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Blacklegged tick, Ixodes scapularis Say (Arachnida: Ixodidae), is a growing health concern for humans as vectors the causative agent of Lyme disease, Borrelia burgdorferi, and many other pathogens. Given the potential health threat I. scapularis entails, and the need to find effective strategies to prevent tick bites, it is pivotal to understand the chemosensory system of ticks and their host-seeking behaviour when exposed to repellents. In this study, we investigated whether the exposure to synthetic and plant-derived repellents impairs the ability of I. scapularis to detect attractants and host volatiles (butyric acid), and ultimately how these repellents interfere with host-seeking behaviour in both wild and lab-reared ticks. Furthermore, we screened wild ticks used in electrophysiology and Y-tube behavioural assays for presence of pathogens ( Borrelia, Anaplasma, and Babesia ) to evaluate if the bacterial infection status would affect the detection of butyric acid under the exposure to repellents. We determined that the exposure to DEET, lemongrass essential oil, citral, and geraniol significantly inhibited the ability of both lab-reared and wild adult female I. scapularis to detect and respond to butyric acid. We found that tick infection status does not significantly impact host-seeking behaviour in adult female I. scapularis . The knowledge gained from our study contributes to advance our understanding of host-seeking behaviour in ticks and the impact that the exposure to repellent has on the tick chemosensory system. These findings will be important for elucidating the mechanism of repellence in ticks and for the development of effective tick repellent management tools., Competing Interests: The authors have no competing interests to declare that are relevant to this article., (© 2024 The Author(s).)

The ANESPSAT, a synthetic spilanthol derivative, and its nanoformulation were evaluated against Rhipicephalus microplus and Amblyomma sculptum ticks. ANESPSAT activity was compared with spilanthol and derivatives (ANESPE and others). The compound was synthesized in a gram-scale by a 2-step process, comprising a direct ester amidation and a Horner-Wadsworth- Emmons reaction. The nanoemulsions were produced by coarse homogenization followed by high-energy ultrasonication, in which hydrodynamic diameter, polydispersity index, and zeta potential remained stable. The spilanthol-eugenol hybrid derivatives did not show significant acaricidal activity. ANESPE killed 83% of the R. microplus larvae at 30 mg.mL -1 , while ANESPSAT killed 97% at 0.5 mg.mL -1 , showing to be the most active compound. Spilanthol and ANESPSAT had similar high mortality rates for tick larvae, with LC 50 values of 0.10 and 0.14 mg.mL -1 for R. microplus larvae, and 0.04 and 0.48 mg.mL -1 for A. sculptum larvae, respectively. The efficacy of spilanthol was lower against R. microplus engorged females when compared with ANESPSAT, which was highly effective (>98%) against R. microplus engorged females. The nanoemulsion with ANESPSAT was effective against tick females, preventing egg laying and achieving 100% efficacy at 2.5 mg.mL -1 . Spilanthol had only 59% efficacy at 10 mg.mL -1 . The results suggest that ANESPSAT, a natural product derivative, could be used in novel formulations for tick management that might be safer and environmentally friendly., Competing Interests: Declaration of Competing Interest The authors declare they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ixodes scapularis ticks are vectors of infectious agents that cause illness in humans, including Lyme disease. Recent years have seen a surge in tick-borne diseases (TBD) resulting in a high demand for tick management products. Plants offer a valuable source of active compounds for the development of novel, eco-friendly tick control products, reducing potential risks to human and animal health. Essential oils (EOs) have emerged as potential acaricides and repellents against ticks providing an alternative to synthetic chemicals and aiding in the prevention of TBD by lowering the risk of tick bites. We investigated the acaricidal activity of EOs from lemongrass (Cymbopogon citratus), geranium (Pelargonium x asperum), savory thyme (Thymus saturejoides), and white thyme (Thymus zygis) on I. scapularis. The interactions (i.e., synergistic, antagonistic, or additive) of their binary mixtures were also evaluated. EO samples were analyzed via gas chromatography-mass spectrometry to determine their chemical composition. The adult immersion test was used to determine the lethal concentration (LC 50 ) of each EO alone and in mixtures. Quantitative assessment of synergistic, additive, or antagonistic effect of the binary mixtures was performed by calculating the combination index. Strong acaricidal activity was recorded for savory thyme and white thyme EOs, with LC 50 values of 28.0 and 11.0 μg/μL, respectively. The LC 50 of lemongrass and geranium EOs were 49.0 and 39.7 μg/μL, respectively. Among the tested EOs, savory thyme and white thyme had a strong acaricidal effect on I. scapularis, which might be linked to the presence of carvacrol (26.05 % ± 0.38) and thymol (53.6 % ± 2.31), main components present in savory thyme and white thyme EOs, respectively. The tick killing efficacy of lemongrass and geranium EOs was lower when mixed than when used separately (LC 50 of 65.3 µg/µL). The same happened with savory thyme and white thyme EOs, except at 9.75 µg/µL where they had a synergistic effect (LC 50 of 58.3 µg/µL). Lemongrass and savory thyme EOs had a synergistic effect at low