In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Studies on glutathione transferase from grasshopper (Zonocerus variegatus)

Glutathione transferase (GST) was purified from the hindgut of grasshopper (Zonocerus variegatus) a polyphagous insect. The purified enzyme had a native molecular weight of 40 kDa and a subunit molecular weight of 19 kDa. The purified enzyme could conjugate glutathione (GSH) with 1-chloro-2,4-dinitrobenzene (CDNB), paranitrobenzylchloride, paranitrophenylacetate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl), and 1,2-dichloro-4-nitrobenzene (DCNB) with specific activities of 3.3 ± 0.3, 0.49 ± 0.10, 0.10 ± 0.002, 1.2 ± 0.2, and 1.7 ± 0.4 μmol/min/mg protein, respectively. CDNB appears to be the best substrate with a specificity constant, k_cat/K_m, of 1.8 ± 0.1 × 10⁻⁴ M⁻¹ S⁻¹. The kinetic mechanism of Z. variegatus GST (zvGST) in the conjugation of GSH with some electrophilic substrates appears complex. Conjugation of GSH with DCNB was inhibited by high DCNB concentration, while with NBDCl, as the electrophilic substrates, different values of K_m were obtained at high and low concentrations of the substrates. Cibacron blue, hematin, S-hexylglutathione, and oxidized glutathione inhibited the enzyme with I₅₀ values of 0.057 ± 0.004, 0.80 ± 0.2, 33 ± 2 μM, and 5.2 ± 0.3 mM, respectively. The nature of inhibition by each of these inhibitors is either competitive or non-competitive at varying GSH or CDNB as substrates. NADH and NAD⁺ inhibited the enzyme with an I₅₀ value of 0.4 ± 0.01 and 11 ± 1 mM, respectively. NADH at a concentration of 0.54 mM completely abolished the activity. As part of its adaptation, the flexible kinetic pathway of detoxication by zvGST may assist the organism in coping with various xenobiotics encountered in its preferred food plants.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.     

Mutagenic, genotoxic and enzyme inhibitory effects of carbosulfan in rainbow trout Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss; 116.88 ± 21.69 g) were exposed to sublethal concentrations (25 μg/L) of carbosulfan for 60 days to test if the long term exposure of fish to carbosulfan affects red blood cells acetylcholinesterase (AChE), δ-aminolevulinic acid dehydratase (ALA-D) and paraoxonase (PON) enzyme activity and induces genotoxic and/or mutagenic effects. The exposure resulted in inhibition of AChE and ALA-D activity of rainbow trout when compared to control fish. The activity of PON was not affected by carbosulfan. Interestingly, carbosulfan was found to induce DNA damage in red blood cells (comet assay) and proved to be mutagenic as revealed by the Ames test. Results indicate that blood AChE and ALA-D of rainbow trout may be a sensitive biomarker for assessment of carbosulfan contaminated water bodies. Furthermore, because the Ames test and comet assay were proven successful to detect the genotoxicity of carbosulfan, we proposed that nonlethal techniques such as blood collection from caudal vein of fish should be used to determine potential toxic effects of other pesticides to surrounding environment.     

Behavioral, morphological deformities and biomarkers of oxidative damage as indicators of sublethal cypermethrin intoxication on the tadpoles of D. melanostictus (Schneider, 1799)

Concerns have been raised that the amphibian larval stages are particularly at risk and may be vulnerable to adverse effects of pesticides. The present study reports acute toxicity of cypermethrin at 24, 48, 72 and 96 h through static renewal bioassay test for Duttaphrynus melanostictus. The LC₅₀ values were 5.15, 4.55, 3.95, and 3.34 μg/L for 24, 48, 72, and 96 h respectively. At sublethal concentration (0.33 μg/L) behavioral, morphological and biochemical changes were studied. The behavioral and morphological anomalies observed in the present study are typical signs of cyano pyrethroid poisoning. Significant changes were observed in total, soluble, and structural proteins. The depletion of all the protein fractions observed in this investigation led to progressive protein oxidation and catabolism of proteins. Decreased protein level has resulted in a marked elevation of free amino acid levels at all time intervals. The induction of catalase, glutathione-S-transferase activities and elevation in the levels of hydrogen peroxide, reduced glutathione, and malondialdehyde eventually lead to oxidative damage of biomolecules, showing that the generation of reactive oxygen species and oxidative stress are involved in the toxicity induced by cypermethrin. Indicating increased susceptibility of tadpoles. Thus, an exposure to cypermethrin at sublethal concentration had catastrophic effect on tadpoles of D. melanostictus.     

