High concentrations of piperonyl butoxide (PBO) enhance toxicity of S-methoprene against the lesser grain borer, Rhyzopertha dominica.

Insect growth regulators (IGRs) have been playing a major role in the effective management of a range of stored product insect pests including species that have developed resistance to major groups of insecticides, such as organophosphates (OPs) and synthetic pyrethroids (SPs). In the present study, we evaluated the efficacy of S-methoprene alone and in combination with piperonyl butoxide (PBO), an adjuvant component of insecticides for synergy, against two strains, Lab-S (susceptible) and Met-R (Methoprene resistant) of an economically important pest species, the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). Adults of both Lab-S and Met-R strains were exposed to wheat treated with multiple concentrations of S-methoprene ranging from 0.001 to 0.01 and 10 to 60 mg/kg, respectively, alone and in combination with PBO. A variety of concentrations, including 0.27, 0.53, 0.80, and 1.07 g/kg, were evaluated for PBO. Mortality of adults and percent reduction in progeny were assessed after 14 and 65 days of treatment, respectively. As anticipated, the adult mortality rates of both strains were not significantly affected by S-methoprene alone. However, the number of progeny produced confirmed that the Met-R strain exhibited a high level of resistance to S-methoprene alone, with over 50 F1 progeny adults surviving in wheat treated with the maximal rate, 10 mg/kg. In contrast, the toxicity of S-methoprene was increased against the same resistant strain (Met-R), by 0.80 or 1.07 g/kg of PBO in combination treatment, resulting in a significant reduction in progeny numbers (25 adults per container). Although the tested concentrations of S-methoprene and PBO were well above the currently registered rate globally, our results highlight the fact that PBO enhances the toxicity of S-methoprene to some extent, reaffirming that the mode of action of the latter involves the inhibition of mixed-function oxidases (MFOs) and highlights the need for further research into developing potential binary or triplet formulations containing these two active ingredients (AIs).

Evaluation of Resistance to Different Insecticides and Metabolic Detoxification Mechanism by Use of Synergist in the Common Bed Bug (Heteroptera: Cimicidae).

Bed bugs have become a common urban pest with consequences on human health and economic costs to the hotel and tourism sectors. Insecticide resistance is considered an important factor in the current bed bug resurgence, and multiple resistance mechanisms could be working in the resistant bed bug populations. In the present study, we determined the resistance profile to four insecticides with a different mode of action in Cimex lectularius L. (Heteroptera: Cimicidae) field-collected colonies from Argentina. Furthermore, the synergism effect of piperonyl butoxide (PBO) with deltamethrin was investigated to explore the contribution of detoxification metabolism to resistance. Our results showed that most of the field-collected colonies are extremely resistant to deltamethrin and propoxur, much more than to azametiphos and imidacloprid. The differences in resistance ratios among field-collected colonies could be associated with different modes of action of insecticides used in control pest and the mechanisms involved in the resistance. PBO pretreatment led to a significantly decreased RR in pyrethroid-resistant colonies, suggesting an upturn of monooxygenase activity for deltamethrin detoxification. However, the high RR detected could involve other mechanisms as part of the whole resistant phenotype in colonies of C. lectularius resistant to pyrethroids.

Single and mixture toxicity of abamectin and difenoconazole to adult zebrafish (Danio rerio).

Concerns have been raised in recent years on the potential risks related with pesticide mixtures that are likely to be present in agricultural edge-of-field waterbodies. Despite the high use of pesticides in tropical countries like Brazil, studies evaluating pesticide mixtures are especially scarce in the tropics. The insecticide abamectin and the fungicide difenoconazole are the main pesticides intensively used in Brazilian strawberry crop and are hence likely to occur simultaneously. The aim of the present study was therefore to evaluate the toxicity of abamectin, difenoconazole and their mixture to the tropical fish Danio rerio. Laboratory toxicity tests with the individual pesticides indicated 48 h-LC50 values of 59 µg L-1 for abamectin and 1.4 mg L-1 for difenoconazole. Mixtures of the two pesticides revealed a synergistic deviation of the independent action model. Implications of study findings for the aquatic risk assessment of pesticide mixtures, especially in tropical countries and indications for future research are discussed.

