Weide Shen

Changes in the activity and the expression of detoxification enzymes in silkworms (Bombyx mori) after phoxim feeding

Silkworm (Bombyx mori) is an economically important insect. However, non-cocoon caused by chemical insecticide poisoning has largely hindered the development of sericulture. To explore the roles of detoxification enzymes in B. mori after insecticide poisoning, we monitored the activity changes of cytochrome P450 monooxygenase, glutathione-S-transferase, and carboxylesterase in B. mori midgut and fatbody after phoxim feeding. At the same time, the expression levels of detoxification enzyme-related genes were also determined by real-time quantitative PCR. Compare to the control levels, the activity of P450 in the midgut and fatbody was increased to 1.72 and 6.72 folds; the activity of GST was no change in midgut, and in fatbody increased to 1.11 folds; the activity of carboxylesterase in the midgut was decreased to 0.69 folds, and in fatbody increased to 1.13 folds. Correspondingly, the expression levels of detoxifying enzyme genes CYP6ae22, CYP9a21, GSTo1 and Bmcce were increased to 15.99, 3.32, 1.86 and 2.30 folds in the midgut and to 3.58, 1.84, 2.14 and 4.21 folds in the fatbody after phoxim treatment. These results demonstrated the important roles of detoxification enzymes in phoxim metabolism. In addition, the detected activities of such enzymes were generally lower than those in cotton bollworms (Helicoverpa armigera), which may contribute to the high susceptibility of B. mori to insecticides. Our findings laid the foundation for further investigations of the molecular mechanisms of organophosphorus pesticide metabolism in B. mori.

Phoxim‐induced damages of Bombyx mori larval midgut and titanium dioxide nanoparticles protective role under phoxim‐induced toxicity

Phoxim (O,O‐diethyl O‐(alpha‐cyanobenzylideneamino) phosphorothioate) is a powerful organophosphorus pesticide with high potential for Bombyx mori larvae of silkworm exposure. However, it is possible that during the phoxim metabolism, there is generation of reactive oxygen species (ROS) and phoxim may produce oxidative stress and neurotoxicity in an intoxicated silkworm. Titanium dioxide nanoparticles (TiO2 NPs) pretreatment has been demonstrated to increase antioxidant capacity and acetylcholinesterase (AChE) activity in organisms. This study was, therefore, undertaken to determine phoxim‐induced oxidative stress and neurotoxicity to determine whether phoxim intoxication alters the antioxidant system and AChE activity in the B. mori larval midgut, and to determine whether TiO2 NPs pretreatment attenuates phoxim‐induced toxicity. The findings suggested that phoxim exposure decreased survival of B. mori larvae, increased malondialdehyde (MDA), carbonyl and 8‐OHdG levels, and ROS accumulation in the midgut. Furthermore, phoxim significantly decreased the activities of AChE, superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione‐S‐transferase (GST), and levels of ascorbic acid (AsA), reduced glutathione (GSH), and thiol in the midgut. TiO2 pretreatment, however, could increase AChE activity, and remove ROS via activating SOD, CAT, APX, GR, and GST, and accelerating AsA–GSH cycle, thus attenuated lipid, protein, and DNA peroxidation and improve B. mori larval survival under phoxim‐induced toxicity. Moreover, this experimental system would help nanomaterials to be applied in the sericulture.

The adverse effects of phoxim exposure in the midgut of silkworm, Bombyx mori

The silkworm is an important economic insect. Poisoning of silkworms by organophosphate pesticides causes tremendous loss to the sericulture. In this study, Solexa sequencing technology was performed to profile the gene expression changes in the midgut of silkworms in response to 24h of phoxim exposure and the impact on detoxification, apoptosis and immune defense were addressed. The results showed that 254 genes displayed at least 2.0-fold changes in expression levels, with 148 genes up-regulated and 106 genes down-regulated. Cytochrome P450 played an important role in detoxification. Histopathology examination and transmission electron microscope revealed swollen mitochondria and disappearance of the cristae of mitochondria, which are the important features in insect apoptotic cells. Cytochrome C release from mitochondria into the cytoplasm was confirmed. In addition, the Toll and immune deficiency (IMD) signal pathways were all inhibited using qRT-PCR. Our results could help better understand the impact of phoxim exposure on silkworm.

Titanium dioxide nanoparticles relieve biochemical dysfunctions of fifth-instar larvae of silkworms following exposure to phoxim insecticide.

Phoxim insecticide is widely used in agriculture, which is toxic to insect pests and nontarget organisms. The phoxim poisoning is hard to prevent for silkworms. TiO(2) NPs have been widely applied in whitening, brightening foods, toothpaste or sunscreens, and orally-administered drugs. However, whether TiO(2) NPs can increase resistance of silkworm to phoxim poisoning has not been reported. The results demonstrated that added TiO(2) NPs significantly decreased reduction of protein, glucose and pyruvate contents, lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase activities, and attenuated increases of free amino acids, urea, uric acid and lactate levels, activities of protease, alanine aminotransferase and aspartate aminotransferase in the hemolymph of silkworms caused by phoxim exposure. From the present study, it is clearly evident that added TiO(2) NPs may relieve toxic impacts of phoxim insecticide on silkworm metabolism, which in turn may result in an increase in silk yield.

Molecular mechanisms of reduced nerve toxicity by titanium dioxide nanoparticles in the phoxim-exposed brain of Bombyx mori.

