Physiological and transcriptomic responses of silkworms to graphene oxide exposure.

The growing use of nanomaterials has sparked significant interest in assessing the insect toxicities of nanoparticles. The silkworm, as an economically important insect, serves as a promising model for studying how insects respond to harmful substances. Here, we conducted a comprehensive investigation on the impact of graphene oxide (GO) on silkworms using a combination of physiological and transcriptome analyses. GO can enter the midguts and posterior silk glands of silkworms. High GO concentrations (> 25 mg/L) significantly (P < 0.01) inhibited larval growth. Additionally, GO (> 5 mg/L) significantly reduced the cocooning rate, and GO (> 15 mg/L) hindered oviduct development and egg laying in silkworms. GO increased the reactive oxygen species content and regulated catalase activity, suggesting that it may affect insect growth by regulating reactive oxygen detoxification. The transcriptome data analysis showed that 35 metabolism-related genes and 20 ribosome biogenesis-related genes were differentially expressed in response to GO, and their expression levels were highly correlated. Finally, we propose that a Ribosome biogenesis-Metabolic signaling network is involved in responses to GO. The research provides a new perspective on the molecular responses of insects to GO.

Studying the role of Bombyx mori molybdenum cofactor sulfurase in Bombyx mori nucleopolyhedrovirus infection.

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture.

A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

Identification and function of the Pax gene Bmgsb in the silk gland of Bombyx mori.

Paired box (Pax) genes are highly conserved throughout evolution, and the Pax protein is an important transcription factor of embryonic development. The Pax gene Bmgsb is expressed in the silk glands of silkworm, but its biological functions remain unclear. This study aimed to investigate the expression pattern of Bmgsb in the silk gland and explore its functions using RNA interference (RNAi). Here, we identified eight Pax genes in Bombyx mori. Phylogenetic analysis showed that the B. mori Pax genes were highly homologous to the Pax genes in other insects and highly evolutionarily conserved. The tissue expression profile showed that Bmgsb was expressed in the anterior silk gland and anterior part of the middle silk gland (AMSG). RNAi of Bmgsb resulted in defective development of the AMSG, and the larvae were mostly unable to cocoon in the wandering stage. RNA-seq analysis showed that the fibroin genes fib-l, fib-h and p25, cellular heat shock response-related genes and phenol oxidase genes were considerably upregulated upon Bmgsb knockdown. Furthermore, quantitative reverse transcription-PCR results showed that the fibroin genes and ubiquitin proteolytic enzyme-related genes were significantly upregulated in the AMSG after Bmgsb knockdown. This study provides a foundation for future research on the biological functions of B. mori Pax genes. In addition, it demonstrates the important roles of Bmgsb in the transcriptional regulation of fibroin genes and silk gland development.

Antifeedant and Antifungal Activities of Metabolites Isolated from the Coculture of Endophytic Fungus Aspergillus tubingensis S1120 with Red Ginseng.

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 µg/cm2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 µg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng.

High mechanical property silk produced by transgenic silkworms expressing the spidroins PySp1 and ASG1.

Spider silk is one of the best natural fibers with excellent mechanical properties; however, due to the visual awareness, biting behavior and territory consciousness of spiders, we cannot obtain spider silk by large-scale breeding. Silkworms have a spinning system similar to that of spiders, and the use of transgenic technology in Bombyx mori, which is an ideal reactor for producing spider silk, is routine. In this study, the piggyBac transposon technique was used to achieve specific expression of two putative spider silk genes in the posterior silk glands of silkworms: aggregate spider glue 1 (ASG1) of Trichonephila clavipes (approximately 1.2 kb) and two repetitive units of pyriform spidroin 1 (PySp1) of Argiope argentata (approximately 1.4 kb). Then, two reconstituted spider silk-producing strains, the AG and PA strains, were obtained. Finally, the toughness of the silk fiber was increased by up to 91.5% and the maximum stress was enhanced by 36.9% in PA, and the respective properties in AG were increased by 21.0% and 34.2%. In summary, these two spider genes significantly enhanced the mechanical properties of silk fiber, which can provide a basis for spidroin silk production.

Scavenger receptor C regulates antimicrobial peptide expression by activating toll signaling in silkworm, Bombyx mori.

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes.

Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication.

