Sensitivity of the mitotic cells of barley (Hordeum vulgare L.) to insecticides on various stages of cell cycle

In this study, the genotoxic effects of insecticides in different phases of cell cycle were investigated in the mitotic cells of barley (Hordeum vulgare L.). The seeds of H. vulgare L. Var. Karan 4 were treated with different concentrations (0.05%, 0.1% and 0.5%) of insecticides alphamethrin (AM) and monocrotophos (MP) for 6 h after synchronization of cells in G₁, S and G₂ phase of cell cycle with the help of various presoaking durations (7 h, 17 h and 27 h.). Positive and negative control was run parallel in the form of ethyl methane sulphonate (EMS) and distilled water, respectively. The data indicate that higher dose of alphamethrin and monocrotophos produce toxicity, chromosomal aberrations and mitotic aberrations in Hordeum vulgare L. The present study indicates the genotoxic potential of the insecticides AM and MP and it also showed that S-phase of cell cycle was more sensitive compared to the G₁ and G₂ phases.     

Comparative sensitivity of barley (Hordeum vulgare L.) to insecticide and fungicide on different stages of cell cycle

In this study, the comparison of cytogenetic effects of insecticide and fungicide in different phases of cell cycle was investigated in the root tip cells of barley (Hordeum vulgare L.). The seeds of H. vulgare L. Var. Karan 16 were treated with different concentrations (0.05%, 0.1% and 0.5%) of insecticide Profenophos (PF) and fungicide Mancozeb (MZ) for 6 h after presoaking durations of 7, 17 and 27 h.The different presoaking durations were used to bring the cells in various phases of cell cycle. Negative control was run parallel in distilled water. Cytogenetic examinations of root meristems exposed to the PF and MZ showed significant inhibition of mitotic index (MI) as well as significant increase in chromosomal aberrations (CAs). These parameters were dependent on the concentrations of insecticide and fungicide. The present study shows that PF and MZ both caused more damage in S phase of cell cycle which indicates that S phase is more sensitive in comparison to other phases.     

Effect of herbicide clomazone on photosynthetic processes in primary barley (Hordeum vulgare L.) leaves

The effect of pre-emergently applied herbicide clomazone on the photosynthetic apparatus of primary barley leaves (Hordeum vulgare L.) was studied. Clomazone application caused a reduction in chlorophyll (a+b) and carotenoid levels that was accompanied by a decline in the content of light harvesting complexes as judged from the increasing chlorophyll a/b ratio. The pigment reduction also resulted in changes in 77 K chlorophyll fluorescence emission spectra indicating lower chlorophyll (Chl) fluorescence reabsorption and absence of the long-wavelength emission forms of photosystem I. The maximal photochemical yield of photosystem II (PSII) and the reoxidation kinetics of the primary quinone acceptor Q_A⁻ were not significantly influenced by clomazone. A higher initial slope of Chl fluorescence rise in the Chl fluorescence induction kinetic indicated an increased delivery of excitations to PSII. Simultaneously, analysis of the Chl fluorescence quenching revealed that clomazone reduced function of the electron transport chain behind PSII. The decrease in the saturation rates of CO₂ assimilation paralleled the decrease of the Chl content and has been suggested to be caused by a suppressed number of the electron transport chains in the thylakoid membranes or by their decreased functionality. The obtained results are discussed in view of physiological similarity of the clomazone effect with changes of photosynthetic apparatus during photoadaptation.     

Metabolism of DDT and related compounds in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of wheat and soybean were incubated with [¹⁴C]-1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT), [¹⁴C]-1,1-dichloro-2,2-bis-(4-chlorophenyl)ethene (DDE), and [¹⁴C]-2,2-bis-(4-chlorophenyl)acetic acid (DDA) under standardized conditions. Polar metabolites were formed in yields of 1–2.5% in the cases of DDT and DDE, and of 56% in the case of DDA. A nonpolar metabolite was only observed in the case of DDT in soybean. This metabolite was identified as DDE on the basis of cochromatography and mass spectroscopy. By the same methods DDA was identified as a major polar DDT metabolite of both soybean and wheat. The further conversion of DDA to hexose esters was demonstrated by chromatographic and mass spectroscopic comparison with synthetic DDA-β-d-glucopyranosyl tetraacetate. These studies suggest the metabolic sequence, DDT → DDA → DDA-hexose ester.     