concentrations, and an antagonistic effect at higher concentrations (LC 50 of 95.4 µg/µL). Lemongrass and white thyme EOs had a synergistic effect against ticks from 15 to 120 µg/µL (LC 50 of 18.5 µg/µL) similar to white thyme EO. Geranium and savory thyme EOs had an antagonistic effect at all concentrations, with an LC 50 of 66.8 µg/µL. Geranium and white thyme EOs also had an antagonistic effect, except at 12.7 µg/µL where they had a synergistic effect (LC 50 of 66.8 µg/µL). The interaction observed when combining selected essential oils suggests promising potential for developing acaricidal formulations aimed at controlling ticks and curbing the transmission of tick-borne disease agents., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Rhipicephalus sanguineus sensu lato 'tropical lineage' (Acari: Ixodidae) is considered a sanitary concern due to its role as a disease vector. Tick strains resistant to synthetic acaricides have caused difficulties in their control, besides synthetic acaricides are harmful to the environment and to the health of non-target animals. The research of plants with acaricidal and repellent properties has proved to be an efficient alternative in tick control. The genus Tagetes spp. excels for its use as traditional pest control in households and plantations and also for its potential as an acaricide against R. sanguineus under laboratory conditions. The first aim of the present study was to evaluate the effect of different doses of Tagetes minuta essential oil (TMEO) on the central nervous system (synganglion) in unfed R. sanguineus adults. The histological analysis of synganglion exposed to the different concentrations of TMEO and amitraz 12.5% (50% of the recommended dose in the package insert) showed a significant effect with signs of cell damage including volume increase, loss of shape, and vacuolization, in addition to chromatin alterations such as condensation, margination, and fragmentation. TMEO were analyzed by gas chromatography coupled with mass spectrometry showing the presence of 21 compounds that according to their chemical structure are classified as terpenoids. Among them (Z)-β-ocimene, ocimene, (Z)-tagetone, and verbenone were found in major quantities., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nanoformulations containing zein nanoparticles (ZN) can promote the stability and protection of molecules with acaricidal activity. The present study sought to develop nanoformulations with ZN associated with cypermethrin (CYPE) + chlorpyrifos (CHLO) + a plant compound (citral, menthol or limonene), characterize them, and verify their efficacy against Rhipicephalus microplus ticks. Additionally, we aimed to assess its safety in nontarget nematodes found in soil at a site subjected to contamination by acaricides. The nanoformulations were characterized by dynamic light scattering and nanoparticle tracking analysis. Nanoformulations 1 (ZN+CYPE+CHLO+citral), 2 (ZN+CYPE+CHLO+menthol), and 3 (ZN+CYPE+CHLO+limonene) were measured for diameter, polydispersion, zeta potential, concentration, and encapsulation efficiency. Nanoformulations 1, 2, and 3 were evaluated in a range from 0.004 to 0.466 mg/mL on R. microplus larvae and caused mortality > 80% at concentrations above 0.029 mg/mL. The commercial acaricide Colosso® (CYPE 15 g + CHLO 25 g + citronellal 1 g) was evaluated also from 0.004 to 0.512 mg/mL and resulted in 71.9% larval mortality at 0.064 mg/mL. Formulations 1, 2, and 3 at 0.466 mg/mL showed acaricidal efficacy of 50.2%, 40.5%, and 60.1% on engorged females, respectively, while Colosso® at 0.512 mg/mL obtained only 39.4%. The nanoformulations exhibited long residual period of activity and lower toxicity to nontarget nematodes. ZN was able to protect the active compounds against degradation during the storage period. Thus, ZN can be an alternative for the development of new acaricidal formulations using lower concentrations of active compounds., Competing Interests: Declaration of Competing Interest The authors warrant that there are no any conflicts of interests among authors and between authors and other people, institutions or organizations., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Haemonchus contortus is the most prevalent and important gastrointestinal nematode (GIN) in small ruminants. Since it reduces the packed cell volume (PCV), causing anemia, early diagnosis can be used for targeted selective treatment (TST) of sheep, reducing antiparasitic drug use and anthelmintic resistance. This study aimed to predict PCV values through near-infrared spectroscopy (NIRS) and to develop a classification and diagnosis model of H. contortus infection using PCV values, eggs per gram of feces (EPG) counts and mean daily weight gain (DWG). A total of 1728 spectra were collected from blood samples of 216 lambs with a portable NIR spectroscope. In parallel, other parameters indicative of infection were measured: PCV by hematocrit, FAffa MAlan CHArt (FAMACHA) scores, EPG and DWG. To evaluate the relationship between NIRS spectra and the evaluated parameters, principal component analysis (PCA) was used for an exploratory analysis, regression by the partial least squares method (PLS) for the prediction of PCV values via NIRS, and PCA linear discriminant analysis (PCA-LDA) as a classification model for diagnosis. The absorption peaks in the NIRS region associated with the excitation of overtones of nitrogen-hydrogen (N-H) functional groups of proteins had a strong impact on the principal components (PCs), indicating that blood proteins, especially hemoglobin, can be estimated by the NIRS technique. The model for