Chunya Bu

Isolation and Identification of Acaricidal Compounds in Inula japonica (Asteraceae)

ABSTRACT We identified acaricidal compounds in Inula japonica Thunberg (Asteraceae) that were effective against carmine spider mite, Tetranychus cinnabarinus (Boisduval). Petroleum-ether extracts from I. japonica were toxic to T. cinnabarinus, with a 50% lethal concentration (LC50) value of 1.18 mg/ml. Silica gel column chromatography was used to separate the acaricidal components. Seventeen of 38 fraction groups had mite mortality rates >50%, nine fraction groups had rates >60%, and three fraction groups had rates >80% at 2 mg/ml. The major volatile compounds in the bioactive fraction groups were identified by gas chromatography-mass spectroscopy, and &bgr;-sitosterol (1), stigmasterol (2), lupeol (3), and &agr;-amyrin (4) were identified and determined to have acaricidal activity against T. cinnabarinus in vitro.

Assessment of the contact toxicity of methyl palmitate on Tetranychus viennensis (Acari: Tetranychidae).

ABSTRACT Previous work demonstrated that methyl palmitate possesses acaricidal activity against Tetranychus viennensis Boisduval (Acari: Tetranychidae) via an unknown mechanism. Here, the symptoms of methyl palmitate toxicity to T. viennensi were studied to determine the acaricidal mechanism of action of this fatty acid methyl ester. Methyl palmitate caused concentration-dependent mortality of T. viennensis, with a moderate concentration (5 mg/ml) eliciting excitement and premature oviposition without spinning shortly after exposure. Tremors of the appendages were subsequently observed, followed by quiescence after ≈5 h. Mites developed dorsal fluid exosmosis at 15–20 h posttreatment with reduced egg production, followed shortly thereafter by death. Some typical neurotoxic symptoms such as excitement and convulsions were observed in methyl palmitateexposed mites, suggesting that methyl palmitate may be a neurotoxin. Compared with other neurotoxic acaricides, methyl palmitate poisoning is a slow process in mites. Transmission electron microscopy revealed serious ultrastructural damage in response to 5 mg/ml methyl palmitate exposure. Autolysis of membranous structures was also observed, especially in the mitochondria, suggesting a novel mode of action for methyl palmitate-induced toxicity.

Pyruvate decarboxylase and alcohol dehydrogenase overexpression in Escherichia coli resulted in high ethanol production and rewired metabolic enzyme networks

Pyruvate decarboxylase and alcohol dehydrogenase are efficient enzymes for ethanol production in Zymomonas mobilis. These two enzymes were over-expressed in Escherichia coli, a promising candidate for industrial ethanol production, resulting in high ethanol production in the engineered E. coli. To investigate the intracellular changes to the enzyme overexpression for homoethanol production, 2-DE and LC–MS/MS were performed. More than 1,000 protein spots were reproducibly detected in the gel by image analysis. Compared to the wild-type, 99 protein spots showed significant changes in abundance in the recombinant E. coli, in which 46 were down-regulated and 53 were up-regulated. Most proteins related to tricarboxylic acid cycle, glycerol metabolism and other energy metabolism were up-regulated, whereas proteins involved in glycolysis and glyoxylate pathway were down-regulated, indicating the rewired metabolism in the engineered E. coli. As glycolysis is the main pathway for ethanol production, and it was inhibited significantly in engineered E. coli, further efforts should be directed at minimizing the repression of glycolysis to optimize metabolism network for higher yields of ethanol production.

Acaricidal Activity of Ethyl Palmitate against Tetranychus cinnabarinus

Isolation and identification of bioactive compounds from medicinal plant of Inula flower is a potential approach for the development of new pesticides against the mite of Tetranychus cinnabarinus. Dry Inula Flower was used as the starting material to obtain active substances with acaricidal activity by organic solvent extraction and bioactivity assay verification. The main component of the active substances in fraction 33 was further identified by GC/MS and proven to be ethyl palmitate. In order to clarify the roles of ethyl palmitate playing against mites of Tetranychus cinnabarinus, the enzymatic activities were assayed regarding to four essential enzymes in the process of either nerve conduction or anti-oxidation reaction of the mites, including assays for Ca2+-ATPase, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). In addition, the substructures of the ethyl palmitate treated adult female mite of T. cinnabarinus were observed by transmission electron microscopy. In the bioassay, ethyl palmitae showed strong activities against the mites with the corrected mortality rates of more than 90% for samples at 2mg/ml concentration. The activity increases of antioxidant enzymes indicate a certain degree of stimulation of the anti-oxidant mechanisms to response for the ethyl palmitate treatment in vivo. The Ca2+-ATPase plays essential role in the maintenance of nerve cell excitability and conduction. Its enzymatic activity was subject to a certain degree of declination, which might cause the death of the mites due to suppression in nerve conduction. The toxicity effect of the mites by ethyl palmitate was further confirmed by TEM observation. The significant destruction of the skin structures and cellular substructures can directly affect the normal function of the cells, and ultimately lead to the death of the mites. Based on the strong acaricidal activity of ethyl palmitate has against the carmine spider mite, it is valuable to be further investigated for a new type of botanical pesticides development.