Effects of a fungicide, an insecticide, and a biopesticide on Tolypothrix scytonemoides

Lower concentrations of Bavistin supported good growth of Tolypothrix scytonemoides with maximum protein and pigment contents. The rates of photosynthetic oxygen evolution declined in cells grown in the pesticide-amended medium but rates of respiratory oxygen consumption were enhanced in cells grown in lower concentrations of Monocrotophos and Nimbicidin. Activities of nitrogenase and glutamine synthetase were affected in all the pesticide treatments but nitrogenase activity was enhanced in the presence of Bavistin. Release of ammonia and carbohydrates was enhanced in cells grown in pesticide-amended medium except for cells grown in medium containing Bavistin where the release of carbohydrates was reduced. New polypeptides of ∼280, 152, and 25 kDa (in 250 ppm Bavistin), ∼58 and 28 kDa (in 0.3 and 0.2-0.4 ppm Monocrotophos, respectively) and ∼31, 28, and 26 kDa (in 0.5 and 1.0 ppm Nimbicidin) were detected in the treated cells.     

Effects of two different neem products on different stages of Nezara viridula (L.) (Heteroptera, Pentatomidae)

In this study, the effects of two different commercial neem insecticides (NeemAzal T/S and Neem Oil) were determined on different stages of Nezara viridula (L.) (Heteroptera: Pentatomidae) under laboratory conditions. Neem Azal and Neem Oil were applied at concentrations of 0.5 % and 2 %, respectively. Mortality was recorded after 3, 7 and 14 days for nymphs and adults; 7 and 14 days for old (4-day-old) eggs; and 14 days for newly laid (one-day-old) eggs. Both products have no significant effect on adults and newly laid eggs. However Neem Oil was found to be more effective than NeemAzal T/S on nymphs and on old laid eggs after 7 and 14 days. It can be concluded that both neem products have potential for insecticidal efficacy (approximately 60 %) against nymphs of N. viridula. at concentrations recommended by manufacturers for registered pests.     

Effects of two different neem products on different stages of Nezara viridula (L.) (Heteroptera, Pentatomidae)

In this study, the effects of two different commercial neem insecticides (NeemAzal T/S and Neem Oil) were determined on different stages of Nezara viridula (L.) (Heteroptera: Pentatomidae) under laboratory conditions. Neem Azal and Neem Oil were applied at concentrations of 0.5 % and 2 %, respectively. Mortality was recorded after 3, 7 and 14 days for nymphs and adults; 7 and 14 days for old (4-day-old) eggs; and 14 days for newly laid (one-day-old) eggs. Both products have no significant effect on adults and newly laid eggs. However Neem Oil was found to be more effective than NeemAzal T/S on nymphs and on old laid eggs after 7 and 14 days. It can be concluded that both neem products have potential for insecticidal efficacy (approximately 60 %) against nymphs of N. viridula. at concentrations recommended by manufacturers for registered pests.     

Impact of chlorfenvinphos, an organophosphate insecticide on human blood mononuclear cells (in vitro)

In the present study, the investigation of the effect of chlorfenvinphos (CFVF) on necrotic and apoptotic changes as well as on selected morphological and biochemical parameters in human blood mononuclear cells were investigated.We analyzed the effect of this compound on proteins damage and free-radical formation in human blood mononuclear cells. Furthermore, changes in the size (FSC-A) and granularity (SSC-A) of human blood mononuclear cells exposed to chlorfenvinphos were assessed. In order to detect apoptosis, two testes were used including analysis of YO-PRO-1 iodide/propidium iodide and Annexin V/propidium iodide staining, which revealed that chlorfenvinphos increased the number of apoptotic cells at its highest concentration of 250 μM. Chlorfenvinphos at the concentrations from 50 and 100 μM increased the size and granularity of the blood mononuclear cells, respectively. Moreover, chlorfenvinphos induced the statistically significant loss of human blood mononuclear cells viability at the concentration of 250 μM. Protein damage (the increase in carbonyl groups content) was provoked by CFVF at concentrations of 100 μM and 250 μM. Furthermore, chlorfenvinphos from relatively low concentrations of 5 μM induced reactive oxygen species formation (ROS).Conclusion: The present findings provide information that chlorfenvinphos only at 250 μM is harmful to human blood mononuclear cells, the concentration which may appear in the organism only as a result of acute or subacute poisoning. Lower concentration (5-50 μM), which caused changes in level of ROS formation can affect human organism as a result of environmental exposure.     

