Behavioural Bioassays and Identification of Cashew Leaf and Stem Volatiles Mediating Attraction to the Stem Girdler, Analeptes trifasciata (Coleoptera: Cerambycidae)

The cashew stem girdler, Analeptes trifasciata Fabricius (Coleoptera: Cerambycidae), damages cashew by its girdling activities in the stem thereby causing huge economic losses. The stem girdler is managed through cultural practice of burning girdled stems and beetles, though this has drawbacks. The objective of this study was to explore the cues mediating attraction to the cashew host plant;hence the role of olfaction in host plant location by A. trifasciata underlying the semio-chemical option for controlling this insect pest. A diffusional Y-tube olfactometer was used to study the behavioural response of A. trifasciata, to freshly cut cashew stem and leaves odour sources. Methanol-extract of these plant tissues was subjected to the coupled gas chromatography-mass spectrometry (GC-MS) analysis. Y-tube olfactometric assays demonstrated that both sexes oriented towards and spent significantly more time in stem odour arm compared to the leaf odour arm in both male (male: t = 2.228, d.f = 11, P = 0.040) and female (t = 2.341, d.f = 11, P = 0.040). A combination of fatty acids, amino acids and carbohydrates were detected in cashew stems. Some of these fatty acids are attractants to other insect pests. It is suspected that these fatty acid blends may possibly be responsible for facilitating host plant location by both sexes. In conclusion, both sexes were independently and strongly attracted to the stem volatiles;this study opens the possibility of utilizing cashew stem volatiles as surveillance and control tools.

Bioassay Determination of Quinclorac Phytotoxicity on Peanut

[ Objective] The phytotoxicity effect of quinclorac on peanut succeeding seedlings was studied. [ Method] Peanut was taken as the indicator crop, plant height and fresh weight were adopted as bioassay indicator, the biological activity of quinclorac on peanut was determined by the method of adding quinclorac in the soil, and the residue dynamic of quinclorac in paddy soil was determined. [Result] The linear correlation equations of peanut plant height and fresh weight with the concentration range of 0.7 -8.0 mg/kg separately were y = 11.235x ＋3.818 6, R^2 = 0.969 1 ; y = 5.973 3x ＋ 6.532 8, R^2 = 0.988 2. There would be no residual phytotoxicity effect when peanut was going to be planted in the same block in the second year. [Condusion] The bioassay method was simple and exact with good repeatability.

Cancer Chemopreventive Retinoids: Validation and Analysis of in Vivo and in Vitro Bioassay Results

Several natural and synthetic retinoids (vitamin-A derived analogies) were examined for their potential anti-cancer activity in both in vivo animal models and a novel in vitro human keratinocyte clonal growth bioassay system. The natural retinoids included all-trans-retinoic (RA), 13-cis-retinoic acid, 4-oxoretinoic acid, and retinol. Among the synthetic retinoids tested were all trans N-(4-hydroxy(phenyl)retinamide, 3-substituted oxoretinoic acids, and 13 cis-N-ethylretinamide. The animal models employed were: 1) vitamin A-deficient hamster tracheal organ assay (HTOC);2) the benzo(α)pyrene-induced squamous metaplasia in a hamster tracheal organ system (BP-HTOC);3) the mouse skin tumor promoter (TPA)-induced ornithine decarboxylase enzyme assay(ODC);4) the mouse skin papilloma (MPA) assay;and 5) a novel retinoid bioassay in which retinoids display IC50 values to inhibit clonal growth of NHK. All-trans-RA, 4-oxoretinoic acid and retinol were consistently more active than any of the synthetic derivatives in all bioassays tested. A statistical model was developed and significant positive correlations were found between: 1) ED50 values in the HTOC system and reduction in TPA-induced ODC enzyme activity;2) tumors per animal in the MPA bioassay and suppression of TPA-induced ODC activity;and 3) a positive correlation between suppression of tumors per animal in the MPA assay, and retinoid inhibition of keratinocyte clonal growth. Test retinoids, were tested for their capacity to inhibit the clonal growth of a squamous carcinoma cell line (SCC-25), which were found to be 2 - 3 logs less sensitive for each tested retinoid than the corresponding activity against NHK cells. Antineoplastic retinoid drugs were reviewed.

Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section （influents） and 9 from post-treatment section （effluents）, were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 17β-estradiol （E2）, estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Pharmacokinetic study with N-Ile^1-Thr^2-63-desulfato-r-hirudin in rabbits by means of bioassay

Aim： To study the pharmacokinetic （PK） properties in rabbits treated with N-Ile^1-Thr^2-63-desulfato-r-hirudin （rH） newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent. Methods： rH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg. Results： The bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows： t1/2p 58.4-59 min. Vd 0.09-0.12 L/kg, CL 0.0035-0.0040 L/（kg·min）; AUC were proportional to the doses, ti/2 and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t1/2p and 13-fold greater.4 UC than normal healthy rabbits. Conclusion： rH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site. The excretion ofrH by kidneys played a very important role in the elimination of rH. The PK ofrH could be described by the two- and one-compartment model after iv and sc dosing, respectively, and folio,wed linear kinetics.

<i>In Vitro</i>Bioassay of Allelopathy in Four Bamboo Species;<i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku;Phyllostachys bambusoides cv. Madake;P. nigra cv. Hachiku;Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

A Bioassay for the Cytotoxicity of Gemcitabine Using the Marine Ciliate Euplotes vannus

This study investigated the cytotoxicity of gemcitabine using the marine ciliate Euplotes vannus as the test organism.The median lethal concentrations(LC50 values)were determined using acute toxicity tests within an exposure time of 30 min with 0,6,12,24,and 48 mg mL^-1 gemcitabine.The median inhibition effect(IC50 value)on the growth of the ciliate cells was examined using chronic toxicity tests within 5 days(120 h)after exposure for 30 min with 0,0.7,3.5,7,and 14 mg mL^-1 gemcitabine.The 30-min LC50value was 10.66-mg mL 1.The LC50 values decreased with increasing exposure times and well fitted to the toxicity curve equation LC50=10.93+28.4e^-0.19t(R2=0.93;P<0.05,t=exposure time).The IC50 value for growth rates was 7.05 mg mL^-1,and the inhibition effect on growth rates well fitted to the model equation r%=0.8681e^-0.0782Cgem(r%means growth rate with inhibition by gemcitabine,Cgem means concentrations of gemcitabine,R^2=0.99 and P<0.05).The LC50 values of a wide range of gemcitabine concentrations could therefore be predicted for any given exposure time.These results suggest that the clinical dose of gemcitabine(20 mg mL^-1)was higher than the 30-min LC50 value,which was almost the same as the 6-min LC50 value(19.88 mg mL^-1)for E.vannus cells.The results also demonstrate that E.vannus can be used as a robust test organism for bioassays of chemotherapeutic drugs during short exposure periods.

Evaluation of Allelochemicals, Abscisic Acid and Coumarin, in Leaf-Origin Suspension Cultured Cells of <i>Prunus yedoensis</i>Using Protoplast Co-Culture Bioassay Method

Dried leaves of Prunus yedoensis and P. lannesiana (50 mg) showed strong inhibitory allelopathic activities, e.g., more than 97% growth inhibition of lettuce seedling using the sandwich method. Similarly, among suspension cultures induced from leaves and peduncles of two Prunus species, we found the strongest inhibitory allelopathic activities of protoplasts of leaf-origin suspension cells of P. yedoensis, when the protoplast co-culture method for bioassay of allelopathy was applied with lettuce as a recipient plant. Effects of two putative allelochemicals, abscisic acid and coumarin, on both protoplast cultures of lettuce and P. yedoensis were investigated. Coumarin inhibited the growth of lettuce protoplasts from low concentrations, while abscisic acid stimulated. Abscisic acid inhibited the protoplast growth of P. yedoensis from low concentrations, while coumarin did not, but inhibited only at a high concentration (1 mM). Contents of abscisic acid in protoplasts were measured using small scale purification and Enzyme Linked Immno Sorbent Assay, and contents of coumarin in leaf-origin susepension cells of P. yedoensis were measured using Gas Chromatography-Mass Spectrometry. Coumarin was more likely the allelochemical causing the strong inhibitory allelopathic activities of P. yedoensis in the protoplast co-culture bioassay. Effectiveness of the protoplast co-culture bioassay method of allelopathy was discussed.