Cloning and Characterization of the Acetylcholinesterase1 Gene of Tetranychus cinnabarinus (Acari: Tetranychidae)

ABSTRACT The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a major agriculture pest. It can be found worldwide, has an extensive host plant range, and has shown resistance to pesticides. Organophosphate and carbamate insecticides account for more than one-third of all insecticide sales. Insecticide resistance and the toxicity of organophosphate and carbamate insecticides to mammals have become a growing concern. Acetylcholinesterase (AChE) is the major targeted enzyme of organophosphate and carbamate insecticides. In this study, we fully cloned, sequenced and characterized the ace1 gene of T. cinnabarinus, and identified the differences between T. cinnabarinus AChE1, Tetranychus urticae Koch AChE1, and human AChE1. Resistance-associated target-site mutations were displayed by comparing the AChE amino acid sequences and their AChE three-dimensional (3D) structures of the insecticide-susceptible strains of T. cinnabarinus and T. urticae to that of a T. urticae-resistant strain. We identified variation in the active-site gorge and the sites interacting with gorge residues by comparing AChE1 3D structures of T. cinnabarinus, T. urticae, and humans, though their 3D structures were similar. Furthermore, the expression profile of T. cinnabarinus AChE, at the different developmental stages, was determined by quantitative real-time polymerase chain reaction; the transcript levels of AChE were higher in the larvae stage than in other stages. The changes in AChE expression between different developmental stages may be related to their growth habits and metabolism characteristics. This study may offer new insights into the problems of insecticide resistance and insecticide toxicity of nontarget species.

Isolation and Identification of the Acaricidal Components Extracted from Stellera chamaejasme

The research of Botanical acaricide is an important aspect of integrated agricultural mite management. Eight kinds of different organic solvents were used for cold-soak extraction from Stellera chamaejasme. Combined with tracking activity, the fraction E7-4 was isolated from ethyl acetate extract using medium-pressure liquid chromatography (MPLC) and preparative HPLC. Using nuclear magnetic resonance, it was determined that fraction E7-4 was Scopoletin. The results showed that the scopoletin in Stellera chamaejasme was the important acaricidal activity ingredient which exhibited contact and systemic activity against Tetranychus cinnabarinus. The Lethal concentrations (LC50) to Tetranychus cinnabarinus were 1.267 mg/mL and 7.388 mg/mL respectively.

Effects of Stigmasterol to Activities of Several Enzymes of Tetranychus cinnabarinus

The acaricidal activity of stigmasterol was carried out on the Tetranychus cinnabarinus, using the slide dip technique. Here the female adult mites were treated by the Stigmasterol extracted from Inula japonica. Bioassay-guided purification of the 12th fraction had higher activity against the T. cinnabarinus, Gas chromatography-mass spectrometry (GC/MS) demonstrated that the chemical composition of the active fraction 12th sample was mainly composed of Stigmasterol. That had not been reported before. And the acaricidal mechanism of Stigmasterol to T. cinnabarinus is unknown. Our results showed that the nervous symptoms of the T. cinnabarinus in response to the Stigmasterol. The protein content, the acetylcholine esterase (AChE) activity, the Calcium enzyme ATP (Ca2+-ATPase) activity and Sodium enzyme ATP (Na+-ATPase) activity of the mites treated with Stigmasterol were assayed. The results showed that the protein content and the activity of AchE in the T. cinnabarinus treated with the Stigmasterol were increased. The activity of Ca2+-ATPase and Na+-ATPase were induced by the Stigmasterol. The effects of the T. cinnabarinus indicated that the central nervous system was the main target of Stigmasterol. The long time over excitation caused the treated mites to die.

Identification of Acarcidal Compounds in Inula japonica Extracts against Tetranychus cinnabarinus

In this study, we aimed to identify acaricidal compounds in Inula japonica against Tetranychus cinnabarinus. Here, I. japonica petroleum-ether extracts were found toxic to T. cinnabarinus with a LC50 value of 1.18 mg/ml. Silica gel column chromatography was used to further separate acaricidal components from I. japonica petroleum-ether extracts. Contact toxicity data showed that 17 out of 38 fractions groups had mite mortality rates above 50%, 9 fraction groups above 60 %, and fractions groups G5, G7 and G15 even above 80 % at a concentration of 2 mg/ml. The major vital compounds in the bioactive fraction groups were identified by GC/MS, and four compounds, namely β-Sitosterol (1), Stigmasterol (2), Lupeol (3), and α-Amyrin (4) were determined to have acaricidal activity to Tetranychus cinnabarinus.

Antifungal Activity of Extracts of Stellera chamaejasme against Botrytis cinerea

The antifungal activity of the extracts from Stellera chamaejasme against Botrytis cinerea was investigated in our work. The active components of Stellera chamaejasme extraction were primary analyzed by bioactive tracking method followed by separation of the highly active components by column chromatography, and then the structure of the final extract was identified by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The results showed that the main antifungal bioactive compound in the eluotropic component 3 was umbelliferone. And a positive correlation between umbelliferone concentration and antifungal activity was observed. The minimal inhibitory concentration (MIC) of umbelliferone was 1.52 mg ml-1. Depending on the analysis of ultrastructure by scanning electron microscope (SEM), it was found that the morphology of Botrytis cinerea spore and mycelium were obviously destroyed by umbelliferone existing in eluotropic component 3.