Central nervous system of Rhipicephalus sanguineus ticks (Acari: Ixodidae): an ultrastructural study

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Parasitol Res DOI 10.1007/s00436-012-2962-2

ORIGINAL PAPER

Central nervous system of Rhipicephalus sanguineus ticks (Acari: Ixodidae): an ultrastructural study Gislaine Cristina Roma & Pablo Henrique Nunes & Patrícia Rosa de Oliveira & Rafael Neodini Remédio & Gervásio Henrique Bechara & Maria Izabel Camargo-Mathias

Received: 12 March 2012 / Accepted: 3 May 2012 # Springer-Verlag 2012

Abstract This study performed the ultrastructural description of the synganglion of Rhipicephalus sanguineus males and females, aiming to contribute to the understanding of the cellular organization of this organ. The results show that the central nervous system of these individuals consists of a mass of fused nerves, named synganglion, from where nerves emerge towards several parts of the body. It is surrounded by the neural lamella, a uniform and acellular layer, constituted by repeated layers of homogeneous and finely granular material. The perineurium is just below, composed of glial cells, which extensions invaginate throughout the nervous tissue. The synganglion is internally divided into an outer cortex, which contains the cellular bodies of the neural cells and an inner neuropile. The neural cells can be classified into two types according to cell size, cytoplasm–nucleus relation, and neurosecretory activity. Type I cells are oval or spherical and present a large nucleus occupying most part of the cytoplasm, which contains few organelles. Type 2 cells are G. C. Roma (*) : P. R. de Oliveira : R. N. Remédio : M. I. Camargo-Mathias Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista “Júlio de Mesquita Filho”, UNESP, Avenida 24 A, 1515, 13506-900 Rio Claro, SP, Brazil e-mail: [email protected] P. H. Nunes Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, USP, Avenida Prof. Orlando Marques de Paiva, 87, 05508-270 São Paulo, SP, Brazil G. H. Bechara Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso Prof. Paulo Castellane, s/n, 14884-900 Jaboticabal, SP, Brazil

polygonal, present a great cytoplasm volume, and their nuclei are located in the cell periphery. The cytoplasm of these cells contains a well-developed rough endoplasmic reticulum, Golgi regions, mitochondria, and several neurosecretory granules. The subperineurium and the tracheal ramifications are found between the cortex and the neuropile. The latter is formed mainly by neural fibers, tracheal elements, and glial cells. The results obtained show that R. sanguineus males' and females' nervous tissue present an ultrastructural organization similar to the one described in the literature for other tick species.

Introduction The ultrastructure of the central nervous system of ticks was already described to Argasidae (Coons et al. 1974; El Shoura 1986) and Ixodidae (Binnington and Obenchain 1982; Ivanov 1983). According to these authors, this system consists of a mass of fused neural cells named synganglion, a set of peripheral nerves, as well as neuroendocrine organs. This structure represents a remarkable evolutive phenomenon, once this organ does not present internal segmentation due to the high level of condensation of the ganglia which form it (Roshdy and Marzouk 1984), which would also occur externally, once these ectoparasites do not present body divisions having fused head, thorax, and abdomen (Storer and Usinger 2000). The tick nervous system is characterized as a vital organ for the biological success of this group as it presents a great diversity of functions, such as control of all the metabolic processes which occur in the organism of these ectoparasites. Thus, morphology, cellular structure, and functional organization of this tissue require intensive investigation to provide information that can help in the understanding of

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the general physiology of Acari, an important group of animals of great medical and veterinary importance (Roshdy and Marzouk 1984). Ticks are characterized as an important group of pathogen vectors in the Arthropoda phylum, being only comparable to mosquitoes (Culicidae) (Marcondes 2009). They are responsible for the transmission of pathogens which affect domestic animals and human beings, including bacteria, helminths, protozoa, and viruses (Jongejan and Uilenberg 2004). Rhipicephalus sanguineus tick (Latreille 1806), popularly known as the brown dog tick, is cosmopolitan of tropical and temperate regions (Walker 1994; Paz et al. 2008) presenting a wide geographic distribution in the Americas, Europe, Africa, Asia, and Australia (Ribeiro et al. 1996). Over the last years, this species has drawn the attention of public health organizations for having become an urban plague (Paz et al. 2008), fact confirmed by the great expansion of the veterinary pharmaceutical industry, concerned about creating new formulations and acaricide substances. Until the early 80s, the manufacturing of commercial acaricides with specific action for the treatment of R. sanguineus in dogs was scarce. Currently, there are tens of products in the market, with different formulations and applications, representing significant profit for the pharmaceutical industries in the veterinary line (Labruna 2004). Several studies have been carried out on ticks aiming to find out an efficient method to control these ectoparasites. However, most of these studies refer to tests on the efficacy of synthetic or natural methods (Taylor 2001; Mencke et al. 2003; Farias et al. 2007, 2009). In this sense, this study aimed to describe, through transmission electron microscopy (TEM), the ultrastructure of the central nervous system (synganglion) of R. sanguineus males and females. As this system is characterized as the target of several acaricides with neurotoxic action, this study will certainly serve as a basis for future research which will need this knowledge to better understand the cellular organization of this organ as well as its physiology in order to improve or find new methods of tick control.

