Studies on some enzymes involved in insecticide resistance in fenitrothion-resistant and -susceptible strains of Musca domestica L. (Dipt, Muscidae)

Abstract: The toxicity of fenitrothion and fenitrothion plus synergists was determined by topical application to adults of fenitrothion-resistant (571ab) and -susceptible (Cooper) strains of Musca domestica L. The strain 571ab was 232-fold resistant to fenitrothion when compared with the Cooper strain. Co-administration of fenitrothion with three synergists, namely piperonyl butoxide (PBO), tributylphosphorotrithioate (DEF) and diethyl maleate (DEM) was investigated, respectively, at 1 : 5, 1 : 5 and 1 : 10 ratio. This co-administration of fenitrothion with PBO, DEF and DEM caused a decrease in the doses which produced 50% lethality (LD₅₀s) in 571ab but had no synergistic effect on fenitrothion toxicity was observed in the Cooper strain. The effect of topical application of fenitrothion alone and in combination with PBO, DEF and DEM at the LD₅₀ level on some enzyme activities in 571ab and Cooper strains was examined. The application of fenitrothion alone and in combination with DEF and DEM at LD₅₀ level caused a significant decrease in activities of total esterases, acetylcholinesterase (AChE) and glutathione S-transferase (GST) in the 571ab strain. The decrease in GST activity was not significant in treated flies of the Cooper strain when compared with GST activity of control flies. A non-significant effect on total cytochrome P450 level was observed with fenitrothion alone and the fenitrothion + PBO treatment. No increase in activity level of total esterases, AChE and GST was found, which might suggest that changes in activity level of these enzymes are not related to fenitrothion resistance in the 571ab strain.     

抗杀螟硫磷二化螟的抗性遗传力研究

应用数量遗传学的方法分析了水稻二化螟(chilo suppressalis)对杀螟硫磷抗药性的遗传力,并就遗传力和杀虫剂的杀死率对抗性发展速率的影响进行了预测。研究结果表明：二化螟对杀螟硫磷抗性的遗传力较低(h²＝0.277),产生高抗性的潜力不大。即使在室内选择的条件下,要获得10倍的抗性约需要6-12代;在野外条件下则需要更长的时间(15-32代)。因此,结合抗性变化动态的监测结果,我们认为在野外条件下二化螟是不致于产生高抗性,杀螟硫磷仍不失为二化螟抗性治理系统中有效品种之一。     

Insecticide applications to soil contribute to the development of Burkholderia mediating insecticide resistance in stinkbugs

Some soil Burkholderia strains are capable of degrading the organophosphorus insecticide, fenitrothion, and establish symbiosis with stinkbugs, making the host insects fenitrothion‐resistant. However, the ecology of the symbiotic degrading Burkholderia adapting to fenitrothion in the free‐living environment is unknown. We hypothesized that fenitrothion applications affect the dynamics of fenitrothion‐degrading Burkholderia, thereby controlling the transmission of symbiotic degrading Burkholderia from the soil to stinkbugs. We investigated changes in the density and diversity of culturable Burkholderia (i.e. symbiotic and nonsymbiotic fenitrothion degraders and nondegraders) in fenitrothion‐treated soil using microcosms. During the incubation with five applications of pesticide, the density of the degraders increased from less than the detection limit to around 10⁶/g of soil. The number of dominant species among the degraders declined with the increasing density of degraders; eventually, one species predominated. This process can be explained according to the competitive exclusion principle using V_max and K_m values for fenitrothion metabolism by the degraders. We performed a phylogenetic analysis of representative strains isolated from the microcosms and evaluated their ability to establish symbiosis with the stinkbug Riptortus pedestris. The strains that established symbiosis with R. pedestris were assigned to a cluster including symbionts commonly isolated from stinkbugs. The strains outside the cluster could not necessarily associate with the host. The degraders in the cluster predominated during the initial phase of degrader dynamics in the soil. Therefore, only a few applications of fenitrothion could allow symbiotic degraders to associate with their hosts and may cause the emergence of symbiont‐mediated insecticide resistance.     

