Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis(Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis(Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins(Cry2 A, Cry1 C, Cry1 Aa, and Cry1 Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride(PMSF), tosyl-L-lysine chloromethyl ketone(TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid(EDTA), PMSF, tosyl phenylalanine chloromethyl ketone(TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane(E-64). EDTA could significantly inhibit the degradation of Cry2 A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1 C and Cry1 Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1 Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.     

Sonme Factors that Affect the Free Radical-scavenging Activity of Tea Extracts

Some factors that affect the free radical-scavenging activety of two tea extracts were studied in vitro.It was found that concentration of tea extract or heating tea extract or treating with activated carbon and diatomite all had obvious effect on the scavenging activety of green tea extract,but heating or treating with diaomite had less effect on the scavenging activity of black tea extract.Ascorbic acid,for having synergic effect with tea extracts,could enhance the scavenging activity of tea extracts markedly,and the contrary was cupric ion.Reducing sugars such as fructose and glucose also had some syncrgic effect to tea extracts.     

Efficiency of Different Methods for dsRNA Delivery in Cotton Bollworm （Helicoverpa armigera）

RNAi trigged by dsRNA not only facilitates the development of molecular biology, but also initiates a new way for pest control by silence of fatal genes. However, one of the key limitations in pest control is lack of the convenient and efficient method for dsRNA delivery. In this study, different dsRNA delivery methods at their own optimum conditions were evaluated comparatively for their efficiency with Helicoverpa armigera as test animal. It was found that the popular one- time injection of larvae with dsRNA could reduce the pupation rate by 43.0% and enhance larva mortality by 11.7%. One- time ingestion of dsRNA did not result in any significant effect on phenotype. Continuous ingestion of in vitro synthesized dsRNA by refreshing the bait diet every day caused 40.4% decrease in successful pupation and 10.0% increase in larval mortality, which was similar as one-time injection. The most efficient method was found to be the continuous ingestion of the bacteria containing dsRNA expressed, which reduced the rate of pupation by 68.7% and enhanced the larval mortality by 34.1%. Further analysis found that dsRNA was degraded faster in midgut juice than in hemolymph. However, the cell of bacteria could protect dsRNA and delay the degradation in the midgut juice of H. armigera. These results throw light on the application of dsRNA in pest management with proper ways.     

A Study on the Activity of Carboxylesterase and the Differential Expression of Its Gene in the Midguts of Bombyx mori Resistant to BmDNV-Z

This study was to discuss the relationship among the change in the activity of Bombyx mori carboxylesterase （BmCarE） in the midguts, the differential expression of BmCarE gene （bmcare） in the midguts, and the ability of Bombyx mori resistant to densonucleosis virus （BmDNV）, and to elucidate the molecular mechanism of resistance to BmDNV-Z. With two silkworm strains, HUABA, which is susceptible to BmDNV-Z, and BC8 （a near isogenic line of HUABA）, which is completely resistant to the same virus, as materials, the activity of BmCarE in the midgut was determined by Bio-Tek Synergy, and the differential expression of bmcare between the two strains was investigated by real-time fluorescence quantitative PCR, both at 12, 36, and 72 h post oral inoculation of the two strains with virus （hereafter referred as inoculation）. While the activity of BmCarE in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculated group, and the HUABA control group by 3.28, 2.26, and 3.02 times, respectively, with the difference being highly significant （P 〈 0.01）, there was no statistical difference among the other groups. The relative expression level of bmcare in the midguts of BC8 inoculation group at 12 h post inoculation was higher than that in the BC8 control group, the HUABA inoculation group, and the HUABA control group by 17.714, 21.76, and 15.09 times, respectively, with the difference being highly significant （P 〈 0.01）, and there was no statistical difference among other groups. The elevation of BmCarE activity and expression level of bmcare in the resistant strain at 12 h post inoculation may relate to the resistant gene （nsd/nsd） and the stimulation of BmDNV-Z. The molecular basis for the elevation of BmCarE activity in the resistant strain BC8 may be the change in the expression level of bmcare.     

