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.

Activation of the ambusher tick Rhipicephalus microplus (Acari: Ixodidae) exposed to different stimuli.

The present study aimed to evaluate the behaviour of larvae of Rhipicephalus microplus exposed to different stimuli. A Y-olfactometer was positioned vertically and R. microplus larvae were exposed to environmental air, CO2 alone, N,N-diethyl-3-methylbenzamide (DEET) alone, and CO2 combined with the repellents DEET and (E)-2-octenal. Tests were also conducted with the olfactometer positioned horizontally; in this case, however, only CO2 was tested. In all tests conducted with the Y-olfactometer positioned vertically, CO2 activated R. microplus larvae even in the presence of DEET and (E)-2-octenal, although activation was lower when these repellents were used. In the absence of CO2 , larval behaviour against DEET was similar to that of the larvae in the control group. In the tests performed with the olfactometer positioned horizontally, the larvae had no significant response to the presence of CO2 . The larvae were not attracted to or repelled by any compound tested in either the vertical or horizontal position of the olfactometer. The lack of horizontal displacement, attraction or repellence may have been a result of the ambush behaviour of this tick species. However, when larvae were exposed to stimuli and the olfactometer was positioned vertically, the interference of attractant and repellent stimuli in larval behaviour was assessed.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) / Primeira avaliação de extratos proteicos de Leucaena leucocephala induzidos por injúria mecânica com atividade carrapaticida sobre Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.(AU)

As plantas respondem a injúria causada por estresse mecânico ou por ataque de herbívoros através da síntese de proteínas de defesa. Não há estudos de proteínas induzidas de plantas contra carrapatos. O objetivo deste estudo foi verificar a atividade acaricida de extratos protéicos de folhas Leucaena leucocephala após injúria mecânica, sobre Rhipicephalus (Boophilus) microplus. Inicialmente foram conduzidos experimentos em diferentes intervalos de tempo para avaliar o impacto da injúria mecânica no conteúdo de proteína, atividade de peroxidase, catalase e inibidor de protease de L. leucocephala. Em seguida foi avaliada a atividade acaricida sobre R. (B.) microplus de um extrato protéico após injúria mecânica. Folhas de L. leucocephala foram artificialmente feridas e após 6, 12, 24 e 48h, as folhas foram coletadas para extração de proteínas. Análises quantitativas e qualitativas das proteínas foram realizadas. A quantidade de proteína e atividades de peroxidase e protease aumentaram 12h após a injúria. O extrato proteico obtido após injúria (12h) reduziu a produção de ovos (8,5%) em comparação com extratos de plantas sem injúria. O extrato reduziu 47,7% a eclosão de ovos e apresentou eficácia geral de 56,3% a 0,1 miligrama de proteína por mL (mgP/mL). Apresentamos que proteínas de defesa de L. leucocephala podem ter atividade sobre R. (B.) microplus.(AU)

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) / Primeira avaliação de extratos proteicos de Leucaena leucocephala induzidos por injúria mecânica com atividade carrapaticida sobre Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Abstract Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

Resumo As plantas respondem a injúria causada por estresse mecânico ou por ataque de herbívoros através da síntese de proteínas de defesa. Não há estudos de proteínas induzidas de plantas contra carrapatos. O objetivo deste estudo foi verificar a atividade acaricida de extratos protéicos de folhas Leucaena leucocephala após injúria mecânica, sobre Rhipicephalus (Boophilus) microplus. Inicialmente foram conduzidos experimentos em diferentes intervalos de tempo para avaliar o impacto da injúria mecânica no conteúdo de proteína, atividade de peroxidase, catalase e inibidor de protease de L. leucocephala. Em seguida foi avaliada a atividade acaricida sobre R. (B.) microplus de um extrato protéico após injúria mecânica. Folhas de L. leucocephala foram artificialmente feridas e após 6, 12, 24 e 48h, as folhas foram coletadas para extração de proteínas. Análises quantitativas e qualitativas das proteínas foram realizadas. A quantidade de proteína e atividades de peroxidase e protease aumentaram 12h após a injúria. O extrato proteico obtido após injúria (12h) reduziu a produção de ovos (8,5%) em comparação com extratos de plantas sem injúria. O extrato reduziu 47,7% a eclosão de ovos e apresentou eficácia geral de 56,3% a 0,1 miligrama de proteína por mL (mgP/mL). Apresentamos que proteínas de defesa de L. leucocephala podem ter atividade sobre R. (B.) microplus.

ABC transporters as a multidrug detoxification mechanism in Rhipicephalus (Boophilus) microplus.

ATP-binding cassette (ABC) transporters are responsible for pumping drugs across membranes and are an important drug detoxification mechanism. Since ABC transporters act on a wide spectrum of chemical compounds, they have been associated with multidrug resistance phenotype in various parasites and cancer cells. Here, we document the presence of a Rhipicephalus (Boophilus) microplus tick population (Jaguar) resistant to four acaricide classes (organophosphates (OP), synthetic pyrethroids (SP), amitraz and macrocyclic lactones (ML)) and reveal that the cattle tick has a multidrug detoxification mechanism based on ABC transporter proteins. Acaricide toxicity was assessed using the larval packet test (LPT), and mortality data were subjected to probit analysis using a susceptible strain (POA) as reference. Larvae were pre-exposed to sub-lethal doses of the ABC-transporter inhibitors, cyclosporin A (CsA) and MK571, and subsequently treated with ivermectin, abamectin, moxidectin, chlorpyriphos, cypermethrin, or amitraz in LPT. Results show that lethal concentrations 50 % (LC(50)) of ivermectin, abamectin, moxidectin (MLs), and chlorpyriphos (OP) were significantly reduced in larvae exposed to CsA and MK571 inhibitors in the Jaguar resistant population, but LC(50) did not change in POA susceptible strain larvae. LC(50) of cypermetrin (SP) and amitraz remained unchanged in inhibitor-exposed larvae, compared to larvae from Jaguar and POA strains not exposed to inhibitor. These results suggest that ABC transporter proteins can protect ticks against a wide range of acaricides and have an important implication in drug resistance development as a multidrug detoxification mechanism.