173 deletion exhibited increased agglutination with M monospecific sera. In a mouse model of chronic infection, the BMEA_B0173 deletion strain displayed increased colonization in the spleen compared to the wild-type pathogen. The iTRAQ assay revealed that 252 proteins were differentially expressed between the BMEA_B0173 deletion and the wild-type strains. In addition, deletion of BMEA_B0173 significantly increased the expression of proteins involved in the denitrification pathway, iron metabolism, and several transcriptional regulators, which might cause increased virulence of the mutant strain. In conclusion, this study preliminary uncovered the function of the pseudogene BMEA_B0173 in Brucella melitensis 63/9 and provided novel insights for studying the pathogenesis of Brucella strains.

YTHDF3 Is Involved in the Diapause Process of Bivoltine Bombyx mori Strains by Regulating the Expression of Cyp307a1 and Cyp18a1 Genes in the Ecdysone Synthesis Pathway.

The variable diapause features of bivoltine silkworm (Bombyx mori) strains regulated by environmental signals in the embryonic stage are closely related to epigenetics. Previously, we showed that the expression of YTHDF3 is significantly different in the pupae of the bivoltine silkworm Qiufeng developed from eggs incubated at a normal temperature (QFHT, diapause egg producer) compared to those from eggs incubated at a low temperature (QFLT, nondiapause egg producer), indicating that the expression of diapause-associated genes is regulated by the m6A modification level. However, how YTHDF3 regulates the expression of diapause-related genes remains unclear. In this study, we observed that the knockdown of B. mori YTHDF3 resulted in delayed embryo development, while the overexpression of YTHDF3 resulted in the transformation of nondiapause-destined eggs into a mixture of diapause and nondiapause eggs. Further studies showed that YTHDF3, as a reading protein, can recognize the m6A site of Cyp307a1 and Cyp18a1 genes in the ecdysone synthesis pathway (ESP), and the overexpression of YTHDF3 affects the diapause traits of the silkworm by decreasing the stabilities of mRNAs of Cyp307a1 and Cyp18a1 and inhibiting their translation. The above results demonstrate that m6A modification mediates YTHDF3 to affect the expression levels of its target genes, Cyp307a1 and Cyp18a1, in the ESP to regulate diapause in bivoltine B. mori. This is the first report of the m6A methylation regulation mechanism in diapause in B. mori and provides new experimental data for clarifying the diapause regulation network.

Bombyx mori miR-2845 represses the expression of fibroin light chain gene both in vitro and in vivo.

To study the regulatory function of Bombyx mori (B. mori) miRNAs (bmo-miR) on the expression of fibroin light chain gene (BmFib-L), the 3'UTR of BmFib-L mRNA was used as the target for online prediction of miRNAs from miRBase using RNAhybrid Software, and miR-2845 was screened out. First, the expression profiles of miR-2845 and BmFib-L in larvae of the 5th instar were analyzed by Real-time quantitative PCR (RT-qPCR). Then recombinant plasmids (pcDNA3.0-pre-miR-2845 and pGL3.0-BmFib-L) were constructed to use for the expression of miR-2845 and BmFib-L 3'UTR, respectively. Cellular-level functional verification of miR-2845 on BmFib-L was carried out using multiple experimental methods (including dual luciferase reporter vectors, artificially synthesized mimics and inhibitors, and target site mutations). Finally, in vivo functional verification was performed by injecting the recombinant vector in 5th instar larvae. BmFib-L expression levels were detected using RT-qPCR in the posterior silk glands (PSG) of the injected larvae. Results showed that the expression of miR-2845 increased between the 1st and 5th day in 5th instar larvae, but began to decline on the 5th day, while the expression of the target gene BmFib-L increased sharply. This suggests that miR-2845 and BmFib-L expression levels show opposing trends, implying a negative regulatory relationship. In BmN cells, miR-2845 significantly down-regulated the expression of BmFib-L; the inhibitory effect of miR-2845 on BmFib-L was disappeared after mutation of the targeting site on 3'UTR of BmFib-L; in individuals, miR-2845 significantly down-regulated BmFib-L expression levels. Our results provide new experimental data for clarifying the molecular regulation mechanism of silk protein expression.

