Protective effects of pretreatment with Fe2+, Cu2+, and Rb+ on phoxim poisoning in silkworm,Bombyx mori

BackgroundPhoxim is a widely used organophosphorus pesticide in agriculture. People are paying more and more attention to its toxicity. At present, there is no appropriate way to solve the phoxim poisoning of silkworm, which severely affected the development of sericulture. Fe²⁺, Cu²⁺, Rb⁺ exerted their biological effects through various forms in vivo.MethodsTo evaluate the effect of Fe²⁺/Cu²⁺/Rb⁺ on phoxim poisoning in silkworm, Bombyx mori were treated with fresh mulberry leaves soaked in 2.5 mg/L phoxim for 2 min with 50 mg/L FeCl₂, 150 mg/L CuCl₂, or 0.5 mg/L RbCl from 5 days of the fifth-instar silkworm.ResultsFe²⁺, Cu²⁺, and Rb⁺ pretreatments significantly inhibited the phoxim-induced reduction of survival rate and alleviated the phoxim-induced poisoning symptoms. The protective effects of Fe²⁺, Cu²⁺, and Rb⁺ on phoxim poisoning might be due to their enhancement of superoxide dismutase (SOD), catalase (CAT), and carboxylesterase (CarE) in the hemolymph and fat body of silkworm. This enhancement might reduce reactive oxygen species (ROS) accumulation and oxidative stress (OS) caused by phoxim poisoning. Thereby it reduced the damage to silkworm tissues and cells.ConclusionsThese results showed that Fe²⁺, Cu²⁺, and Rb⁺ treatments protected the silkworm from phoxim poisoning by directly enhancing the activity of SOD, CAT, and CarE enzymes and reducing oxidative stress, but not dependent on the high expression of CYP genes. The use of Fe²⁺, Cu²⁺, and Rb⁺ to enhance the activity of SOD, CAT, and CarE enzymes may be an underlying effective way to solve phoxim poisoning in the silkworm industry.     

BmNPV Resistance of Silkworm Larvae Resulting from the Ingestion of TiO2 Nanoparticles

Bombyx mori nucleopolyhedrovirus (BmNPV) causes infection in the silkworm that is often lethal. The infection is hard to prevent, partly because of the nature of the virus particles and partly because of the different strains of B. mori. Titanium dioxide nanoparticles (TiO₂ NPs) have been demonstrated to have antimicrobial properties. The present study investigated whether TiO₂ NPs added to an artificial diet can increase the resistance of B. mori larvae to BmNPV and examined the molecular mechanism behind any resistance shown. The results indicated that ingested TiO₂ NPs decreased reactive oxygen species and NO accumulation in B. mori larvae under BmNPV infection, which in turn led to a decrease in their growth inhibition and mortality. In addition, the TiO₂ NPs significantly promoted the expression of resistance-related genes, including those encoding superoxide dismutase, catalase, glutathione peroxidase, acetylcholine esterase, carboxylesterase, heat shock protein 21, glutathione S transferase o1, P53, and transferring and of genes encoding cytochrome p302 and nitric oxide synthase. These findings are a useful addition to the understanding of the mechanism of BmNPV resistance of B. mori larvae in response to TiO₂ NPs addition. Such information also provides a theoretical basis for the use of TiO₂ NPs in sericulture.     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

