Effects of acaricides on the activities of monooxygenases in bovine liver microsomes.

Organophosphates (OPs), pyrethrins and fipronil, are acaricides commonly used in cattle, mainly as pour on formulations. Scant information is available on their potential interactions with hepatic xenobiotic metabolizing enzymes. This work aimed to evaluate in vitro the potential inhibitory effects of widely employed acaricides on catalytic activities mediated by hepatic cytochrome P450 (CYP) and flavin-monooxygenase (FMO) enzymes in cattle. Bovine (n = 4) liver microsomes were incubated in the absence (control assays) and in presence of different OPs (fenthion, chlorpyrifos, ethion, diazinon and dichlorvos), fipronil and cypermethrin at 0.1-100 µm. Five oxidative enzyme activities were assayed by spectrofluorimetric or HPLC methods: 7-ethoxyresorufin O-deethylase (for CYP1A1), methoxyresorufin O-demethylase (for CYP1A2), benzyloxyresorufin O-debenzylase (for CYP2B), testosterone 6-beta hydroxylase (for CYP3A) and benzydamine N-oxidase (for FMO). All acaricides, particularly phosphorothionate-containing OPs, inhibited to some extent more than one enzyme activity. The most frequent inhibitor was fenthion, which inhibited (p < .05) all enzyme activities tested (from 22% at 1 µm to 72% at 100 µm). However, low inhibitory potencies (IC50s higher than 7 µm) of all acaricides studied were observed against the catalytic activities assayed. Therefore, the risk of in vivo metabolic interactions due to inhibition of monooxygenases would be low under common husbandry conditions.

Impact of environmental temperatures on resistance to organophosphate insecticides in Aedes aegypti from Trinidad.

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and ß-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.

Impact of environmental temperatures on resistance to organophosphate insecticides in Aedes aegypti from Trinidad / Repercusión de las temperaturas ambientales sobre la resistencia de Aedes aegypti a los insecticidas organofosforados en Trinidad

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and β-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.

OBJETIVO: Examinar los efectos del aumento de las temperaturas de desarrollo larvario sobre el estado de resistencia a los insecticidas organofosforados de las poblaciones de Aedes aegypti en Trinidad. MÉTODOS: En 2007 y 2008 se llevaron a cabo ensayos biológicos y bioquímicos en larvas de A. aegypti recogidas en el 2006 de ocho áreas geográficamente separadas en Trinidad (Trinidad y Tabago). Las poblaciones larvarias se desarrollaron en cuatro temperaturas (28 ± 2 ºC, 32 ºC, 34 ºC y 36 ºC) antes de los ensayos biológicos con insecticidas organofosforados (fentión, malatión y temefós) y los análisis bioquímicos para las enzimas de esterasa. RESULTADOS: La mayoría de las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC fueron susceptibles al fentión (mortalidad > 98%) pero resistentes al malatión y al temefós (mortalidad < 80%). Se encontró una asociación positiva entre la resistencia a los insecticidas organofosforados y la mayor actividad de αy β-esterasas en las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC. Aunque las poblaciones larvarias que se desarrollaron a temperaturas mayores mostraron variaciones en la resistencia a los organofosforados, hubo un aumento general de la sensibilidad. Sin embargo, los aumentos o las disminuciones en los niveles de actividad de las enzimas no siempre se correspondieron con un aumento o disminución en la proporción de individuos resistentes desarrollados a las temperaturas más altas. CONCLUSIONES: Aunque el recalentamiento del planeta puede causar un aumento de la transmisión del dengue, según los resultados de este estudio el uso de insecticidas para la prevención y el control del dengue todavía puede ser eficaz si las temperaturas aumentan según lo proyectado.

Developing a systems approach for Sternochetus mangiferae (Coleoptera: Curculionidae) in South Africa.

