Silkworm Larvae Research Articles

BmGr4 responds to sucrose and glucose and expresses in tachykinin-related peptide-secreting enteroendocrine cells

The regulatory hormones known as tachykinin-related peptides (TRPs) are identified as brain-gut peptides in insects. Dietary components from mulberry leaves, including glucose, induce secretion of TRPs from Bombyx mori midgut. However, the sensory molecules that recognize these compounds are still unknown. Here, we identified the gustatory receptor, BmGr4, as a sucrose and glucose receptor using Ca2+ imaging. Immunostaining revealed BmGr4 expression not only in the midgut, but also in the brain. In addition, BmGr4 expression was found to co-localize with TRP-expressing cells in both midgut enteroendocrine cells (EECs) and brain neurosecretory cells (NSCs). Furthermore, dietary nutrients after food intake result in an increase of TRP-level in hemolymph of silkworm larvae. These results provide significant circumstantial evidence for the involvement of the sucrose and glucose receptor, BmGr4, in the elicitation of TRP secretion in midgut EECs and brain NSCs.

Biosynthesis optimization of silver nanoparticles (AgNPs) using Trichoderma longibranchiatum and biosafety assessment with silkworm (Bombyx mori)

Silver nanoparticles (AgNPs) have raised public concern due to their widespread application in the field of agriculture, medicine, and environment and their potential toxic effects on humans and the environments. In this study, biosynthesis of nanosilver particles mediated by Trichoderma longibranchiatum using orthogonal experimental design (OED) was optimized. Silkworm larvae were exposed via the mulberry leaves to AgNPs to evaluate their toxic effects. The results showed that 2 mmol/L silver nitrate and 55 °C of reaction temperature at pH 7.0 for 24 h were the optimum values for AgNPs biosynthesis with the synthesis amount and antifungal activity of AgNPs as the indices. The characterization of the biosynthesized AgNPs was conducted using electron microscopy, energy dispersive X-ray analysis (EDS), UV/visible spectrophotometry, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The crystalline structured nanoparticles were spherical or polyhedral with a mean size ranging about 5–50 nm. FTIR showed that many functional group moieties (–OH, –CH 3 , –C–O, etc.) involved as a capping and reducing agent in AgNPs biosynthesis. After the larvae were fed with 50 mg/mL AgNPs, there were no obvious adverse effects on the growth of larvae and cocoon quality. Further supplement of AgNPs-B could promote the weight of larvae and the cocoon shell ratio. The data presented herein provided valuable information on a reliable eco-friendly, simple, low-cost biosynthesis of AgNPs and its biosafety evaluation which may contribute to its increased application in the future.

BmCH25H, a vertebrate interferon-stimulated gene(ISG) homolog, inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori.

Cholesterol-25-hydroxylase (CH25H) has been identified as an interferon-stimulated gene (ISG) in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol (25HC). However, invertebrates lack an antiviral system homologous to vertebrate interferons (IFNs) because the genomes of invertebrates do not encode IFN-like cytokines. Nevertheless, CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates. In this study, the Bombyx mori CH25H (BmCH25H) gene, of which the encoded protein has high homology with other lepidopteran species, was identified and located on chromosome 9. Interestingly, we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus (BmNPV) -infected BmN cells and silkworm (B.mori) larvae at the early infection stage. The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC. More importantly, we demonstrated that during BmNPV infection, BmCH25H expression was increased through the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, similar to the induction of ISGs following virus infection in vertebrates. This is the first report that CH25H has antiviral effects in insects; the study also elucidates the regulation of its expression and its mechanism of action.

Screening and risk assessment of biological control agents against bacterial wilt of ginger

Bacterial wilt caused by Ralstonia pseudosolanacearum race 4 is a highly destructive disease in ginger. To screen for biological control agents of the disease, 118 bacteria were isolated from different cultivating soils. First, the suppressive activity of soil bacteria on lesion development by R. pseudosolanacearum was evaluated using ginger leaves. Subsequently, a tobacco hypersensitive reaction test was performed for risk assessment in plants. After the clonality test by repetitive element sequence-based PCR, 12 screened isolates were inoculated into silkworm larvae as a preliminary risk assessment in animals, and we observed that five isolates did not cause a lethal effect. Finally, based on the results of the disease suppression test in infested soil, Pseudomonas sp. 008 was selected as the most suitable isolate for biological control. To ascertain the taxonomic position of the isolate, biochemical and physiological tests, multilocus sequence analysis, and average nucleotide identity with related species analysis were performed. Our results suggest that Pseudomonas sp. 008 is a novel species belonging to the P. mandelii subgroup of the P. fluorescens group. Risk assessment and taxonomic approaches with robust resolution demonstrated in the present study are expected to contribute to the efficient screening of biological control agents.