Bioassay of Clostera anastomosis Granulosis virus

The second-instar healthy larvae of Clostera. anastomosis were collected in the artificial woodland of poplar in Shuangcheng Town, Heilongjiang Province, China. The dead larvae of C. anastomosis infected by granulosis virus （GV） of Clostera anastomosis were grinded to obtain GV. The GV viral pesticide was diluted to seven concentrations, 1.58×10^3PIB·mL^-1, 1,58×10^4PIB·mL^-1, 1.58×10^5PIB·mL^-1 1.58×10^6PIB·mL^-1, 1,58×10^7PIB·mL^-1; 1.58×10^8PIB·mL^-1 and 1.58×10^9PIB·mL^-1 and the fresh poplar leaves were dipped in the seven concentrations liquids to feed the larvae. After nine days the mortality of larvae was investigated. The minimum corrected mortality （7.32%） of larvae was observed at concentrations of 1.58×10^3PIB·mL^-1 and the maximal mortality （97,36%） was observed at concentration of 1.58×10^9PIB·mL^-1. The regression equation between the logarithm of the virus concentration and the mortality was y= 1.946＋0.558x The LC50 was 2.97×10^5PIB·mL^-1. The LT50 for the virus concentration of 1.58×10^5, 1.58×10^6, 1.58×10^7, 1,58×10^58, 1.58×10^9 PIB·mL^-1 were 8.55d, 6.89d, 5.9d, 4.65d, and 4.08d, respectively, shorting gradually with the concentration increasing, It is concluded that the toxicity of Clostera anastomosis GV is very strong and as a kind of insecticides it has big potential in practical application.

Evaluation of mouse bioassay results in an inter-laboratory comparison for paralytic shellfish poisoning toxins

An inter-laboratory comparison of the AOAC mouse bioassay for paralytic shellfish poisoning (PSP) toxicity in shellfish was carried out among 25 Chinese laboratories to examine the overall performance for PSP testing in China, and to analyze the main factors affecting the performance of this method. The toxic scallop Patinopecten yessoensis collected from coast of Bohai Sea, China, was used as a test sample in the comparison study. The results were reported and evaluated using robust statistical methods. The z scores showed that 80%, 8%, and 12% of laboratories reported satisfactory results, unsatisfactory results, and questionable results, respectively. This evaluation demonstrates that the PSP mouse bioassay is an appropriate method for screening and testing PSP toxicity in shellfish. However, it was found that the experience and skill of technicians, as well as the body weight and health status of mice being used significantly affected the accuracy of the method.

A New Bioassay Method for Evaluation Allelopathic Potential of Rice Germplasm

Allelopathy was defined in 1996 by IAS （international allelopathy society） as any process involving secondary metabolites produced by plant, algae, bacteria and fungi that influences the growth and development of agricultural and biological systems. Rice allelopathy against weeds was reported since 1989, which offers an integrated weed management with substantially reduced herbicide usage. Application of allelopathic rice cultivars is thought a resources conservation and environmental friendly way of weed bio-control, and could promote the sustainable development of agriculture. Screening or evaluating the allelopathic potential rice variety is the first step. In this paper, a new bioassay method was set up by the allelopathic potential of 9 rice lines on the target weed barnyardgrass （Echinochloa crusgalli）, and comparing with bioassay methods such as relay seeding in filter paper （RSF） and relay seeding in agar （RSA）. The results indicated that three methods had a same tendency in evaluating the allelopathic potential of rice; there existed a significant difference among different bioassay methods, and an interaction between bioassay methods and rice lines. The method of root exudates （RE） with the highest value and a correlation efficiency of 0.98 was considered as ideal bioassay method for evaluation of allelopathic potential.

Development and validation of microbial bioassay for quantification of Levofloxacin in pharmaceutical preparations

The aim of this study was to develop and validate a simple,sensitive,precise and cost-effective onelevel agar diffusion（5＋1） bioassay for estimation of potency and bioactivity of Levofioxacin in pharmaceutical preparation which has not yet been reported in any pharmacopoeia.Among 16 microbial strains.Bacillus pumilus ATCC-14884 was selected as the most significant strain against Levofioxacin.Bioassay was optimized by investigating several factors such as buffer pH,inoculums concentration and reference standard concentration.Identification of Levofioxacin in commercial sample Levoflox tablet was done by FTIR spectroscopy.Mean potency recovery value for Levofioxacin in Levoflox tablet was estimated as 100.90%.A validated bioassay method showed linearity（r^2 = 0.988）,precision（Interday RSD=1.05%,between analyst RSD=1.02%） and accuracy（101.23%,RSD=0.72%）.Bioassay was correlated with HPLC using same sample and estimated potencies were 100.90% and 99.37%.respectively.Results show that bioassay is a suitable method for estimation of potency and bioactivity of Levofioxacin pharmaceutical preparations.

