Thymol and eugenol against Rhipicephalus sanguineus sensu lato engorged females: Biological, histopathological and bioinformatic analysis.

The aim of this study was to evaluate the reproductive biology and histopathology of the ovaries of engorged females of Rhipicephalus sanguineus sensu lato exposed to combinations of thymol and eugenol, as well as to evaluate in silico the possible interactions of thymol and eugenol in tick cell membranes. To evaluate the tick reproductive biology, the adult immersion test (AIT) was performed, in which the engorged females were immersed in solutions of thymol and eugenol, combined or alone, at concentrations of 2.5 and 5.0 mg/mL. Two control groups (water and 3% DMSO) were also performed. The ticks were kept in a controlled chamber (B.O.D - 27 ± 1 °C and 80% ± 5% RH) to evaluate egg production and viability. To perform the ovaries histopathological evaluation, females were immersed in combination of thymol and eugenol (each at 2.5 and 5.0 mg/mL) and control (water and 3% DMSO) solutions. After immersion, the females were kept in B.O.D (27 ± 1 °C and RH of 80% ± 5%) for four days, they were dissected and the ovaries processed for histological analysis. In addition, an in silico analysis was performed using PASS online® software to predict probability activity (PA) of thymol and eugenol in cell membranes. The treatment with the combination of thymol and eugenol (each at 5.0 mg/mL) caused a reduction (p < 0.01) in oviposition, while the treatments with thymol (5.0 mg/mL) and combination of thymol and eugenol (5.0 mg/mL) reduced (p < 0.05) the egg viability. The treatment with combination of thymol and eugenol (5.0 mg/mL) resulted in a control percentage of 99.9%, while in the other treatments, control percentages below 56% were observed. Oocytes from the females exposed to the combinations of thymol and eugenol (each compound at 2.5 mg/mL) showed histopathological changes, except on oocyte V, while those treated with these compounds alone at 2.5 mg/mL, did not reveal any change. Changes in the shape of the oocyte, presence of vacuoles in the cytoplasm and germinal vesicle, reduction and fusion of yolk granules and rupture of some oocytes were observed. In silico analysis, showed that these compounds can act as membrane permeability inhibitors, membrane permeability agonists, membrane integrity antagonists and apoptosis agonists. We conclude that the combination of thymol and eugenol causes changes in the reproductive biology and morphophysiology of engorged females oocytes. The in silico analysis using thymol and eugenol revealed the possibility of disorganization in the cell membranes, a fact that may explain the histopathological alterations observed.

Effect of the formulation with fipronil and fluazuron on the reproductive biology and ovaries histopathology of Rhipicephalus microplus engorged females.

This study aimed to evaluate the effects of a commercial formulation containing fipronil and fluazuron on the reproductive biology and the morphology of ovaries from Rhipicephalus microplus engorged females. To carry out the study, three calves were artificially infested every 3 days with approximately 5000 larvae. On day 0, the animals were treated with a commercial formulation containing fipronil (1.25 mg/kg) + fluazuron (2.5 mg/kg). Before the application of the acaricide, engorged females of R. microplus were collected to constitute the control group (10 for biology analyses and 20 for histology analyses). After applying the commercial formulation, naturally detached engorged females were recovered on days + 5, + 10, and + 20 (10 engorged females/day) to evaluate their reproductive biology, and on days + 4, + 12, and + 20 (20 engorged females/day) for histological evaluation of the ovaries. Females from the treated groups produced smaller amounts of eggs, exhibiting lower viability when compared to eggs from the control group (p < 0.05). The ovaries of females from all treated groups (+ 4, + 12, and + 20) showed morphological changes, including: cytoplasmic disorganization, cytoplasmic degradation, irregular shape of the oocyte and germinal vesicle, reduction and vacuolization of yolk granules and oocyte disruption. Oocytes were observed in smaller numbers in all stages of development (I, II, III, IV, and V) and greater numbers of indeterminate oocytes were verified in the ovaries of the treated groups when compared to the control group. Therefore, results showed that the commercial formulation containing fipronil and fluazuron affected the reproductive biology, caused morphological changes in the ovaries, and reduced the number of oocytes in R. microplus engorged females.

