Effects of carvacrol on oocyte development in semi-engorged Rhipicephalus sanguineus sensu lato females ticks (Acari: Ixodidae).

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.

Morphological alterations in salivary glands of Rhipicephalus sanguineus ticks (Acari: Ixodidae) exposed to neem seed oil with known azadirachtin concentration.

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.

Fipronil-induced cell death in salivary glands of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged females.

The tick Rhipicephalus sanguineus is currently considered an urban plague. For this reason many studies are intended to find methods to control these ectoparasites. Thus, the present study analyzed the ultrastructural modifications of the salivary glands cells of semi-engorged females of R. sanguineus resulting from their exposition to Fipronil (active ingredient of Frontline®). The studied individuals were divided into four groups. Group 1 was exposed to distilled water (control) and groups 2, 3 and 4 were exposed to 1, 5 and 10 ppm of Fipronil, respectively. The salivary gland of ticks subjected to the acaricide showed accelerated process of cell death by atypical apoptosis, as well as augmented cell damages as the concentration of the chemical compound was increased. The acaricide toxicity at cellular level was demonstrated by remarkable changes of elements of the cytoskeleton and spherocrystals (extremely hard inorganic structures). However, tick defense mechanisms, such as the observed autofagic vacuoles proved the cells attempt to preserve their integrity and minimize the devastating action of this chemical compound on the salivary glands.

Morpho-histochemical characterization of salivary gland cells of males of the tick Rhipicephalus sanguineus (Acari: Ixodidae) at different feeding stages: description of new cell types.

This study describes the changes undergone by cells of the salivary glands of unfed and feeding (at day two and four post-attachment) Rhipicephalus sanguineus males, as well as new cell types. In unfed males, types I and II acini are observed with cells "undifferentiated", undefined 1 and 2 (the latter, with atypical granules), a, c1 and c3; type III is composed of cells d and e; and type IV present cells g. In males at day two post-attachment, type I acini exhibit the same morphology of unfed individuals. An increase in size is observed in types II, III, and IV, as cells are filled with secretion granules. Some granules are still undergoing maturation. In type II acinus, cells a, b and c1-c8 are observed. Cells c7 and c8 are described for the first time. Cells c7 are termed as such due to the addition of polysaccharides in the composition of the secretion granules (in unfed individuals, they are termed undefined 1). Type III acini exhibit cells d and e completely filled with granules, and in type IV, cells g contain granules in several stages of maturation. In males at day four post-attachment, type I acini do not exhibit changes. Granular acini exhibit cells with fewer secretion granules, which are already mature. In type II acini, cells a, b, c1-c5 are present, type III exhibit cells d and e, and type IV contain cells g with little or no secretion. This study shows that in the salivary glands of R. sanguineus males, cells a, c1, and c3 of type II acinus, and cells d and e of type III do not exhibit changes in granular content, remaining continuously active during the entire feeding period. This indicates that during the intervals among feeding stages, gland cells reacquire the same characteristics found in unfed individuals, suggesting that they undergo reprogramming to be active in the next cycle.

Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.

Cytoplasmic RNA and nuclear changes detected cytochemically during the degeneration of salivary glands of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae).

The present study reports cytochemistry data about salivary glands of females (unfed, engorged, and at day three post-engorgement) and males (unfed, at day seven post-attachment, and at days three and seven post-detachment from the host) of the tick Rhipicephalus sanguineus. The results revealed nuclear changes in engorged females and at day three post-engorgement, and in males in all stages (except unfed). These changes were more prominent in females. Cytoplasmic changes were also observed in cells of all acini of males and females. In types II and III acini of engorged females, nuclear changes were observed in the shape (irregular, with blebs, fragmenting or fragmented), size (enlarged or reduced), and arrangement and condensation level of chromatin (marginal or as blebs). Changes were also detected in nucleoli, regarding their shape (fragmenting or fragmented), size (enlarged), and location (central, marginal or as blebs). Some nucleoli were also compacted or disorganized. In females at day three pos-engorgement, all acini exhibited similar changes to those observed in engorged females. RNA staining was stronger in cells of engorged females than those at day three post-engorgement. In males at day seven post-attachment, cells of types II, III, IV acini presented changes in the size of the nucleus and condensation level of chromatin similar to those of females. The shape of the nucleus was round, irregular or undergoing fragmentation, and the chromatin was located at the margin or throughout the nucleus. The changes in the nucleolus were similar to those of females, regarding size and organization, although round-shaped and in the central location. In males at day three post-detachment, cells of all acini exhibited nuclear changes similar to those of males at day seven post-attachment, in addition to the fragmentation of the nucleolus. At day seven post-detachment, changes were detected in all acini similar to the observed in males at day seven post-attachment. Regarding cytoplasmic RNA, staining was prominent in males at day seven post-attachment and weak in those at day seven post-detachment from the host. In females as well as males, different RNA staining patterns in the cytoplasm and nuclear changes characterized apoptotic cell death.