Prevalencia y riesgo de malformación congénita en mujeres gestantes expuestas a plaguicidas. en el Hospital Regional de Ica, Perú / Prevalence and risk of pregnant women in congenital malformation exposed to pesticides. Regional Hospital Ica, Peru

Objetivo: Determinar la prevalencia y asociación entre exposición a plaguicidas antes y durante el primer trimestre de la gestación y el riesgo de malformación congénita. Material y métodos: Estudio observacional, transversal retrospectivo, analítico de casos y controles. Los casos (n = 57) se seleccionaron por muestreo no probabilístico y los controles (n= 114) por muestreo aleatorio simple de una población hospitalaria de la Región Ica, durante el periodo de 3 años comprendido entre el 1 de enero del 2005 al 31 de diciembre del 2007. Los casos, se definieron como neonatos con malformaciones congénitas y los controles, recién nacidos sin malformaciones. Se consideró exposición a cualquier contacto con plaguicidas. Se evaluaron otros factores de riesgo conocidos para malformación congénita: in gesta de medicamentos, drogadicción, alcoholismo y anemia materna severa, como factores de confusión. Resultados: Se reportaron 26 malformaciones de la cara, boca y paladar, 11 del sistema cardiovascular, 11 de las extremidades, 7 del sistema genitourinario, 6 del sistema nervioso central y 12 otras malformaciones. Las gestantes expuestas tuvieron un alto riesgo de procrear hijos malformados OR 2,85 (IC 95% 1,46 ­ 5,54 p < 0,05). Los riesgos más elevados a malformaciones fueron: exposición por acudir a campos fumigados OR: 3,82 (IC 95% 1,92 ­ 7,60 p< 0,05) y vivir cerca de campos fumigados OR: 3,07 (IC 95% 1,59 ­ 5,92 p < 0.05) . Conclusiones: Se muestra evidencia epidemiológica de la relación causal entre exposición a plaguicidas y malformaciones congénitas en una muestra de mujeres gestantes en el hospital regional de Ica. (AU)

Objective: To determine the prevalence and association between pesticide exposure before and during the first trimester of pregnancy and the risk of congenital malformation in the systems: cardiovascular, genitourinary, central nervous malformations of the face, mouth, palate and limbs. . Materials and Methods: Observational, transversal retrospective analytical case-control study. The cases (n = 57) were selected by non-probability sampling and controls (n = 114) by simple random sampling from a hospital population of Ica region during the three years period from 1 January 2005 to 31 December 2007. The cases were defined as infants with congenital malformations and controls infants without malformations. Exposures to any contact with pesticides are considered. In quest of drugs, drug addiction, alcoholism and severe maternal anemia, as confounding factors: other known risk factors for congenital malformation they were evaluated. Results: 26 were reported defects of the face, mouth and palate, cardiovascular system 11, 11 of the limbs 7 of the genitourinary system, central nervous system 6 and 12 other malformations. Exposed pregnant women had a higher risk of bearing children malformed OR 2,85 (95% CI 1,46 to 5,54 p < 0,05). The highest risk of malformations were fumigated exposure attend camps OR : 3,82 ( 95% CI 1,92 to 7,60 p < 0,05 ), living near sprayed fields OR : 3,07 ( IC 95 % 1,59 to 5,92 p < 0,05) and living with a spouse working in fields sprayed OR: 2,40 (95% CI 1,21 to 4,78 p<0,05). Conclusions: Epidemiological evidence of a causal relationship between pesticide exposure and birth defects. (AU)

Effects of piperonyl butoxide on the toxicity of the organophosphate temephos and the role of esterases in the insecticide resistance of Aedes aegypti.

INTRODUCTION: The effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. METHODS: A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02 mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. RESULTS: At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. CONCLUSIONS: The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae.

Effects of piperonyl butoxide on the toxicity of the organophosphate temephos and the role of esterases in the insecticide resistance of Aedes aegypti

Introduction The effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. Methods A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. Results At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. Conclusions The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae. .

In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics.

