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Due to planarian regeneration capacity, researchers have made efforts in studying its gene regulation, cell and tissue responses. Despite such advances, many aspects of planarian biology and histology, especially for Girardia tigrina species, remain unknown. Thus, in this study, we aimed at analyzing the potencial of G. tigrina as a biological model for ecotoxicological research by firstly characterizing the morphology and histology of healthy individuals and then evaluating the sublethal effects of Malathion, a widely used insecticide in Brazil. We exposed individuals of G. tigrina to two sublethal concentrations of Malathion (0.08 mg L-1 and 0.8 mg L-1) insecticide for 96h, sectioned them, and then exposed the regenerating portions for another 72h. We submitted all material to scanning electron microscopy and to different histological/histochemical light microscopy techniques. Several changes in the treatment groups were present when compared to the control group. In external morphology, the following alterations were noteworthy: malformation and underdevelopment of the blastemas, pores dilation in the epidermis and loss of ventral cilia. Our main results regarding histology, histochemistry and cytochemistry were the disruption of several tissues, characterized by cytoplasmic changes, such as vacuolation, histochemical and nuclear changes, including chromatin marginalization. Thus, we concluded that Malathion, mainly in the lowest concentration tested, has acted on reproductive strategies of the tested organism, in addition to promoting necrosis in different tissues.

This study presents the morphological and physiological characterization of the salivary glands of semi-engorged Amblyomma triste females. Unfed individuals were placed on New Zealand White rabbits for feeding and the females, after 4 days, were collected, dissected and the salivary glands were submitted to the application of histological (hematoxylin–eosin technique) and histochemical tests for the detection of protein (bromophenol blue technique, polysaccharides (periodic acid–Schiff technique), lipid (Nile blue technique) and calcium (von Kossa technique). The histological results show that the glandular tissue is composed by a system of ducts and three types of acini (I, II and III). The acini I are formed by a large central cell surrounded by several smaller agranular peripheral cells. Acini II are formed by cells a , c1 , c2 , c3 and c5 , which are full of secretion granules. Acini III are constituted by cells d , e and f ; the former two contain secretion granules, the latter is agranular. The glandular histochemical composition was also verified. Data obtained here will certainly help in the understanding of the cellular morphology and of the general physiology of these organs in this specie, providing important information for the creation of scientific bases which will contribute for the development of more specific and efficient methods of control.

Ticks of Rhipicephalus sanguineus species have great medical and veterinary importance for being a vector of various diseases. In an attempt to minimize their action on the host, people have resorted to chemical control by using various acaricides, such as selamectin. Although previous studies have demonstrated its toxic action in domestic animals, no studies focused on the detection of cell death when exposed to selamectin. For this reason, the technique for detecting autophagic cell death was used in order to demonstrate the responses of rabbits' skin tissues pre-infested with R. sanguineus and exposed to different concentrations of selamectin. The obtained results when exposed to 100 and 80% concentrations of selamectin showed a strong mark of acid phosphatase on the cells of the connective tissue of the dermis and hair follicles, whereas the ones exposed to the 50% concentration had a weak mark on the cells of the connective tissue of the dermis and moderate staining in hair follicles. It became clear that, when used at high concentrations (100 and 80%), selamectin is capable to induce a large scale occurrence of the autophagic cell death process. On the other hand, the concentration of 50% causes minor morphophysiological changes in the skin of rabbit hosts when evaluated the cell death process. Therefore, the data confirms that selamectin is a powerful dose-dependent toxic agent causes increased activity of the enzyme acid phosphatase.

Actually, the most used method to control ticks is synthetic acaricides with neuro- toxic action. However, the use of these methods presents inconveniences, such as the contamina- tion of the environment and risks to the host's health due to the residual effects. Thus, several studies have been developed aiming to fi nd alternative ways to control these ectoparasites, such as the use of natural compounds with active ingredients, which act controlling some species of plagues in addition to presenting medicinal properties that are beneficial to humans. The pres- ent study aimed to analyze the action of andiroba oil (Carapa guianensis) on the synganglion of Rhipicephalus sanguineus semiengorged females through morphological and cytochemical tech- niques aiming to verify if this natural product have neurotoxic action as the numerous synthetic acaricides. The results showed that andiroba oil interferes in the synganglion through structural and enzymatic changes, which lead the nervous tissue to apoptotic death involving autophagy. Among these changes was observed the emergence of large empty spaces between the perineu- rium and the cortical region, vacuolated cortex cells and with cell swelling, neural cells with pic- notic nuclei or in initial stage of chromatin margination and neuropile with high structural disorganization. Considering these data, it can be concluded that andiroba seed oil can be used as an alternative method in the control of R. sanguineus ticks due to its neurotoxic action. Microsc. Res. Tech. 00:000-000, 2013. V C 2013 Wiley Periodicals, Inc.