predicting PCV by PLS presented a standard error of prediction of 2.53, root-mean-square error of 2.48, and coefficient of determination of 0.84, indicating good correlation between the PCV values predicted by the model and the PCV obtained by hematocrit. The PCA-LDA model presented 93.33% sensitivity and 82.18% accuracy, both higher than those of the FAMACHA method, as was expected for resilient Morada Nova lambs. The multivariate models associated with the NIRS technique reported here can be used in the future as a quick and versatile tool for H. contortus infection diagnosis and TST application in lambs., (Copyright © 2023 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The study analyzed the chemical composition and the acaricide effect of Egletes viscosa Less (macela-da-terra) and Lippia schaueriana Mart. (lipia-da-serra) essential oils (EOs) on Rhipicephalus sanguineus s. l. (Acari: Ixodidae) engorged females. The chemical analysis (GC-MS and GC-FID) identified 27 components in E. viscosa EO and 18 in L. schaueriana EO, which comprise more than 98% of its constituents. The effects of the oils on the reproductive biology of R. sanguineus ticks were assessed by adult immersion test. Both EOs significantly reduced (p < 0.05) the egg production index when the females were exposed to 25 and 50 mg/mL, also affecting the egg viability. During the laying process, the eggs produced by the females exposed to the EO showed several morphological alterations such as dehydrated, darkened, and disaggregated, and these alterations were more severe as the concentrations increased. The mortality percentages were 58.9%, 70.8% and 92.7% when the ticks were exposed to 12.5, 25 and 50 mg/mL of E. viscosa oil, respectively. In the same concentrations, the efficacy of L. schaueriana was 39.3%, 53.4%, and 84.6%. Therefore, it can be concluded that the essential oils of E. viscosa and L. schaueriana have acaricidal effect in females of R. sanguineus s.l ticks., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest with respect to the publication of the present study., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nanocarriers of acaricidal compounds improve the bioavailability, absorption, and tissue distribution of active ingredients, releasing them in a slow, targeted way and protecting them against premature degradation. Thus, this study aimed to develop formulations from solid lipid nanoparticles (SLN), or nanostructured lipid carriers (NLC) associated with cypermethrin (cip) + chlorpyrifos (chlo) and vegetable compounds (citral, menthol, or limonene). Particles were then characterised, and their efficacy was verified on R. microplus in comparison to nanoformulations without the plant-based compounds. Six different formulations were developed and characterised by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Formulations 1 (SLN+cyp+chlo+citral), 2 (SLN+cyp+chlo+menthol), 3 (SLN+cyp+chlo+limonene), 4 (NLC+cyp+chlo+citral), 5 (NLC+cyp+chlo+menthol) and 6 (NLC+cyp+chlo+limonene) had mean diameters from 286 to 304 nm; polydispersion from 0.16 to 0.18; zeta potential from -15.8 to -20 mV, concentration from 3.37 ± 0.24 × 10 13 to 5.44 ± 0.18 × 10 13 particles/mL and encapsulation efficiency (EE) > 98.01 % for all active ingredients. All formulations were evaluated for their acaricidal potential by the larval packet test (LPT) and compared with nanoformulations without the plant-based compounds. Formulations were also compared with positive (Colosso® at 512 µg/mL) and negative controls (distilled water and nanoparticles without active ingredients). The SLN (1, 2 and 3) and NLC (4, 5 and 6) formulations, at 7 µg/mL, resulted in 90.4 % , 75.9 % , 93.8 % , 100 % , 95.1 % and 72.7 % mortality. The data demonstrated that the addition of citral, menthol or limonene in the formulations improved their acaricide action against tick larvae. Except for formulation 4, for which it was not possible to determine lethal concentrations (LC). Formulations, 1, 2, 3, 5 and 6 reached LC 50 and LC 90 values of 3.3 and 7.2, 5.4 and 9.2, 4.0 and 8.1, 2.3 and 5.4 as well as 5.5 and 9.4 µg/mL, respectively. It was possible to encapsulate the active ingredients and characterise the lipid carrier systems. SLN and NLC protected the active ingredients against degradation in solution and increased the overall stability. A stabile solution is necessary for synthesizing commercial acaricidal products. It is hoped that these findings may contribute to new studies focused on the use of nanocarriers in tick formulations. By reducing the amount or concentration of active ingredients within commercial products, the risk of residues presents in food of animal origin or remaining in the environment is reduced. Nanocarriers help prevent these challenges, while still maintaining effective parasitic control. Utilizing a combination of natural and synthetic products can be part of integrated management solutions and can help overcome widespread acaricide resistance in populations of cattle ticks., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate a method of targeted selective treatment (TST) of Morada Nova lambs, based on the average daily weight gain (DWG). For this, 114 lambs in the rainy and 102 in the dry season were randomized into three treatments: control (CT), routine (RT, treated every 42 days), and targeted selective (TST, treated according to DWG). Packed cell volume (PCV) and eggs per gram of feces (EPG) tests were performed. The anthelmintic resistance of parasites was assessed by the EPG count reduction test (FECRT), RESISTA-Test © , and molecular test. For CT, RT, and TST groups, the