Comparative effects of two plant growth regulators; indole-3-acetic acid and chlorogenic acid on human and horse serum butyrylcholinesterase

Butyrylcholinesterase (BChE), a major detoxification enzyme found abundantly in many tissues and organisms, constitutes the first line defense in the serum of higher organisms and is a marker for toxic exposure. In this study, the interaction of two plant growth regulators, indole-3-acetic acid (IAA) and chlorogenic acid (CA) with purified human and horse serum BChE is investigated. The time dependent interaction of IAA with the two enzyme species was concentration dependent and rapid. Through kinetic studies, IAA was found to be linear-mixed type inhibitor for human serum BChE, and uncompetitive inhibitor for the horse serum enzyme. For the human BChE, α and the K_i value was calculated as 2.15 ± 1.09 and 3.09 ± 0.98 mM, respectively, and for the horse enzyme the K_i value was calculated as 1.05 ± 0.09 mM. The time dependent interaction of CA with the two enzyme species was biphasic. At low CA concentrations, CA stimulated the activities of both enzyme species whereas at high CA concentrations, inhibition was observed. At high concentrations, the inhibition kinetics for both enzymes fitted the non-competitive inhibition model. The K_i values calculated for human and horse BChE were 2.75 ± 0.14 and 0.96 ± 0.07 mM, respectively. The differences in the interaction of these two growth regulators with the two enzymes species arises from the structural differences between the human and horse serum BChE which can be considered as a triple human mutant BChE.     

Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.     

Cytogenetic effects of commercially formulated atrazine on the somatic cells of Allium cepa and Vicia faba

In the present study cytogenetic effects of atrazine herbicide, were examined on the root meristem cells of Allium cepa and Vicia faba. Test concentrations were chosen by calculating EC₅₀ values of formulated atrazine against both the test systems which determined to be 30 mg l⁻¹ for A. cepa and 35 mg l⁻¹ for V. faba, respectively. For cytogenetic effects root meristem cells of A. cepa were exposed to 15, 30 or 60 mg l⁻¹ whereas V. faba to 17.5, 35 or 70 mg l⁻¹ for 4 or 24 h. Roots exposed for 4 or 24 h, after sampling, were left in water for 24 h recovery and sampled at 24 h post-exposure. A set of onion bulbs or seedlings of V. faba exposed to DMSO (0.3%) was run parallel for negative control. Treatment of atrazine significantly and dose-dependently inhibited the mitotic index (MI) and induced micronucleus formation (MN) chromosome aberrations (CA) and mitotic aberrations (MA) in both the test systems at 4 or 24 h. Root meristem cells examined at 24 h post-exposure also revealed significant (p < 0.001) frequencies of MN, CA or MA despite considerable decline. Chromosome breaks and fragments were found to be major CA whereas C-metaphase, chromosome bridges and laggards were prevalent MA. Results of our study, indicate that atrazine may produce genotoxic effects in plants. Further, both the plant bioassays found to be sensitive indicators for the genotoxicity assessment as the outcome of majority of in vivo/in vitro mammalian tests are comparable.     

An in vitro assay for the assessment of the effects of an organophosphate, paraoxon, and a triazine, atrazine, on the heart of the gilthead sea bream (Sparus aurata)

The effects of paraoxon and atrazine on the spontaneously beating auricle, isolated from the heart of Sparus aurata, were assessed. Paraoxon, 5 μM, eliminated the atria contraction within 28.4 ± 2.8 min, an effect which was fully reversed by 15 μM atropine, an antagonist of muscarinic cholinergic receptors. The IC₅₀ was estimated to be 3.2 ± 1.5 μΜ. Atrazine, 50 and 100 μM, induced a 22.5 ± 3.2 and 32.9 ± 2.3% increase in the force of auricle contraction, caused by excitation of sympathetic synaptic terminals releasing adrenaline. This effect was reversed by 50 μM propranolol, a blocker of β-adrenoreceptors. The results have shown that both sympathetic and parasympathetic nerve terminals are activated by atrazine. Also, the auricle contraction is mainly under sympathetic control, while the frequency is dominated by cholinergic system. Finally, the detailed parameters of the auricle contraction estimated during exposure to specific pesticides, force, frequency, time-response curves and electromechanical coupling can be further used to assess and compare the toxic effects of other compounds, anticholinesterases for example, on the heart of the fish.     