KEY WORDS ticks; natural product; germ cells; cytotoxicity; reproductive efficiency indexABSTRACT Because of the increasing medical-veterinary importance of ticks, the developmentof alternative control methods, less aggressive to the host and the environment has become the tar-get of several researches. In this sense, the present study analyzed the action of different concen-trations (5, 10, and 20%) of andiroba seed oil (Carapa guianensis) on the reproductive system ofRhipicephalus sanguineus females, through histochemical techniques and the quantification of thereproductive efficiency index. The results showed that andiroba oil is a potent natural agent, ableto cause several changes in the oocytes of this species, impairing the reproductive success, oncethis natural product induces great physiological changes in the oocytes in all development stages,such as drastic reduction in proteins, polysaccharides, and lipids in these cells, and these compo-nents are essential for the viability of the embryo. In addition, it was observed that this productstimulate the oviposition, mainly at the concentration of 20%. This higher production of eggs repre-sents a defense mechanism developed by the organism in order to ensure the reproductive successof the species, even in the presence of the toxic agent. However, the results obtained suggested thatthe laid eggs would not be viable, due to the great changes undergone by the oocytes. Thus, thepresent study showed that the use of this vegetal product would be an alternative way to controlthe ticks, bringing benefits similar to the ones obtained through the use of synthetic acaricides;however, with less damage to nontarget organisms and the environment as well. Microsc. Res.Tech. 00:000–000, 2012.

Made available in DSpace on 2013-09-27T14:53:29Z (GMT). No. of bitstreams: 1 WOS000305307000004.pdf: 224251 bytes, checksum: ad832f37dbd1804429de3fe43307203c (MD5) Previous issue date: 2012-06-01 Made available in DSpace on 2013-09-30T18:47:15Z (GMT). No. of bitstreams: 1 WOS000305307000004.pdf: 224251 bytes, checksum: ad832f37dbd1804429de3fe43307203c (MD5) Previous issue date: 2012-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:56:03Z No. of bitstreams: 1 WOS000305307000004.pdf: 224251 bytes, checksum: ad832f37dbd1804429de3fe43307203c (MD5) Made available in DSpace on 2014-05-20T13:56:03Z (GMT). No. of bitstreams: 1 WOS000305307000004.pdf: 224251 bytes, checksum: ad832f37dbd1804429de3fe43307203c (MD5) Previous issue date: 2012-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) As recent studies have shown that ecdysteroids may play a major role in the regulation of vitellogenesis in Ixodidae, the present study quantified, by means of a radioimmunoassay, the levels of ecdysteroids present in the hemolymph of semi-engorged females of Rhipicephalus sanguineus ticks obtained from control females (exposed to distilled water) and those exposed to increasing concentrations of permethrin. The levels of ecdysteroids decreased significantly as the concentration of permethrin increased, suggesting that this compound could be an inhibitor of ecdysteroids secretion, and consequently interfering with the reproductive ability of these ticks, since this hormone is responsible for the synthesis and incorporation of vitellogenin by oocytes. This study complements the previous results with R. sanguineus semiengorged females, showing that permethrin is a potent agent causing major morphological changes in tick oocytes, such as the appearance of large vacuoles in the cytoplasm, reduction in the amount of yolk granules and a decrease in oocyte size, thus culminating in cell death and consequently reducing or preventing reproduction in treated females. The findings that permethrin leads to a decrease in ecdysteroid titers could represent an entry step into this scenario. (C) 2012 Elsevier B.V. All rights reserved. Univ Estadual Paulista, UNESP, Dept Biol, Inst Biociencias, BR-13506900 Rio Claro, SP, Brazil UNESP, Fac Ciencias Agr & Vet, Dept Patol Vet, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Paulista, UNESP, Dept Biol, Inst Biociencias, BR-13506900 Rio Claro, SP, Brazil UNESP, Fac Ciencias Agr & Vet, Dept Patol Vet, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 07/57809-5 FAPESP: 07/59020-0 FAPESP: 09/54125-3 CNPq: 308733/2006-1

Ticks have great medical and veterinary importance because they transmit several pathogens to their hosts, including humans; moreover, they cause skin lesions and favour secondary infections and blood loss. Selamectin, a macrocyclic lactone, has been used as a potent antiparasitic and is commercialized in Brazil as a product called Revolution® manufactured by Pfizer Animal Health (Guarulhos, Sao Paulo, Brazil). There is little information on the method of action of this chemical compound on tick physiology so this study presents an analysis of the toxic potential of selamectin on engorged females of the tick Rhipicephalus sanguineus. For the toxicity study of selamectin, the adult immersion test was performed, in which individual ticks were exposed to different concentrations of the chemical compound. The percentage of dead females was submitted to probit analysis to determine the median lethal concentration (LC50) of selamectin. Bioassays showed that R. sanguineus females are sensitive to the chemical co...