Sternochetus mangiferae (F.) (Coleoptera: Curculionidae) is a quarantine pest of mango, Mangifera indica L., in South Africa. The aim of the study was to test cultural and chemical control measures in a systems approach for risk mitigation of S. mangiferae in South Africa. A large-scale experiment, using a randomized block design with three replicates, was undertaken on 'Tommy Atkins' mangoes in South Africa. Five treatment combinations were applied over two seasons: (1) untreated control; (2) sanitation and physical removal of egg-infested fruit (P+S); (3) thiamethoxam, P+S; (4) thiamethoxam, fipronil, P+S; and 5) thiamethoxam, two fenthion applications, P+S. A weekly random sample of eight fruit from 40 trees per orchard was taken from 5 wk to harvest over two harvest periods. In total, 57,600 fruit were sampled. In a separate experiment, the same variety was used to test the efficacy of covering fruit with paper bags over one season in a completely randomized design. In both trials, all the pests were recorded from internal and external fruit inspections. In trial 1, results after the first season showed >98% control in the three chemical combination treatments. In the second season, treatment 2 provided 19% and treatment 3 99.9% control, whereas no fruit were infested with S. mangiferae in treatments 4 and 5. Trial two showed that fruit bagging significantly reduced S. mangiferae infestation. The data showed that a systems approach for S. mangiferae control in South Africa can be effective and that bagging can be an effective organic control option.

Temephos-induced resistance in Aedes aegypti and its cross-resistance studies to certain insecticides from India.

A temephos-induced resistance in Aedes aegypti that was developed for 24 generations exhibited 20.3-fold resistance as compared to susceptible strain. The diagnostic dose of temephos 0.02 mg/l exhibited gradual decrease in larval mortality with the progression of generations. An operational dose (1 mg/l) exhibited the LT(50) value of 41.42 min in the susceptible population, whereas the value of the resistant population increased to 72.62, 108.86, 122.34, 182.03, 244.82 and 304.86 min in the fourth, eighth, 12th, 16th, 20th and 24th generation, respectively. The study carried out showed 120 min as the cut-off limit for differentiation between susceptible and resistant A. aegypti. Cross-resistance studies showed a varied degree of cross-resistance to fenthion, chlorpyrifos, malathion and DDT, whereas comparatively higher cross-resistance was observed to chlorpyrifos. Study on diagnostic doses of insecticides to A. aegypti females indicated a gradual decrease in adult mortality at every eighth generation as compared to susceptible population when exposed to malathion 0.8%, fenthion 0.25%, DDT 4%, permethrin 0.25% and temephos 6.5%, whereas little or no change in mortality when exposed to lambda-cyhalothrin 0.03% and propoxur 0.1%, was observed. Thus, the expression of larval resistance was observed in adult stages also.

Susceptibility of immature stages of Aedes (Stegomyia) aegypti; vector of dengue and chikungunya to insecticides from India.

Susceptibility of Aedes aegypti to some insecticides in different geographic areas was conducted during dengue and chikungunya outbreak. At present, the only method of preventing dengue and chikungunya is to control the vector, which is the weakest link in vector-borne diseases. In our study, the susceptibility of A. aegypti collected from urban areas of Delhi, Mumbai, Jodhpur, Chennai and Coimbatore was evaluated against temephos, fenthion, malathion and DDT. The A. aegypti from different locations exhibited 0.33-7.11, 0.36-3.00, 0.65-2.84 and 2.16-20.8 fold more lethal concentration value of 50% (LC50) to temephos, fenthion, malathion and DDT, respectively, compared to susceptible reference strain. The result reveals that A. aegypti from various locations studied are still susceptible to temephos, fenthion and malathion, whereas low level of DDT resistance was noticed in field-collected A. aegypti. Amongst the insecticides tested, temephos was found to be relatively more effective in controlling A. aegypti, followed by fenthion, malathion and DDT.

Synthesis of fenthion sulfoxide and fenoxon sulfoxide enantiomers: effect of sulfur chirality on acetylcholinesterase activity.

Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the organophosphate pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were transformed into chiral fenoxon sulfoxides using a two-step protocol. The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (>82%) from the parent sulfide. Single recrystallizations afforded chiral fenthion sulfoxides with >99% ee, measured by chiral HPLC analysis. The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis of the (S)-(-)-antipode. Inhibition of human recombinant (hrAChE) and electric eel (eeAChE) acetylcholinesterase were assayed with fenthion, fenoxon, and the racemates and enantiomers of fenthion sulfoxide and fenoxon sulfoxide. Results revealed stereoselective inhibition with (R)-(+)-fenoxon sulfoxide when compared with that of (S)-(-)-fenoxon sulfoxide (IC50 of 6.9 and 6.5 microM vs 230 and 111 microM in hrAChE and eeAChE, respectively). Fenthion sulfoxide (R or S enantiomers) did not present anti-AChE properties. Although the stereoselective sulfoxidation of fenthion to (R)-(+)-fenthion sulfoxide by FMO represents a detoxification pathway, the results of this study support the notion that subsequent oxidative desulfuration of (R)-(+)-fenthion sulfoxide (in vivo) may represent a critical bioactivation pathway, resulting in the production of (R)-(+)-fenoxon sulfoxide, a potent AChE inhibitor.