Involvement of two plasmids in fenitrothion degradation by Burkholderia sp. strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.     

Fenitrothion: an alternative insecticide for the control of deltamethrin‐resistant populations of Triatoma infestans in northern Argentina

Deltamethrin‐based campaigns to control Triatoma infestans (Klug) (Hemiptera: Reduviidae) have decreased in success as a result of the development of insecticide resistance. We compared the in vitro effects of the pyrethroid deltamethrin and two doses of the organophosphate fenitrothion, presented on different materials, on T. infestans from La Esperanza, Argentina. Laboratory tests demonstrated a decrease in susceptibility to deltamethrin in the field population [LD₅₀: 30.32 nanograms per insect (ng/i)] compared with the reference population (LD₅₀: 0.13 ng/i), giving a high resistance ratio of 233.42. By contrast, similar susceptibility to fenitrothion was assessed in both the field and reference populations (LD₅₀: 21.65 ng/i and 21.38 ng/i, respectively). The effectiveness of the formulated insecticides varied according to the surfaces to which they were applied. The application of fenitrothion formulations to glass or brick resulted in mortality of 90–100%. The application of fenitrothion formulations to wood or mud caused mortality in the range of 6.7–56.7%. Resistant insects presented low mortality when exposed to the deltamethrin formulation and high mortality when exposed to fenitrothion formulations. Moreover, the insecticides demonstrated residual activity only when applied to glass. The present work demonstrates that fenitrothion is an alternative to pyrethroids for the management of deltamethrin‐resistant insects in La Esperanza. However, this effectiveness is not sustained over time.     

Baseline responses of adult Alphitobius diaperinus (Coleoptera: Tenebrionidae) to fenitrothion and susceptibility status of populations in Queensland and New South Wales, Australia

Traditionally in Australia, regular applications of insecticide to the floors and lower walls of broiler houses after cleanout periods have been used in an attempt to control lesser mealworm, Alphitobius diaperinus (Panzer). The Australian chicken meat industry has been concerned in recent years with the failure to control A. diaperinus in its broiler houses by using this method and with large beetle populations breaching farm biosecurity. Resistance to fenitrothion was suspected to be responsible for these recent control failures. In response, beetles from 13 poultry facilities were compared with an insecticide-susceptible reference population by using a topical application method. Generally, strong resistance to fenitrothion (up to 79 times that of the susceptible at the LC50) occurred in populations of A. diaperinus in long-established broiler growing areas of southeastern Queensland, where fenitrothion had been used continuously for up to 20 yr. In newly established broiler growing areas, where considerably less fenitrothion had been used (i.e., approximately 5 yr), much weaker or no resistance occurred. In addition, dose-mortality data generated for the susceptible reference beetle population over a range of fenitrothion concentrations showed that 0.15% fenitrothion at a LC(99.9) level could be used as a convenient topical dose concentration to discriminate between susceptible and resistant individuals. Using this method, the susceptibility of 27 field populations of A. diaperinus was determined. Of this total, 23 populations did not exhibit complete mortality against the discriminating concentration (mortality range 0-98.7%). Application of fenitrothion in Australian broiler houses for control of A. diaperinus has now ceased.     

Effects of chemical and botanical insecticides used for locust control on Metarhizium anisopliae var. acridum conidia after short- to medium-term storage at 30°C

The short- to medium-term viability and growth of Metarhizium anisopliae var. acridum conidia were investigated when combined with six insecticides, at three different concentrations. All of the insecticides used in this study were suitable for immediate spraying with M. anisopliae var. acridum conidia except for fenitrothion. Fipronil, teflubenzuron, and fenitrothion formulations significantly reduced conidial viability over time. The 10% teflubenzuron treatment caused loss of viability relatively quickly with 9.9% germination after 28 days. Mycelial growth was affected by all the treatments except fenitrothion.     