Made available in DSpace on 2013-09-27T14:53:29Z (GMT). No. of bitstreams: 1 WOS000289820600010.pdf: 1255797 bytes, checksum: 270053e2c620250e62891abd53746f5c (MD5) Previous issue date: 2011-06-01 Made available in DSpace on 2013-09-30T17:56:51Z (GMT). No. of bitstreams: 1 WOS000289820600010.pdf: 1255797 bytes, checksum: 270053e2c620250e62891abd53746f5c (MD5) Previous issue date: 2011-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:55:02Z No. of bitstreams: 1 WOS000289820600010.pdf: 1255797 bytes, checksum: 270053e2c620250e62891abd53746f5c (MD5) Made available in DSpace on 2014-05-20T13:55:02Z (GMT). No. of bitstreams: 1 WOS000289820600010.pdf: 1255797 bytes, checksum: 270053e2c620250e62891abd53746f5c (MD5) Previous issue date: 2011-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Because of the medical and veterinary importance of ticks and the wide use of synthetic chemical substances such as permethrin (active ingredient of Advantage(R) Max3 - Bayer)for their control, this study evaluated the effects of different concentrations (206, 1031 and 2062 ppm) of the acaricide on the salivary glands of Rhipicephalus sanguineus semi-engorged females. Results showed that permethrin is a potent substance that acts morpho-physiologically in the tick glandular tissue, causing changes in the acini shape intense vacuolation in acinar cells, and disruption of the tissue by cell death process, with subsequent formation of apoptotic bodies, especially at higher concentrations, thus precluding the accurate identification of different types of acini. Importantly, it is demonstrated that permethrin acts on salivary gland tissue, as well as affecting the nervous system, accelerating the process of glandular degeneration, and interfering with the engorgement process of female ticks, preventing them from completing the feeding process. (C) 2011 Elsevier B.V. All rights reserved. Univ Estadual Paulista, UNESP, Inst Biociencias, Dept Biol, BR-13506900 Rio Claro, SP, Brazil UNESP, Fac Ciencias Agr & Vet, Dept Patol Vet, BR-14884900 Jaboticabal, SP, Brazil Univ Estadual Paulista, UNESP, Inst Biociencias, Dept Biol, BR-13506900 Rio Claro, SP, Brazil UNESP, Fac Ciencias Agr & Vet, Dept Patol Vet, BR-14884900 Jaboticabal, SP, Brazil FAPESP: 09/13854-4 FAPESP: 07/57809-5 FAPESP: 07/59020-0 CNPq: 308733/2006-1

Given the wide use of synthetic chemicals to control ticks, this study evaluated the effects of the permethrin pyrethroid on oocytes of Rhipicephalus sanguineus fully engorged females in order to examine whether this compound, in addition to the proven neurotoxic effect, also acts directly on germ cells. The results revealed that permethrin effectively inhibits and/or interrupts the reproductive process of R. sanguineus. Exposed oocytes exhibited prominent structural changes such as altered shape of cells and germ vesicle (oocyte nucleus), cytoplasmic vacuolation, and decrease of yolk granules. The composition of the latter, however, was not altered. These findings confirm those already reported by Roma et al. (Food Chem Toxicol 48:825-830, 2010) demonstrating that permethrin acts on germ cells of R. sanguineus via direct absorption from the hemolymph by pedicel cells, or by the oocyte plasmic membrane. On the other hand, these results contradict studies reporting that acaricides act exclusively on the nervous systems of ticks and that all the changes in other organs are a result from the indirect action of these chemical compounds, because blocking of the nervous system would compromise the normal metabolism of other organs (dependent on sensory information).

To understand the morphological and histological aspects of internal systems of ticks has become important matter since these arthropods have an impact in the areas of the economy and public health. In this context, this study has provided morphological data on female germinative cells of Rhipicephalus sanguineus ticks, ectoparasites of dogs that maintain a close relationship with human on a daily basis. Oocytes of engorged females were analyzed, through the PAS reaction (detection of polysaccharides) counterstained by methyl green (detection of RNA) revealing information that allowed to infer for the first time the presence of Cajal bodies, in the germinal vesicles (nuclei) of developing oocytes, as well as showing how the RNA and the polysaccharides are involved in the dynamics of the vitellogenesis in this species.

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.