Avermectin exposure induces apoptosis in King pigeon brain neurons

The effect of avermectin was studied on King pigeon brain nerve cells by cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT] and apoptosis [acridine orange/ethidium bromide (AO/EB) assay, transmission electron microscope (TEM) evaluation, measurement of mitochondrial membrane potential (Δψm), phosphatidylserine (PS) exposure, caspases activities, DNA fragmentation, reactive oxygen species (ROS) and caspase-3 mRNA expression] within the 2.5–10 μg L⁻¹ concentration-range. The results revealed that within the concentrations of 2.5–10 μg L⁻¹, avermectin showed obvious cytotoxicity and induced apoptosis in a dose-dependent manner to neurons of King pigeon in vitro. Cell viability were 99.93 ± 8.52%, 82.02 ± 4.99% and 78.23 ± 5.67% after 24 h of treatment with avermectin at the concentrations of 0, 2.5 and 5 μg L⁻¹, which decreased to 56.36 ± 2.17% of 10 μg L⁻¹. Treated cells showed typical apoptosis morphological changes including cytoplasmic vacuolation, chromatin condensation, unclear nuclear membrane and decreased/swollen mitochondria. Typical biochemical hallmarks of apoptosis including Δψm loss, PS exposure, activations of caspase-3, caspase-8 and caspase-9, DNA fragmentation were observed too. Moreover, the levels of ROS in the avermectin treatment groups increased significantly compared to control group. Furthermore, the caspase-3 mRNA levels increased significantly following AVM treatment. In conclusion, our experimental results show that avermectin has cytotoxicity to brain neurons of King pigeon in vitro and the mechanism of neurotoxicity induced by avermectin is closely related to apoptosis.     

Effect of dihydrodillapiole on pyrethroid resistance associated esterase inhibition in an Indian population of Spodoptera litura (Fabricius)

Resistance in Spodoptera litura (Fabricius) has been attributed to enhanced detoxification of insecticides by increased levels of esterases, oxidases and/or glutathione S-transferases. Enzyme inhibiting insecticide synergists can be employed to counter increased levels of such enzymes in S. litura. Dihydrodillapiole induced synergism of pyrethroid toxicity was examined in the laboratory-reared third instar larval population of S. litura collected in Delhi (susceptible), and Guntur (resistant) region of Andhra Pradesh, India. The Guntur population was found to be 7.04 and 10.19 times resistant to cypermethrin and lambdacyhalothrin, respectively. The activity of cypermethrin, lambdacyhalothrin and profenophos against susceptible and resistance populations of S. litura, was gradually increased when used along with a plant-derived insecticide synergist dihydrodillapiole. The α-naphthyl acetate hydrolysable esterase activity in Delhi population was less as compared to the Guntur population. Resistance associated esterases in Delhi population were inhibited by pre-treatment with dihydrodillapiole. The esterase level in insect was instantly reduced initially, sustained for about 3 h and equilibrated at 4 h post treatment. The esterase activity of Guntur population was increased to 1.28 μmoles/mg/min at 2 h post treatment and subsequently reduced to lower than 0.70 μmoles at 4-12 h post treatment. The variation in esterase activity is suggestive of its homeostatic regulation in test populations. Dihydrodillapiole thus caused significant reduction of resistance in S. litura to cypermethrin, lambda cyhalothrin and profenophos.     

Single nucleotide polymorphism detection at the Hypothenemus hampeiRdl gene by allele-specific PCR amplification with Tm-shift primers