A study was conducted to evaluate the efficacy of cypermethrin, amitraz, and piperonyl butoxide (PBO) mixtures, through in vitro laboratory bioassays and in vivo on-animal efficacy trials, for the control of resistant Rhipicephalus (Boophilus) microplus on cattle in the Mexican tropics. Also, to examine mechanisms of resistance to cypermethrin in this tick population, the frequency of a mutated sodium channel gene (F1550I) was determined using a PCR assay. Results of laboratory bioassays using modified larval packet tests revealed that cypermethrin toxicity was synergized by PBO (from 46.6-57.0% to 83.7-85.0% larval mortality; P<0.05). The cypermethrin and amitraz mixture showed an additive effect (from 46.6-57.0% to 56.0-74.3% larval mortality). Strong synergism was observed with the mixture of cypermethrin+amitraz+PBO and this mixture was the most effective killing resistant tick larvae in vitro (96.7-100% of larval mortality). Tick larvae surviving exposure to cypermethrin or mixtures either with amitraz and PBO in vitro showed 2.9-49.6 higher probability to present the mutated allele than those killed by acaricide treatment (P<0.05). In the in vivo trial, the mixtures containing cypermethrin+PBO (80.6-97.3%), and cypermethrin+amitraz (87.0-89.7%) were more efficacious than cypermethrin alone (76.3-80.5%). The highest level of efficacy was obtained with the mixture of cypermethrin+amitraz+PBO, which yielded >95% control that persisted for 28 days post-treatment against R. microplus infesting cattle when tested under field conditions in the Mexican tropics. Although this mixture is a potentially useful tool to combat pyrethroid resistance, a product based on an acaricide mixture like the one tested in this study has to be used rationally.

Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control.

Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases.

Mechanisms of resistance to DDT and pyrethroids in Patagonian populations of Simulium blackflies.

Mixed populations of the pest blackflies Simulium bonaerense Coscarón & Wygodzinsky, S. wolffhuegeli (Enderlein) and S. nigristrigatum Wygodzinsky & Coscarón (Diptera: Simuliidae) are highly resistant to DDT and pyrethroids in the Neuquén Valley, a fruit-growing area of northern Patagonia, Argentina. As these insecticides have not been used for blackfly control, resistance is attributed to exposure to agricultural insecticides. Pre-treatment with the synergist piperonyl butoxide (PBO) reduced both DDT and fenvalerate resistance, indicating that resistance was partly due to monooxygenase inhibition. Pre-treatment with the synergist tribufos to inhibit esterases slightly increased fenvalerate toxicity in the resistant population. Even so, biochemical studies indicated almost three-fold higher esterase activity in the resistant population, compared to the susceptible. Starch gel electrophoresis confirmed higher frequency and staining intensity of esterase electromorphs in the resistant population. Incomplete synergism against metabolic resistance indicates additional involvement of a non-metabolic resistance mechanism, such as target site insensitivity, assumed to be kdr-like in this case. Glutathione S-transferase activities were low and inconsistent, indicating no role in Simulium resistance. Knowing these spectra of insecticide activity and resistance mechanisms facilitates the choice of more effective products for Simulium control and permits better coordination with agrochemical operations.

Plasma achiral and chiral pharmacokinetic behaviour of intravenous oxfendazole co-administered with piperonyl butoxide in sheep.

Co-administration of piperonyl butoxide (PB) potentiates fenbendazole (FBZ) in small ruminants. The resultant increase in bioavailability of FBZ and its metabolite oxfendazole (OFZ) has important implications for the efficacy of these drugs against benzimidazole (BZD)-resistant strains of Teladorsagia circumcincta. This study evaluated the racemic (achiral) and enantiomeric (chiral) plasma disposition kinetics of OFZ and its metabolites after the co-administration of PB and OFZ in sheep. Six 6-8-month-old, parasite-free, female Dorset sheep (30-40 kg) were used in a two-phase crossover experiment. In phase I, three sheep received 30 mg/kg PB orally, followed by a single intravenous (i.v.) injection of OFZ at 5 mg/kg. The other three animals were treated similarly except that 5 mL of water replaced PB. In phase 2, treatments for the two groups were reversed and were given 14 days after the initiation of phase I. Three analytes OFZ, FBZ and fenbendazole sulphone (FBZSO(2)) were recovered in plasma up to 48 h post-treatment in both experimental groups. Achiral and chiral pharmacokinetic (PK) profiles for OFZ, after the co-administration of PB, were characterized by a significantly greater area under the concentration--time curve (AUC) and a longer mean residence time (MRT). Chiral OFZ distribution ratios were comparable in both treatment groups. Piperonyl butoxide treatment markedly influenced the plasma PK profiles for FBZ and FBZSO(2) following OFZ administration. Production of FBZ was enhanced as reflected by increased (> 60%) AUC, delayed T(max) and a significantly delayed (> 45%) elimination (t(1/2)(el)). Although AUC values for FBZSO(2) were not significantly different between groups, this metabolite was depleted more slowly from plasma (t(1/2)(el) > 60% and MRT > 42%) following PB treatment. This study demonstrated that PB co-administration is associated with an inhibition of OFZ biotransformation, as evidenced by the significantly higher plasma concentrations of OFZ and FBZ, and this could have important implications in terms of anti-parasite therapy against BZD-resistant parasite strains.