Pulmonary Toxicity in Mice Following Exposure to Cerium Chloride

The widespread application of lanthanoids (Lns) in manufacturing industries has raised occupational and environmental health concerns about the possible increased health risks to humans exposed to Lns in their working and living environments. Numerous studies have shown that exposures to Ln cause pulmonary injury in animals, but very little is known about the molecular mechanisms of the pulmonary inflammation caused by cerium chloride (CeCl₃) exposure. In this study, we evaluated the oxidative stress and molecular mechanism underlying with the pulmonary inflammation associated with chronic lung toxicity in mice treated with nasally instilled CeCl₃ for 90 consecutive days. Our findings suggest that significant cerium accumulated in the lung, leading the obvious increase of the lung indices, significant increases in inflammatory cells and levels of lactate dehydrogenase, alkaline phosphate, and total protein, overproduction of reactive oxygen species and peroxidation of lipids, reduced antioxidant capacity, and pulmonary inflammation. CeCl₃ exposure also activated nuclear factor κB, increased the expression of tumor necrosis factor α, cyclooxygenase-2, heme oxygenase 1, interleukin 2, interleukin 4, interleukin 6, interleukin 8, interleukin 10, interleukin 18, interleukin 1β, and CYP1A1. However, CeCl₃ reduced the expression of nuclear factor κB (NF-κB)-inhibiting factor and heat shock protein 70. These findings suggest that the pulmonary inflammation caused by CeCl₃ in mice is closely associated with oxidative stress and inflammatory cytokine expression.     

Enzyme activities in the sheep placenta during the last three months of pregnancy

In order to assess the extent to which metabolism within the sheep placenta may influence the transfer of metabolites between mother and foetus at different stages of gestation the activities of enzymes concerned with some aspects of carbohydrate, amino acid and ketone body metabolism were determined in placental cotyledons resected from ewes during the last three months of pregnancy.The activities of pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37), ATP citrate (pro-3S)-lyase (EC 4.1.3.8), citrate (si)-synthase (EC 4.1.3.7), acetyl-Co A synthetase (EC 6.2.1.1), acetyl-CoA acetyltransferase (EC 2.3.1.9) and 3-keto acid CoA-transferase (EC 2.8.3.5) per gram wet weight cotyledon do not change during the period studied. The activities of alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42), ornithine-oxoacid aminotransferase (EC 2.6.1.13) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) show an increase in activity between the third and fourth months of pregnancy whilst the activities of arginase (EC 3.5.3.1) and possibly pyruvate carboxylase (EC 6.4.1.1) show an increase in activity between the fourth and final months of pregnancy. Ornithine decarboxylase (EC 4.1.1.17) activity declines to one tenth of its activity during this later period. The absence of detectable activities of phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and ornithine carbamoyltransferase (EC 2.1.3.3) indicate that gluconeogenesis and urea synthesis from ammonia do no occur in the sheep placenta.It appears that the ability of the placenta to metabolise several substrates is achieved by the time the placenta reaches its maximum size at approximately 90 days.     

N-acetyl cysteine and selenium protects mercuric chloride-induced oxidative stress and antioxidant defense system in liver and kidney of rats: A histopathological approach

Mercury exposure is second-most common cause of metal poisoning which is quite stable and biotransformed to highly toxic metabolites thus eliciting biochemical alterations and oxidative stress. The aim of present study describes the protective effect of selenium either alone or in combination with N-acetyl cysteine (NAC) against acute mercuric chloride poisoning. The experiment was carried out in male albino Sprague Dawley rats (n = 30) which was divided into five groups. Group 1 served as control. Groups 2–5 were administered mercuric chloride (HgCl₂: 12 mol/kg, i.p.) once only, group 2 served as experimental control. Animals of groups 3, 4 and 5 were received N-acetyl cysteine (NAC: 0.6 mg/kg, i.p.) and selenium (Se: 0.5 mg/kg, p.o.) and NAC with Se in combination. Acute HgCl₂ toxicity caused significant rise in serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, albumin, bilirubin, γ-glutamyl transpeptidase, cholesterol, triglycerides, protein, urea, creatinine, uric acid and blood urea nitrogen content. Animals also showed significantly higher mercury content in liver and kidney, significant rise in lipid peroxidation level with concomitant decrease in reduced glutathione content and the antioxidant enzyme activities of superoxide dismutase and catalase after HgCl₂ exposure. Results of the present investigation clearly showed that combination therapy with NAC + Se provide maximum protection against mercury toxicity than monotherapy (alone treated groups) by preventing oxidative degradation of biological membrane from metal mediated free radical attacks.     