The resistant Rdl allele for dieldrin insecticide was detected on the Hypothenemus hampei populations from Colombia using conventional PCR methods. Based on this sequence, a melting temperature (T_m) shift genotyping method that relies on allele-specific PCR is described for insecticide resistance-associated single nucleotide polymorphism (SNP) at the H. hampeiRdl gene. The method reported here uses GC-rich tails of unequal length attached to allele-specific primers containing 3′ terminal bases that correspond to SNP allelic variants. Specific PCR products are identified by inspection of a melting curve on a real-time PCR thermocycler using SYBR Green DNA binding dye. Resistant and susceptible alleles resulted in specific PCR products with T_m of 83.3 ± 0.1 °C and 86.0 ± 0.2 °C, respectively. The RdlT_m-shift genotyping method is a new method to identify the Rdl gene in the coffee berry borer H. hampei, the principal pest of coffee that in general show low genetic diversity and very few genetic strategies for control of this pest have been developed. The method supplies a high-throughput tool for dieldrin resistance-associated SNP diagnostic in the coffee berry borer which will be useful for resistance-management strategies and as genetic marker in the colombian insect populations for genetics research.     

Ultra-structural changes in the integument induced by the use of three of six forms of allylisothiocyanate tested against Plutella xylostella and Pieris rapae larvae

The insecticidal activity of four forms of Hong Jing (HJ) allylisothiocyanate (AITC), AITC + cypermethrin (HJ_A, HJ_B, and HJ_C) with ratio of (1:1, 4:1, and 2:1), pure AITC (HJ_D), and two forms of Hong Du (HD) AITC, AITC + chlorpyrifos (HD_A and HD_B) with ratio of (2:1 and 2:1), respectively, were studied on the major cruciferous insect larvae Plutella xylostella (L.) and Pieris rapae (L.) by combining both spraying and dipping methods. The P. rapae was more susceptible than P. xylostella larvae. The LC₅₀ values 72 h after treatment of AITC forms (HJ_B, HJ_A, HJ_C, HJ_D, HD_B, and HD_A) on the P. rapae were; 0.07, 0.08, 0.16, 0.83, 0.26, 1.08 gL⁻¹, and 0.69, 0.26, 5.45, 0.93, 3.01, 5.98 gL⁻¹ on the P. xylostella, respectively. The toxicity of some of the AITC forms was very close to or better than that of the commercial contact insecticides such as chlorpyrifos (LC₅₀ = 0.03 and 0.04 gL⁻¹ on P. rapae and P. xylostella, respectively), and cypermethrin (0.65 and 0.78 gL⁻¹, respectively, against P. rapae and P. xylostella). The ultrastructural studies on the integument of the third larval instar of P. xylostella treated by sub-lethal concentration (LC₂₀) of HJ_B, HJ_D, and HD_B were carried out by using transmission electron microscope. The more pronounced alterations in the hypodermis and mitochondria cells. They exhibited changes in all treated samples. The hypodermis was almost completely destroyed, and the mitochondria exhibited morphological alterations, represented by enlargement, matrix rarefaction and vacuolization of the mitochondria matrix, quantity of cristae reduced, and density electron matrix lessened. These AITC forms have potential as contact insecticides, and the ultra structural observations confirm the insecticidal efficiency of different AITC forms on P. rapae and P. xylostella.     

The toxic effects of pyrethroid deltamethrin on the common carp (Cyprinus carpio L.) embryos and larvae

Deltamethrin, a synthetic pyrethroid pesticide contaminating aquatic ecosystems as a pollutant, was investigated in the present study for toxic effects on embryos and larvae of common carp, Cyprinus carpio as a model. The control and five test experiments were repeated five times. The water temperature in the experimental units was kept at 24 ± 1 °C. The number of dead embryos significantly increased in response to deltamethrin concentrations 0.005, 0.05, 0.5, 5, 25, and 50 μg L⁻¹ (p<0.05 for each cases). Dose-response decreases in hatching success were recorded as 75.2, 64.6, 47.4, 26.0, 14.4, and 9.0%, respectively. The lowest concentration of deltamethrin (0.005 μg L⁻¹) produced a significantly decrease in number of dead larvae compared to control group (p<0.05). With increasing deltamethrin concentrations, the larvae exposed duration 1-48 h significantly increased the number of dead larvae (p<0.05 for each cases). The 48 h LC₅₀ values (with 95% confidence limits) of deltamethrin for common carp embryos and larvae were estimated as 0.213 (0.103-0.404) and 0.074 (0.011-0.260) μg L⁻¹, respectively. The results provide evidence that deltamethrin pollution may have an adverse effect on the reproduction and development of carp, which should be considered when this chemical is used in agricultural areas near aquatic ecosystems.     