Antiproliferative effect of azadirachtin A on Spodoptera litura Sl-1 cell line through cell cycle arrest and apoptosis induced by up-regulation of p53

In order to illustrate the mechanism of cell cycle arrest and apoptosis induced by botanical insecticide, the effects of cell proliferation inhibition and apoptosis induction by azadirachtin A were investigated in Spodoptera litura cell line Sl-1 in vitro. The cell proliferation, cell cycle division, mitotic and apoptotic figure of nucleus, and the expression of related proteins were detected by a variety of methods, including MTT assay, flow cytometry, fluorescence microscope observation, and Western blot technique. Sl-1 cells grew in a dose-dependent inhibition manner and the proliferation inhibition rates were 18.95%, 33.45% and 41.00%, after 48 h of treatment with azadirachtin A at the concentrations of 0.1, 0.5 and 1.0 μg/mL, respectively. Sl-1 cell cycle was arrested in G2/M phase with lower expression of cyclin B1. The apoptosis rates were found to be 19.71%, 23.40% and 24.91% after 48 h of treatment with 0.1, 0.5, and 1.0 μg/mL of azadirachtin A, which increased with the activities of caspase-3. The up-regulation of p53 was observed before apoptosis and cell cycle arrest occurred. These findings suggest that p53 protein was involved in cell cycle arrest, apoptosis induction and cell proliferation inhibition when the insect cultured cells were treated with azadirachtin A.     

Effect of terbutryn at environmental concentrations on early life stages of common carp (Cyprinus carpio L.)

The aim of this study was to assess the toxicity of terbutryn to early developmental stages of common carp (Cyprinus carpio). Based on accumulated mortality in the experimental groups, lethal concentrations of terbutryn were estimated at 36 day LC50 = 3.06 mg l⁻¹ terbutryn. Based on inhibition of growth in the experimental groups, lowest observed-effect concentration (LOEC) = 0.005 mg l⁻¹ terbutryn; and no observed-effect concentration (NOEC) = 0.0007 mg l⁻¹ terbutryn. Fulton’s condition factors were significantly lower in fish exposed to 2 mg l⁻¹ compared with controls. By day 30, fish exposed to 0.00002 mg l⁻¹ - real environmental concentration in Czech rivers - 0.02, 0.2, and 2 mg l⁻¹ terbutryn showed significantly lower mass and total length compared with controls. No significant negative effects on hatching or embryo viability were demonstrated at the concentrations tested, but significant differences in early ontogeny among groups were noted. Fish from the two highest tested concentrations (0.2 and 2 mg l⁻¹) showed a dose-related delay in development compared with the controls. At concentrations of 0.02, 0.2, and 2 mg l⁻¹ damage to caudal kidney tubules when compared to control fish was found.     

不同叶龄鸭跖草对咪唑乙烟酸的耐药性

鸭跖草是黑龙江省大豆田危害严重的恶性杂草,而且其叶龄愈大愈难防除。作者通过盆栽试验及细胞显微技术研究不同叶龄鸭跖草对咪唑乙烟酸的耐药性差异及其与叶片显微结构的关系。结果显示,鸭跖草随着叶龄增大,对咪唑乙烟酸的耐药性增强,并在3叶龄时出现拐点;随着叶龄的增大,叶片表皮气孔密度显著下降;叶片厚度、叶片上下表皮厚度、栅栏组织及海绵组织厚度显著增大;10个栅栏组织细胞长度显著减小,栅栏组织排列由稀疏不规则向紧密规则的方向发展。以上变化是鸭跖草对咪唑乙烟酸耐药性增强的原因之一。     