The insect brain is the central part of the neurosecretory system, which controls morphology, physiology, and behavior during the insect's lifecycle. Lepidoptera are holometabolous insects, and their brains develop during the larval period and metamorphosis into the adult form. As the only fully domesticated insect, the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process. Hormonal regulation in insects is a key factor in multiple processes. However, how juvenile hormone (JH) signals regulate brain development in Lepidoptera species, especially in the larval stage, remains elusive. We recently identified the JH receptor Methoprene tolerant 1 ( Met1) as a putative domestication gene. How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin's theory on domestication. Here, CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain, unlike precocious pupation of the cuticle. At the whole transcriptome level, the ecdysteroid (20-hydroxyecdysone, 20E) signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain. Pathways related to cell proliferation and specialization processes, such as extracellular matrix (ECM)-receptor interaction and tyrosine metabolism pathways, were suppressed in the brain. Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B. mori, which decreased transcriptional binding activity. The B. mori MET1 protein showed a changed structure and dynamic features, as well as a weakened co-expression gene network, compared with B. mandarina. Based on comparative transcriptomic analyses, we proposed a pathway downstream of JH signaling (i.e., tyrosine metabolism pathway) that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

Mechanism of the growth and development of the posterior silk gland and silk secretion revealed by mutation of the fibroin light chain in silkworm.

Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms.

The effects of quercetin combined with nucleopolyhedrovirus on the growth and immune response in the silkworm (Bombyx mori).

Flavonoids are secondary metabolites that help plants resist insect attack. It can resist insect attack by inhibiting insect immune defense, and pathogens can also inhibit insect immune defense. It is speculated that the combination of flavonoids and pathogens may inhibit the immune defense and have stronger toxicity to silkworm. In this study, the combined treatment of quercetin with Bombyx mori nuclear polyhedrosis virus (BmNPV) had significant negative effects on the growth and survival of silkworm compared with BmNPV group. The detoxifying enzyme activity of BmNPV group was significantly increased at 96 h, while the activity of the combined treatment group was significantly decreased with the increase of quercetin exposure time (72 or 96 h). The activity of antioxidant enzymes also showed a similar trend, that was, the activity of antioxidant enzymes in the combined treatment group also decreased significantly with the increase of quercetin exposure time, which led to the increase of reactive oxygen species content. The silkworm cells would produce lipid peroxidation, malondialdehyde content was significantly increased, so that the expression of immune-related genes (the antimicrobial peptide, Toll pathway, IMD pathway, JAK-STAT pathway, and melanin genes) were decreased, leading to the damage of the immune system of silkworm. These results indicated that quercetin combined with BmNPV could inhibit the activities of protective enzymes and lead to oxidative damage to silkworm. It can also affect the immune response of the silkworm, and thus resulting in abnormal growth. This study provides the novel conclusion that quercetin accumulation will increase the susceptibility of silkworm to pathogens.

Mutations in a ß-group of solute carrier gene are responsible for egg and eye coloration of the brown egg 4 (b-4) mutant in the silkworm, Bombyx mori.

The brown egg 4 (b-4) is a recessive mutant in the silkworm (Bombyx mori), whose egg and adult compound eyes exhibit a reddish-brown color instead of normal purple and black, respectively. By double digest restriction-site associated DNA sequencing (ddRAD-seq) analysis, we narrowed down a region linked to the b-4 phenotype to approximately 1.1 Mb that contains 69 predicted gene models. RNA-seq analysis in a b-4 strain indicated that one of the candidate genes had a different transcription start site, which generates a short open reading frame. We also found that exon skipping was induced in the same gene due to an insertion of a transposable element in other two b-4 mutant strains. This gene encoded a putative amino acid transporter that belongs to the ß-group of solute carrier (SLC) family and is orthologous to Drosophila eye color mutant gene, mahogany (mah). Accordingly, we named this gene Bmmah. We performed CRISPR/Cas9-mediated gene knockout targeting Bmmah. Several adult moths in generation 0 (G0) had totally or partially reddish-brown compound eyes. We also established three Bmmah knockout strains, all of which exhibit reddish-brown eggs and adult compound eyes. Furthermore, eggs from complementation crosses between the b-4 mutants and the Bmmah knockout mutants also exhibited reddish-brown color, which was similar to the b-4 mutant eggs, indicating that Bmmah is responsible for the b-4 phenotypes.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Identification of potential allergens in larva, pupa, moth, silk, slough and feces of domestic silkworm (Bombyx mori).