Selection of single-chain variable fragment antibodies against fenitrothion by ribosome display

A single-chain variable fragment (ScFv) complementary DNA (cDNA) library against fenitrothion was constructed, and ScFvs specific for fenitrothion were selected by ribosome display from the library. After three rounds of ribosome display, the ScFv genes were cloned into Escherichia coli for expression. The expressed ScFvs of 160 clones were analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Of these, 40 clones produced antibodies with relatively high activity against fenitrothion, and 3 were selected for Biacore and ELISA analysis. These 3 antibodies-ScFv-AF50, ScFv-AF93, and ScFv-AF132-had IC(50) values of 1.6, 3.4, and 2.2 ng/ml, respectively. Cross-reactivity with other organophosphorus (OP) pesticides was below 0.1% except for parathion-methyl (≤2.8%). The IC(50) values and cross-reactivity were lower than achieved previously with polyclonal or monoclonal antibodies against fenitrothion. The equilibrium dissociation constant (K(D)) values determined by Biacore analysis were 4.56×10(-10)M for ScFv-AF50, 1.42×10(-9)M for ScFv-AF93, and 2.66×10(-10)M for ScFv-AF132. These results demonstrate that the ribosome display has great potential in selection of ScFvs against pesticides. Recoveries of fenitrothion from fortified rice and cucumber were in the range 80.6 to 108%, indicating that the ELISAs with the isolated ScFvs can accurately determine fenitrothion in food samples after the simple and rapid extraction procedure.     

An ectomycorrhizal fungus,Cenococcum geophilum,in a coastal pine forest has a high tolerance for an insecticide used to control pine wilt disease

Cenococcum geophilum Fr., one of several ectomycorrhizal species associated with black pine (Pinus thunbergii Parl.), is dominant in the coastal forests of Japan, even under adverse abiotic environmental conditions. In these forests, many tonnes of Sumipine® (fenitrothion) are applied every year to protect P. thunbergii from pine wilt disease, which is transmitted by a beetle. Here, we examined the effect of this insecticide on the species of fungi found as ectomycorrhizae on naturally regenerated P. thunbergii seedlings collected from coastal forest sites that had or had not been sprayed with fenitrothion. The proportion of C. geophilum ectomycorrhizae on black pine root tips was significantly higher in areas where fenitrothion had been applied than in areas where it had not. We measured the in vitro mycelial growth of C. geophilum as well as other ectomycorrhizal fungi of coastal black pine, Rhizopogon roseolus (Corda) Th. Fr. and Pisolithus arhizus (Scop.) Rauschert, at three levels of fenitrothion (density: 1.32 g/cm³), i.e., 0, 0.1 and 0.2 mL L^?1. The growth of all three species decreased significantly as the fenitrothion dosage increased. However, the reduction of mycelial growth in response to fenitrothion was lower in C. geophilum than in the other two species. These results suggest that C. geophilum has a high tolerance for fenitrothion, which may explain its dominance over other ectomycorrhizal species in coastal forests in Japan where fenitrothion is routinely sprayed.

     

Changes in leucine transport activity in Chironomus riparius larvae after short-term exposure to potassium dichromate and fenitrothion

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.     

Highly selective fluorescent sensing of fenitrothion using per-6-amino-β-cyclodextrin:Eu(III) complex

A unique, efficient, highly sensitive and selective fluorescent chemosensor for fenitrothion has been reported for the first time using per-6-amino-β-cyclodextrin:Eu(III) complex. Among the various pesticides, the sensitivity response is found to be in the order, fenitrothion>quinalphos>methylparathion>parathion>methylparaoxon>paraoxon>fenchlorphos>profenofos>malathion. A detection limit as low as 1 × 10(-12)M for fenitrothion sensing is realized with a 2.4% relative standard deviation (RSD) of three consecutive runs. The per-6-amino-β-cyclodextrin:Eu(III):pesticide complexes and their sensing mechanism are evidenced from emission, NMR, FT-IR, binding constant measurement, Job's plot, ICD spectra, ESI-MS, lifetime measurements and molecular modeling studies. The proposed sensing is a consequence of Absorption Energy Transfer Emission (AETE) process as a result of better encapsulation of fenitrothion inside the cavity of per-6-amino-β-cyclodextrin:Eu(III) complex. The remarkable sensitivity and selectivity of fenitrothion compared to other OPs, is attributed to a more deeper binding and tighter fit of fenitrothion inside the CD cavity, which is evident from binding constant values and molecular modeling studies. This tighter fit ensures the replacement of two coordinating water molecules on Eu(III) ion, which may have contributed to the more selective sensing of fenitrothion.     