Potential risk of pathogen transmission by acaricide-poisoned ticks.

Many aspects of tick poisoning with acaricides have yet to be elucidated. One of them is the influence of acaricide poisoning on tick infectivity to their hosts. To clarify this problem, we should know how tick poisoning develops after acaricide application. Data obtained during more than three decades of work with ticks of various species and with acaricides of different groups are presented in the paper. The first important phenomenon found was the gradual and progressive development of toxic symptoms after acaricide application, with death of the treated ticks delayed for days or even weeks ('slow-death syndrome'). The development of symptoms was much faster after the application of fenthion, an organophosphorous acaricide, when compared to DDT, a chlorinated hydrocarbon. The larger the adult ticks of a particular species, the more refractory they were to acaricide action. The duration of the development of toxic symptoms directly correlates with the degree of species-specific refractoriness. A special index T(LD(50)) (T(LC(50)) was introduced for comparing the duration of the poisoning development between different tick species, populations etc. The index determines the time when the final mortality is reached after using the LD(50) of a particular acaricide. Another index , T(LD(90)), was used for practical purposes. The values of these indices decreased with increasing age of tick populations. The prolonged duration of poisoning was also observed in nymphal ticks of species with a prolonged life-span and the ability to over-winter (Ixodes, Haemaphysalis) but not in ticks characterized by a short life-span (Dermacentor). During the entire period of poisoning, from acaricide application until tick death, the individual tick passes through six stages defined by its locomotor capabilities. The stages are the same for nymphs and adults of both hard and soft ticks after treatment with various acaricides. When ticks are at the initial stages of poisoning (1st to 3rd stage for DDT or 1st to 2nd for fenthion), they can attach to hosts and imbibe blood. The average body weight of such ticks after repletion corresponded to that of control ticks. Engorged nymphal ticks normally molted to adults, engorged females normally oviposited, and their progeny did not differ from the progeny of control females. This second important phenomenon, called 'overcoming the poisoning', was observed in all studied species of ticks from several genera. Thus, the slow development of tick poisoning creates a potential for ticks to attach to hosts and to gorge blood, and for infected ticks to transmit pathogens to those hosts, while the ability to overcome the poisoning allows the ticks to survive and makes possible the subsequent trans-stadial and transovarial passage of pathogens. These data can be considered as strong circumstantial evidence of the risk that ticks can present to humans and animals at the initial stages of poisoning after acaricide treatment.

Laboratory bio-assay of temephos and fenthion against some vector species of public health importance.

Laboratory studies carried out using Temephos and Fenthion, two commonly used larvicides under Urban Malaria Scheme (NMEP) and NFCP for the control of mosquito larvae. The results revealed that the LC50 and LC90 values for temephos against Aedes aegypti 0.0177 and 0.0559, Anopheles stephensi 0.0148 and 0.0472, Culex quinquefasciatus 0.0157 & 0.0480 and for Culex vishnui group of mosquitoes 0.043 & 0.0118 ppm respectively. The results obtained revealed that there is a 62.8 & 94.12 times increase in the LC50 & LC90 of Cx. Quinquefasciatus which indicates that the species has developed resistance to temephos. There were 6.32 & 8.34 fold increase in Ae. aegypti and 2.27 & 2.34 fold increase in LC50 & LC90 values of An. stephensi are indicative of development of tolerance against temephos. Similarly LC50 and LC90 values estimated for fenthion against Ae. aegypti 0.0173 & 0.0392, An. stephensi 0.0274 & 0.0992 and Cx. quinquefasciatus 0.03 & 0.0469 respectively. The slope values were found to be higher in fenthion as compared to temephos. It was recorded to be 2.72 times higher in Cx. quinquefasciatus and 1.54 times against An. stephensi. However, much difference was not observed in the slope values of temephos and fenthion in Ae. aegypti.