following results were obtained: mean EPG values were 4665.1, 3063.5, and 3462.1 in the rainy season and 4475.1, 1341.7, and 2863.4 in the dry season, respectively; mean PCV values were 32.1, 33.4, and 32.3% in the rainy season and 33.9, 36.0, and 35.1% in the dry season; mean DWG readings were 0.087, 0.101, and 0.094 kg in the rainy season and 0.102, 0.113, and 0.112 kg in the dry season; efficacies of levamisole in FECRT were 66.4, 24.1, and 76.4% in the rainy and 90.7, 12.4, and 64.8% in the dry season, respectively; in the RESISTA-Test © , the LC 50 values were 0.482, 1.926, and 0.117 µg.mL -1 in the rainy and 0.437, 0.851, and 0.045 µg.mL -1 in the dry season, respectively; the frequencies of the homozygous-resistant genotype were 57.1, 71.4, and 40.0% in the rainy and 47.8, 55.9, and 41.9% in the dry season. In conclusion, TST reduced the development of resistance through refugia maintenance, without productive losses, contributing to the sustainability of sheep breeding., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14 th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick., (Copyright © 2022. Published by Elsevier GmbH.)

Studies seeking control methods for infestation of Riphicephalus sanguineus sensu lato (s.l.) ticks (dog ticks) have been carried out in order to minimize damage to both the tick's hosts and the environment, the latter due to the misuse of acaricide products. In this regard, carvacrol has been used as a natural alternative against ticks as it displays several properties including acaricidal. In this context, the present study aimed to evaluate the ultramorphology and morphohistochemistry of the integument of semi-engorged R. sanguineus s.l. females exposed to different carvacrol concentrations. The findings indicate that the integument surface of females exposed to the highest carvacrol concentration (25 μl/ml) became wrinkled, suggesting dehydration or the result of integument cuticular and epithelial layer disorganization in response to the toxic product. Morphohistochemical integument layer alterations were more significant and intense in females exposed to the highest carvacrol concentration (25 μl/ml), confirming dose-dependent carvacrol action. Among other cell and tissue alterations, changes in epithelial cell shape, size, and arrangement (epidermal layer) were noted, alongside altered and pyknotic-shaped nuclei, suggesting a death process for these cells. This epithelium changed from simple cubic to stratified, also in response to the presence of the evaluated bioactive compound. Thus, the findings reported herein demonstrate that carvacrol may be an alternative for an efficient and more sustainable tick control in the near future., (© 2021 Wiley Periodicals LLC.)

Tick cell lines have already proved to be a useful tool for obtaining more information about possible vector species and the factors governing their ability to transmit a pathogen. Here, we established and characterized a cell line (RBME-6) derived from embryos of Rhipicephalus microplus from Brazil. Primary tick cell cultures were prepared in L-15B medium supplemented with 20% fetal bovine serum and 10% tryptose phosphate broth. The cell monolayers were subcultured when they reached a density of approximately 8 × 10 5 cells/mL (95% viability). Only after the sixth subculture were cells thawed from storage in liquid nitrogen successfully. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect total polysaccharides and total protein, respectively . No DNA from Anaplasma spp., Anaplasma marginale, Babesia spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Rickettsia spp. or Mycoplasma spp. was detected in the cells through PCR assays. In addition, we performed chromosomal characterization of the tick cell line and confirmed the R. microplus origin of the cell line through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In conclusion, we established and characterized a new cell line from a Brazilian population of R. microplus, which may form a useful tool for studying several aspects of ticks and tick-borne pathogens., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rhipicephalus sanguineus sensu lato (s. l.) ticks are targets of acaricidal treatments due to their great veterinary and medical importance. Acetylcarvacrol stands out as a promising acaricidal substance for its increased biocidal activity and stability. Additionally, its toxicity to mammals is reduced when compared to the parent molecule, carvacrol. The present study aimed to evaluate the effects of acetylcarvacrol on the morphology of ovaries of engorged R. sanguineus s. l. ticks. The animals were subjected to the Adult Immersion Test to calculate the lethal concentrations (LC 50 and LC 90 ) of acetylcarvacrol. Subsequently, the surviving ticks were dissected for collection of the ovaries. The samples were processed through routine histological techniques. The histological sections were stained with hematoxylin and eosin, bromophenol blue (protein detection) and PAS (polysaccharide detection). The alterations found in the ovaries were evaluated by morphometric and semiquantitative analysis. The LC 50 and LC 90 were 17.805 and 26.164 μL/mL, respectively. The most severe morphological alterations were disappearance of the female germ cells (oocytes), nucleolus vacuolization, thicker and irregular chorion, and decrease in size of the oocytes. Also, the content of proteins and carbohydrates in the oocytes were heavily affected by the chemical, as evidenced by a non-homogeneous staining pattern. The group exposed to the highest concentration of acetylcarvacrol (20 μL/mL) exhibited a statistically