Material and methods R. sanguineus ticks Unfed adults (males and females) of R. sanguineus were supplied by the tick colony maintained at the Brazilian Central of Studies on Ticks Morphology (BCSTM) at the Biosciences Institute of São Paulo State University, Rio Claro, SP, Brazil. The ticks were kept under controlled conditions (28±1°C, 80 % relative humidity, and 12 h photoperiod) in a Biological Oxygen Demand incubator and blood fed on New Zealand

White rabbits. Details on feeding and maintenance of R. sanguineus ticks on the hosts are given by Bechara et al. (1995). This study was approved by the Ethics Committee in the Animal Use, CEUA, UNESP, Rio Claro, SP, Brazil, Protocol no. 4093. Transmission electron microscopy (TEM) R. sanguineus males and females were dissected on Petri dishes containing phosphate-buffered saline (PBS) solution (NaCl 0.13 M, Na2HPO4 0.017 M, KH2PO4 0.02 M, pH 7.2). Synganglia were removed, fixed in 2.5 % glutaraldehyde fixative solution in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h. Two 15-min washes in cacodylate buffer followed this process. Postfixation was performed in 1 % osmium tetroxide in 0.1 M in the same buffer (pH 7.2) for 2 h in darkness, followed by another two 15-min washes in the same buffer. To increase the contrast, material was immersed in a solution of 2 % uranyl acetate in 10 % acetone, for 4 h in darkness. Dehydration was performed in a graded acetone series (50, 70, 90, 95, and 100 %) for 5 min each. Then, the material was embedded in Epon resin diluted in acetone (1:1) for 12 h, following pure Epon resin, and incubated at 60°C for 24 h. Semithin sections were obtained with Sorvall-Porter Blum MT2-B ultramicrotome and stained with azur II (1 %) and methylene blue (1 %). Grids containing ultrathin sections were contrasted with uranyl acetate and lead citrate (Reynolds 1963) for 45 min and 10 min, respectively. Then, they were analyzed and photographed in a Philips CM 100 TEM from the Biology Department at the Biosciences Institute, UNESP, campus of Rio Claro, SP, Brazil.

Results The central nervous system of R. sanguineus males and females consists of a mass of fused nerves, named synganglion, from where nerves emerge towards several parts of the body. The esophagus runs through the synganglion (Figs. 1a and 3l) and divides it into two regions: (1) supraesophageal, the smaller of the two regions, consisting of a protocerebrum, a single dorsal ganglion located anterodorsally to the esophagus and (2) subesophageal, which consists in most part of the synganglion, being located in the ventral and posterior regions of the esophagus. The nervous tissue of these ectoparasites consists of an outer cortex and an inner neuropile, both surrounded by a neural lamella or neurilemma, a uniform and acellular layer. This structure is enclosed by a cavity named periganglionic sinus, which continues to the aorta and heart of the ticks (Figs. 1a–b, 2a–c, and 3a).

Parasitol Res Fig. 1 Schematic drawing of the central nervous system (synganglion) of R. sanguineus ticks. a General view and b detail of synganglion of males and females. nl neural lamella, p perineurium, c cortex, sp subperineurium, np neuropile, g ganglion, es esophagus, gc glial cells, e extensions of glial cells, n nucleus, m mitochondria, I type I neural cells, II type II neural cells, rer rough endoplasmic reticulum, Gr Golgi region, ng neurosecretory granules, tr tracheal elements, nf neural fibers, nt neurotubules