Ground deposition impact of aerially applied fenthion on the fiddler crabs, Uca pugilator.

Caged fiddler crabs, Uca pugilator, were exposed to field ULV applications to measure the impact of fenthion. Two nozzle systems, conventional flat-fan nozzles (Tee Jet 8002SS) and high-pressure hydraulic nozzles (1/8 MIS), were compared using single spray swaths. Fenthion residues were detected throughout the 4.83-km test zone for both systems. Heavy ground deposits (650-1,670 microg/m2) of fenthion were found within 1 km using the flat-fan nozzle systems, which resulted in 80% fiddler crab mortality. Less than 100 microg/m2 fenthion ground deposits were detected during the high-pressure nozzle trials. No fiddler crab mortality was observed within the first 1-km zone following 3 single swath applications repeated during 3 consecutive nights. We found also that when the fiddler crabs were exposed to 700-800 microg/m2 fenthion, mortality occurred. Significant crab mortality (>50%) was observed when residues exceeded 1,000 microg/m2.

Ultrastructural changes in testis of gobiid fish Glossogobius giuris (Ham) induced by fenthion.

Pesticides are known to affect the testis in fishes and cause cytomorphological changes or gross physiological alterations due to changes in its histology, retard gonadotrophic secretions thereby causing regression of the gonads. Light and ultrastructural studies were made on control and fenthion treated testis, interstitial gland and sperms of G. giuris during spawning phase after exposing them to different sub-lethal concentrations (0.05, 0.25 and 0.5 ppm) fenthion for a short-term period (24, 48, 72 and 96 hr). The results indicated extensive cytotoxic damages in the testis including atrophy of testis, reduction in lipoidal material in the interstitial cell and degeneration of sperms. The alteration in histology of testis, interstitial cell and sperms progressively increased with the increase in the sub-lethal concentration of pesticides.

Comparison of ivermectin SR bolus, benzimidazole anthelmintics, and topical fenthion on productivity of stocker cattle from grazing through feedlot.

A study was conducted to determine the effect of different parasite control programs on weight gain and other measurements for stocker beef calves during the grazing season and subsequent feedlot phase of production. One hundred eighty recently weaned beef steers were purchased from a Mississippi sale barn and were allocated by restricted randomization on pretreatment weight to three treatments: (1) no anthelmintic treatment; treated only with a topical organophosphate (OP) during processing into the feedlot; (2) one benzimidazole (BZD) treatment at initiation of grazing, and a second given at the time of processing upon arrival at the feedlot, along with a topical OP; and (3) ivermectin sustained-release (SR) bolus administered at initiation of grazing, with no further treatment given at the feedlot. The cattle grazed separately by treatment for 125 days, with six replicated pastures per treatment; then were penned according to the same groupings after entry into the feedlot on day 127. Cattle were individually weighed at approximately 2-month intervals, and feed consumption was measured during the 167 days in the feedlot. Fecal nematode egg counts were individually monitored for all animals during both phases of the trial. Carcass weight, quality grade, yield grade, and the incidence of liver abscesses were recorded for each animal at slaughter. Cattle treated with the ivermectin SR bolus gained significantly (P < .05) more weight through the grazing period and from the start of grazing through the end of the feedlot phase than the controls or the cattle treated with BZD products. There were no significant differences in feed efficiency between any of the groups. Mean carcass weight for cattle treated with boluses was significantly (P < .05) greater than that of the controls and the group treated with BZD. Dressing percentage and quality grade were significantly (P < .05) higher for the BZD and bolus groups, and yield grade was slightly (but not significantly) better for each of these groups than for controls. Significantly (P < .05) fewer livers of cattle treated with boluses had abscesses at slaughter than did livers of controls or cattle treated with BZDs. During both phases of the trial, fecal egg counts were significantly (P < .05) lower for the group treated with boluses than for the untreated group or the group treated with BZD. These data indicate that treatment with boluses for parasite control at the beginning of the grazing period had beneficial effects on weight gain as compared to no anthelmintic treatment or treatment with a BZD at the start of grazing and again at the time of introduction into the feedlot. These bolus benefits were sustained through the feedlot phase of production and provided further improvements with significantly (P < .05) increased carcass weights. The bolus is a tool that can significantly increase the efficiency of production for cattle producers who retain ownership into the feedlot phase of production or feedlot operators who graze stockers before feedlot entry.

Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.

The Pto gene was derived originally from the wild tomato species Lycopersicon pimpinellifolium and confers resistance to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. The Fen gene is also derived from L. pimpinellifolium and confers sensitivity to the insecticide fenthion. We have now isolated and characterized the alleles of Pto and Fen from cultivated tomato, L. esculentum, and designated them pto and fen. High conservation of genome organization between the two tomato species allowed us to identify the pto and fen alleles from among the cluster of closely related Pto gene family members. The pto and fen alleles are transcribed and have uninterrupted open reading frames that code for predicted proteins that are 87 and 98% identical to the Pto and Fen protein kinases, respectively. In vitro autophosphorylation assays revealed that both the pto and fen alleles encode active kinases. In addition, the pto kinase phosphorylates a previously characterized substrate of Pto, the Pto-interacting Pti1 serine/threonine kinase. However, the pto kinase shows impaired interaction with Pti1 and with several previously isolated Pto-interacting proteins in the yeast two-hybrid system. The observation that pto and fen are active kinases and yet do not confer bacterial speck resistance or fenthion sensitivity suggests that the amino acid substitutions distinguishing them from Pto and Fen may interfere with recognition of the corresponding signal molecule or with protein-protein interactions involved in the Pto- and Fen-mediated signal transduction pathways.

The Pto bacterial resistance gene and the Fen insecticide sensitivity gene encode functional protein kinases with serine/threonine specificity.

The catalytic activity and amino acid specificity of the tomato Pto and Fen kinases were investigated. The Pto and Fen genes were fused to the carboxyl terminus of the maltose-binding protein and expressed in Escherichia coli. Incubation of the purified fusion proteins with [gamma-32P]ATP in an in vitro assay showed that both proteins were capable of autophosphorylation. Mutant fusion proteins in which the conserved lysine residue of subdomain II was changed to a glutamine were unable to autophosphorylate. Phosphoamino analysis of the active fusion proteins indicated that both kinases phosphorylate serine and threonine residues but not tyrosine.

The disease-resistance gene Pto and the fenthion-sensitivity gene fen encode closely related functional protein kinases.

Resistance to bacterial speck in tomato is governed by a gene-for-gene interaction in which a single resistance locus (Pto) in the plant responds to the expression of a specific avirulence gene (avrPto) in the pathogen. Disease susceptibility results if either Pto or avrPto are lacking from the corresponding organisms. Leaves of tomato cultivars that contain the Pto locus also exhibit a hypersensitive-like response upon exposure to an organophosphorous insecticide, fenthion. Recently, the Pto gene was isolated by a map-based cloning approach and was shown to be a member of a clustered multigene family with similarity to various protein-serine/threonine kinases. Another member of this family, termed Fen, was found to confer sensitivity to fenthion. The Pto protein shares 80% identity (87% similarity) with Fen. Here, Pto and Fen are shown to be functional protein kinases that probably participate in the same signal transduction pathway.

A member of the tomato Pto gene family confers sensitivity to fenthion resulting in rapid cell death.

Leaves of tomato cultivars that contain the Pto bacterial resistance locus develop small necrotic lesions within 24 hr after exposure to fenthion, an organophosphorous insecticide. Recently, the Pto gene was isolated and shown to be a putative serine/threonine protein kinase. Pto is one member of a multigene family that is clustered within a 400-kb region on chromosome 5. Here, we report that another member of this gene family, termed Fen, is responsible for the sensitivity to fenthion. Fen was isolated by map-based cloning using closely linked DNA markers to identify a yeast artificial chromosome clone that spanned the Pto region. After transformation with the Fen gene under control of the cauliflower mosaic virus (CaMV) 35S promoter, tomato plants that are normally insensitive to fenthion rapidly developed extensive necrotic lesions upon exposure to fenthion. Two related insecticides, fensulfothion and fenitrothion, also elicited necrotic lesions specifically on Fen-transformed plants. Transgenic tomato plants harboring integrated copies of the Pto gene under control of the CaMV 35S promoter displayed sensitivity to fenthion but to a lesser extent than did wild-type fenthion-sensitive plants. The Fen protein shares 80% identity (87% similarity) with Pto but does not confer resistance to Pseudomonas syringae pv tomato. These results suggest that Pto and Fen participate in the same signal transduction pathway.