Effects of the synergists piperonyl butoxide and S,S,S-tributyl phosphorotrithioate on propoxur pharmacokinetics in Blattella germanica (Blattodea: Blattellidae).

Effects of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on propoxur pharmacokinetics were examined in the German cockroach, Blattella germanica (L.). Treatment of adult male German cockroaches with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased propoxur toxicity by 2- and 6.8-fold, respectively, implicating hydrolysis as a major detoxification route of propoxur in the German cockroach. However, significant hydrolytic metabolism could not be demonstrated conclusively in vitro resulting in a conflict between in situ bioassay data and in vitro metabolic studies. In vitro propoxur metabolism with NADPH-fortified microsomes produced at least nine metabolites. Formation of metabolites was NADPH-dependent; no quantifiable metabolism was detected with cytosolic fractions. However, microsomal fractions lacking an NADPH source did produce a low, but detectable, quantity of metabolites (1.6 pmol). PBO inhibited NADPH-dependent propoxur metabolism in a dose-dependent fashion, implicating cytochrome P450 monooxygenases as the enzyme system responsible for the metabolism. Interestingly, DEF also inhibited the NADPH-dependent metabolism of propoxur, albeit to a lower extent. Treatment with PBO or DEF also caused a significant reduction in the cuticular penetration rate of propoxur. The data demonstrate that unanticipated effects are possible with synergists and that caution must be exercised when interpreting synergist results.

Thiols and polyamines in the potentiation of malathion toxicity in larval stages of the toad Bufo arenarum.

Treatment with exogenous spermidine enhanced acute malathion toxicity during larval development of the toad Bufo arenarum Hensel. The polyamine was rapidly incorporated in the larvae with a subsequent metabolization to putrescine and spermine, which were excreted to the media. Endogenous polyamine levels were not changed by either spermidine or malathion treatments. However, 0.5-mM spermidine modified malathion uptake and bioavailability increasing the concentration of the xenobiotic in the larvae. The amount of reduced thiols was decreased by both compounds, but the depletion was insufficient to induce cytotoxicity. The oxidative degradation of polyamines competes for the pool of reduced glutathione used in the conjugation of malathion in the larvae, thus leading to the reported potentiation of toxicity. Our results suggest that exposure to thiols-depleting agents may induce alteration of organophosphate degradation in amphibian larvae.

Toxicity of synthetic piperonyl compounds to leaf-cutting ants and their symbiotic fungus.

The development of Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens was inhibited in vitro by synthetic compounds containing the piperonyl group. In addition, worker ants that were fed daily on an artificial diet to which these compounds were added had a higher mortality rate than the controls. The inhibition of the fungal growth increased with the size of the carbon side chain ranging from C1 through C8 and decreasing thereafter. 1-(3,4-Methylenedioxybenzyloxy)octane (compound 5) was the most active compound and inhibited the fungal development by 80% at a concentration of 15 micrograms ml-1. With worker ants the toxic effects started with compound 5 and increased with the number of carbons in the side chain. Thus, for the same concentration (100 micrograms ml-1) the mortality rates observed after 8 days of diet ingestion were 82%, 66% and 42%, for 1-(3,4-methylenedioxybenzyloxy)decane, 1-(3,4-methylenedioxybenzyloxy)dodecane and compound 5, respectively, whereas with commercial piperonyl butoxide the mortality was 68%. The latter compound, which is known as a synergist insecticide, was as inhibitory to the symbiotic fungus as the synthetic compound 5. The possibility of controlling these insects in the future using compounds that can target simultaneously both organisms is discussed.

Cypermethrin pour-on synergized with piperonyl butoxide: effects on Haematobia irritans (Diptera: Muscidae) natural populations resistant to cypermethrin.

Development of pyrethroid resistance in Haematobia irritans in Santa Fe province, Argentina, resulted in an increased use of pyrethroid insecticides, probably due to lack of suitable alternative treatments. We explored the efficacy of mixtures of cypermethrin and piperonyl butoxide (PBO) against pyrethroid-resistant H. irritans. Groups of 25 Holstein cows each, naturally infested with cypermethrin resistant H. irritans were assigned to treated or control groups in April, September, October and December 1997. Cattle in treated groups were medicated with pour-on oil formulations of 5% cypermethrin (dose = 4 mg per kg of body weight) with 5% or 10% PBO in April, and with a mixture containing 5% of both components thereafter. Efficacy was tested for 21 days after treatment. A treatment of 5% cypermethrin pour-on without PBO was evaluated in October 1997. Samples of horn flies were obtained before September, October and December treatments and exposed for 2 h to filter papers impregnated with different cypermethrin concentrations to determine the 50% lethal concentration (LC50). No difference in efficacy was found between cypermethrin pour-on formulations with 5% or 10% of PBO (more than 94% efficacy on day 21 after treatment). Efficacy of 5 % cypermethrin-5% PBO mixture decreased rapidly in the successive treatments (less than 40% efficacy was observed on day 21 after the December treatment), and the period after treatment with an efficacy higher than 95% was 14 days for the treatment carried out in April, 10 days in September; 7 days for the treatment performed in October and 4 days for the December treatment. The LC50 of cypermethrin was 36.6 microg per cm2 in September and increased to 116.6 and 226.1 microg per cm2 in October and December, respectively. It is concluded that the addition of PBO to cypermethrin did not provide a treatment that would give a long term control of pyrethroid resistant-horn flies.

Effect of GSH depletion mediated by l-BSO on the insecticidal activity of DDT and fenitrothion in Triatoma infestans (Hemiptera).

l-BSO injected at a sublethal dose in nymph V of Triatoma infestans produces a transient GSH depletion in the abdomen. A depletion of the total content of GSH was observed in nymph II fed with a sublethal concentration of l-BSO. Either ingestion or injection pretreatments of T. infestans nymph V with sublethal concentrations of l-BSO produced a slight synergism on the toxicity of fenitrothion. Feeding administration of a sublethal concentration of l-BSO to nymphs II of T. infestans resulted in a weak potentiation of the acute toxicity of DDT.

Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control.

The susceptibility of field collected Aedes aegypti larvae was evaluated in terms of median lethal time (LT50) and final mortality, when treated with temephos, Bacillus thuringiensis var israelensis as well as mixtures of these two agents. Third instar larvae were shown to be more susceptible than early and late fourth instar ones to the entomopathogen. Survival of some individuals when exposed to temephos suggest possible resistance. Temporal synergism in early fourth instar larvae was detected when they were exposed to mixtures of Bti-temephos. The possibility of this integrated treatment is commented on.

Susceptibility of Aedes aegypti larvae to temephos and Bacillus thuringiensis var israelensis in integrated control

A susceptibilidade de larvas de Aedes aegypti coletadas no campo foi avaliada em termos do tempo letal mediano (TL50) e da mortalidade final, quando tratadas com temephos, Bacilus thuringiensis var israelensis ou misturas desses dois agentes. As larvas de terceiro estádio mostraram-se mais susceptíveis ao patógeno do que aquelas no início ou no fim do quarto estádio. A sobrevivência de alguns indivíduos aos tratamentois com temephos permite sugerir a possibilidaqde de resistência. Foi detectada a existência de sinergismo temporal, quando larvas no início do quarto estádio foram tratadas com as misturas do Bti com o temephos. A possibilidade do tratamento integrado é comentada