Thymol is a monoterpene with proven acaricide action for several tick species. In addition to killing these ectoparasites, thymol can also reduce oviposition and egg hatch rate. However, the effects of thymol on the morphophysiology of tick ovaries are still unknown. Thus, the aim of this study was to evaluate the morphophysiological changes caused by this active principle in ovaries of Rhipicephalus sanguineus after a 6-day feeding period, through the application of morphohistochemical techniques. After the feeding period, a total of 50 females were divided into five groups and immersed in the following solutions: (I) distilled water (control), (II) 30% ethanol (control), (III) 1.25 mg/mL thymol, (IV) 2.5 mg/mL thymol, and (V) 5.0 mg/mL thymol. The experimental groups were kept in a climatic chamber (27 ± 1 °C; RH 80 ± 10%) for 5 days. After this period, morphological (hematoxylin/eosin) and histochemical (von Kossa) techniques were applied after remotion of the ovaries. The morphological results revealed large vacuoles in germ cells at different developmental stages and invaginations that represent deformations in the chorionic membrane. From the results obtained in this study, it was concluded that thymol interfered with the development of oocytes, which showed degeneration signs. The treatment containing 5.0 mg/mL thymol affected more accentuately the morphological development. Moreover, thymol also altered the calcium content of yolk granules, which generally showed an intense staining for this element.

This study evaluated the effects of one or two injection of differents concentrations (0.2 and 0.04μg/μL) of extracts obtained from the salivary glands of female ticks Rhipicephalus sanguineus (Acari: Ixodidae) (Latreille, 1806) also called “brown dog tick” fed for 2 days on the rabbit hosts on the morpho-physiology of the leg musculature of female Wistar rats inoculated with Walker 256 tumor cells. The number of leukocytes and the creatinine levels were quantified for individuals from all groups. The results of both histology and electron microscopy revealed that a single, low-concentration injection of the extract (0.04μg/μL) was more effective in containing tumor invasion and caused less ‘collateral damage’ to the muscle tissue, which was the object of this study. The results also revealed that creatinine levels were higher in rats subjected to both one and two injections of the extract at a concentration of 0.04μg/μL than in those subjected to one and two injections of the extract at the higher concentration (0.2μg/μL), suggesting that in the first group, injection of the extract contributed to maintaining the integrity of the muscle tissue. With regard to the number of leukocytes, the results suggested that in all the inoculated rats (Walker 256 cells), there was a significant increase in the total number of leukocytes. The inoculated rats that received both one and two injections of the extract at a concentration of 0.2μg/μL experienced a significant increase in the number of leukocytes compared with those inoculated but not exposed to the extract; this result can be explained by the fact that beyond the tumor cells, the extract itself acted to boost the defense response at this concentration. However, inoculated rats subjected to injections (one and two) of the extract at a concentration of 0.04μg/μL showed a significant decrease in the total number of leukocytes compared with rats that were only inoculated and those inoculated and injected with the extract at a concentration of 0.2μg/μL. These results reinforced that the extract at the 0.04μg/μL concentration not only acted more effectively to inhibit Walker 256 tumor cells but also did not act as a stressor, because the number of leukocytes was lower. Therefore, the data obtained here indicated that the same molecules or a pool of molecules produced by the salivary glands of ticks belonging to this species have the ability to inhibit tumor growth, while minimizing ‘collateral damage’ to the body.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:16Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:28:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-01-31 Rhipicephalus sanguineus are bloodsucking ectoparasites, whose main host is the domestic dog, thus being present in urban areas and closely located to people. Eventually, this tick species parasitize humans and can become a potential vector of infectious diseases. Methods to control this type of pest have been the focus of many research groups worldwide. The use of natural products is increasingly considered nowadays, due to the low toxicity levels to the host and low waste generation to the environment. This study tested the effect of ricinoleic acid esters from castor oil (as an potential acaricide) on the reproductive system of R sanguineus females, more specifically on the vitellogenesis process. For this, two groups were established: the control group (CG) and the treatment group (TG) with five rabbits in each (New Zealand White), used as hosts. NaCl and ester were added to rabbits' food and offered to the hosts. After full engorgement, the females were collected and had their ovaries extracted. The ticks ovaries were submitted to histochemical techniques so the effects of esters could be observed over polysaccharides, proteins and lipids yolk. Changes in the deposition of yolk components were observed. This caused modifications on elements of polysaccharide origin and on glycoprotein compounds, interfering in the final yolk synthesis and compromising the development of the future embryo. © 2012. UNESP, 24th Av., 1515, PO Box 199, Rio Claro 13506-900, SP UNESP, 24th Av., 1515, PO Box 199, Rio Claro 13506-900, SP

