Biology of the non-parasitic phase of the cattle tick Rhipicephalus (Boophilus) microplus in an area of Amazon influence.

BACKGROUND: Rhipicephalus (Boophilus) microplus is the most important tick species affecting cattle in the world. Under field conditions, the non-parasitic phase of R. (B.) microplus is unknown in the Amazon biome, including Brazil. The present study aimed to evaluate the non-parasitic phase of R. (B.) microplus in field (grass plots) and laboratory conditions. METHODS: The study was conducted from September 2020 to April 2022 in an Amazonian region (Maranhão State, Brazil). We evaluated the biological parameters of R. (B.) microplus under laboratory and field conditions. Engorged females were exposed to experimental conditions every 14 days, totaling 20 months of study. The following biological parameters were observed: pre-oviposition period, egg mass incubation period, and maximum larval survival period. RESULTS: Abiotic data (e.g., temperature and humidity) varied little throughout the year. Precipitation was the factor that varied the most throughout the year (dry ~ 30 mm3 and rain 400 mm3), and the parameters of pre-oviposition and pre-hatching are longer during the rainy season. A possible negative effect of the dry season on the percentage of hatched eggs was observed. Larval longevity in the plots of both control and free females was short (mean ~ 50-60 days), below that recorded for larvae under controlled conditions (mean ~ 95 days). CONCLUSIONS: Rhipicephalus (Boophilus) microplus was able to complete its non-parasitic phase by producing host-seeking larvae in the pasture during all months of the study. The results indicate that R. (B.) microplus can complete up to six generations per year in biome Amazon. To our knowledge, this is the highest number of annual generations for R. (B.) microplus in Latin America.

Effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of Rhipicephalus microplus (Acari: Ixodidae).

The present study was conducted to evaluate the effects of carvacrol and thymol on the antioxidant and detoxifying enzymes of larvae from two populations of R. microplus: Jaguar (tick population resistant to six classes of acaricides) and Porto Alegre (susceptible tick population). Carvacrol and thymol were tested at concentrations ranging from 0.14 to 5.0 mg mL-1 in both populations to determine the LC50. In addition, the LC1, LC25, and LC75 were estimated using the LC50 and HillSlope of each compound. Larvae of both populations of R. microplus were then treated with the LC1, LC25, LC50, and LC75 of each monoterpene, and those that survived were processed to evaluate the effects of the compounds on the antioxidant and detoxifying systems of larvae; these effects were assessed by determining the activity of the enzymes, glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX). Larvae from the Jaguar population treated with different lethal concentrations of carvacrol and thymol displayed a dose-dependent increase in CAT, GPX, SOD, and GST after treatment with LC25. Further, larvae treated with the LC75 had the highest levels of enzyme activity for carvacrol (1.76 mg mL-1) and thymol (1.32 mg mL-1). CAT, GPX, SOD, and GST activity in Porto Alegre population larvae treated with carvacrol and thymol also increased significantly up to the LC50 of each monoterpene. However, at the LC75 of carvacrol and thymol, a decrease in the activity of all enzymes was observed for this tick population. These findings indicate that carvacrol and thymol induced increased activity of all evaluated enzymes at different lethal concentrations in R. microplus larvae from two populations. Such findings unveil the possible mechanisms of action of these natural acaricides.

Development and validation of software that quantifies the larval mortality of Rhipicephalus (Boophilus) microplus cattle tick.

Rhipicephalus (Boophilus) microplus is controlled almost exclusively using synthetic acaricides, and reports of resistant populations have been described worldwide. Several time-consuming and laborious toxicological in vitro tests have been used to diagnose acaricidal resistance, especially those that require differential counting of live and dead larvae. Larval mortality is currently done manually and subjectively, which can limit the performance of a large number of tests and comparing results between different laboratories. The present study aimed to develop and validate a new automatic counting method to evaluate tick larval mortality. A software for differentiation of live and dead larvae was developed using different steps: obtaining videos; image segmentation using the firefly algorithm; detection of larvae with the fast radial symmetry transform technique (FRST); and tracking of the larvae at a given time. Larval immersion tests with ivermectin, cypermethrin, and fipronil were performed to validate the developed software. The larval mortality evaluation was performed by (1) recording for 60 s for each package and (2) manual counts of the same sample using three different analysts, each responsible for counting one replicate of each test. All videos obtained were copied and cut at 60, 40, and 20 s for later analysis in the counting software. The median lethal doses (LD50) of the different compounds in each test were calculated for each method (automatic and manual) for different video times. There was no statistical difference in LD50 between manual and automatic count techniques for ivermectin and fipronil. The LD50 of cypermethrin calculated with manual evaluation was up to 2.2 times lower than that of automatic evaluation. The acquisition time of the videos was 2.9-4.4 times faster than the manual evaluation. The average processing time for each video was 5.73 min, regardless of their duration. Thus, the method developed for automatic counting of tick larvae was validated, and although it still has points to be optimized, it can be considered a viable alternative for determining the percentage of tick larvae mortality and could be applied to toxicological in vitro tests with acaricides, assisting in the diagnosis of resistant tick populations and studies of novel acaricide development.

Combination of cypermethrin and thymol for control of Rhipicephalus microplus: Efficacy evaluation and description of an action mechanism.

The cattle tick Rhipicephalus microplus is one of the most important ectoparasites in the tropical and subtropical regions of the world. Synthetic pyrethroids are widely used to control this tick, and the selection of resistant populations is a huge problem worldwide. The activity of thymol, a natural monoterpene, free or in combination with other compounds, has been demonstrated against different species of ticks. However, the mode of action is not fully understood. The present study aimed to evaluate the efficacy and the potential mode of action of the combination of cypermethrin and thymol on ticks from two populations with different levels of susceptibility to cypermethrin (low and high susceptibility). The isolated acaricidal activity of cypermethrin and thymol on larvae was carried out in different concentrations. The combination with different concentrations of cypermethrin and fixed concentrations of thymol (1300 µg/mL for the low susceptibility population; 690 µg/mL for the high susceptibility population) were performed. Adult engorged females were divided into five experimental groups (n = 20): 1) Control group untreated; 2) Control group: 2.0% (v/v) DMSO; 3) Thymol group: 1300 µg/mL thymol; 4) Cypermethrin group: 3700 µg/mL cypermethrin; 5) Association of cypermethrin (3700 µg/mL) + thymol (1300 µg/mL). A subgroup was used to study the efficacy of the reproductive parameters and another subgroup, with ten adults from each treatment, was used to quantify thymol and cypermethrin by HPLC chromatographic analysis. All compounds tested were effective on larvae from both populations, and the combination with thymol decreased the LC50 of cypermethrin (232.4 to 52.7 µg/mL) on the low-susceptibility population. The combination of thymol and cypermethrin was effective in both populations of R. microplus (reproductive performance of engorged females) when compared to the untreated control group, even with higher percent control values (pop. 1: 93.5 ± 5.6% and pop. 2: 92.7 ± 1.1%) than the group treated only with cypermethrin (pop. 1: 87.3 ± 7.3% and pop. 2: 83.5 ± 1.2%). From the HPLC analyzes, a higher concentration of cypermethrin (pop. 1: 30.3 ± 6.9 and pop. 2: 45.4 ± 17.7 ng/mg) was detected in the tissues of engorged females treated with the combination compared to analyte concentrations in groups treated with cypermethrin only (pop. 1: 12.4 ± 4.4 pop. 2: 25.5 ± 9.4 ng/mg). This was the first study to investigate the acaricidal efficacy of the combination of thymol + cypermethrin on R. microplus and demonstrate that the presence of thymol increases the concentration of cypermethrin in the internal tissues of engorged females through a possible mechanism for increasing the penetration of cypermethrin at the cuticular level.