No differences between the ultrastructure of R. sanguineus males' and females' synganglia were found in this study. Thus, this organ will be described in the same way for the ticks here analyzed. Ultrastructurally, the neural lamella consists of repeated layers of homogeneous and finely granular material which encloses the synganglion as well as the esophageal channel and the peripheral nerves which emerge from this organ (Figs. 1a–b, 2a–c, and 3a–c). The periganglionic membrane or perineurium is just below, formed by a series of glial cells, which extensions invaginate throughout the nervous system (Figs. 1a–b, 2a–d, and 3a–c). These cells are characterized by their elongated nucleus as well as by their irregular membrane (Figs. 1b, 2c–d,

and 3b–c). Several mitochondria are found in the cytoplasm (Figs. 1b, 2d and 3c). The synganglion is internally covered by an outer cortical region named cortex which contains neural cell bodies or neurons (Figs. 1a–b; 2a–c, e; and 3a). Two cell types can be distinguished in the cortex of R. sanguineus males and females according to the size of the cell, cytoplasm–nucleus relation, and neurosecretory activity (Figs. 1b; 2e—f; and 3a, d). Type I cells are most frequently found in all the extensions of the cortex. These cells are spherical or oval and present a large nucleus compared to the cytoplasm, which contains few organelles, such as mitochondria (Figs. 1b; 2e–g; and 3a, d–e). The nuclei of type I cells

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ƒFig. 2

Electron micrograph of synganglion of males of Rhipicephalus sanguineus ticks. a–b General view of the synganglion showing wall of the blood sinus (w), neural lamella (nl), perineurium (pn), extensions of glial cells (arrow) into the cortex (c), subperineurium (sp), neuropile (np), and ganglion (g). c General view and d detail of perineurium (pn) formed by glial cells (gc) exhibiting mitochondria (m) and elongated nucleus (n), besides extensions of glial cells (arrow). e General view of cortex (c) showing the extensions of glial cells (arrow) forming extracellular spaces between glial cells and nervous cells of the cortex. Observe types I (I) and II (II) neural cells. f General view of types I (I) and II (II) neural cells, showing these cells are separated by extensions of glial cells (arrow). g Detail of type I (I) neural cell; observe nucleus (n) and few organelles. h Type II (II) neural cell exhibiting nucleus (n) and neurosecretory granules (ng); observe the direct contact between cell membranes of adjacent neural cells (asterisk) or separated by extensions of glial cells (arrow). i Detail of great amount of neurosecretory granules (ng) in the cytoplasm of type II neural cells. j–k Detail of the cytoplasm of type II neural cells showing rough endoplasmic reticulum (rer), Golgi region (Gr), vesicles (ve), mitochondria (m), nucleus (n), and neurosecretory granules (ng). l Detail of subperineurium (sp) between cortex (c) and neuropile (np); observe tracheal elements (tr). m Detail of neuropile exhibiting the neural fibers (nf), neurotubules (nt), mitochondria (m), and glial cells (gc)

present condensed chromatin accumulation adjacent to the nuclear envelope (Figs. 1b and 3e). Neural cells type II are polygonal and are distributed among type I cells. They present a great volume of cytoplasm in comparison with the other cell type as well as nucleus located in the periphery of the cell and with disperse chromatin (Figs. 1b; 2e–f, h; and 3a, d, and f). The cytoplasm of these cells contains a greater number of organelles in comparison with the type I cell, with well-developed rough endoplasmic reticulum, Golgi regions, vesicles, mitochondria, and several neurosecretory granules (Figs. 1b; 2 h–k; and 3f–h). The cortex cells can be in direct contact with each other through the junction of the cellular membranes and/or separated by the extensions of the perineurium glial cells (Figs. 1b; 2a, e–f, h; and 3d, f). The subperineurium is found between the cortex and the neuropile and presents the same ultrastructural characteristics of the perineurium; however, branches of the tracheas are found here (Figs. 1a–b; 2a–b, e, l; and 3a, i). The neuropile, inner structure of the synganglion, is constituted by ramifications of the neural cells, also called neural fibers, by the tracheal elements which penetrate the neuropile through the subperineurium and glial cells (Figs. 1a–b; 2a–b, e, l–m; and 3a, i, j–k). The neural fibers are the most common elements found in the neuropile. These structures are mainly formed by mitochondria, neurofilaments, and neurotubules surrounded by the extensions of the glial cells (Figs. 1b; 2 l–m; and 3j–k).