This study presents the histochemical analysis of the glycoprotein, lipoprotein, enzyme (acid phosphatase) and mineral (calcium) contents of the secretion produced by salivary glands of Rhipicephalus sanguineus females fed for 2, 4 and 6 days on hosts (New Zealand White rabbits), which had been previously immunized with glandular extract obtained from 2-day fed females (SGE2). The results revealed that such glands present alterations in the secretory cycle, which occurred: a) by the inactivity of some c1 cells (in the glands of the 2-day fed females); c1 and c5 (in the glands of the 4-day fed females) and c1 and d (in the glands of the 6-day fed females), and b) by the modification in the constitution of secretion in relation to the glyco/lipoprotein, enzyme and mineral contents in the glands of 2 to 6-day fed females. Data clearly show that the most pronounced histochemical modifications were detected in 2-day fed females, since their hosts were inoculated with SGE2 extract, and therefore, developed resistance to the components of the glandular secretion present in this extract. In addition, it was verified that over the studied period (2-6 days), some cells underwent greater alterations due to the resistance acquired by the hosts, such as the f cells, demonstrating the great immunogenic capacity of the secretion synthesized by such cells, as well as the importance of the secretion in the modulation of the immune-inflammatory and hemostatic system of the host. Considering all the data obtained, this study clearly demonstrates that the modifications in the intensity of staining in the elements analyzed occurred because of different immunogenic capacities of the components present in SGE2 extract, which indicated that the greater the immunogenic capacity of the molecules present in SGE2, the greater the alterations in the cells responsible for the synthesis of such component. This study also demonstrated that the histochemical alterations here detected modified the salivary secretion, making it increasingly efficient over the process of modulation of the host, suggesting a possible compensation, or even an alternative route to overcome the resistance developed by the hosts, enabling the ectoparasites’ fixation, permanence and feeding.

Tick control has been accomplished through the use of synthetic acaricides, which has created resistant individuals, as well as contaminating the environment and nontarget organisms. Substances of plant origin, such as oils and extracts of eucalyptus and neem leaves, have been researched as an alternative to replace the synthetic acaricides. Ricinoleic acid esters from castor oil have recently been shown as a promising alternative in eliminating bacterial contamination during ethanol fermentation, by acting as an effective biocide. The same positive results have been observed when these esters are added to the food given to tick-infested rabbits. This study tested the effect of these substance on the reproductive system of Rhipicephalus sanguineus females, added to rabbit food, more specifically on oogenesis. For this, four groups were established: four control groups (CG1, CG2, CG3, and CG4) and four treatment groups (TG1, TG2, TG3, and TG4) with one rabbit in each (New Zealand White), used as hosts. After full 4 days feeding (semi-engorgement), the females were collected and had their ovaries extracted. In this study, it was observed that R. sanguineus females exposed to esters had their ovaries modified, which was demonstrated through transmission electron microscopy techniques. The addition of ricinoleic esters to the diet of tick-infested rabbits revealed how toxic such substances are for the cytoplasmic organelles of oocytes and pedicel cells. These compounds can change the morphophysiology of germ and somatic cells, consequently influencing their viability and, therefore, confirming that the ricinoleic acid esters from castor oil are a promising substance in the control of R. sanguineus.

This study analyzed the histopathology of rabbit skin, previously immunized with SGE2, SGE4, and SGE6 gland extracts prepared from salivary glands of Rhipicephalus sanguineus female with 2, 4, and 6 days of feeding, at the region of the R. sanguineus female feeding lesion 2, 4, and 6 days after tick attachment. In this work, infestation-naive New Zealand White rabbits were inoculated either with the extracts (test group (TG)) or with phosphate buffer and complete Freund’s adjuvant mixture (control group 2 (CG2)). Each extract-inoculated- (TG and CG2) and non-inoculated (CG1) rabbit was subsequently infested with R. sanguineus. Skin biopsies were collected from the rabbit at the tick feeding lesion at 2, 4, and 6 days of feeding. Results revealed that rabbit immunization with gland extracts induced acquisition of resistance against this species. It should be stated that the SGE4 extract was the most effective in developing an immune–inflammatory response against ectoparasites, being this process characterized by the presence of an early and intense inflammatory cell infiltrate. On the other hand, SGE6 extract caused a later appearance of resistance with less infiltrate occurrence and intense edema at the feeding lesion site. As to the inflammatory process deriving from SGE2 extract inoculation, it was the less intense. It was concluded that immunization with different extracts from R. sanguineus female salivary glands did not change microscope features of the inflammatory process, although an earlier or more intense and later response, which was also dependent on the inoculate extract, was noticed.