greater score in the semiquantitative analysis when compared to the other groups. The morphological changes in the ovaries may reduce the ticks' offspring production or generate descendants that will struggle to carry out essential biochemical processes during their lives. Thus, acetylcarvacrol may be a promising alternative to control tick infestation by impairing the reproduction of this parasite., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study investigated the effects of different concentrations of Acmella oleracea extract on the germinative cells and digestive processes of semi-engorged Rhipicephalus sanguineus females. For this experiment, 150 ticks were divided into five groups (30 individuals each). The animals were immersed for 5 min in different concentrations of the extract, distilled water, or ethanol 50%/DMSO 1%, dried, and kept in biological oxygen demand incubator for 7 days. The alterations were associated with the size of germinative cells and yolk granules; presence, size, and location of vacuoles in the cytoplasm of germinative cells; nuclear modifications in the germinative cells; damages to the nucleus and cytoplasm of the midgut generative cells; size of digestive cells; number of captured blood elements; accumulated digestive residues and digestive vacuoles in the digestive cells cytoplasm; and the number and distribution of proteins and polysaccharides in all the cells of both organs. The concentrations used in this study prevented an efficient and complete blood digestion by the midgut epithelial cells of the treated animals, resulting in the absence of the necessary nutrients to maintain the physiological events in the ectoparasites. In advanced stages, This can lead the ectoparasite to death. The germinative cells were highly impaired and probably not able to advance developmental stages (I-V) or complete vitellogenesis to be released during ovulation, which would prevent the females from originating a new individual. Thus, it can be concluded that the effects of A. oleracea are similar to those caused by chemical products widely recognized as effective to control ticks., Competing Interests: There are no conflicts of interest.

Currently, the most commonly used method to control ticks is the use of synthetic acaricides. However, these compounds are potentially harmful to hosts and the environment, in addition to causing the selection of resistant individuals. Therefore, several studies have been conducted to find sustainable methods to control ticks, such as Rhipicephalus sanguineus sensu lato, the most important vectors of pathogens for dogs. There has been increasing research on natural products with acaricidal action, especially with regard to plant-derived compounds as carvacrol, an aromatic monoterpene with several biological properties, including repellent and acaricidal activities, besides exerting cytotoxic effects on the exposed ticks. The objective of the present study was to evaluate the action of different carvacrol concentrations on the ovaries morphophysiology in semi-engorged R. sanguineus s.l. females to determine its effect on oocyte development. The results showed the occurrence of significant morphological alterations in the shape of oocytes (from round-shaped to irregular) and in the germinal vesicles, in addition to extensive cytoplasmic vacuolation.These effects were observed after the application of carvacrol at a concentration of 20 μL/mL. The most significant alterations were observed at the highest concentration (100 μL/mL), at which the oocytes could not develop further than stage II (total absence of oocytes III, IV and V). These data showed that even though carvacrol was unable to kill all ticks at these concentrations, surviving females could have had an altered reproduction, which would hinder the generation of new individuals, resulting in a long-term control. Data regarding the inhibition of oocyte development are unprecedented and indicate the use of carvacrol as a natural product with the potential to control R. sanguineus s.l. ticks., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The present study analyzed the effects of different concentrations of Acmella oleracea crude ethanolic extract (EEAO) on the development of germ cells from semi-engorged Amblyomma cajennense females in order to evaluate the potential of this natural chemical as a strategy to control these important ectoparasites. A hundred semi-engorged females were divided into five groups (duplicates) (10 animals/group): Control 1 (distilled water); Control 2 (solvent ethanol 50% and DMSO 1%); and Treatment I to III (3.1, 6.2, and 12.5 mg/mL of EEAO, respectively). For the exposure of the ticks to the extract was used the Adult Immersion Test. After the exposition, the ovaries were removed and submitted to histological analysis using Harris hematoxylin and aqueous eosin. The histochemical tests were performed using PAS and Bromophenol blue staining techniques, for the detection of total polysaccharides and total protein, respectively. The extract caused significant alterations in the oocytes, including changes in the shape of the cells, disorganization, and cytoplasmic vacuolation, decrease in the number of yolk granules and germ vesicle fragmentation. These alterations were more intense in the oocytes in initial developmental stages (I and II). The results obtained in this study confirm the cytotoxic potential of the ethanolic extract of A. oleracea on the germ cells of A. cajennense females, opening up the possibility to use this extract as an alternative to control these ectoparasites., (© 2018 Wiley Periodicals, Inc.)