Determination of genotoxicity of Fenaminosulf by Allium and Comet tests

Genotoxic effects of Fenaminosulf, fungicide and micro-biocide, were examined by using mitotic index (MI), mitotic phase, and Comet assay on the root meristem cells of Allium cepa. In the Allium root growth inhibition test, EC₅₀ value was firstly determined as 25 ppm and, 12.5 (0.5 × EC₅₀), 25 (EC₅₀) and 50 (2 × EC₅₀) ppm concentrations of Fenaminosulf were introduced to onion tuber roots. Distilled water was used as a negative control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance (ANOVA) were employed and p < 0.05 was accepted as significant value. While MI (except in 24 h at 12.5 and 50 ppm) and prophase index increased, metaphase, anaphase and telophase indexes decreased in all concentrations compared to control at each exposure time. A significant increase in DNA damage was also observed at the concentration of 25 ppm in 24 h, 25 and 50 ppm in 96 h by Comet assay.     

Effects of dietary pyridoxine on growth and biochemical responses of Labeo rohita fingerlings exposed to endosulfan

A sixty-day experiment was carried out to study the effect of dietary pyridoxine (PN) on growth performance, RNA/DNA ratio and some biochemical parameters of Labeo rohita fingerlings exposed to sub-lethal dose of endosulfan (1/10th of 96 h static non-renewal LC₅₀ = 0.2 ppb) to assess the role of pyridoxine in ameliorating the negative effects of endosulfan. Two hundred seventy fingerlings (6.5 ± 0.26 g) were randomly distributed into six treatments in triplicates (15 fish/tank). Five iso-nitrogenous (35.45-35.75% crude protein) purified diets were prepared with graded levels of pyridoxine. Six treatment groups were T₀ (10 mg PN + without endosulfan), T₁ (0 mg PN + endosulfan), T₂ (10 mg PN + endosulfan), T₃ (50 mg PN + endosulfan), T₄ (100 mg PN + endosulfan) and T₅ (200 mg PN + endosulfan). Weight gain (%), specific growth rate (SGR), tissue glycogen, and protease activity were significantly (P < 0.05) higher in pyridoxine fed groups compared to their non-pyridoxine fed counterpart. Protease activity was positively correlated (R² = 0.931) with (%) weight gain. Glucose-6-phosphate dehydrogenase (G6PDH) activity was significantly (P < 0.05) higher in non-pyridoxine fed group and decreased in pyridoxine fed counterparts. There were no significant (P > 0.05) effect of dietary pyridoxine on feed conversion ratio (FCR), protein efficiency ratio (PER), survival, gastro-somatic index (GSI), hepato-somatic index (HSI) and liver and muscle DNA levels of L. rohita fingerlings. RNA levels, both in liver and muscle, increased significantly (P < 0.05) in pyridoxine fed groups. A positive correlation was observed between growth and RNA levels, both in liver (R² = 0.91) and muscle (R² = 0.88). RNA/DNA ratio showed a third order polynomial relationship with dietary pyridoxine, both in liver (Y = −0.014x³ + 0.1613x² − 0.5333x + 0.7933, R² = 0.987) and muscle (Y = −0.0407x³ + 0.4763x² − 1.6358x + 2.4667, R² = 0.9345). The overall results obtained in present study indicated that dietary pyridoxine supplementation at 100 or 200 mg PN/kg diet ameliorates the negative effects of endosulfan and restores optimal growth of L. rohita fingerlings.     

Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab.

An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC₅₀) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed the highest insecticidal activity at 6 μM concentration within 48 h. The nutritional indices were also significantly affected by the 6 μM concentration (P < 0.05). Food consumption, efficiency of conversion of ingested and digested food, relative growth rate, and consumption values declined significantly while approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an effective biocide and alternative to Bt toxin.     