Ticks are ectoparasites of great medical and veterinary importance around the world and synthetic chemicals such as permethrin have been used for their control. This study provides a cytochemistry analysis of both degenerative and cell death processes in salivary glands of the brown dog tick Rhipicephalus sanguineus semi-engorged females exposed to 206, 1,031, and 2,062 ppm of permethrin. The results presented herein demonstrate that permethrin is a potent chemical acaricide that would act on the glandular tissue's morphophysiology in this tick species by eliciting severe changes in the acinus shape, intense vacuolation of the acinar cells' cytoplasm, marked glandular tissue disorganization, culminating in an advanced degenerative stage with consequent formation of many apoptotic bodies (cell death). In addition, permethrin induced major changes in the acinar cells' nucleus, such as a change both in its shape and size, chromatin marginalization, nuclear fragmentation, and appearance of picnotic nuclei, especially when the highest concentrations of the product were used. Thus, permethrin induced early degeneration of this tissue characterized by significant changes in the structure of acinar cells and production of enzymes related to the cell death process, in addition to interfering directly in the genetic material of these cells. Microsc. Res. Tech. 75:1012–1018, 2012. © 2012 Wiley Periodicals, Inc.

Rhipicephalus sanguineus is a widely distributed tick species that has adapted to the urban environment, and the dog is its main host. This species is also known as a vector and reservoir of diseases caused by bacteria, protozoa, and viruses. Currently, acaricides of synthetic chemical origin have been widely and indiscriminately used, leading to the development of resistance to these products by ticks and causing damage to the environment. Thus, these issues have made it necessary to seek other forms of controlling these ectoparasites. R. sanguineus was artificially infested in host New Zealand White rabbits, which were divided into four treatment groups: control (CG1 and CG2) and treatment (TG1 and TG2) groups. TG1 and TG2 hosts were provided with feed supplemented with esters of ricinoleic acid from castor oil at a concentration of 5 g/kg of feed for 7 and 15 days. Afterward, the ovaries of the female ticks were removed for analysis by transmission electron microscopy. The results showed ultrastructural changes in the somatic and germ cells of ovaries from TG1 and TG2 females, particularly with respect to chorion deposition, a protective membrane of the oocyte, as well as in the transport process of vitellogenic materials via the hemolymph and pedicel cells. Moreover, the mitochondria were less electron-dense and had cristae that were more disorganized than the mitochondria from CG1 and CG2 individuals. Thus, this study demonstrated the action of esters on the ovaries of R. sanguineus, signaling the prospect of a way to control this ectoparasite without affecting nontarget organisms or the environment.

Currently the most effective method of tick control is the use of acaricides, among which stands out permethrin (active ingredient of acaricide Advantage(®) Max3, Bayer), a neurotoxic pyrethroid. However, assessments of their effects on other tick systems such as glandular are still scarce. Thus, this study provides information, through histochemical techniques, about the toxic effect of this pyrethroid on the morphophysiology of salivary glands of semi-engorged Rhipicephalus sanguineus females exposed to different concentrations of permethrin (206, 1031, and 2062 ppm). The results showed that permethrin caused significant changes in the salivary gland metabolism accelerating the process of glandular degeneration, an event which would occur naturally and with great intensity only in the final engorgement stage. Furthermore, this study pointed out that permethrin reduces the salivary gland secretion ability through a drastic reduction of proteins, lipids, and polysaccharides in acinar cells. These changes impair the females to finalize the feeding process, what indirectly affects the reproductive process.

The present study histologically analyzed the salivary glands of Rhipicephalus sanguineus females fed for 2, 4, and 6 days in hosts which had been previously immunized with glandular extracts obtained from females from this same species in different periods of feeding, having as main objective verify the action of these extracts in the secretor cycle of these glands. For this, glandular extract of females fed for 2 days (SGE2), glandular extract of females fed for 4 days (SGE4), and glandular extract of females fed for 6 days (SGE6) extracts were obtained from salivary glands of R. sanguineus females fed for 2, 4, and 6 days respectively. Then, New Zealand White naive rabbits were inoculated either with extracts (test group = TG), or with a mixture of phosphate buffer and Freund's complete adjuvant (control group 2 = CG2). Each inoculated rabbit (TG and CG2) and non-inoculated (control group 1 = CG1) was posteriorly infested with 15 couples of fasting R. sanguineus from which the salivary glands had been collected from females fed for 2, 4, and 6 days. The results revealed that the resistance the hosts had acquired by the immunization with the extracts affected differently the secretory activity of the glandular cells. It was verified that the resistance to SGE2 and SGE4 extracts acted in the cells of acini II and III, being c1 and c5 from II and d from III inactivated due to the action of SGE2 and c1 and c4 from II and f from III inactivated by the action of SGE4. As for the resistance to SGE6 the effect was only on cells of acini II (c1, c3 e c4), which were also inactivated. In addition, the hosts’ resistance to SGE2–SGE6 extracts made the degenerative process earlier in comparison to CG1. On the other hand, the resistance to the extracts did not influence the characteristics of the degenerative process normally found in salivary glands. The assynchronism of the degenerative process was maintained—acini III were always the most affected and I the less affected. The structural cell alterations, such as cytoplasmic vacuolation, nuclear alterations and formation of apoptotic bodies which characterize the occurrence of atypical apoptosis were also maintained in the glands of individuals from TG making it clear that the immunization of the hosts with glandular extracts SGE2–SGE6 had influenced the glandular physiology of R. sanguineus, which is an important piece of information in the search for a way to control these ectoparasites.