Degradation of Cry1Ab Protein Within Transgenic Bt Maize Tissue by Composite Microbial System of MC1

Environmental safety issues involved in transgenic plants have become the concern of researchers, practitioners and policy makers in recent years. Potential differences between Bt maize（ND1324 and ND2353 expressing the insecticidal Cry1Ab protein） and near-isogenic non-Bt varieties（ND1392 and ND223） in their influence on the composite microbial system of MC1 during the fermentation process were studied during 2011-2012. Cry1Ab protein in Bt maize residues didn＇t affect characteristics of lignocellulose degradation by MC1, pH of fermentation broth decreasing at initial stage and increasing at later stage of degradation. The quality of various volatile products in fermentation broth showed that no signifi cant difference of residues fermentation existed between Bt maize and non-Bt maize. During the fermentation MC1 efficiently degraded maize residues by 83%-88%, and cellulose, hemicelluloses and lignin content decreased by 70%-72%, 72%-75% and 30%-37%, respectively. Besides that, no consistent difference was found between Bt and non-Bt maize residues lignocellulose degradation by MC1 during the fermentation process. MC1 degraded 88%-89% Cry1Ab protein in Bt maize residues, and in the fermentation broth of MC1 and bacteria of MC1 Cry1Ab protein was not detected. DGGE profi le analyses revealed that the microbial community drastically changed during 1-3 days and became stable until the 9th day. Though the dominant strains at different fermentation stages had signifi cantly changed, no difference on the dominant strains was observed between Bt and non-Bt maize at different stages. Our study indicated that Cry1Ab protein did not infl uence the growth characteristic of MC1.     

Effect of Exogenous MJA Treatment of Tea Plants on the Growth of Geometrid Larvae

The effect of tea plant Camellia sinensis induced by exogenous methyl jasmonate （MJA） on lipoxygenase （LOX）, polyphenol oxidase （PPO） and proteinase inhibitor （PI） activity in the leaves of tea plants, as well as the growth and midgut proteinase activity of the geometrid Ectropis obliqua larvae were studied. MJA significantly induced LOX, PPO and PI activity in leaves of tea plants. When geometrid larvae have fed on leaves of tea plants treated with MJA, the activities of the high alkaline trypsin-like enzyme and chymotrypsin-like enzyme in their midgut were significantly inhibited, but the activities of the low alkaline trypsin-like enzyme in their midgut were unaffected, leading to imbalance between different types of proteinase activity in the midgut of the larvae and in turn, the growth were inhibited. These chains of response may be an important mechanism of the direct resistance induced by MJA-treatment of tea plant on geometrid larvae.     

Changes of Nitrate Reductase Activity in Cucumber Seedlings in Response to Nitrate Stress

In China, nitrogen fertilizer application rates in intensive agricultural systems have increased dramatically in recent years, especially in protected vegetable production systems. This excessive use of nitrogen fertilizer has resulted in soil secondary salinization, which has become a significant environmental stress for crops such as cucumber, in the protected farmland of China. So it is necessary to illuminate how crops respond to nitrate stress. The objective of this work was to examine the effects of increased nitrate concentration [14 （CK） and 140 mmol L^-1 （T）] on NO3- concentration, and in vitro and in vivo nitrate reductase activities in the roots and leaves of cucumber （Cucumis sativus L. cv. Xintaimici） seedlings with hydroponic culture. The results showed that the NO3- concentration in the roots and leaves of T seedlings significantly increased over treatment course, and at 12 d increased by 1.08 and 1.72 times with respect to CK seedlings, respectively; in vitro nitrate reductase activity of T was increased dramatically to 1.74 times of CK in the roots at 2 d and 1.56 times of CK in the leaves at 6 d, and then decreased. At 12 d, in vitro activity was still 24.3% higher in the roots and only 9.9% lower in the leaves than CK. Compared with in vitro nitrate reductase activity, in vivo activity responded differently to the increase of treatment time. At the beginning, in vivo nitrate reductase activity in the roots and leaves of T had no significant difference from CK, whereas with the increase of treatment duration, the activity decreased. At 12 d, in vivo activity in the roots and leaves of T lowered by 20.1 and 52.8% with respect to CK, respectively. This evidence suggests that posttranslational activation of nitrate reductase in cucumber seedlings may be seriously inhibited by nitrate stress.     

Effect of increasing levels of nitrogen on yield of tea in different agro-ecological conditions of Pakistan

Field experiment was conducted on mature tea bushes of Qi-Men variety at two different altitudes from sea level i.e. at National Tea Research Institute Shinkiari （1000 m） and on farmer＇s field in Konsh valley at village Battal （1500 m）. Different levels of nitrogen with constant dose of phosphorus and potassium were applied to observe the optimal dose of nutrients for highest yield under local edaphic conditions. The experiment was repeated for three consecutive years. The highest yield of fresh tea leaves were obtained by the application of 420 kg ha^-1 N followed by the application of 360 kg ha^-1 N at 1000 masl. The highest dose of nitrogen also reduced significantly the soil pH in the upper （0-15cm） layer depth during 1998 and 1999. During the year 2000, the reduction in soil pH was observed by the application of highest dose of nitrogen but was non-significant at 5% level. The lower soil depth （15-30 cm） showed the reduction in pH with the highest dose of nitrogen during the experimental year but was statistically non significant. At the higher altitude of 1500 masl the highest yield of fresh tea leaves was obtained by the application of 420 kg ha^-1 during the experimental years but the increase was significant during the year 1999 and 2000. The yield of the fresh tea leaves was positively correlated with the amount of nitrogen application at both the altitudes. The increasing amount of nitrogen decreased the soil pH at both the depths of 0-15cm and 15-30cm. Unlike the lower altitude （1000 m） there was no any significant reduction in the soil pH during 1998 and 1999. A significant reduction in soil pH was found in the year 2000.     