Discussion Over the last five decades, acaricides with different active ingredients have been used; however, different mechanisms of resistance have already been developed by the ticks as strategies to survive. The selection of resistant tick strains mainly occurs due to the incorrect use of acaricides (Häuserman et al. 1992). This is a relevant fact, once it is an irreversible process. For this reason and for the problems caused by the contamination of nontarget organisms and the environment, the search for less toxic and environment-impacting acaricide products has been intensified. However, to make this happen, morphophysiological studies on the different tick organs are necessary to better study the biology and physiology of these specimens; once, without this knowledge, it is not possible to develop viable alternatives to control ticks. In this sense, this study performs the ultrastructural description of the central nervous system (synganglion) of R. sanguineus males and females, since this organ is the target of neurotoxic acaricides. As described for ticks in general (Obenchain and Oliver 1976; Roshdy and Marzouk 1984; Binnington 1986; El Shoura 1989; Sonenshine 1991; Oliver et al. 1992; Prullage et al. 1992; Lees and Bowman 2007), the central nervous system of R. sanguineus males and females consists in a mass of fused nerves named synganglion, from where nerves emerge towards different parts of the body. The synganglion of R. sanguineus is found inside a cavity named periganglionic sinus, which is continuous to the aorta and heart. This location allows the synganglion to secrete and/or continuously absorb several essential substances from the hemolymph in order to maintain metabolism. This data corroborates Coons and Alberti (1999) on their studies about the internal morphology of ticks. In this study, the ultrastructural analysis did not show differences between the central nervous systems of R. sanguineus males and females, confirming preliminary light microscopy data by Roma et al. (2012). According to Coons et al. (1974), Ivanov (1983), El Shoura (1986), Sonenshine (1991), and Coons and Alberti (1999), the synganglion of Argasidae and Ixodidae ticks would be surrounded by the neural lamella, constituted by several layers of finely granular homogeneous material. The results obtained here corroborate these authors' findings, suggesting that this structure would have the function to support the tissues located in its interior, in addition to resisting to the hydrostatic pressure and acts as a selective permeability barrier for several nutrients and ions from the hemolymph. The perineurium or periganglionic membrane is just below the neural lamella, and it is formed by glial cells, which

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ƒFig. 3

Electron micrograph of synganglion of females of Rhipicephalus sanguineus ticks. a–b General view of the synganglion showing neural lamella (nl), perineurium (pn), cortex (c) with types I (I) and II (II) neural cells, subperineurium (sp), and neuropile (np). Observe in b the extensions (arrow) of glial cells (gc) of perineurium into the cortex, besides the great amount of neurosecretory granules (ng) in type II (II) neural cells. c Detail of neural lamella (nl) and perineurium (pn), showing the glial cell (gc) with many mitochondria (m), elongated nucleus (n), and extensions (arrow). d General view of types I (I) and II (II) neural cells, showing these cells are separated by extensions of glial cells (arrow). e Detail of type I (I) neural cells exhibiting nucleus (n) and few organelles, such as mitochondria (m). f Type II (II) neural cell exhibiting nucleus (n), neurosecretory granules (ng), mitochondria (m), Golgi region (Gr), and rough endoplasmic reticulum (rer). Observe the direct contact between cell membranes of adjacent neural cells (asterisk) or separated by extensions of glial cells (arrow). g–h Detail of the cytoplasm of the type II (II) neural cell showing neurosecretory granules (ng), mitochondria (m), Golgi region (Gr), vesicles (ve), besides well-developed rough endoplasmic reticulum (rer). i Detail of subperineurium (sp) between cortex (c) and neuropile (np); observe tracheal elements (tr). j General view and k detail of neuropile exhibiting the neural fibers (nf), neurotubules (nt), and mitochondria (m); observe the glial cells (gc) among the neural fibers. l Esophagus (es) into the neuropile (np)

extensions penetrate throughout the nervous tissue. These invaginations form extracellular spaces among the cortex cells. These results corroborate the findings of Coons et al. (1974) and Binnington and Obenchain (1982) for Argas arboreus and Boophilus microplus, respectively. According to these authors, these extracellular spaces would function as a reservoir of cations which would be used during the regulation of the ionic concentration of the cortex cells, which would certainly be occurring here, once several mitochondria were found in the glial cells of the perineurium of R. sanguineus males and females. These organelles would probably be involved in the production of the necessary energy for this process to occur (through the synthesis of ATP). However, these spaces were not observed in the nervous tissue of Amblyomma americanum larvae (El Shoura 1989). In this study, it is also suggested that the extensions of the glial cells would increase the contact area between these cells and those found in the cortex, allowing the exchange of substances between them. According to Sonenshine (1991), the glial cells would play a fundamental role in the metabolic regulations of the neural activities. Thus, it is suggested that these cells in R. sanguineus males and females would be involved in the transportation of elements from the hemolymph (absorbed via neural lamella, according to Marzouk et al. 2001) to the cortex cells, a function which is essential for the maintenance of the physiological processes which are inherent to this organ. Thus, the elements synthetized by the cortex neural cells would be secreted in the periganglionic sinus through the transportation to the perineurium and posteriorly to the neural lamella, which due to its selective permeability