The ultrastructure of the salivary glands of Rhipicephalus (Boophilus) microplus females is described during feeding. In beginning of feeding, individuals show acini I with many mitochondria and wide basal labyrinth in peripheral cells; glycoprotein granules only in b and c3 cells (acini II); and epithelial interstitial cells with developed basal labyrinth between f cells (acini III). Semi-engorged females show cells in degeneration, with autophagic vacuoles, lysosomes, myelin figures, and irregular, condensed, and/or fragmented nuclei, in addition to apoptotic bodies. R. B. microplus points to apoptosis in these organs before the detachment from the host, in contrast to others tick species.

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.

This study showed the interference of esters extracted from Ricinus communis in the secretory cycle of salivary glands of Rhipicephalus sanguineus ticks, which consequently caused collateral effects on their feeding process. Ticks attached on hosts which were fed with commercial feed containing different concentrations of R. communis oil esters suffered damages such as cytoplasmic changes in their salivary glands, notably in the acinar cells, impairing the functioning of the acini and accelerating the organs degeneration as a whole. It was found that esters interfered with the activity of cellular secretion by changing the glycoprotein of salivary composition especially in acini II cells. It was also shown that the damages caused by esters in the salivary glands cells of these ectoparasites increased in higher concentrations of the product and degenerative glandular changes were more pronounced.

This study examines the effects of ricinoleic acid esters from Ricinus communis castor oil on the vitellogenesis of Rhipicephalus sanguineus ticks attached to hosts that were fed with commercial rabbit food containing these esters. The oocytes of ticks from the treatment group (TG) showed cytoplasmic changes that inhibited the development of oocytes I and II to the advanced stages (IV and V) in addition to preventing the maturation of oocytes V, resulting in small ones. In addition, sperm was not observed in ampoules. Our findings confirm the acaricide potential of ricinoleic acid esters.

The permethrin, active ingredient of the Advantage® Max3 – Bayer, has been widely used in the chemical control of ticks. These ectoparasites are one of the most important animal groups that cause serious damage to their hosts. This study evaluated the toxic effects of permethrin in oocytes of Rhipicephalus sanguineus semi-engorged females subjected to four treatments: group I (control – distilled water), group II (206 ppm of permethrin), group III (1031 ppm of permethrin) and group IV (2062 ppm of permethrin). Results demonstrated that permethrin is a potent chemical agent causing major structural changes in oocytes, such as emergence of large vacuolated cytoplasm regions, reducing the amount of yolk granules and decreasing the size of oocytes, culminating with cell death. As reported in the literature, these oocytes changes, besides affect the tick nervous system, also drastically reduce or prevent the reproduction process in females of R. sanguineus ticks subjected to this compound.

The present study analyzed the effects of the chemical compound fipronil, active ingredient of Frontline® (acaricide and insecticide), on the salivary glands of unfed and semi-engorged female Rhipicephalus sanguineus tick. Unfed females were only exposed to the concentration of 1 ppm of fipronil, while semi-engorged females were treated with fipronil in three concentrations: 1 ppm, 5 ppm, and 10 ppm (distilled water was used as control). The histological and histochemical results revealed significant changes caused by this compound in the morphology and physiology of the gland tissue of unfed and semi-engorged females. In unfed females, the morphological changes in type I acini were characterized by an increase in size and diameter of the lumen. These changes are probably associated with the excretory function, indicating that type I acini might be responsible for eliminating this xenobiotic from the system of the parasite. In semi-engorged females, fipronil did not interfere in the cell death, which in these individuals occurred by apoptosis. However, it accelerated salivary gland degeneration, as the extent of damage increased along with the concentrations of the product. Our results clearly showed that fipronil interferes with the process of engorgement in females that consequently is reflected in the reproductive process, decreasing or even halting egg laying, and resulting in less blood losses for the hosts and reducing the transmission of pathogens through these glands.

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.

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.