Rhipicephalus microplus cystatin as a potential cross-protective tick vaccine against Rhipicephalus appendiculatus.

Rhipicephalus appendiculatus, the brown ear tick, is an important disease vector of livestock in eastern, central and southern Africa. Rhipicephalus appendiculatus acaricide resistance requires the search for alternative methods for its control. Cystatins constitute a superfamily of cysteine peptidase inhibitors vital for tick blood feeding and development. These inhibitors were proposed as antigens in anti-tick vaccines. In this work, we applied structural and biochemical approaches to characterize a new cystatin named R. appendiculatus cystatin 2a (Racys2a). Structural modeling showed that this new protein possesses characteristic type 2 cystatin motifs, besides conservation of other structural patterns along the protein. Peptidase inhibitory assays with recombinant Racys2a showed modulation of tick and host cathepsins involved in blood digestion and immune system responses, respectively. A heterologous tick challenge with R. appendiculatus in rabbits immunized with recombinant Rhipicephalus microplus cystatin 2c (rBmcys2c) was performed to determine cross-reactivity. Histological staining showed that rBmcys2c vaccination caused damage to the gut, salivary gland and ovary tissues in R. appendiculatus. Furthermore, cystatin vaccine reduced the number of fully engorged adult females in 11.5 %. Consequently, strategies to increase the protection rate are necessary, including the selection of two or more antigens to compose a vaccine cocktail.

Comparative analysis of germ cells and DNA of the genus Amblyomma: adding new data on Amblyomma maculatum and Amblyomma ovale species (Acari: Ixodidae).

Among tick species, members of the subfamily Amblyomminae have received special attention, since they serve as vectors for pathogens such as Rickettsia spp. and display cryptic species complexes that make their taxonomical classification challenging. Amblyomma ovale, Amblyomma maculatum, and other species of the genus Amblyomma have shown a long history of taxonomic controversies. Spermiotaxonomy has proved to be a valuable tool in the solution of systematic conflicts in Metazoa that can aid molecular and external morphological analyses in ticks and, overall, provide more robust analyses and results. With this in mind, this study included histological analyses of the reproductive system of the species A. ovale and A. maculatum, as well as the description of morphohistological characters of the male reproductive system of ticks of the genus Amblyomma, in order to evaluate these characters within the current clustering proposals. In addition, 16S rDNA and COI (mitochondrial) molecular markers were used to study the genetic relationships of the species. The results show that the tick male reproductive system and its germ cells contain useful candidate characters for taxonomical analyses of Ixodida.

Chemical composition and acaricidal activity of the essential oil of Baccharis dracunculifolia De Candole (1836) and its constituents nerolidol and limonene on larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae).

Baccharis dracunculifolia DC (common name "alecrim-do-campo" in Brazil) is a plant with widespread distribution in South America that is the botanical origin of green propolis. The aim of this study was to evaluate the chemical composition and acaricidal activity of the essential oil of B. dracunculifolia and its constituents nerolidol and limonene on unengorged larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). The essential oil yield was 0.8% of dry mass and the major constituents were nerolidol (22.3%), germacrene D (7.2%), limonene (6.9%), ß-pinene (6.7) and bicyclogermacrene (6.5%). The acaricidal activity of the essential oil and the pure compounds nerolidol and (R)-(+)-limonene were assessed in the laboratory through the modified larval packet test (LPT) and the female immersion test (FIT). In the LPT, the essential oil and nerolidol were both active, causing more than 90% mortality at concentrations from 15.0 and 10.0 mg mL(-1), respectively, whereas (R)-(+)-limonene was not active. In the FIT, the oil and nerolidol caused reduction in the quantity and quality of eggs produced, with control percentages of 96.3% and 90.3% at concentrations of 60.0 and 50.0 mg mL(-1), respectively. It can be concluded that the essential oil obtained from the aerial parts of B. dracunculifolia and its major component nerolidol have high activity on R. microplus larvae and engorged females.