Development of An ICR Mouse Bioassay for Toxicity Evaluation in Neurotoxic Poisoning Toxins-Contaminated Shellfish

Objective To develop an ICR （female） mouse bioassay （MBA） for toxicity confirmation and evaluation of neurotoxins （brevetoxins）-contaminated shellfish. Methods Brevetoxins （BTX-B） as a causative agent of neurotoxic shellfish poisoning （NSP） under different shellfish matrices were intraperitoneally injected at different doses into mice to study their toxic effects and to differentiate the range of lethal and sublethal dosages. Their sensitivity and specificity were analyzed with 2 competitive ELISA kits for quantitative determination of standard BTX-B and dihydroBTX-B under different shellfish matrix-diluent combinations. Detection rates of MBA and two antibody-based assays for BTX-B from field NSP-positive shellfish samples were compared. Results BTX-B could be detected in shellfish tissues at concentration of 50-400 μg/100 g under shellfish matrix-Tween-saline media, which were appropriate to identify toxic shellfish at or above the regulatory limit （80 μg/100 g shellfish tissues）. The LD 50 identified was 455 g/kg for BTX-B under general shellfish matrices （excluding oyster matrices） dissolved in Tween-saline. The presence of shellfish matrices, of oyster matrices in particular, retarded the occurrence of death and toxicity presentation in mice. Two antibody-based assays, even in the presence of different shellfish matrix-diluent combinations, showed acceptable results in quantifying BTX-B and dihydroBTX-B well below the regulatory limit. Conclusion The two ELISA analyses agree favorably （correlation coefficient, r 0.96; Student＇s t-tests, P〉0.05） with the developed bioassay.

Preparative Purification and Bioassay of Bt Toxin from Cry1Ab Transgenic Rice

A method of extracting and purifying Cry1Ab protein(Bt toxin) from Cry1Ab transgenic rice was established. Most of the Bt toxin present in the tissue of Cry1Ab transgenic rice was extracted effectively with a solution of 50 mmol/LNa_2CO_3 and NaHCO_3. The crude protein containing Bt toxin was obtained after the pretreatment of Cry1Ab transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCl buffer+1.0 mmol/L EDTA, pH=8.0) mixed with 0.1, 0.3, 0.5 and 0.8 mol/L NaCl in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.

Evaluation on the Joint Action Between Chlorsulfuron and Haloxyfop-R by Bioassay

The joint action between chlorsulfuron and haloxyfop R was evaluated by bioassay with wheat and corn,respectivly.The dose response curve derived from wheat bioassay showed that the inhibition of haloxyfop R to wheat root growth wasn't affected by the increasing rate of chlorsulfuron.It indicated that chlorsulfuron had no antagonism to haloxyfop R.Meanwhile,the variation analysis of corn bioassay indicated that these two herbicides had joint action on inhibition to corn primary root growth.The joint action was evaluated as additive action by using Isobole Method.So chlorsulfuron and haloxyfop R could be used as tank mixture.

Improvement of Teaching Experimental Design of Pesticide Bioassays

For mistakes taken in pesticide bioassays, teaching experimental design is improved in the paper, so as to let students explore and analyze in teaching experiments to get a deeper understanding of theoretical knowledge, thereby effectively avoiding frequently-taken mistakes in pesticide bioassays.