Studies on Amylase and Degradation of Starch and Pigment of Tobacco Leaf During Process of Flue-Curing

The changes in the activity of amylase and amylase-isoenzyme and the degradation of starch and pigment of tobacco leaf during flue-curing were studied by using the electricheated flue-curing barn designed and made by the Henan Agricultural University. The temperature and humidity of the barn were controlled automatically. The results indicated that starch in tobacco leaf decreased rapidly and leveled off after 48h of cutting, in the meantime, the content of soluble sugar increased accordingly and reached a peak at the stage of color-fixing. Both of them had a rapid-changing stage in the first 36 hours of yellowing. The changes of starch and soluble sugar contents had highly significant negative-correlation at 1% level (rNC89=-0.8962**, rYY85=-0.9704**). The activity of amylase increased with the proceeding of curing and reached a peak after 36 hours of curing, then decreased. But the activity of amylase kept at a high level when the humidity of curing-environment was very low, even if the tobacco leaf had been dried. The rapid degradation of starch showed a significantly negative correlation with the increase of activity of amylase at 5 % level (rNC89=-0.8495*, rYY8*=-0.7839*). The degradation of starch and pigment had the same regulation and had highly significant correlation at 1% level (rNC89=0.9649**, rYY85=0.9428**). There were mainly three amylase-isoenzyme bands -A, B, C respectively, in tobacco leaf during flue curing. They were identified as α-AMY, -AMY, R-AMY, and the activity of β-AMY was the highest. The changes in amylase activity and contents of starch and pigment were affected by the tobacco leaf moisture and environmental humidity during curing.     

Alpase of Follicles during the Period before and after Ovulation in Superovulated Rabbits

The Alpase of follicles was studied systematically during the period before and after ovulation in superovulated rabbits.The locations and changes of follicular Alpase varied with the four kinds of physiological status before and after ovulation.Alpase activity was the highest at oestrus and started to drop at the beginning of ovulation,then it increased 15h later and decreased slightly 39h afterwards the beginning of ovulation.Alpase was found on the follicular basement membrane at ocstrus,The synthesis of fiber in fiberblasts was conncted with Alpase activity.     

Adsor ption and Degradation of Bensu—lfuron—methylin Paddy Field

This paper studied adsorption and deyradation of bensulfuron-methyl(BSM) by bioassay method.The results showed that adsorption rate of BSM increased with increasing organic matter content.Degradation velocity of BSM changed fast with pH increasing and temperature decreasing.There were different degradation of BSM between in water and soil.BSM was degraded rapidly in initial period.Afterward the degradation of BSM was slow dow.With increasing organic matter content deyradation of BSM in water was faster than that in soil.     

Polycalin is involved in the action mechanism of Cry2Aa toxin in Helicoverpa armigera(Hübner)

Receptor proteins on the brush border membrane of the insect midgut epithelium are involved in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis(Bt). Polycalin has been identified as a binding protein of the Bt Cry1 Ac toxin in several Lepidoptera including Helicoverpa armigera, but its role in the action mechanism of Cry2 Aa is still unclear. In this study, we investigated the binding characteristics of polycalin from the midgut of H. armigera with Cry2 Aa and its role in the toxicity of Cry2 Aa. The results demonstrated that heterologously expressed H. armigera polycalin peptide could bind with Cry2 Aa with high affinity(K_d=32 nmol L~(–1)). The toxicity of Cry2 Aa decreased by 27% after H. armigera larvae ingested polycalin antisera. These results suggested that polycalin could be a potential functional receptor for Cry2 Aa, and it plays an important role in the susceptibility of H. armigera to Cry2 Aa.     