Involvement of Two Plasmids in Fenitrothion Degradation by Burkholderia sp. Strain NF100

A bacterium capable of utilizing fenitrothion (O,O-dimethyl O-4-nitro-m-tolyl phosphorothioate) as a sole carbon source was isolated from fenitrothion-treated soil. This bacterium was characterized taxonomically as being a member of the genus Burkholderia and was designated strain NF100. NF100 first hydrolyzed an organophosphate bond of fenitrothion, forming 3-methyl-4-nitrophenol, which was further metabolized to methylhydroquinone. The ability to degrade fenitrothion was found to be encoded on two plasmids, pNF1 and pNF2.     

Genotoxic effects of some organophosphorous pesticides. I. Induction of micronuclei in bone marrow cells in rat

The genotoxic effects of 4 organophosphorous pesticides, i.e. ekatin, fenitrothion, methylparathion and phorate, were examined employing the micronucleus test in bone marrow cells of the rat. Methylparathion and phorate were found to be mutagenic, while ekatin was weakly mutagenic. The frequency of micronuclei induction by fenitrothion did not differ significantly from that noticed in negative control.     

A microcosm study on bioremediation of fenitrothion-contaminated soil using Burkholderia sp. FDS-1

Fenitrothion, a toxic organophosphorus pesticide, can build up the concentration of nitrophenolic compound in soils and hence needs to be removed. Burkholderia sp. FDS-1, a fenitrothion-degrading strain, was used in this work to study factors affecting its growth, and then evaluated for its capacity to degrade fenitrothion in soil microcosms. Minimal salt medium containing 1% (w/v) glucose was found to be a suitable carbon source for inoculum preparation. Various factors, including soil pH, temperature, initial fenitrothion concentration, and inoculum size influenced the degradation of fenitrothion. Microcosm studies performed with varying concentrations (1–200 mg kg⁻¹) of fenitrothion-spiked soils showed that strain FDS-1 could effectively degrade fenitrothion in the range of 1–50 mg kg⁻¹ soil. The addition of Burkholderia sp. FDS-1 at 2×10⁶ colony forming units g⁻¹ soil was found to be suitable for fenitrothion degradation over a temperature range of 20–40 °C and at a slight alkaline pH (7.5). The results indicate that strain FDS-1 has potential for use in bioremediation of fenitrothion and its metabolite-contaminated sites. This is a model study that could be used for decontamination of sites contaminated with other compounds.     

Mutagenicity studies on fenitrothion in bacteria and mammalian cells

The mutagenicity of fenitrothion was determined in strains of Salmonella typhimurium and Escherichia coli. Fenitrothion was found to be non-mutagenic in Salmonella typhimurium strains of TA98, TA1535 and TA1537 and in Escherichia coli WP2uvrA both with and without S9 mix, while weak mutagenicity was observed only in Salmonella typhimurium TA100 and enhanced by the addition of S9 mix. The mutagenicity observed in the TA100 strain was not expressed in a nitroreductase-deficient strain, TA100 NR, and decreased in a transacetylase-deficient strain, TA100 1,8-DNP6. The mutagenicity of fenitrothion was also examined by a gene mutation assay using the gene for hypoxanthine-guanine phosphoribosyltransferase (hgprt) in V79 Chinese hamster lung cells. Fenitrothion did not induce any increment of 6-thioguanine-resistant mutant cells at doses ranging from 0.01 to 0.3 mM regardless of the presence or absence of S9 mix. These results suggest that reduction of fenitrothion by a bacterial nitroreductase of TA100 to an active form is essential for the expression of the mutagenicity of fenitrothion in TA100 and that a bacterial transacetylase of TA100 also has an important role in the process of mutagenic activation.     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