The present study examined the salivary glands of Rhipicephalus sanguineus males at days 0, 3, and 7 post-detachment from the host. Degeneration of this organ occurred in the three stages and it advanced as time away from the host progressed. Thus, characteristics of degeneration were more prominent in males at day 7 post-detachment than in males at day 0 post-detachment. In males at day 0 post-detachment, type I acini were intact; while in other stages these acini exhibited signs of degeneration. In type II acini of individuals at day 0 post-detachment, cells a, c1–c5, c8, and indeterminate were identified. Only c1 and c8 were intact. The remaining cell types were undergoing degeneration, as well as all cells d–f in type III acini, and all g in type IV acini. In males at day 3 post-detachment from the host, all cells (a, c1–c5, c8 and indeterminate) of type II acini, cells d and e in type III acini, and g in type IV were undergoing degeneration. In some Indeterminate acini, the boundaries of cells still could be distinguished, while in others, only a cytoplasmic mass was observed. At day 3 post-detachment, apoptotic bodies were present. In males at day 7 post-detachment from the host, the degeneration process progressed. All cells a, c1, c3–c5, c8 and indeterminate in type II, and d and e in type III acini were undergoing degeneration. Type IV acini still contained remnants of secretion and in Indeterminate acini, only a cytoplasmic mass could be observed. At this stage, apoptotic bodies were also present. The present study still revealed that cells of salivary glands of R. sanguineus males when degenerating undergo the following changes: (a) decrease in secretion production with or without granule breakage, (b) changes in nuclear morphology, (c) cytoplasm shrinkage, (d) loss of cell shape, (e) loss of cell boundaries, and (e) cytoplasmic vacuolation. Together, these changes result in cell fragmentation with release of apoptotic bodies.

A histochemical analysis was performed in this study in order to detect proteins, polysaccharides, lipids, acid phosphatase and calcium in the secretion produced by the salivary glands of Rhipicephalus sanguineus females (Latreille, 1806) (Acari, Ixodidae) fed for 2, 4 and 6 days in New Zealand White rabbits which had been previously immunized with glandular extract obtained from females fed for 4 days (SGE4). The results revealed that such glands presented alterations in their secretory cycle, which occurred: a) by the inactivity of some c1 cells (in the glands of females fed for 2 days) and c4 (in those fed for 4 days) and b) by the modification in the constitution of the secretion of females fed for 2-6 days. It was verified that, in the glands of females fed for 2 days, there was an increase in proteins and calcium; a reduction in lipids and the contents of polysaccharides and acid phosphatase remained unaltered. In those fed for 4 days there was an increase in proteins, calcium and acid phosphatase; reduction in the lipids, and the content of polysaccharides remained unaltered. In the females fed for 6 days an increase in the components was observed; however, there was a reduction in lipids and acid phosphatase. In addition, it was verified that, in a decrescent order of histochemical alterations, the most affected cells were: f; c2, c3, c5; a, d and c4, e in the glands of females fed for 2 days; c5; a, c2, c3, d and c1, e in the glands of females fed for 4 days and a, c1, c2, d and e, c3 in the glands of females fed for 6 days. The data here obtained clearly show that the most pronounced histochemical modifications were detected in the glands of females fed for 2 and 6 days; however, the modifications observed in the females fed for 4 days must also be considered, once they were significant as well. This has probably occurred because the hosts were inoculated with SGE4, obtained from the salivary glands of females fed for 4 days, intermediate stage of the glandular cycle, which contains antigens that are common to glandular tissues of females fed for 2 and 6 days.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:43Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:30:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-23 Studies on the molecular bases of the neurotoxic action of acaricides are found in the literature; but there are no studies of this action on the nervous system of ticks at the cellular level. The present study describes the morphological and cytochemical changes in the synganglion of Rhipicephalus sanguineus semi-engorged females exposed to different concentrations of permethrin, a pyrethroid with recognized neurotoxic action. Permethrin induced the degeneration of the synganglion through a process of apoptosis involving autophagy, characterized by the condensation and margination of the chromatin, formation of blebs in the nuclear envelope and fragmentation of the nucleus, loss of shape of neural cells and integrity of cellular membrane, cytoplasmic shrinkage, and lower levels of acid phosphatase in the nervous tissue as the concentration of permethrin increased. This study provided further evidence of the neurotoxic action of permethrin, which impairs the metabolism of R. sanguineus nervous systems, and consequently the physiology of other systems, dependent on the neural control. These results provide cytochemical and histological confirmation of the neurotoxic action of permethrin, previously inferred from molecular and tick behavioral evidence. © 2013 Elsevier B.V. UNESP - Universidade Estadual Paulista, SP UNESP - Universidade Estadual Paulista, SP