Changes of the gene expression in silkworm larvae and Cordyceps militaris at late stages of the pathogenesis

Cordyceps militaris is an entomopathogenic fungus that forms its fruiting body. The gene expression change in C. militaris and silkworm larvae were analyzed using RNA-seq to investigate the relationship of C. militaris with the host, silkworm larvae before the death by mycosis. At 144 h after the injection of C. militaris conidia, genes encoding proteases, protease inhibitors, and cuticle proteins in the fat body of silkworm larvae were upregulated, but genes encoding lipoproteins and other proteins in hemolymph were downregulated. On the other hand, at 168 h after the injection of C. militaris conidia, genes encoding amino acid and oligopeptide transporters and permeases in C. militaris were upregulated, suggesting that C. militaris may use peptides and amino acids in silkworm larvae as a nutrient to grow in vivo.Additionally, one gene cluster composed of genes putatively involved in the degradation of phenolic substrates was also upregulated. The addition of 4,5-dichlorocatechol, an inhibitor of catechol 1,2-dioxygenase, inhibited the in vivo growth of C. militaris, Beauveria bassiana and Metarhizium anisopliae. These results also suggest that the expression of the gene cluster may be crucial for the in vivo growth of C. militaris and entomopathogenic fungi. This study will clarify how C. militaris grows in insect hosts by avoiding host's immune systems.

Host instar susceptibility and stage specific predatory potential of stink bug Eocanthecona furcellata on tasar silkworm Antheraea mylitta

Aim: The study was conducted to find out the susceptible stage of tasar silkworm larvae to predation by stink bug and stage dependent feeding potential of stink bug over different instars of tasar silkworm. Methodology: Susceptibility of silkworms was studied by recording the daily larval mortality of all silkworm stages (1st to 5th instar) due to predation by E. furcellata. Stage specific predatory potential was estimated by recording the number of larvae required to complete each developmental stages (2-5th Nymphal instars and adult male and female). Results: Experimental results revealed that 1st and 2nd instar silkworms were most susceptible towards the attack of both nymphal and adult stages of stink bug whereas 3rd instar silkworms were moderately susceptible, however, third instar onwards susceptibility of tasar silkworm towards E. furcellata was negligible. Further stage specific cumulative predatory potential revealed that adult females of predatory bug were significantly more damaging to first instar silkworms (F= 125.20; df=5, 24; P= ≤ 0.0001) as compared to other stages by recording more larval consumption. Observations were also made on the second instar silkworms and it was observed that female adults were again superior over other stages of predatory stink bug (F= 135.30; df=5, 24; P= ≤ 0.0001). Similar trend of feeding potential was also recorded on 3rd instar silkworms; however, the rate of feeding was comparatively lesser than the first and second instar silkworms. Interpretation: Early instar silkworms were less defensive to predators attack, and were highly vulnerable to stink bug, however, as silkworm reached third instar, larvae started depending themselves by rapid head/thoracic movement to ward off the stink bugs. Significant difference in the feeding potential between different developmental stages was observed and adults being superior in feeding indicates their higher nutritional requirements as they need to be fit for reproductive performance to continue their progeny. Key words: Eocanthecona furcellata, Larval susceptibility, Predatory potential, Tasar silkworm

Studies on the amylase activity in the haemolymph of sex-limited silkworm <em>Bombyx mori</em> L. breeds for cocoon colour