Isolation of Fenitrothion-degrading Strain Burkholderia sp. FDS-1 and Cloning of mpd Gene

A short rod shaped, gram-negative bacterium strain Burkholderia sp. FDS-1 was isolated from the sludge of the wastewater treating system of an organophosphorus pesticides manufacturer. The isolate was capable of using fenitrothion as the sole carbon source for its growth. FDS-1 first hydrolyzed fenitrothion to 3-methyl-4-nitrophenol, which was further metabolized to nitrite and methylhydroquinone. The addition of other carbon source and omitting phosphorus source had little effect on the hydrolysis of fenitrothion. The gene encoding the organophosphorus hydrolytic enzyme was cloned and sequenced. The sequence was similar to mpd, a gene previously shown to encode a parathion-methyl-hydrolyzing enzyme in Plesiomonas sp. M6. The inoculation of strain FDS-1 (10⁶ cells g⁻¹) to soil treated with 100 mg fenitrothion emulsion kg⁻¹ resulted in a higher degradation rate than in noninoculated soils regardless of the soil sterilized or nonsterilized. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment. Zhonghui Zhang, Qing Hong: Both authors contributed equally to this work     

二化螟对杀螟硫磷产生抗性的机理

本文就二化螟Ckilo suppressalis Walker对杀螟硫磷抗性发生的机理进行了较为全面的研究。活体增效作用研究表明,脱叶磷(DEF)和氧化胡椒基丁醚(PBO)对抗性种群可分别增效1.3和34.8倍。这表明了多功能氧化酶(MFO)活性增强,而似乎与水解酶的活性无关。对寓体解毒酶的进一步研究表明,抗性和敏感种群的非特异性酯酶活力无明显差异,但羧酸酮酶(CarE,加入10^-4mol/L的毒扁豆碱)和MFO的活力在抗性种群中有所增高。R种群幼虫CarE活力分别是s的2.42和2.92倍(以a-和β-乙酸萘酯为底物);R种群幼虫MFO的O-脱甲基作用的活力为s幼虫的1.43倍(以对硝基茴香醚为底物);R幼虫乙酰胆碱酯酶(AChE)对乙酰胆碱的活力是s的1.3倍,两者相差不大。但从米氏常数K_m)和最大反应速度(V_max)看,R幼虫是s幼虫的1.9和1.6倍;以对氧磷为抑制剂的R幼虫150是s幼虫的2倍。这些表明了R和s的AChE是不同的。研究结果还表明,二化螟对杀螟硫磷抗性的机理至少包括：(1)CarE活性的增高;(2)MFO的O-脱甲基活力的增高和(3)AChE敏感性的降低等。     

Mannich Bases Derived from 5-Substituted-1,3,4-oxadiazol-2-thiones

The organophosphate (OP)-resistant Daisan Yumenoshima (3-Y_M) housefly strain, which had been reared under the pressure of malathion, was further selected by fenitrothion (3-Y_F) and by diethyl fenitrothion (3-Y_E). The 3-Y_F and 3-Y_E selected strains were highly resistant to the chemical used in the selection, and their acetylcholinesterases were insensitive to the corresponding OP-oxon. The 3-Y_F and 3-Y_M strains had a high activity for glutathione (GSH)-dependent degradation of fenitrothion, whereas that of the 3-Y_E strain was lower. Salithion was relatively effective against both the 3-Y_F and 3-Y_E strains as well as against 3-Y_M. K-1 and K-2, salioxon analogs as the inhibitors of malathion carboxylesterase, were synergistic with fenitrothion against these strains by inhibiting GSH-dependent detoxication.     

A generalized approach to detection of organophosphate resistance in mosquitoes

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.