Nitrogen metabolism-related enzymes in <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> after <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

We compared C₃ and CAM (crassulacean acid metabolism) states in Mesembryanthemum crystallinum, a facultative CAM species, with respect to the involvement of phosphoenolpyruvate carboxylase (PEPC) and nitrogen metabolismrelated enzymes in plant response to Botrytis cinerea infection. The enzyme activities were monitored both in pathogeninoculated 2^nd leaf pair and non-inoculated 3^rd leaf pair. The control activities of most studied enzymes were dependent on the mode of photosynthesis. Compared to C₃ plants, those performing CAM exhibited higher PEPC, nitrate reductase (NR), and deaminating glutamate dehydrogenase (NAD-GDH) activities but lower glutamine synthetase (GS) and alanine aminotransferase (ALT) activities. Regardless of the mode of photosynthetic carbon assimilation, the plants responded to infection with enhancement of PEPC and inhibition of NR activities in the inoculated leaves. Whereas the activity of GS remained unaffected, those of all glutamate-yielding enzymes, namely ferredoxin-dependent glutamate synthase (Fd-GOGAT), aspartate aminotransferase (AST), ALT, and aminating glutamate dehydrogenase (NADHGDH) were altered after infection. However, the time-course and extent of the observed changes differed in C₃ and CAM plants. In general, CAM plants responded to infection with an earlier increase in PEPC and Fd-GOGAT activities as well as later inhibition of NR activity. Contrary to C₃ plants, in those performing CAM the activities of PEPC, Fd-GOGAT, NADH-GDH, and AST in the non-inoculated 3^rd leaf pair were similarly influenced by infection as in leaves directly inoculated with the pathogen. This implies that the local infection induced an alteration of carbon/nitrogen status in healthy upper leaves. This reprogramming resulting from changes in PEPC and nitrogen metabolism-related enzymes was C₃- and CAM-specific.     

Cloning and Characterization of Bombyx mori PP-BP a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12₇₈₂) was obtained from the hemolymph RNA pools. Using 5′-RACE with a specially designed primer based on the FDD study, a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.     

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.     

Proteomic analysis of nucleopolyhedrovirus infection resistance in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Silkworm hemolymph is an important defense tissue to resist bacteria and virus infections. To study the response of silkworm hemolymph in the resistance of Bombyx mori L. nucleopolyhedrovirus (BmNPV), we constructed a near-isogenic silkworm line with BmNPV resistance using highly resistant and highly susceptible parental strains. In this paper, two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Desorption/Ionization (MALDI)-mass spectrometry were employed to investigate the differences of protein patterns in the hemolymph of the highly resistant, highly susceptible and near-isogenic silkworm strains after BmNPV was administrated to the larvae. A comparison between the proteomes of these three silkworm strains led us to identify two differentially expressed proteins, beta-N-acetylglucosaminidase 2 and aminoacylase. The expression levels of these proteins were higher in the BmNPV resistant strains.     

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.     

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

The wild silkworm B. mandarina is living in the natural environment has a strong stress resistance and adaptability after harsh natural selection. The indoor rearing or domestication of the wild silkworm under artificial custody for long period deteriorates stress resistance and ecological adaptability. Therefore, we aimed to investigate the effects of artificial domestication and evolutionary pressure on the gut bacterial diversity of B. mandarina and B. mori. The intestinal content of 6th day of fifth instar B. mandarina and B. mori larvae were analyzed by sequencing of the 16S rRNA gene through Illumina miseq sequencing technology. The outcome of the study revealed that abundance of predominant bacteria of phylum Firmicutes were respectively 81.40% and 81.85% in the late fifth instar silkworm larvae (6th day) of B. mandarina and B. mori. In Firmicutes, abundance of predominant bacterial genus Enterococcus in B. mandarina (69.73%) was comparatively higher than B. mori (48.99%). The genus Advenella belongs to phylum Proteobacteria was recorded only in B. mandarina (11.54%). The abundance of Unclassified_Peptostreptococcaceae, Methanobrevibacter, Ignatzschineria, Petrimonas and Proteiniphilum in B. mandarina were between 0.12 and 0.17%, nevertheless, these bacterial genera were not detected in B. mori. The abundance of genera Lactococcus, Bacillus and Pseudomonas in B. mori (17.73%, 5.02%, and 1.61%) were remarkably higher than B. mandarina (0.15%, 0.54% and 0.45%). These results indicated that substantial difference was observed between the intestinal bacteria of B. mori and B. mandarina population, and structure of the intestinal bacteria could be affected by the artificial domestication and evolutionary pressure.     