Comparative metabolism of atrazine and EPTC in proso millet (Panicum miliaceum L.) and corn

The purpose of this study was to examine the differential activities of proso millet (Panicum miliaceum L.) and corn (Zea mays L.) with respect to atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine] and EPTC (S-ethyldipropyl thiocarbamate) metabolism. GSH-S-transferase was isolated from proso millet shoots and roots. When assayed spectrophotometrically using CDNB (1-chloro 2,4-dinitrobenzene) as a substrate, the shoot enzyme had only 10% of the activity of corn shoot enzyme while the root enzyme had 33% the activity of corn root enzyme. However, when proso millet shoot GSH-S-transferase was assayed in vitro using ¹⁴C-ring-labeled atrazine, it degraded the atrazine to water-soluble products at the same rate as the corn shoot enzyme. Incubation of excised proso millet and corn roots with [¹⁴C]EPTC indicated that uptake of EPTC was similar in both plants. However, proso millet metabolized the EPTC to water-soluble products at only half the rate of corn. Glutathione levels of proso millet roots were 35.9 μg GSH/g fresh wt, compared with 65.4 μg GSH/g fresh wt for corn. However, a 2.5-day pretreatment with R-25788 (N,N-diallyl-2-2-dichloroacetamide) elevated proso millet GSH levels to 62.7 μg GSH/g fresh wt. R-25788 did not elevate the activity of proso millet GSH-S-transferase, in contrast to its effects on corn. We conclude that differences in response to atrazine and EPTC in proso millet and corn are a result of their differential metabolism.     

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.)

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b₅ in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.     

田间小菜蛾抗药性监测及毒理机制研究

田间监测结果表明，福州地区小菜蛾Plutella xylostella(L.)已对生产上常用的各类杀虫剂产生不同程度的抗性，从9月至次年5年期间，田间小菜蛾表现出较高水平的抗性，而在8月份则表现出明显低的抗性水平，短期高温和长期低温的环境条件可明显抑制小菜蛾幼虫的AChE和CarE活性，但短期低温的环境条件对小菜蛾幼虫的AChE和CarE活性影响不大，饲养温度的变化不会影响小菜蛾幼虫AChE和Ki值，但在较高的酶与杀虫剂的反应温度下，有机磷对小菜蛾、菜青虫和黄曲条跳甲AChE的抑制作用显著增高，此外，探讨了田间影响小菜蛾抗性水平的环境因素及毒理学机制。     

Induction time of Fe-SOD synthesis and activity determine different tolerance of two Desmodesmus (green algae) strains to chloridazon: A study with synchronized cultures

Cells of two Desmodesmus armatus strains (276-4a and 276-4d) grown asynchronously in batch cultures after 24-h treatment revealed different tolerance to chloridazon (photosynthetic herbicide) applied at a concentration of 3.45 mg L⁻¹. To find time- and cell cycle-dependent biochemical reasons leading to such a difference, a population of young autospores of both strains synchronized by a light/dark (14/10) regime were exposed to chloridazon at the initiation of the light period. Chloridazon reduced the growth and number of divisions of cell strain 276-4d. In consequence, at the end of the dark phase the number of released autospores was reduced by 50% compared with the control. In contrast, the growth and reproductive processes of cell strains 276-4a was unaffected. Moreover, chloridazon treatment speeded up cell development, as a result of which the release of autospores took this process observed in the control cells over. There is a relationship between photosynthetic activity response to chloridazon and time-dependent changes in Fe-SOD content and activity. The energy trapped in the reaction centre (RC) was similar in both strains, but the amount of energy absorbed by RCs was twice as high in strain 276-4d as in 276-4a. In consequence, non-photochemical energy dissipation occurring in the cells of 276-4d strain far exceed the value obtained for 276-4a strain. The control cells of both strains differed significantly in the content of FSD 1 and FSD 2 proteins, whereas the differences in Fe-SOD isoforms activities were slight. The 8-fold increase in SOD content in CHD treated cells of strain 276-4a was associated with the transience of photosynthetic efficiency impairment. In CHD treated cells of strain 276-4d, neither activity of Fe-SOD nor FSDs protein content was instantly affected. Different response of developing cells of two Desmodesmus strains to CHD is influenced by the inherent features of cells; the short time required to induce stress adaptive mechanism involving chloroplastic Fe-SOD activity and biosynthesis in the cells of CHD tolerant strain 276-4a seems to play the key role, being an overriding on the high, but not induced in response to stress, FSD protein level and activity in cells of strain 276-4d.     