Enzymes play a very significant role in growth and physiology of silkworm (Bombyx mori L.). The growth of the silkworm during larval stage is enormous, increase in growth by size and weight necessitates involvement of various enzymes. Amylase is a hydrolytic enzyme involved in digestion and metabolism of carbohydrates present in the mulberry leaves in the form of starch. Amylase activity is determined by calculating the reducing component volume being released from the soluble starch. The haemolymph amylase was selected for the present study, since it is the only carrier for transport of every substance including amylase and other enzymes. Three different breeds viz., Pure Mysore (Polyvoltine), APS45 (Bivoltine) and SLFC27 (Sex-limited breed for cocoon colour) were selected for estimation of amylase activity. The haemolymph of the selected strain at their 5th instar starting from first day to seventh day was considered to determine the amylase activity. The haemolymph of both the sexes was used for the assay. Increased trend of amylase activity was noted in the haemolymph of all the selected breeds from first day to fifth day which was from a low range of 127.35 µg/20 µL in SLFC27 on first day to a higher range of 427 µg/20 µL in APS45 on fourth day. Amylase activity of PM is highly significant on day 5 when compared to SLFC27 and Amylase activity of SLFC27 is significantly high on the 5th day when compared to APS45 (* p<0.05, ** p<0.01) indicating that there is a gradual decrease in the amylase activity on day six by 22-25% than fifth day in all the selected breeds as this may be due to maturation of silkworm larvae and leaf consumption getting reduced as they approach for spinning process. The study specifies that, all the breeds showed high enzyme activity during larval stage along with positive correlation with respect to cocoon parameters. The higher amylase activity was recorded during the larval stage of the silkworm and decreased gradually towards spinning stage.

ε-Poly-l-lysine-modified natural silk fiber membrane wound dressings with improved antimicrobial properties

Many complex diseases, such as bacterial infections, frequently accompany cutaneous wound healing, adding to the difficulty of clinical wound management. Consequently, in addition to displaying strong biocompatibility and actively promoting wound healing, an optimal wound dressing should also possess antimicrobial qualities to address issues with bacterial infection. This paper developed natural silk fiber (SF) membranes (also known as a flat silk cocoon (FSC)) with antimicrobial properties as a dressing for skin wounds. By changing the spinning tools and environment of silkworm larvae, a novel natural SF membrane with a cocoon structure and controllable size was prepared. The functional SF membranes were obtained via a hot press process and grafted with ε-Poly-l-lysine (EPL). The results showed that the SF membrane dressing was adjustable in size with a similar structure to the extracellular matrix (ECM), displaying inherent mechanical properties, excellent antimicrobial qualities, and biocompatibility. In vivo experiments using a full-thickness skin defect model indicated that EPL-modified SF membranes significantly promoted the rate of wound healing, exhibiting thicker granulation tissue and higher collagen disposition than commercial dressings (Tegaderm™ film). Therefore, the excellent mechanical qualities and cytocompatibility of the antimicrobial EPL-modified SF membranes substantially promote their potential application as a chronic wound dressing.

Impact of imidazolium salts to reduce the infection of Bombyx mori caterpillars by Beauveria bassiana

The silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is susceptible to several entomopathogens, including the fungus Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales), which causes white muscardine. Aiming at effective alternatives for disease control, the effect of one pyridinium salt (PS) and seven imidazolium salts (IS) on biological parameters of this fungus – conidia germination, colony-forming units (CFU), vegetative growth, and conidia production – were evaluated in vitro. Then, the in vivo action of the IS on silkworm larvae and pupae was verified. Preventive and curative actions of selected IS for disease suppression in B. mori caterpillars were evaluated either via oral or integumentary administration. When administered daily via feeding sheets, the IS 1,3-di-n-decyl-2-methylimidazolium chloride at 100 μg ml−1 was effective in reducing the mortality of caterpillars by the fungal infection. In addition, it did not show any negative effects on developing B. mori larvae. Therefore, 1,3-di-n-decyl-2-methylimidazolium chloride can be an effective tool in the management of white muscardine in silkworm rearing houses.

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate.