Cordyceps cicadae polysaccharides inhibit human cervical cancer hela cells proliferation via apoptosis and cell cycle arrest.

The present study presented the extraction and purification of polysaccharides from artificially cultured Cordyceps cicadae and wild Cordyceps cicadae by pre-soaking ultrasonic water extraction. The effects of different concentrations of polysaccharides on proliferation and cytotoxicity of Hela cells were detected by MTT and LDH methods. The results showed that the proliferation of Hela cells was inhibited by polysaccharides treatment (25 µg/mL-1600 µg/mL). The results of flow cytometry further confirmed that polysaccharides blocked the cell cycle in the S phase and promoted apoptosis. RT-qPCR and Western Blot were used to study the mRNA and protein expression of genes related to cell cycle and apoptosis signaling pathway. The results showed that polysaccharides treatment inhibited the expression of Cyclin E, Cyclin A and CDK2 and up regulated the expression of P53. Further, activation of Caspase cascade reaction, up regulation of death receptor, and the ratio of pro-apoptotic factor/anti-apoptotic factors, thus caused the cell cycle arrest and induced the apoptosis. The above research results lay a foundation for extending the anti-cancer effects of natural plant resources with low toxicity and high efficiency.

18 Additional Amino Acids of the Signal Peptide of the Bombyx mori Nucleopolyhedrovirus GP64 Activates Immunoglobulin Binding Protein (BiP) Expression by RNA-seq Analysis.

GP64 is the key membrane fusion protein of Group I baculovirus, and while the Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 contains a longer n-region (18 amino acid) of the signal peptide than does the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the function of the n-region has not been determined. In this study, we first showed that n-region is required for membrane protein localization in BmN cells, then the transcriptome sequencing was conducted on proteins guided by different signal peptide regions, and the results were analyzed and validated by quantitative PCR and luciferase assays. The results indicated that 1049 differentially expressed genes (DEGs) were identified among the different region of signal peptides and the control. With the n-region, the protein export pathway was upregulated significantly, the Wnt-1 signaling pathway was downregulated, and BiP was significantly activated by the GP64 full-length signal peptide. Furthermore, RNA interference on BiP efficiently increased luciferase secretion. These results indicate that the GP64 n-region plays a key role in protein expression and regulation.

A 14-amino acids deletion in BmShadow results to non-moult on the 2nd instar in the bivoltine silkworm, Bombyx mori.

The Bombyx mori Shadow gene (BmShadow) belongs to the superfamily of cytochrome P450 genes. To elucidate the function of the BmShadow gene and its association with diapause, we employed the CRISPR/Cas9 system to knock out the BmShadow gene in the bivoltine strain Qiufeng. The mutant (BmShadow-/-) was obtained in G2, exhibiting a 42-base deletion corresponded exactly to the amino acids regions from positions 155 to 168. The larvae of BmShadow-/- cannot moult at the pre-moulting stage of the 2nd instar. When the BmShadow-/- larvae were fed with 20E analogue at the late stage of the 2nd instar, they were rescued and developed into the 3rd instar. Rescue experiments indicated that the 20E concentration of BmShadow-/- larvae was significantly lower than that in WT larvae, and the 20E concentration of BmShadow-/- larvae which fed 20E analogue was restored to normal levels. Interestingly, the BmShadow-/- larvae could not moult on the 1st instar when they hatched from eggs after being stored at 5 °C for 40 days or after hibernation, suggesting that the 20E transported from the mother was partially consumed in the diapause maintenance phase. Our study confirmed that BmShadow is involved in 20E synthesis and a 14-amino acids region from position 155 to 168 was essential for its function, also there appears to be no other compensation pathway in vivo, which offered an important potential target locus for the control of silkworm development and the biological control of agricultural and forestry pests.