苏云金杆菌对抗性及敏感小菜蛾的拒食活性

调查了苏云金杆菌对抗性和敏感小菜蛾的拒食活性。选择性和完全拒食试验表明,Bt对抗性和敏感小菜蛾种群都有一定的拒食作用,且拒食活性随Bt浓度的增加而显著增强。相对于抗性种群,小菜蛾敏感种群对Bt杀虫剂表现了更强的拒食趋势,其拒食中浓度AFC50(0.089 3 μg/mL)显著低于抗性种群(0.148 1 μg/mL)。试验结果表明,抗性种群对有毒叶片的行为避害能力弱于敏感种群,小菜蛾对Bt杀虫剂的抗性产生应与其行为机制无关。     

Physiological basis for antagonism of clethodim by imazapic on goosegrass (Eleusine indica (L.) Gaertn.)

Greenhouse and laboratory experiments were conducted to determine the effect of imazapic on the herbicidal activity of clethodim on goosegrass. Imazapic did not affect absorption of [¹⁴C]clethodim by goosegrass. Averaged across the two treatments of clethodim alone and clethodim plus imazapic, absorption was 36 and 89% of applied [¹⁴C]clethodim at 0.5 and 96 h, respectively. The majority of [¹⁴C]clethodim (79% of applied) was absorbed by 24 h. Translocation of ¹⁴C was not affected by imazapic, and 3.6% of applied ¹⁴C had translocated into the portion of the shoot below the treated leaf at 96 h after treatment. Metabolism of clethodim was not affected by the presence of imazapic. Three major metabolites of clethodim were detected in treated tissue at all harvest intervals. The majority (58%) of [¹⁴C]clethodim was converted to a relative polar metabolite form 96 h after treatment, whether clethodim was applied alone or in the presence of imazapic. One day after treatment, the photosynthetic rate in plants treated with imazapic decreased below the rate in the non-treated check, and was less for 8 days, the duration of the study. These data suggest that the antagonism of clethodim by imazapic may be caused by imazapic reducing the photosynthetic rate of goosegrass and therefore the sensitivity of ACCase to clethodim.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Spread of potato virus Y in potato cultivars (Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato (Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y^NTN (PVY^NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY^NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.     

Brusatol isolated from Brucea javanica (L.) Merr. induces apoptotic death of insect cell lines

Brucea javanica (L.) Merr. is a medicine plant distributed widely throughout Asia where its bitter fruits have been used traditionally in medicine for treating various ailments and controlling some pests. In recent years, concerns over the potential impact of synthetic pesticides on human health and environment have now become more pressing to develop environmentally friendly pesticides. In this paper, brusatol, a quassinoid, was isolated from the fruit of B. javanica, and identified using X-ray crystallographic analysis. Results showed that brusatol has potent contact toxicity (LD₅₀, 2.91 μg/larva, 72 h) and anfieedant activity (AFC₅₀, 17.4 mg/L, 48 h) against the third-instar larvae of Spodoptera exigua. Brusatol demonstrated cytotoxic effects to the tested insect cell lines, IOZCAS-Spex-II and Sf21, in a time- and dose-dependent manner. After brusatol treatment, apoptotic cell death with the DNA fragmentation, activation of caspase-3 and release of cytochrome c was preliminarily observed in both IOZCAS-Spex-II and Sf21. These results indicated the existence of apoptotic death with the mitochondrial-dependent pathway induced by brusatol in Sf21 and IOZCAS-Spex-II cell lines. Our studies will provide important knowledge to understand mechanisms of action of brusatol and to develop brusatol and its derivatives as insecticides.