In this study, silkworm larvae were used for expression of porcine rotavirus A (KS14 strain) inner capsid protein, VP6, and outer capsid protein, VP7. Initially, VP6 was fused with Strep-tag II and FLAG-tag (T-VP6), and T-VP6 was fused further with the signal peptide of Bombyx mori 30k6G protein (30k-T-VP6). T-VP6 and 30 k-T-VP6 were then expressed in the fat body and hemolymph of silkworm larvae, respectively, with respective amounts of 330 μg and 50 μg per larva of purified protein. Unlike T-VP6, 30k-T-VP6 was N-glycosylated due to attached signal peptide. Also, VP7 was fused with PA-tag (VP7-PA). Additionally, VP7 was fused with Strep-tag II, FLAG-tag, and the signal peptide of Bombyx mori 30k6G protein (30k-T-ΔVP7). Both VP7-PA and 30k-T-ΔVP7 were expressed in the hemolymph of silkworm larvae, with respective amounts of 26 μg and 49 μg per larva of purified protein, respectively. The results from our study demonstrated that T-VP6 formed nanoparticles of greater diameter compared with the ones formed by 30k-T-VP6. Also, higher amount of VP6 expressed in silkworm larvae reveal that VP6 holds the potential for its use in vaccine development against porcine rotavirus with silkworm larvae as a promising host for the production of such multi-subunit vaccines.

Effects of Naphthaleneacetic Acid, Indole-3-Butyric Acid and Zinc Sulfate on the Rooting and Growth of Mulberry Cuttings

The mulberry tree (Morus alba) is a perennial and fast-growing tree distributed worldwide under different climatic conditions. Most of the world’s silk production (&gt;90%) is facilitated by the feeding of silkworm larvae on the leaves of mulberry (Morus alba L.) varieties. Therefore, exploration of the protocol for improving the propagation efficiency and increasing the reproductive capacity of M. alba varieties could be of great significance. This study aimed to determine the effect of four concentrations (0, 100, 200 and 400 mg L−1) each of naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and zinc sulfate (0, 100 and 200 mg L−1), supplied separately or combined, on the rooting and growth of mulberry cuttings. M. alba cuttings were immersed for 5 s in each solution using the quick-dip method and subsequently, the cuttings were dried and planted in plastic pots and maintained in a greenhouse for 60 days. The number of leaves (NL), longest root size (LRS), longest stem size (LSS), number of rooted cuttings (NRC), number of stems per tree (NSP), rooting percentage (RP), wet root weight (WRW), dry root weight (DRW), wet stem weight (WSW), dry stem weight (DSW), wet leaf weight (WLW) and dry leaf weight (DLW) were evaluated. The results obtained showed an increase in all growth parameters of the mulberry cuttings. Treatments of hormones (IBA and NAA) and Zn sulfate were effective on LSS, LRS and WSW. The highest values of LSS were obtained for the treatments T5, T6, T14, T15, T16 and T18. Moreover, T5, T12 and T10 showed the highest values of LRS. The highest value of WSW was observed for T18, T5, T14, T15 and T16. The highest values of WLW and DLW were observed in T20 and T14. Dry stem weight (DSW) was high in T18 and T14. The application of NAA (at 200 mg L−1), IBA (200 and 400 mg L−1) and Zn sulfate (200 and 400 mg L−1), either alone or in double combination, can be a suitable and reliable method for mulberry propagation.

DNA G-quadruplex structure participates in regulation of lipid metabolism through acyl-CoA binding protein.

G-quadruplex structure (G4) is a type of DNA secondary structure that widely exists in the genomes of many organisms. G4s are believed to participate in multiple biological processes. Acyl-CoA binding protein (ACBP), a ubiquitously expressed and highly conserved protein in eukaryotic cells, plays important roles in lipid metabolism by transporting and protecting acyl-CoA esters. Here, we report the functional identification of a G4 in the promoter of the ACBP gene in silkworm and human cancer cells. We found that G4 exists as a conserved element in the promoters of ACBP genes in invertebrates and vertebrates. The BmACBP G4 bound with G4-binding protein LARK regulated BmACBP transcription, which was blocked by the G4 stabilizer pyridostatin (PDS) and G4 antisense oligonucleotides. PDS treatment with fifth instar silkworm larvae decreased the BmACBP expression and triacylglycerides (TAG) level, resulting in reductions in fat body mass, body size and weight and growth and metamorphic rates. PDS treatment and knocking out of the HsACBP G4 in human hepatic adenocarcinoma HepG2 cells inhibited the expression of HsACBP and decreased the TAG level and cell proliferation. Altogether, our findings suggest that G4 of the ACBP genes is involved in regulation of lipid metabolism processes in invertebrates and vertebrates.