The Rhipicephalus sanguineus is considered a species of medical and veterinary importance. The feeding process of these animals occurs due to the combined action of their mouthparts and the saliva produced by the salivary glands, vital organs for the biological success of the ticks. In addition, these glands act as storage sites for the pathogens transmitted to the host through the inoculation of the saliva. In this sense, the present study had the objective to analyze the behavior of male Wistar rat hepatic cells submitted to in vivo application of the salivary gland extract (SGE) obtained from R. sanguineus female ticks. The study involved five groups (four male adults each): CG (non-inoculated individuals); PBS1 (one phosphate buffer saline injection); PBS2 (two PBS injections); SGE1 (one injection of SGE at 0.04 μg/μL) and SGE2 (two injections of SGE at 0.04 μg/μL). After the exposures, the livers were removed and submitted to the following histological and histochemical stains: HE, toluidine blue, Xylidine Ponceau, alcian blue/PAS, and osmium-imidazole. The results showed that both the PBS and the SGE caused hepatic moderate alterations, such as: (a) emergence of lipid plaques among the hepatic cords; (b) cytoplasmic vacuolation of the hepatic cells; (c) hepatocytes showing pyknotic nuclei; (d) presence of homogeneous or granular secretion in the cytoplasm of the hepatocytes. Despite the slight morphological alterations observed in the hepatic cells and tissue, the latter did not show signs of disorganization after the exposure to the extracts., (© 2018 Wiley Periodicals, Inc.)

The present study evaluated through morphohistological and histochemical techniques the effects of different concentrations of crude ethanolic extract of A. oleracea (EEAO) (Jambu) on the male reproductive system of Amblyomma cajennense sensu stricto (s.s.) ticks. The toxicity of this natural chemical was stablished, signalizing the promising potential of the compound as a strategy to control ectoparasites in the near future. For the experiment, 100 males fed on host rabbits with homogeneous weight (p>0.05) were used. The ticks were divided into five groups (10 animals each): Control 1-exposed to distilled water; Control 2-exposed to ethanol 50% and DMSO 1%; Treatment 1-3-exposed to the concentrations of 6.2, 12.5 and 25mg/mL of the EEAO, respectively, diluted in ethanol 50% and DMSO 1%, with exposure by immersion. After exposure, the males were dissected for the removal of the reproductive system and subjected to routine histological analysis with HE staining and histochemical techniques (PAS for the detection of neutral polysaccharides and Bromophenol blue to detect total proteins). The exposed individuals showed alterations in the glandular complex cells; however, the testes remained intact. The secretory cells of the multilobulated accessory glands presented intense cytoplasmic vacuolation. Additionally, the synthesis and secretion were reduced in the secretion granules, mainly concerning the polysaccharides, glyco- and lipoprotein elements, substances that will constitute the seminal fluid and enable the capacitation of spermatozoa in the female genital tract and also necessary for the formation of the spermatophore, which will encapsulate the mature spermatids. The alterations were dose-dependent, i.e., more intense and severe as the concentration of the product increased. .This experiment confirmed the cytotoxic potential of A. oleracea ethanolic extract in the concentrations of 6.2, 12.5 and 25mg/mL on the reproductive system of A. cajennense s.s. male ticks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study demonstrated the effects of dinotefuran (active ingredient of the acaricide Protetor Pet ® ) on the ovary and midgut cells of semi engorged R. sanguineus females exposed to different concentrations of this chemical. For this, 120 semi-engorged females were divided into four treatment groups with 30 individuals each: group I or control (distilled water), group II (5000ppm), groups III (6250ppm) and group IV (8334ppm of dinotefuran). All the ticks were immersed in the different concentrations of dinotefuran or in distilled water for 5min and then dried and kept in BOD incubator for 7days. The results showed alterations mainly regarding the damaged cell structures, such as yolk granules, organelles and the plasma membrane of the germ cells. In addition, structures related with defense mechanisms were found, such as vacuoles, cytoskeletal filaments, and myelin figures in the germ cells. Damages in the generative cells of the midgut, alterations in the size of digestive cells, the number of endosomes, digestive vacuoles, digestive residues, lipid drops and organelles in the cytoplasm of the digestive cells and the presence of microvilli in the plasma membrane of these cells also demonstrate the progressive damages caused by the action of dinotefuran in the midgut and germ cells of R. sanguineus semi-engorged females. The concentrations applied partially impaired the digestive processes; and, without proper nutrition, all the ectoparasite's physiologic events are prevented from occurring, leading the individual to death. The germ cells were also damaged, and probably would not be able to advance in their development (I-V) and complete the vitellogenesis, which would affect the fertility of the female and consequently impede the formation of a new individual., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The use of synthetic acaricides is currently the main method to control ticks. However, the indiscriminate use of these chemicals can lead to the selection of resistant individuals and in the accumulation of chemical residues in the environment, contaminating the soil and water streams, consequently affecting the flora, fauna, and the human beings as well. In this sense, the objective of this study was to investigate the acaricidal effect of crude ethanolic