Biotransformation of clomazone in rice (Oryza sativa) and early watergrass (Echinochloa oryzoides)

Rice (Oryza sativa), a relatively tolerant species, and early watergrass (Echinochloa oryzoides; EWG), a relatively susceptible species, were exposed to ¹⁴C-labeled clomazone to determine accumulation, biotransformation, and mass balance. On a total mass basis, rice absorbed more clomazone than EWG (p < 0.05), but on a nmol/g basis, there was no significant difference between the two species (p > 0.05). Rice contained more extractable ¹⁴C residues (7.7 ± 0.5 vs. 4.8 ± 0.5 nmol in rice vs. EWG, respectively; p < 0.5), but the concentration in EWG was significantly higher (4.2 ± 0.5 vs. 1.8 ± 0.1 nmol/g in EWG vs. rice, respectively; p < 0.01). More metabolized residue was measured in EWG compared to rice (84.1% vs. 67.9%; p < 0.01). Both species produced hydroxylated forms, β-d-glucoside conjugates, and several other unidentified polar metabolites, but EWG generally produced higher metabolite concentrations. The concentration of the suspected active metabolite, 5-ketoclomazone, was significantly higher in EWG vs. rice (21 ± 2 vs. 5.7 ± 0.5 pmol/g, respectively; p < 0.01). Differences in sensitivity to clomazone between rice and EWG appear to be due to differential metabolism, but in this case the more susceptible EWG qualitatively and quantitatively metabolized more clomazone than the more tolerant rice. This is consistent with the action of a metabolically activated herbicide. This metabolic difference could be exploited to develop herbicide safeners for use with clomazone.     

Effect of waterborne benomyl on the hematological and antioxidant parameters of the Nile tilapia, Oreochromis niloticus

The present study was conducted to determine the 96 h-LC₅₀ of benomyl to the Nile tilapia, Oreochromis niloticus and to investigate the biochemical or hematological indices of blood and the alterations in the antioxidant enzymes of this fish in response to sublethal concentrations of benomyl. Fish weighing 71.61 ± 12.05 g were used in this study; they were subjected to fasting for 4 weeks before treatment. An aqueous solution of benomyl (0, 0.5, 1, 2, 4, 8, and 16 mg L⁻¹) was administered for 96 h to determine the LC₅₀. The 96 h-LC₅₀ value of benomyl was 4.39 (3.23-5.60) mg L⁻¹ in the present study. For 5 weeks, the aqueous solution of benomyl (0, 100, 200, and 400 μg L⁻¹) was administered to investigate its effect on the hematological parameters and antioxidant enzymes. The predominant hematological findings in fish exposed to benomyl were as follows: no significant change in the Hb (g dL⁻¹) level, MCV (μm³), MCH (pg) and MCHC (%) as compared to the control. Benomyl exposure led to greater increases in the GPT, GOT (Karmen-unit), LDH (Wroblewski unit), total cholesterol, Fe, and Ca (mg dL⁻¹) values, whereas the levels of ALP (KA unit), total protein, triglyceride, albumin, and Mg (mg dL⁻¹) did not increase. Benomyl increased the in vivo HSI (%), GST (nmol min⁻¹ mg protein⁻¹), and SOD (U mg protein⁻¹) values in the fish livers in the test group, unlike those in the control group for 5 weeks. At concentrations higher than 100 μg L⁻¹, benomyl affected the GST and SOD levels of Nile tilapia in a dose- and time-dependent manner. The present findings suggest that the in vivo hepatotoxicity associated with benomyl may, in part, result from the hematological index, and antioxidants may provide limited protection against benomyl toxicity.     

The effects of Artemisia annua L. and Achillea millefolium L. crude leaf extracts on the toxicity, development, feeding efficiency and chemical activities of small cabbage Pieris rapae L. (Lepidoptera: Pieridae)

The biological effects of two important medicinal plants, Artemisia annua L. and Achillea millefolium (L.) (viz, mortality, growth, and feeding indices as well as enzyme and non-enzymatic activities) were studied on small white Pieris rapae L a deleterious pest of cruciferous plants under controlled conditions (16:8 h L:D at 25 ± 1 °C and 65 ± 5% RH). The LC₅₀ and LC₂₅ values were 9.387% and 3.645% for A. annua L. and 4.19% and 1.69% for A. millefolium (L.), respectively. At the lowest concentration (0.625%), the deterrency was 29.826% and 44.185% for A. annua L. and A. millefolium (L.), respectively. Feeding indices were variously affected with changes in a number of parameters and an increase in larval and pupal duration. The activity level of alkaline phosphatase increased sharply while alanin and aspartate aminotransferases showed a sharp decrease. For non-enzymatic compounds, the amount of glucose and uric acid increased, but total protein and cholesterol decreased. These results indicate that these two medicinal plants might possess potential secondary metabolites that may be useful for controlling potential insect pests.