Effect of plant extracts on activity of some defense enzymes of apple fruit in interaction with Botrytis cinerea

The efficacy of seven plant extracts(neem,fennel,lavender,thyme,pennyroyal,salvia and asafetida) in controlling postharvest of apple(caused by Botrytis cinerea) was evaluated in vitro and in vivo.In vitro,all plant extracts treatments inhibited spore germination.Inhibitory rates of pore germination was 17.41 and 20.83% for neem extract treatment(methanolic and aqueous extracts,respectively) with significant difference compared to control(73.6 and 85.33%) for aqueous and methanol extracts.In the storage conditions,the application of aqueous extract of neem(at concentration of 25%) resulted in 89.11% reduction of disease severity compared with the untreated control.Results of enzymes activity showed the plant extracts can increase the activity of peroxidase,phenylalanine ammonia-lyase,β-1,3-glucanase and polyphenol oxidase in the presence of pathogens,in apple fruits.However,the results of this research revealed that application of neem extracts was more effective than the application of other plant extracts.According to this study,it could be concluded that plant extracts may be useful to control postharvest disease as a safe alternative option to chemical fungicides.     

Studies on some characteristics of nitrate reductase from sugar beet （Beta vulgaris L.）leaves

Some characteristics of nitrate reductase from sugar beet leaves shown in this paper were as follows:The nitrate reductase from sugar beet leaves required NADH as an electron donor.Accordingly,the nitrate reductase was classified as NADH-dependent(E.C.1.6.61).The Km value of the nitrate reductase for NADH and NO3^- were 0.86m mol and 0.18μ mol respectively.The optimum pH in reaction mixture solution for nitrate reduction activity was 7.5.The effect of variable concentrations of inorganic phosphorus in the reaction buffer on nitrate reductase activity was investigated.When the inorganic phosphorus concentration was below 35m mol,the nitrate reductase activity was increased with increase of inorganic phosphorus concentration.Conversely,when the inorganic phosphorus concentration was over 35m mol,the nitrate reductase activity was inhibited.The nitrate reductase activity assayed in vitro was 3.2 and 5.6times of that assayed in vivo under the condition of exogenous and endogenous ground substance respectively.     

Effect of Hydropqinone on Ruminal Urease in the Sheep and its Inhibition Kinetics in Vitro

Effect of hydropuinone (HQ) on rumen urease acivity was studied. Hydroquinone at concentration of 0. 01 mg· L-1 , 1 mg· L-1and 10 mg · L-1 inhibited urease of intact rumen microbes in vitro by 25%, 34%,55% and 64% respectively. In the present of low concentration of βmercaptoethanol, rumen urease could be solubilized and partially purified. The Km for the enzyme was 2 × 10-3 mol · L-1 with Vmax of 319. 144μmoles/mg/min. The kinetics of inhibition with partially purified rumen urease was investigated. The result showed that the inhibitory effect was not eliminated by increasing urea concentration indicating a noncompetitive in nature with inhibition constant 1.2 × 10-5mol · L-1. Hydropuinone at a concentration that produced 64% urease inhibition did not affect ruminal total dehydrogenase, proteolytic enzyme( P ＞ 0. 05) but increased cellulase activity by 28% ( P ＜ 0. 05 ) in vitro. These results demonstrated that hydropuinone was a specific inhibitor of rumen urease and could delay urea hydrolysis effectively without negative effect. The inhibitor appeared to offer the potential to improve nitrogen utilization by ruminants fed diets containing urea.     

Application of Natural Antioxidants from Strawberry Tree （Arbutus unedo L.） and Dog Rose （Rosa canina L.） to Frankfurters Subjected to Refrigerated Storage

The effect of the addition of natural antioxidants from strawberry tree （Arbutus unedo L.; AU） and dog rose （Rosa canina L.; RC）, in frankfurters elaborated with or without the addition of antioxidant additives （sodium ascorbate and nitrite） was studied. Six different types of experimental frankfurters were prepared depending on the addition of phenolic-rich extracts from RC and AU and the presence （P） or absence （C） of antioxidant additives. Thiobarbituric acid reactive substances （TBARS）-numbers signiifcantly increased during chilled storage of C-frankfurters while additives and fruit phenolics inhibited lipid oxidation in P-frankfurters. The amount of protein carbonyls signiifcantly increased in all treatments except in P-AU frankfurters. The discoloration process that occurred during the chilled storage was reduced by the addition of substances with proven antioxidant activity （P-frankfurters）. Texture characteristics as hardness, springiness, cohesiveness and gumminess also suffered a signiifcant deterioration in C-frankfurters. The use of phenolic fruit extracts in combination with traditional antioxidant additives is a successful strategy to enhance the oxidative stability of frankfurters without modifying their color and texture properties.     