extract of Acmella oleracea (L.) R.K. Jansen aerials parts at different concentrations on fed males and semi-engorged females of A. cajennense s.s. An in vitro bioassay (Adult Immersion Test) was carried out to determine the lethal concentration 50 (LC 50 ) of ethanolic extract, calculated by Probit analysis. The results showed that the fed males were sensitive to all the concentrations of A. oleracea ethanolic extract, and mortality rate progressively increased (15-65%) in higher ethanolic extract concentrations. However, semi-engorged females were not sensitive to all the concentrations used here. In the highest concentration (100mg/mL), a mortality rate of 100% was observed after 72h of exposure, indicating that the acaricidal effect would probably be dose-dependent. The LC 50 values obtained for the fed A. cajennense s.s males and semi-engorged females were 29.4534mg/mL (limits: 24.4467-41.3847mg/mL) and LC 50 =17.6335mg/mL (limits: 5.2506-23.5335mg/mL), respectively., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The present study analyzed the effects of different concentrations of the hexane extract of A. oleraceae (HEAO) (Jambú) on the germ cells of semi-engorged Rhipicephalus microplus female ticks, through a morpho-histological study, evaluating the effectiveness of the extract in the genesis of the individuals. To perform this analysis, 100 semi-engorged females were divided into five groups with 20 individuals each: groups I and II, respectively constituted by distilled water control and 50% ethanol + 1% DMSO, and groups III, IV, and V constituted by treatment with HEAO in the concentrations of 12.5, 25.0 and 50.0 mg/mL, respectively. All the ticks were immersed in the different concentrations of the extract or in distilled water for 5 minutes, dried and conditioned in BOD incubator for 7 days. The individuals of the treatment groups revealed the action of this extract showing alterations in the germ cells of the females from the different groups when compared with those from the groups I and II (control groups). These alterations were mainly related to the size and shape of the oocytes; number of yolk granules; presence, number, size and location of vacuoles in the cytoplasm of all the germ cells; and the presence of nuclear alterations in these cells as well. Thus, it was demonstrated that the concentrations of HEAO affected the germ cells of R. microplus ticks. The effects of the extract are similar to those caused by renowned and efficient chemical products used to control these ticks. Microsc. Res. Tech. 79:744-753, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Neem (Azadirachta indica) has attracted the attention of researchers worldwide due to its repellent properties and recognized effects on the morphology and physiology of arthropods, including ticks. Therefore, this study aimed to demonstrate the effects of neem seed oil enriched with azadirachtin on salivary glands of Rhipicephalus sanguineus ticks, targets of great veterinary interest because of their ability to transmit pathogens to dogs. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil, with known azadirachtin concentrations (200, 400 and 600ppm). After dissection, salivary glands were collected and evaluated through morphological techniques in light microscopy, confocal scanning laser microscopy and transmission electron microscopy, so that the possible relation between neem action and further impairment in these ectoparasites feed performance could be established. Neem oil demonstrated a clear dose-dependent effect in the analyzed samples. The agranular (type I) and granular acini (types II and III) showed, particularly in individuals treated with the highest concentrations of the product, cells with irregular shape, intense cytoplasmic disorganization and vacuolation, dilation of rough endoplasmic reticulum lumen, besides alterations in mitochondrial intermembrane space. These morphological damages may indicate modifications in salivary glands physiology, demonstrating the harmful effects of compounds present in neem oil on ticks. These results reinforce the potential of neem as an alternative method for controlling R. sanguineus ticks, instead of synthetic acaricides., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The present study demonstrated the effects of dinotefuran (active compound of the Protetor Pet® acaricide) in germ cells and the digestive processes of semi-engorged females of R. sanguineus exposed to different concentrations of the chemical. For this purpose, 120 semi-engorged females were divided into four treatment groups with 30 individuals each: group I or control (distilled water), group II (5000 ppm), group III (6250 ppm), and group IV (8334 ppm of dinotefuran). All ticks were immersed in different concentrations of dinotefuran or in distilled water for 5 min and then were dried and stored in biological oxygen demand (BOD) incubator for 7 days. The results show the action of this compound, exhibiting morphohistologic and histochemical changes in the oocytes and the midgut cells of individuals of different groups, which were compared with those of group I (control). The alterations occurred mainly in relation to the size of the germ cells and yolk granules; presence, quantity, size, and location of vacuoles found in the cytoplasm of these germ cells; the damage occurred in the generative cells of the midgut; the size of the digestive cells; the quantity of blood elements captured, accumulated digestive wastes and digestive vacuoles found in the cytoplasm of the digestive cells of the midgut, as well as the amount and distribution of proteins, polysaccharides, lipids of all cells in both organs. So, it has demonstrated the effectiveness of dinotefuran in the reduction of fertility and digestive processes of semi-engorged females of R. sanguineus, data that points the possibility of employing this chemical to control these ectoparasites.