Gamma Irradiation Enhanced Leaf Bioactive Components and Bioassay Parameters in M₅Mulberry (<i>Morus</i>Sp.) Mutant

Gamma radiation is an effective tool for inducing genetic variation in plant characters. In the present experiment, M5 mulberry variety juvenile twigs were subjected to source Co60 gamma irradiation (1 kR - 10 kR) and mutants grown in triplicates in randomized block design to raise M1 and M2 generation. In M2 generation plants were subjected to phytochemical and bioassay tests. Silkworm rearing parameters and commercial characters of cocoons were recorded by feeding cross breed silkworms. Results show that M5 mutant leaves revealed significant variations in phytochemical constituents and moisture content. Bioassay tests recorded significant differences compared to control in M2 generation. Commercial characters like cocoon weight (1.41 g), shell weight (0.24 g), shell percentage (16.29 %), filament length (821.00 mts), renditta (8.2), denier (2.24) and effective rate of rearing (92.14 %) were increased. It is concluded that, gamma rays treatment enhances the mulberry plants leaf bioactive components, silkworm rearing and cocoon parameters and shows beneficial variants in M2 generation.

A Simple Bioassay Using Fluorescent Microbeads and Daphnia magna

The amount of microbeads ingested by Daphnia magna decreases on exposure to toxic materials; tins oenawor was used to develop a toxicity test. To determine the toxicity of seven metals, D. magna were collected and homogenized, and the fluorescence intensity of the microbeads ingested by D. magna was measured. The amount of ingestion was determined from fluorescence intensity. The fluorescence intensity was half of that of the controls which was measured as the 30 min-FI50, and these data correlated well with those from an acute immobilization method （24 h-EC50）. An advantage of the method using fluorescent beads is that an estimate of the 24 h-EC50 can be obtained.

Global parameter estimation of the Cochlodinium polykrikoides model using bioassay data

Cochlodinium polykrikoides is a notoriously harmful algal species that inflicts severe damage on the aquacultures of the coastal seas of Korea and Japan. Information on their expected movement tracks and boundaries of influence is very useful and important for the effective establishment of a reduction plan. In general, the information is supported by a red-tide（a.k.a algal bloom） model. The performance of the model is highly dependent on the accuracy of parameters, which are the coefficients of functions approximating the biological growth and loss patterns of the C. polykrikoides. These parameters have been estimated using the bioassay data composed of growth-limiting factor and net growth rate value pairs. In the case of the C. polykrikoides, the parameters are different from each other in accordance with the used data because the bioassay data are sufficient compared to the other algal species. The parameters estimated by one specific dataset can be viewed as locally-optimized because they are adjusted only by that dataset. In cases where the other one data set is used, the estimation error might be considerable. In this study, the parameters are estimated by all available data sets without the use of only one specific data set and thus can be considered globally optimized. The cost function for the optimization is defined as the integrated mean squared estimation error, i.e., the difference between the values of the experimental and estimated rates. Based on quantitative error analysis, the root-mean squared errors of the global parameters show smaller values, approximately 25%–50%, than the values of the local parameters. In addition, bias is removed completely in the case of the globally estimated parameters. The parameter sets can be used as the reference default values of a red-tide model because they are optimal and representative. However, additional tuning of the parameters using the in-situ monitoring data is highly required.As opposed to the bioassay data, it is necessary because the bioassay data have limitations in terms of the in-situ coastal conditions.

Comparison between Two Bioassays in Monitoring and Evaluation of Water Quality

[ Objective] The aim was to compare the application of two bioassays to monitoring and evaluation of water quality. [ Method ] By using two bioassays （micronucleus detecting technology based on Vicia faba root tip cells and the bioassay by luminous bacteria）, we monitored and evaluated seven kinds of single solutions respectively added different pollutants （ Hg, Cd, As, Cr6 ＋ , Pb, I_A8 and CODc,） and a mixed solution added those seven pollutants. Afterwards, we compared their results under the same pollutant and concentration, so as to study the two bioassays＇ sensitivity and sensitive concentration to the seven pollutants. [ Result] Under the same pollutant and concentration, micronucleus detecting tech- nology based on Vicia faba root tip cells had responses to Hg, Cd, As and Cr6＋ , but there was no response to Pb, LAS and CODer. However, the bioassay by luminous bacteria had responses to most pollutants except Cr6＋. Comparing the sensitive degree and concentration to each pollutant, they can complement each other. For these seven pollutants, the bioassay by luminous bacteria was better than micronucleus detecting technology based on Vicia faba root tip cells. Meanwhile, from the testing result of the mixed solution, the combined toxicity of several pollutants in lower con- cantrations was serious. At the same time, contrasted to normal chemical methods, bioassays were fast and effective. [ Conclusion] The research could provide theoretical references for the correlation study of bioassays.