The Evaluation of the Biological Effects of Melanin by Using Silkworm as a Model Animal.

Melanin has been reported to have potential applications in industries such as cosmetics and food due to its anti-UV and antioxidative qualities. However, the corresponding data on its safety evaluation or biological consequences are fairly limited; such data are critical given its widespread use. The effect of different concentrations (1, 2, 3, and 4%) of melanin on growth status (larvae length and weight, cocoon weight, and morphology), the microstructure of the various tissues (fat body, silk gland, and midgut), and silk properties was studied by using the silkworm (bombyx mori) as the model organism. The weight and length of silkworm larvae fed with melanin were lower than the control, indicating that melanin appears to have a negative effect on the growth status of silkworms; however, the histophysiology analysis indicates that the cell morphologies are not changed, the XRD and FTIR spectra indicate that the secondary and crystalline structures of silks are also well preserved, and the thermogravimetric analysis and tensile test indicate that the thermal stability and mechanical properties are well maintained and even improved to some extent. Generally, it indicates that melanin has a certain inhibitory effect on the growth of silkworm larva but causes no harm to the cell microstructures or silk properties; this demonstrates that the safety of melanin as a food addictive should be considered seriously. The increase of thermal stability and mechanical properties shows that melanin may be a good chemical modifier in textile industries.

In vivo enzymatic digestion of HRV 3C protease cleavage sites-containing proteins produced in a silkworm-baculovirus expression system.

Baculovirus expression vector system (BEVS) has been recognized as a potent protein expression system in engineering valuable enzymes and vaccines. Various fusion tags facilitate protein purification, leaving the potential risk to influence the target protein's biological activity negatively. It is of great interest to consider removing the additional tags using site-specific proteases, such as human rhinoviruses (HRV) 3C protease. The current study validated the cleavage activity of 3C protease in Escherichia coli and silkworm-BEVS systems by mixing the cell or fat body lysates of 3C protein and 3C site containing target protein in vitro. Further verification has been performed in the fat body lysate from co-expression of both constructs, showing remarkable cleavage efficiency in vivo silkworm larvae. We also achieved the glutathione-S-transferase (GST) tag-cleaved product of the VP15 protein from the White spot syndrome virus after purification, suggesting that we successfully established a coinfection-based recognition-and-reaction BEVS platform for the tag-free protein engineering.

Effects of exposure to trace pyriproxyfen on the intestinal bacterial diversity and immune signal pathways of silkworm (Bombyx mori) larvae

Silkworm (Bombyx mori) is a widely used lepidopteran model insect. Pyriproxyfen is an insect growth regulator (IGR) that can be used to control pests such as lepidoptera and diptera, and there is no research about its influence on the intestinal bacterial diversity and immune signal pathway of non-target lepidopteran insect larvae. In this study, we treated fifth-instar silkworms with trace pyriproxyfen (10−4 mg/L). The results showed that the abundance and diversity of silkworm larvae intestinal flora at the level of phylum, class, order, family and genus were all changed. The function prediction result showed that the functional changes mainly focused on transcription, amino acid transport and metabolism, carbohydrate transport and metabolism, signal transduction mechanisms, energy production and conversion et al. after exposure to trace pyriproxyfen. Furthermore, qRT-PCR results showed that trace pyriproxyfen exposure activated the transcriptions of antimicrobial peptides (AMPs) genes regulated by Toll and IMD pathways. This research can lay the foundation for the study on the resistance of lepidopteran insects to pyriproxyfen and other juvenile hormone pesticides.

Evaluation of biosafety and skin-care efficacy for “Four white” medicinal materials used in cosmetics