Bmo-miR-2780a regulates the expression of the sericin-1 gene of Bombyx mori.

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.

Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP1,2 expression in BmN cells, EBOV-GP1,2 with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid via transposition. The infectivity of the recombinant bacmids was investigated after transfection, expression and localization of EBOV-GP were investigated, and cell morphological changes were analyzed by TEM. The GP64 signal peptide, but not the GP1,2 native signal peptide, caused GP1,2 localization to the cell membrane, and the differentially localized GP1,2 proteins were cleaved into GP1 and GP2 fragments in BmN cells. GP1,2 expression resulted in dramatic morphological changes in BmN cells in the early stage of infection. However, GP1,2 expression did not rescue GP64 deficiency in BmNPV infection. This study provides a better understanding of GP expression and processing in BmN cells, which may lay a foundation for EBOV-GP expression using the BmNPV baculovirus expression system.

Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is closely related to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with over ~93% amino acid sequence identity. However, their host ranges are essentially nonoverlapping. The mechanism of BmNPV entry into host cells is completely different from that of AcMNPV, and whether the entry mechanism difference relates to the host range remains unclear. BmNPV produces an abortive infection in nonhost cells due to virion nuclear transportation failure. Here, we performed a detailed study by increasing BmNPV infection in Sf21 cells with the aid of methyl-beta-cyclodextrin (MßCD). We found that low-concentration MßCD incubation efficiently activates membrane ruffling in Sf21 cells, which mediates the increase in BmNPV infection. Interestingly, MßCD incubation after virion internalization also increases the infection, which suggests that macropinocytosis is involved in BmNPV infection in Sf21 cells after virion internalization. Further study revealed that clathrin-mediated endocytosis (CME) is employed by BmNPV to facilitate entry into Sf21 cells, and chlorpromazine application abolishes BmNPV infection in cells incubated both with and without MßCD. Based on these studies, we show that BmNPV enters Sf21 cells via CME and that parallel induction of macropinocytosis facilitates BmNPV infection in Sf21 cells. This study reveals the mechanism of BmNPV entry into Sf21 cells and provides clues for improving BmNPV infections in nonpermissive cells.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

The silkworm (Bombyx mori) neuropeptide orcokinin is involved in the regulation of pigmentation.

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-lp) and brown quail-like (q-lb) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-lp mutant also presents a developmental abnormality. In this study, genes controlling q-lp and q-lb 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.

Transport Via Macropinocytic Vesicles is Crucial for Productive Infection with Bombyx Mori Nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen in the sericulture industry and enters host cells via macropinocytic endocytosis; however, the current understanding of the BmNPV entry mechanism remains limited. To confirm whether direct membrane fusion (DMF) results in productive BmNPV infection, DMF infectivity induced by low pH during BmNPV infection was investigated, and the infectious viral particle was traced using an eGFP-labeled virion. We found that BmNPV infection efficiently induced fluid uptake, which allowed BmNPV to bypass the cell membrane barrier via macropinocytosis. However, DMF induced by a low pH abolished the infection. While low pH is an essential condition for membrane fusion triggering, it is not sufficient for productive BmNPV infection, and DMF results in failure to transport the nucleocapsid into the nucleus. These results indicate that transport via macropinocytic vesicles facilitates BmNPV entry into the nucleus and contribute to our understanding of the BmNPV entry mechanism.

Preincubation with a low concentration of methyl-ß-cyclodextrin enhances baculovirus expression system productivity.

OBJECTIVES: To enhance the productivity of foreign protein in culture cells using baculovirus expression system. RESULTS: A low concentration of MßCD, with the optimal application concentration of 0.25 mM and the appropriate preincubation time range from 10 to 120 min, can efficiently enhance expression levels in both the AcMNPV and BmNPV expression systems. CONCLUSIONS: Preincubation with a low concentration MßCD enhance baculovirus infection and foreign protein expression productivity.