The effect of the desert cobra (Walterinnesia aegyptia) crude venom and its protein fractions on the metabolic activity of cultured human fibroblasts

Fibroblast cultures were used to study the effect of crude venom and six venom protein fractions (F₂–F₇) fromWalterinnesia aegyptia) on their metabolic activity. This was done by incubation of six fibroblast cultures with 10 g of crude venom for 3 h at 37°C. The activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase were significantly lowered upon incubation with all fractions except F₂. Glycogen phosphorylase activity was significantly increased, leading to a significant concurrent drop of glycogen content. This effect was only seen for fractions F₃ and F₅. Creatine kinase activity and cellular ATP levels rose significantly upon incubation with all venom proteins except fractions F₂ and F₇. Increases were seen for aspartate and alanine amino-transferases by all venom proteins except fractions F₂ and F₄. Incubation of cell sonicates with all the venom proteins did not significantly alter activities of any of the parameters. Thus, fibroblasts in culture under such conditions appear to mobilize glycogen, phosphocreatine, and protein for ATP production to compensate for decreased glucose.Abbreviations ALT alanine aminotransferase - AST aspartate aminotransferase - ATP adenosine 5-triphosphate - CS citrate synthhase - GP glycogen phosphorylase - LDH lactate dehydrogenase - PFK phosphofructokinase     

Permethrin- and malathion-induced macromolecular abnormalities in adult Tribolium castaneum (herbst)

In Tribolium castaneum adults, sublethal doses of 1 and 2 ppm permethrin and 300 ppm malathion led to significant changes in amylase, trehalase, acid phosphatase, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and isocitrate dehydrogenase activities. Malathion at 150 ppm did not affect phosphatases and lactate dehydrogenase. Both malathion and permethrin significantly increased cholinesterase activity. Mixing of the two insecticides resulted in antagonistic action with reference to various enzymatic activities. Glucose and glycogen contents were at first mobilized for energy supply under insecticidal stress conditions followed by lipid and cholesterol. Soluble protein, total protein, free amino acids, and urea contents remained unaltered under all experimental conditions.     

Enzymes in intracellular organelles of adult and developing rat brain

Eighty percent of the hexokinase and about a half of the lactate dehydrogenase, pyruvate kinase, and aldolase activities of adult rat cerebral homogenates is particulate, associated to a large extent, with the sediment (P₂) obtained by centrifugation at 17,000g. Centrifugation of P₂ into sucrose gradients shows that all four enzymes are associated with synaptosomes: their peak concentration coincides with that of glutamate decarboxylase rather than with those of mitochondrial enzymes, glutamate dehydrogenase, and aspartate aminotransferase. After hypoosmotic shock and high-speed centrifugation considerable portions of synaptosomal enzymes are recovered in the supernatant phase; the composition of this fluid, as indicated by the higher specific activity of several enzymes, is different from that of the soluble fraction of whole homogenates.The concentration of the seven enzymes studied is considerably lower in fetal than in adult brain and, in general, a larger fraction of the total is soluble. Preferential accumulation with age in the particulate fraction is especially striking in the case of hexokinase. Between fetal and adult life there are changes in the enzymic composition as well as increases in the amount of the total protein attributable to the synaptosomal fraction. Glutamate decarboxylase and lactate dehydrogenase are the synaptosomal enzymes to rise first (before or at birth), followed by hexokinase and, in the third postnatal week, by aldolase and pyruvate kinase. The upsurge of mitochondrial enzymes (that of glutamate dehydrogenase at term and of aspartate aminotransferase 10 days later) is accompanied by insignificant or small increases in the total protein content of the same fraction. The results indicate that the maturation of subcellular organelles involves a stepwise enrichment with various enzymes; some signs of biochemical differentiation precede and others coincide with the development of cerebral functions known to occur in 2- to 4-wk-old rats.     

Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx mori

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314?bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg²⁺ and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).