The silkworm (Bombyx mori) is an important economic insect that can be used as food in many countries in Asia. However, silkworms and their metabolites are an important source of allergens, which can induce severe allergic reactions. So far, there are no systematic studies on the potential allergens in silkworm and its metabolites. These studies have important guiding significance for the prevention, diagnosis, and treatment of silkworm allergy. The aim of this study was to identify the potential allergens from larva, pupa, moth, silk, slough and feces of silkworm and analyze the sequence homology of silkworm allergens with other allergens identified in the Allergenonline database. We have found 45 potential allergens in silkworm. The results of the homology comparison suggested that silkworm allergens likely cross-react with those of Dermatophagoides farinae, Aedes aegypti, Tyrophagus putrescentiae, Triticum aestivum and Malassezia furfur.

Bombyx mori Apolipophorin-III inhibits Beauveria bassiana directly and through regulating expression of genes relevant to immune signaling pathways.

Insect Apolipophorin-III is a multifunctional protein and also plays an important role in insect innate immunity. Early transcriptome and proteome studies indicated that the gene expression level of Bombyx mori Apolipophorin-III (BmApoLp-III) in silkworm larvae infected with Beauveria bassiana was significantly up-regulated. In this study, BmApoLp-III gene was cloned, its expression patterns in different larval tissues investigated, the BmApoLp-III protein was successfully expressed with prokaryotic expression system and its antifungal effect was verified. The results showed that the BmApoLp-III gene was expressed in all the tested tissues of the 5th instar larvae infected by B. bassiana, with the highest expression in fat body. The fungistatic zone test showed that the recombinant BmApoLp-III has a significant antifungal effect on B. bassiana. Injecting purified BmApoLp-III to the larvae delayed the onset and death of the infected larvae. Conversely, silencing BmApoLp-III gene by RNAi resulted in early morbidity and death of the infected larvae. At the same time, injecting BmApoLp-III up-regulated the expression of genes including BmßGRP4 and BmMyd88 in the Toll signaling pathway, BmCTL5 and BmHOP in the Jak/STAT signaling pathway, serine proteinase inhibitor BmSerpin5, and antimicrobial peptide BmCecA, but down-regulated the expression of BmTak1 of Imd signaling pathway; while silencing BmApoLp-III gene down-regulated the expression of BmßGRP1 and BmSpaetzle, BmCTL5 and BmHOP, BmSerpin2 and BmSerpin5, BmBAEE and BmPPO2 of relevant pathways and BmCecA, but up-regulated the expression of BmPGRP-Lc and BmTak1 of Imd pathway. These results indicate that the BmApoLp-III could not only directly inhibit B. bassiana, but also participate in regulation of the expression of immune signaling pathway related genes, promote the expression of immune effectors, and indirectly inhibit the reproduction of B. bassiana in the silkworm.

Morus alba: Host reaction for Meloidogyne javanica, biological nematicides assessment and study of these relationships with yield and quality of leaves, cocoon and health of the silkworm.

Root-knot nematodes cause damage to several crops and the importance of each species can vary according with the crop and the agricultural region. In Brazil, Meloidogyne javanica is one of the most important nematode species parasitizing mulberry. To define management strategies, it is important to know if the crop species is damaged by the parasitism of the nematode and the best choices for control, as the use of nematicides. Biological nematicides have been extensively used in Brazil, but no information regarding its efficiency to control M. javanica in mulberry is available. Besides, it is not known if biological nematicides could improve the quality of leaves or if they alter the nutrient composition of leaves, which could interfere in the development of the silkworms that are feed with these leaves or in the quality of the silk produced. With the aim to address these questions, we propose a study that will start in the phenotyping of the main Brazilian mulberry cultivars to Meloidogyne species, passing through the test of efficiency of biological nematicides in the control of M. javanica in mulberry cultivar Miura, evaluation of the amount and quality of leaves produced and, using these leaves to feed silkworms, in the analyzes of the impact of these diet in the health of silkworms, and in the production and quality of the silk.

Trypsin-type serine protease p37k hydrolyzes CPAP3-type cuticle proteins in the molting fluid of the silkworm Bombyx mori.