Mutagenesis of Arachidonic Acid-producing Mortierella Isabellina and Analyses of △6-desaturase Role by qPCR

Arachidonic acid （AA or ARA）, an essential to-6 polyunsaturated fatty acid （PUFA）, can be produced by Mortierella isabellina. Mutagenesis on Mortierella isabellina As3.3410 was induced to raise ARA production. The mutant strain of YZ-124 had the highest ARA of 4.72 g. L-1, which was 5.5 times higher than that of the original strain 3.3410. mRNA expression level of △ 6- desaturase was determined in five different kinds of ARA-producing Mortierella isabellina after cultured for 7 days, and in the mutant strain YZ-124 over a 3-8 day time-course. In addition, the desaturase activity and ARA content were measured at the selected time points. The lowest expression of △6-desaturase was observed in the original strain and the highest expression in the mutant strain YZ-124, which increased with increasing time in culture. Furthermore, a positive correlation was observed between the expression levels of △6-desaturase and ARA content. Based on this, △6-desaturase played a significant role in ARA synthesis pathway in Mortierella isabellina.     

Toxicity of Bacillus thuringiensis Crystal Toxins to Field Populations of Rice Leaf Folder, Cnaphalocrocis medinalis （Guenee） and Establishment of Baseline Susceptibility to Cry1Ab

Eight insecticidal crystal proteins of Bacillus thuringiensis, CrylAa, CrylAb, CrylAc, CrylB, Cry2Aa, CrylC, CrylDa and Cry 1Ea were assessed for toxicity against 1 st instar larvae of rice leaf folder, Cnaphalocrocis medinalis （Guenee） at 48 HAT and 72 HAT. Bioassay results depicted CrylAa was the most toxic （LCso 2.35 ppm） followed by CrylBa （LCso 8,50 ppm） and CrylAb （LCso 8.73 ppm） at 48 HAT, whereas, at 72 HAT CrylAb proved to be highly toxic （LC50 0.50 ppm） followed by CrylAa （LCso 4.07 ppm）, CrylAc （LCso 4,84 ppm） and CrylBa （LCso 6.42 ppm）. Toxins Cry2Aa, CrylCa, CrylDa and CrylEa did not resulted in any mortality at 48 HAT and 72 HAT, respectively. Baseline estimates for CrylAb against 1st instar larvae of C. medinalis sampled from seven geographical locations revealed variation in LC50＇s from 0.37 ppm to LC50 16.25 ppm at 48 HAT and LC50 0.50 ppm to LC50 6.49 ppm 72 HAT, respectively with relative resistance ratios of 44-fold and 13-fold at 48 HAT and 72 HAT over the susceptible population.     

Effects of Different Compound Trace Element Premixed Materials on Beef Cattle Production Performance and Anti-oxidation Capacity

In this study, the effects of two types of premixed materials with different combinations of trace elements on the production performance and antioxidant capacity of simmental beef cattle were examined.Fifteen healthy simmental beef cattle of similar weight(approximately 330 kg), the same age(12 months), without castration, and a good physique were divided into three groups, with five beef cattle in each group.Food of GroupⅠ beef cattle was supplemented with a commercially marketed 5% compound trace element premixture for fine beef cattle.Food of GroupⅡbeef cattle was supplemented with a 5% compound microelement premixture for beef cattle that was designed to address local nutrient deficiencies and surpluses.In the blank control group, the beef cattle were not fed a premixture.The pretest period was 15 days, and the test period was divided into prefattening(45 days) and postfattening(45 days) stages.Body weight and body size indices were recorded at 1, 2 and 3 months, and blood samples were collected regularly.In GroupⅠ, the daily weight gain increased significantly by 15.7% compared with that of the control group.The largest daily weight gain was in Group Ⅱ, which increased by 31.6% compared with that in the control.During the test period of 90 days, the body size indices of the three different groups increased in different months, with significant increases in the indices for both test groups compared with those of the control.In GroupⅠ, the activity of CP, the total activity of SOD and Cu-Zn-SOD increased significantly(p0.05) compared with those in the control group, with a highly significant increase observed in GSH-PX activity(p0.01).In GroupⅡ, the increases in the activity of CP and the total activity of SOD and Cu-Zn-SOD were highly significant compared with those in the control group(p0.01).In addition, a significant increase was observed in GSH-PX activity(p0.05).Based on pretest results, the concentrations of Cu, Zn, Mo, Mn, Se and Co in the blood of experimental beef cattle were lower than those of the normal range.After feeding for 90 days, the concentrations of Cu, Zn, Mo, Mn, Se and Co in the blood of GroupsⅠ andⅡ were significantly higher than those in the control group(p0.05).The concentrations of elements in the blood of Group II were close to the appropriate levels.Thus, the effects of the specifically designed compound microelement premixture on the prevention of nutrient imbalances and control of beef cattle nutrition metabolism and the production of fattened beef cattle were significant.