Bombyx mori (B. mori), silkworm, is one of the most important economic insects in the world, while phoxim, an organophosphorus (OP) pesticide, impact its economic benefits seriously. Phoxim exposure can damage the brain, fatbody, midgut and haemolymph of B. mori. However the metabolism of proteins and carbohydrates in phoxim-exposed B. mori can be improved by Titanium dioxide nanoparticles (TiO2 NPs). In this study, we explored whether TiO2 NPs treatment can reduce the phoxim-induced brain damage of the 5th larval instar of B. mori. We observed that TiO2 NPs pretreatments significantly reduced the mortality of phoxim-exposed larva and relieved severe brain damage and oxidative stress under phoxim exposure in the brain. The treatments also relieved the phoxim-induced increases in the contents of acetylcholine (Ach), glutamate (Glu) and nitric oxide (NO) and the phoxim-induced decreases in the contents of norepinephrine (NE), Dopamine (DA), and 5-hydroxytryptamine (5-HT), and reduced the inhibition of acetylcholinesterase (AChE), Na+/K+-ATPase, Ca2+-ATPase, and Ca2+/Mg2+-ATPase activities and the activation of total nitric oxide synthase (TNOS) in the brain. Furthermore, digital gene expression profile (DGE) analysis and real time quantitative PCR (qRT-PCR) assay revealed that TiO2 NPs pretreatment inhibited the up-regulated expression of ace1, cytochrome c, caspase-9, caspase-3, Bm109 and down-regulated expression of BmIap caused by phoxim; these genes are involved in nerve conduction, oxidative stress and apoptosis. TiO2 NPs pretreatment also inhibited the down-regulated expression of H+ transporting ATP synthase and vacuolar ATP synthase under phoxim exposure, which are involved in ion transport and energy metabolism. These results indicate that TiO2 NPs pretreatment reduced the phoxim-induced nerve toxicity in the brain of B. mori.

Cerium chloride improves protein and carbohydrate metabolism of fifth-instar larvae of Bombyx mori under phoxim toxicity

The organophosphorus pesticide poisoning of the silkworm Bombyx mori is one of the major events causing serious damage to sericulture. Added low-dose rare earths are demonstrated to increase resistance in animals. However, very little is known about whether or not added CeCl3 can increase resistance of silkworm to phoxim poisoning. The present findings suggested that added CeCl3 to mulberry leaves markedly increased contents of protein, glucose and pyruvate, and carbohydrate metabolism-related enzyme activities, including lactate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, and attenuated free amino acids, urea, uric acid and lactate levels and inhibited the protein metabolism-related enzymes activities, such as protease, alanine aminotransferase and aspartate aminotransferase in the haemolymph of B. mori, under phoxim toxicity. These findings suggest that added CeCl3 may improve protein and carbohydrate metabolisms, thus leading to increases of growth and survival rate of B. mori under phoxim stress.

Transcription level of messenger RNA per gene copy determined with dual-spike-in strategy

To quantify the transcription level of a gene, we have conceived a novel concept, transcription level of messenger RNA (mRNA) per gene copy, which was determined with a dual-spike-in strategy. In this strategy, an exogenous DNA was added as the spike reference for target DNA in addition to the exogenous RNA as the reference for target RNA. After the mRNA-to-DNA ratio of a target gene was estimated by real-time polymerase chain reaction (PCR), it was first normalized with the mRNA-to-DNA ratio of the exogenous reference. The normalized ratio was multiplied by the ratio of exogenous RNA to exogenous DNA to obtain the transcription level of mRNA per gene copy. This quantified transcription value allows one to compare the expression of a target gene in different tissues or the expression in a specified tissue under different conditions.

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.

Analysis of reference gene expression for real-time PCR based on relative quantitation and dual spike-in strategy in the silkworm Bombyx mori

In general, for real-time quantitative polymerase chain reaction (qPCR), normalization strategies use a reference gene as a control and to avoid the introduction of experimental errors expression of this gene should not vary in response to changing conditions. However, the expression of many reference genes has been reported to vary considerably and, without appropriate normalization, the expression profile of a target gene can be misinterpreted. In this study, the expression levels of seven commonly used reference genes (ACT, GAPDH, 28srRNA, RPL3, α-tubulin, UBC, and TBP) were detected at different development time points and in response to treatment with 20-hydroxyecdysone (20E) and with rutin. The expression stability was analyzed using geNorm and NormFinder software. Significant variations were found among normal tissues and between experimentally treated tissues. The dual spike-in strategy also revealed significant variations of the expression levels of the reference genes among normal tissues and between experimentally treated tissues. Glutathione-S-transferase sigma 1 (GSTs1), which has a high expression level in fat body and is related to the mechanism of resistance, was used as a target gene to validate the feasibility and difference of these two approaches.

Comparative analysis of two acetylcholinesterase genes of Bombyx mandarina and Bombyx mori

Medicinal plants are important elements of indigenous medical system that have persisted in developing countries. Many of the pharmacological principles currently used as anticancer agents were first isolated from plants. However, some important anticancer agents are still extracted from plants because they cannot be synthesized chemically on a commercial scale due to their complex structures that often contain several chiral centers. The aim of this study was to test different extracts from the leaves of Moringa or drumstick tree ( Moringa oleifera ) for activity against leukemia and hepatocarcinoma cells in vitro . The extracts could kill majority (70 - 86%) of the abnormal cells among primary cells harvested from 10 patients with acute lymphoblastic leukemia (ALL) and 15 with acute myeloid leukemia (AML) as well as a culture of hepatocarcinoma cells (75% death), but most significantly by the hot water and ethanol extracts. In conclusion, M. oleifera may have potential for use as source of natural treatment for diseases such as cancer.