ObjectiveTo verify the antioxidant, whitening and anti-inflammatory effects of “Four white” extracts in cosmetic applications. MethodsThe extracts of the Chinese herbal medicines “Four white” (Silkworm Larva, Japanese Ampelopsis root, Largehead Atractylodes rhizome. White peony root) were prepared by an alcoholic extraction method. The safety and irritation of the extracts were assessed by the erythrocyte hemolysis test and MTT. The antioxidant ability of the extracts was determined by measuring their capabilities to scavenge hydroxyl and DPPH radicals, reduce iron ions and cellular antioxidant assays. The whitening capability was determined by the ability of tyrosinase inhibition. Its anti-inflammatory capacity was determined by measuring the inhibiting rates against hyaluronidase and lipopolysaccharide (LPS)-induced NO production in RAW264.7 cells. ResultsThe results of the irritation test showed that the “Four white” extract has low irritation and high biosafety. It can scavenge different free radicals and has protective effect against oxidative damage. The tyrosinase inhibition test showed that the extracts had whitening ability. In terms of anti-inflammatory activity, it can inhibit hyaluronidase activity to a certain extent and reduce the production of NO by LPS-stimulated cells. Conclusion“Four white” extract has high biosafety and skin-care efficiency such as antioxidant, whitening and anti-inflammatory properties.

DNA hypermethylation level of ACTL6A may promote BmNPV infection in B. mori

• ACTL6A DNA methylation level and its gene expression was up-regulated upon BmNPV infection. • Decrease of DNA methylation level of ACTL6A inhibited the expression of ACTL6A. • Knockdown of ACTL6A suppressed BmNPV replication. • The first report on DNA methylation role in BmNPV-silkworm interaction. Bombyx mori nucleopolyhedrosis virus (BmNPV) can cause acute virus disease in silkworm, seriously endangering the sustainable development of sericultural industry. DNA methylation is an extensive regulation mode of gene expression in epigenetics, which plays an important role in a series of life processes such as genomic imprinting, embryo development, aging and viral infection. Previously, we found the DNA hypermethylation of actin-like protein 6A gene ( ACTL6A ) in BmNPV-infected midgut of silkworm larvae by whole genome bisulfite sequencing. In this study, firstly, we validated the enhanced transcription level and the DNA methylation level of ACTL6A in the midgut upon BmNPV infection by qRT-PCR and BS-PCR, respectively. Then, treated the BmN cells with DNA methylation inhibitor (Zebularine), both the DNA methylation level and the transcription level of ACTL6A were declined. Further, knock down of ACTL6A by siRNA inhibited the proliferation of BmNPV. The results showed that BmNPV infection may induce the DNA hypermethylation of ACTL6A in Bombyx mori , which promoted the transcription level of ACTL6A , and then affected the proliferation of BmNPV. Our results may provide an insight into exploring the role of DNA methylation in the interaction between BmNPV and silkworm.

Biochemical toxicity and transcriptome aberration induced by dinotefuran in Bombyx mori

Dinotefuran is a third-generation neonicotinoid pesticide and is increasingly used in agricultural production, which has adverse effects on nontarget organisms. However, the research on the impact of dinotefuran on nontarget organisms is still limited. Here the toxic effects of dinotefuran on an important economic species and a model lepidopteran insect, Bombyx mori, were investigated. Exposure to different doses of dinotefuran caused physiological disorders or death. Cytochrome P450, glutathione S-transferase, carboxylesterase, and UDP glycosyl-transferase activities were induced in the fat body at early stages after dinotefuran exposure. By contrast, only glutathione S-transferase activity was increased in the midgut. To overcome the lack of sensitivity of the biological assays at the individual organism level, RNA sequencing was performed to measure differential expressions of mRNA from silkworm larvae after dinotefuran exposure. Differential gene expression profiling revealed that various detoxification enzyme genes were significantly increased after dinotefuran exposure, which was consistent with the upregulation of the detoxifying enzyme. The global transcriptional pattern showed that the physiological responses induced by dinotefuran toxicity involved multiple cellular processes, including energy metabolism, oxidative stress, detoxification, and other fundamental physiological processes. Many metabolism processes, such as carbon metabolism, fatty acid biosynthesis, pyruvate metabolism, and the citrate cycle, were partially repressed in the midgut or fat body. Furthermore, dinotefuran significantly activated the MAPK/CREB, CncC/Keap1, PI3K/Akt, and Toll/IMD pathways. The links between physiological, biochemical toxicity and comparative transcriptomic analysis facilitated the systematic understanding of the integrated biological toxicity of dinotefuran. This study provides a holistic view of the toxicity and detoxification metabolism of dinotefuran in silkworm and other organisms.