The Brazilian forests have one of the world's biggest biodiversities. Achyrocline satureioides (macela) and Acmella oleracea (jambu) are native species from Brazil with a huge therapeutic potential, with proved anti-inflammatory and anesthetic action, respectively. The jambu's crude extract after depigmentation with activated charcoal and macela's essential oil were incorporated in a film made with hydroxyethyl cellulose. Those films were evaluated by mechanical test using a texturometer and anti-inflammatory and anesthetic activities by in vivo tests: wound healing and antinociceptive. The film containing the highest concentration of depigmented jambu's extract and macela's essential oil obtained an anesthesia time of 83.6 (±28.5) min longer when compared with the positive control EMLA®; the same occurred with the wound healing test; the film containing the highest concentration had a higher wound contraction (62.0% ± 12.1) compared to the positive control allantoin and the histopathological analysis demonstrated that it increases collagen synthesis and epidermal thickening. The results demonstrate that the films have a potential use in skin wounds, pressure sore, and infected surgical wounds treatment.

The concern about the harmful effects caused by synthetic pesticides has led to the search for safe and ecological alternatives for pest control. In this context, the neem tree (Azadirachta indica) stands out due to its repellent properties and effects on various arthropods, including ticks. For this reason, this study aimed to demonstrate the potential of neem as a control method for Rhipicephalus sanguineus ticks, important vectors of diseases in the veterinary point of view. For this, R. sanguineus semi-engorged females were subjected to treatment with neem seed oil enriched with azadirachtin, its main compound, and ovaries were assessed by means of morphological techniques in conventional light microscopy, confocal laser scanning microscopy, and transmission electron microscopy. Neem demonstrated a clear dose-dependent effect in the analyzed samples. The observed oocytes presented, especially in the groups treated with higher concentrations of neem oil, obvious signs of cytoplasmic disorganization, cellular vacuolization, nuclear and nucleolar irregularity, dilation in mitochondrial cristae, alterations in mitochondrial matrix, and swelling of rough endoplasmic reticulum. Intracellular microorganisms were observed in all analyzed groups, reinforcing the importance of ticks in the transmission of pathogens. A greater quantity of microorganisms was noted as the concentration of neem increased, indicating that the damaged oocytes may be more susceptible for their development. Such morphological alterations may promote future damages in reproductive performance of these animals and demonstrate the potential of neem seed oil for the control of R. sanguineus ticks, paving the way for new, cheaper, and safer methods of control.

Currently, the necessity of controlling infestation by ticks, especially by Rhipicephalus sanguineus, has led researchers and public health managers around the world to search for new and more efficient control methods. This way, we can highlight neem (Azadirachta indica A. Juss) leaf, bark, and seed extracts, which have been very effective on tick control, and moreover causing less damage to the environment and to the host. This study showed the potential of neem as a control method for R. sanguineus through morphological and morphometric evaluation of the integument and synganglion of females, in semiengorged stage. To attain this, routine techniques of optical microscopy, scanning electron microscopy and morphometry of the cuticle and subcuticle of the integument were applied. Expressive morphological alterations were observed in both organs, presenting a dose-dependent effect. Integument epithelial cells and nerve cells of the synganglion showed signs of cell vacuolation, dilated intercellular boundaries, and cellular disorganization, alterations not previously reported in studies with neem. In addition, variations in subcuticle thickness were also observed. In general, the effects of neem are multiple, and affect the morphology and physiology of target animals in various ways. The results presented in this work are the first evidence of its effects in the coating and nervous system of ticks, thus allowing an indication of neem aqueous extracts as a potential control method of the brown dog tick and opening new perspectives on acaricide use., (© 2014 Wiley Periodicals, Inc.)

The intestinal epithelial cells of ticks are fundamental for their full feeding and reproductive success, besides being considered important sites for the development of pathogens. Rhipicephalus sanguineus ticks are known for their great medical and veterinary importance, and for this reason, the knowledge of their intestinal morphology may provide relevant subsidies for the control of these animals, either by direct acaricidal action over these cells or by the production of vaccines. Therefore, this study aimed to describe the midgut morphology of male and female R. sanguineus ticks in different feeding stages, by means of histological analysis. Significant differences were observed between the genders, and such alterations may refer mainly to the distinct demands for nutrients, much higher in females, which need to develop and carry out the egg-laying process. In general, the midgut is coated by a thin muscle layer and presents a pseudostratified epithelium, in which two basic types of cells can be observed, connected to a basal membrane-generative or stem and digestive cells. The latter was classified as follows: residual, deriving from the phase anterior to ecdysis; pinocytic, with vesicles containing liquid or pre-digested components of blood; phagocytic, with entire cells or remnants of nuclear material inside cytoplasmic vesicles; and mature, free in the lumen. Digestion is presumably intracellular and asynchronous and corresponds to a process which starts with the differentiation of generative cells into pinocytic digestive cells, which subsequently start to phagocytize intact blood cells and finally detach from the epithelium, being eliminated with feces.