Cuticular proteins analogous to peritrophin 3 (CPAP3)-type cuticle proteins constitute a family of proteins with three chitin-binding domains (CBDs) that play an important role in cuticle formation by associating with chitin. In our previous study, we identified CPAP3-type cuticle proteins in the silkworm genome, of which we characterized CPAP3-A2 (BmCBP1), a protein highly expressed in the epidermis. In this study, to elucidate the digestion mechanism of CPAP3-type cuticle proteins, we incubated CPAP3-A2 with molting fluid in vitro and found that its hydrolysis, which was inhibited by serine and cysteine protease inhibitors, produced two major bands with a molecular weight of approximately 22 kD and 11 kD. A trypsin-type serine protease, p37k, was presumed to be responsible for hydrolyzing CPAP3-A2 based on liquid chromatography-tandem mass spectrometry analysis of naturally purified molting fluid. To verify this, p37k was subsequently expressed in Sf9 cells using the Bac-to-Bac baculovirus expression system. In its active form, the recombinant protease could successfully hydrolyze CPAP3-A2. Finally, we analyzed the CPAP3-A2 molting fluid digestion site. When arginine 169 of CPAP3-A2 was mutated to alanine, a weaker hydrolysis of mutant CPAP3-A2 was observed compared to that of normal CPAP3-A2. Collectively, we identified a trypsin-type serine protease that is involved in the degradation of CPAP3-type cuticle proteins, including CPAP3-A2, suggesting that this protease plays an important role during molting in Bombyx mori. These findings provide the basis for further elucidation of the mechanisms underlying insect molting and metamorphosis.

Identification and location of sericin in silkworm with anti-sericin antibodies.

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.

Identification of the in vitro antiviral effect of BmNedd2-like caspase in response to Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture, and the underlying antiviral mechanism in silkworm is still unclear. Bombyx mori Nedd2-like caspase (BmNc) has been identified as a candidate antiviral gene from previous transcriptome data, since it is differentially expressed in the midgut of differentially resistant silkworm strains following BmNPV infection. However, the molecular mechanism by which BmNc responds to BmNPV is unknown. In this study, the relationship between BmNc and BmNPV was confirmed by its significantly different expression in different tissues of differentially resistant strains after BmNPV infection. Moreover, the antiviral role of BmNc was confirmed by the significantly higher fluorescence signals of BV-eGFP after knockdown of BmNc in BmN cells, and a reduced signal after overexpression. This was further verified by the capsid gene vp39 expression, DNA copy number, and GP64 protein level in the RNAi and overexpression groups. Furthermore, the antiviral phenomenon of BmNc was found to be associated with apoptosis. In brief, BmNc showed a relatively high expression level in the metamorphosis stages, and the effect of BmNc on BmNPV infection following RNAi and overexpression was eliminated after treatment with the inducer, Silvestrol, and the inhibitor, Z-DEVD-FMK, respectively. Therefore, it is reasonable to conclude that BmNc is involved in anti-BmNPV infection via the mitochondrial apoptosis pathway. The results provide valuable information for elucidating the molecular mechanism of silkworm resistance to BmNPV infection.

Septin homologs cooperating in the Proliferative Stage of Microsporidia Nosema bombycis.

The single-celled pathogen Nosema bombycis, that can infect silkworm Bombyx mori and other lepidoptera including Spodoptera, is the first identified Microsporidia which has diplokaryotic nuclei throughout the life cycle. Septin proteins can form highly ordered filaments, bundles or ring structures related to the cytokinesis in fungi. Here, three septin proteins (NbSeptin1, NbSeptin2 and NbSeptin3) from Nosema bombycis CQ I are described. These proteins, appear to be conserved within the phylum Microsporidia. NbSeptins transcripts were detected throughout the pathogen developmental cycle and were significantly enhanced from second days of infection, which lead to our hypothesis that NbSeptins play a role in merogony. Immunofluorescence assay (IFA) revealed a broad distribution of NbSeptins in meronts and partly co-localization of NbSeptins. Interestingly, in some of meronts, NbSeptin2 and NbSeptin3 showed localization between the nuclei of the diplokaryon. Yeast two-hybrid and co-immunoprecipitation analysis verified that NbSeptins can interact with each other. Our findings suggest that NbSeptins can cooperate in the proliferation stage of Nosema bombycis and contribute towards the understanding of the rols of septins in microsporidia development.

Bombyx mori nucleopolyhedrovirus ptp and egt genes are dispensable for triggering enhanced locomotory activity and climbing behavior in Bombyx mandarina larvae.

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors.