In vivo and in vitro apoptosis induced by new acaricidal ethyl-carbamates in Rhipicephalus microplus.

The ability of ethyl-4-bromophenylcarbamate (LQM 919) and ethyl-4-chlorophenylcarbamate (LQM 996) to induce in vivo apoptosis of Rhipicephalus microplus ovarian cells and in vitro apoptosis of tick and mammalian cell culture was evaluated. The ovaries of engorged females treated with 1 mg mL-1 LQM 919 or LQM 996 presented more (p < 0.001) peroxidase-TUNEL-positive labeled cells (apoptotic cells) in situ than their respective control groups, and this increase was time-dependent (p < 0.001). The majority of apoptotic cells were observed in the epithelium and ovarian pedicel. HepG2, Vero and Rm-sus cells, as well as cells from primary cultures of R. microplus salivary glands, intestine and ovaries were exposed to different concentrations of the ethyl-carbamates. Both ethyl-carbamates induced a concentration-dependent reduction in the viability of all cell types (p < 0.001). Exposure to the ethyl-carbamates increased caspase 3 activity (p < 0.01) in primary cultures and cell lines, except in HepG2 cells. Fluorescent TUNEL-positive cells were observed in all cell types treated with 600 µM LQM 919 or LQM 996. These results indicate that both ethyl-carbamates induce apoptosis of the ovarian, intestinal and salivary glands cells in R. microplus and strongly suggest that this is their main mechanism of acaricidal action.

The action of two ethyl carbamates on acetylcholinesterase and reproductive organs of Rhipicephalus microplus.

The effects produced by the new synthetic carbamates ethyl-(4-bromophenyl) carbamate and ethyl-(4-chlorophenyl) carbamate on the acetylcholinesterase (AChE) activity, egg structure and reproductive organs of two Rhipicephalus microplus strains were evaluated. Inhibition kinetic parameters showed that the studied carbamates are weak inhibitors and have a low affinity for R. microplus AChE. Histologically, in oocytes from carbamate-treated engorged female ticks, a loss of shape, cytoplasmic vacuoles, decreased chorion deposition, alterations in cytoplasmic granularity and irregular membranes were observed. In oocyte germinal vesicles, a loss of shape, nucleolar fragmentation and membrane alterations with degenerative signs were observed. The ovarian epithelium was vacuolated, flattened, eroded and contained pyknotic nuclei. These alterations were observed from the first day and persisted and increased in severity until day 7 post-treatment. The ovaries from carbamate-treated ticks had fewer stage IV-V oocytes and more stage I-II oocytes. Additionally, eggs produced by the treated ticks had a modified appearance, decreased size, a reduced superficial waxy layer and a loss of viability. The results of this study show that the effects of carbamates on R. microplus were independent of AChE inhibition and show that the morphological alterations in the reproductive organs were due to carbamate actions on the vitellogenesis and viability of the ovarian cells.

Effect of new ethyl and methyl carbamates on Rhipicephalus microplus larvae and adult ticks resistant to conventional ixodicides.

The effects of six new synthetic carbamates on fully engorged females of four Rhipicephalus microplus strains (one reference strain susceptible to conventional ixodicides, two strains multiresistant to ixodicides and one tick field isolate) were compared. In addition, the effect of two other new synthetic carbamates was tested on larvae from the same strains. The first six tested carbamates reduced egg laying and inhibited egg hatching in the four studied strains (P<0.05). Compared with untreated females, the eggs produced by the treated engorged female ticks of all strains had a dark, dry, opaque appearance and were less adherent. The remaining two tested carbamates induced larval mortality in all of the evaluated strains. The three studied R. microplus strains displayed 50% resistance ratios (RR50) of less than 2 when compared to the susceptible reference strain. These results demonstrate that both carbamates with a larvicidal effect and carbamates that inhibit egg laying and embryo development are efficacious against tick strains that are resistant to commercial ixodicides, no cross resistance was observed.

Effect of new ethyl and methyl carbamates on biological parameters and reproduction of the cattle tick Rhipicephalus microplus.

The effect of carbamates on engorged female Rhipicephalus microplus ticks and larvae was evaluated using the adult immersion test (AIT) and the larval packet test (LPT), respectively. Seventeen synthetic carbamates different from current commercial acaricides were synthesised at the National Autonomous University of Mexico. None of the carbamates had an effect on the percentage of females laying eggs. Six of the compounds inhibited egg laying up to 65.4% and inhibited egg hatching by up to 100% (p<0.05). Compared to untreated females, eggs produced by treated females had a dark, dry, opaque appearance and were less adherent. Carbamates LQM 934 and LQM 938 had an effect on larval mortality (p<0.05). Carbamate LQM 934 showed lethal concentrations (LC) of LC90=0.76% and LC99=0.87%, while LQM 938 showed concentrations of LC90=0.267% and LC99=0.305%. The compounds were distributed into three classes of acaricidal activity using the AIT or the LPT. These three classes were as follows: (1) compounds having no apparent effect; (2) compounds that inhibit egg laying and embryo development or (3) compounds that exhibit acaricidal activity to larval ticks.