Dual display hemagglutinin 1 and 5 on the surface of enveloped virus-like particles in silkworm expression system

Rous sarcoma virus-like particles (RSV-LPs) displaying hemagglutinins of H1N1 (A/New Caledonia/20/99) (H1) and H5N1 (A/Vietnam/1194/2004) (H5) of the influenza A virus were produced. The H1 has its transmembrane domain, but the H5 was fused with the transmembrane domain of glycoprotein 64 (BmGP64) from Bombyx mori nucleopolyhedrovirus (BmNPV). H1 and RSV Gag protein were coexpressed in the hemolymph of silkworm larvae, copurified, and confirmed RSV-LP displaying H1 (VLP/H1). Similarly, the RSV-LP displaying H5 (VLP/H5) production was also achieved. Using fetuin agarose column chromatography, RSV Gag protein-coexpressed H1 and H5 in silkworms were copurified from the hemolymph. By immuno-TEM, H1 and H5 were observed on the surface of an RSV-LP, indicating the formation of bivalent RSV-LP displaying two HAs (VLP/BivHA) in the hemolymph of silkworm larvae. VLP/H1 induced the hemagglutination of red blood cells (RBCs) of chicken and rabbit but not sheep, while VLP/H5 induced the hemagglutination of RBCs of chicken and sheep but not rabbit. Additionally, VLP/BivHA allowed the hemagglutination of RBCs of all three animals. Silkworm larvae can produce RSV-LPs displaying two HAs and is a promising tool to produce the bivalent enveloped VLPs for the vaccine platform.

A Comparative Analysis of Bombyx mori (Lepidoptera: Bombycidae) β-fructofuranosidase Homologs Reveals Different Post-Translational Regulations in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

Simple SummaryThe β-fructofuranosidase (β-FFase) encoding gene BmSuc1 regulates the glycometabolism of silkworm larvae, and it participates in the resistance of mulberry alkaloids. However, there is no molecular or biochemical information available about the mulberry pest Glyphodes pyloalis Walker β-FFase homologs. In this paper, we have obtained five β-FFase homologous genes in G. pyloalis and characterized the expression and the localization of GpSUC1a in the midgut. The β-FFase activity in the midgut of G. pyloalis larvae and GpSUC1a were both confirmed, while recombinant GpSUC1a displayed little activity as compared with the higher activity of BmSUC1. Some putative N-glycosylation sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a. The results indicate that such post-translational modifications (PTMs) are differentially supporting that β-FFase are active in these two mulberry feeding caterpillars, and the activation of GpSUC1a may be controlled by a more complex post-translational regulatory system in G. pyloalis larvae. This is the first report on the characterization of β-FFase genes from G. pyloalis and the first comparison of expression regulation between two mulberry feeding insects B. mori and G. pyloalis. Moreover, this research may provide new ideas for the management of mulberry borers.The silk-spinning and Lepidopteran model insect Bombyx mori (Bombycidae) is a mulberry specialist. The BmSuc1 gene is the first β-fructofuranosidase (β-FFase) encoding gene identified in animals, and β-FFase acts as an essential sucrase for glycometabolism modulation in the silkworm larvae, involved in resistance to mulberry alkaloids. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is an important mulberry pest leading to heavy economic loss of sericulture. However, no molecular or biochemical information is available about G. pyloalis β-FFase homologs. In this study, five β-FFase homologous genes in G. pyloalis were obtained. The genes GpSuc1a and GpSuc2c were expressed in the midgut; GpSuc2c encodes a truncated polypeptide. The expression and the localization of GpSUC1a in the midgut was characterized. Whereas recombinant GpSUC1a expressed in both Escherichia coli and BmN cells displayed little activity as compared with higher activity of BmSUC1, β-FFase activity in the larval midgut of G. pyloalis and GpSUC1a purified from the midgut were both confirmed. The data suggested that the activation of GpSUC1a is probably controlled by a more complicated post-translational regulation system in G. pyloalis larvae than that of BmSUC1 in B. mori. To study post-translational modifications (PTMs), GpSUC1a and BmSUC1 were purified from larval midguts using immunoprecipitation and subjected to LC-MS to perform PTMs analysis. Some putative N-glycosylated sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a, indicating that such PTMs were supporting the differential β-FFases activities in these two mulberry feeding caterpillars.