(Marzouk et al. 2001) would eliminate the neurosecretion directly in the hemolymph, controlling the general metabolism of all the organs and tissues of the ticks. Coons and Alberti (1999) reported that deposits of glycogen would be found in the glial cell cytoplasm of engorged tick females and used during the oviposition of these ectoparasites. However, this element was not found in the glial cells of R. sanguineus females, probably due to the fact that these females were fasting, not having started the engorgement phase. The synganglion of R. sanguineus males and females, as of ticks in general, is covered by an outer region named cortex which contains neural cell bodies, called neurons by some authors (Sonenshine 1991; Coons and Alberti 1999). Two types of cells can be identified in this region, corroborating the studies of Coons et al. (1974), Ivanov (1983), and El Shoura (1986) for other species of ticks. Type I neural cells are oval or spherical and present a large nucleus occupying most part of the cytoplasm. According to these data, these cells would be similar to the motor neurons described by El Shoura (1989) and Coons and Alberti (1999) for other species of ticks and would be involved in the transmission of impulses from the central nervous system to the target organs. In the cytoplasm of these cells, both in R. sanguineus males and females, few organelles were observed, such as mitochondria, suggesting that type I neural cells would not be related to the synthesis of neurosecretory elements, and the organelles would be involved in the maintenance of the cell's metabolism. The hypothesis that type I cells would not be neurosecretory are confirmed in the results by Coons et al. (1974) and Ivanov (1983) for other species of ticks. However, El Shoura (1986) reported that Ornithodoros erraticus females and A. americanum larvae would play an important role in the synthesis of neurosecretory granules. Moreover, according to Sonenshine (1991), some authors believe that the neurosecretory activity capacity of the neural cells would be a phase of the cellular cycle which is not limited to a certain type of cell. According to Coons and Alberti (1999), type II neural cells would be distributed among type I cells, forming small groups, the neurosecretory centers. They are characterized by a great volume of cytoplasm and nucleus located in the periphery of the cell. These data corroborate the ones found for the synganglion of R. sanguineus males and females. A greater number of organelles were observed in the cytoplasm of these cells in comparison with type I cell, such as, well-developed rough endoplasmic reticulum and Golgi regions, vesicles, mitochondria, and several deposits of neurosecretory granules. These results suggest that type II cells would be

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secretory, corroborating descriptions by Ivanov (1983) and El Shoura (1986) while studying the synganglion of Argasidae and Ixodidae. El Shoura (1989) described a third type of neural cells (type III) in A. americanum larvae, which would have a large nucleus and narrow cytoplasm with few organelles; however, the function of these cells would not be known yet. It should be stressed that this type of cell was not identified in this study. Considering the results here obtained, it can be suggested that the communication between the neural cells of the cortex (types I and II) would happen through the direct contact between cells, once in some regions of the membrane of adjacent cells would be fused. These membranes could be acting as a barrier of selective permeability, controlling the transportations of material in both directions (intra- and extracellular). On the other hand, the union of the membranes of adjacent cells could also be related with the provision of greater cohesion to this tissue. However, in some regions, the neural cells are separated by the extensions of the perineurium glial cells, which, as described above, would control all the metabolism of the tick central nervous system. Between the cortex and the neuropile of R. sanguineus synganglion, as described for ticks in general (Coons and Alberti 1999), is the subperineurium, which presents the same ultrastructural characteristics of the perineurium. However, branches of tracheas were found in this region, which would be providing the necessary oxygen supply to this tissue of high energetic consumption, considering the functions performed. The neuropile is the most complex region of the central nervous system of the ticks (El Shoura 1989). In R. sanguineus males and females, as well as in other ticks’ species (Sonenshine 1991; Coons and Alberti 1999), it is formed by the ramifications of types I and II nervous cells by the tracheal elements and glial cells. Most neural fibers are surrounded by the extensions of glial cells, which can come from two sources: (1) the cells located in the neuropile itself or (2) the extensions of the glial cells of the subperineurium. Thus, considering data here described, it can be concluded that the central nervous system of R. sanguineus males and females presents an ultrastructural organization similar to the one described in literature for other species of ticks. Moreover, the importance of this kind of study for the improvement and discovery of new efficient methods to control ticks without harming hosts and the environment is emphasized here. Acknowledgments The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (grant nos. 2010/519428, 2011/06865-8, and 2011/10427-6) for financial support. Conflict of interest The authors declare that there are no conflicts of interest.

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