Parasitol Res DOI 10.1007/s00436-012-3173-6
ORIGINAL PAPER
Rhipicephalus sanguineus (Acari: Ixodidae) female ticks exposed to castor oil (Ricinus communis): an ultrastructural overview B. R. Sampieri & K. C. S. Furquim & P. H. Nunes & M. I. Camargo-Mathias
Received: 25 September 2012 / Accepted: 8 October 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract 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. B. R. Sampieri : K. C. S. Furquim : P. H. Nunes : M. I. Camargo-Mathias (*) Laboratório de Histologia, UNESP, Av. 24-A, 1515, Jardim Bela Vista, PO Box 199, 13506-900 Rio Claro, São Paulo, Brazil e-mail:
[email protected]
Introduction Currently, several studies with ticks are being carried out to develop new ways to control these ectoparasites. Finding substances that generate minimal waste to the environment and are harmless to nontarget organisms, while acting effectively on the parasite, is the main focus of such studies (Denardi et al. 2010; Roma et al. 2011). Many recent studies have brought important data on the ecology and life cycle of ticks of medical and veterinary importance, in order to control these animals (Labruna 2004; Leal et al. 2003), mainly on species that parasite small and large ruminants, which represents the most important animal groups to livestock in development countries (Sajid et al. 2011). However, little has been studied about the internal morphology and physiology of ticks, which hinders the understanding of their systems regarding to acaricide exposure. The Brazilian Center of Studies on Ticks Morphology has sought to modify this scenario, bringing to light knowledge on the morphophysiology of the principal organs of tick species with medical and veterinary importance. Such studies aim to clarify how and when acaricides act in these ectoparasites, as well as generating useful information for the development of new products to combat this pest and/or new methods to control it, as the resistance was develop by ticks when hosts are exposed (previously to the infestation) to ticks saliva extract (Furquim et al. 2011). Research on substances of plant origin with potential pesticide activity against both agricultural and urban pests has been promising, despite the lack of interest from the world’s major pesticide manufacturers in subsidize studies with this class of products (Chagas et al. 2002). Oils and plant extracts, such as oil and leaf extracts of neem (Meliacea), showed encouraging results because they caused morphological changes in Rhipicephalus sanguineus
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ovaries (Denardi et al. 2010) and high mortality rates in Dermacentor variabilis (Landau et al. 2009). More recently, ricinoleic acid esters from castor oil (Ricinus communis) emerged as a potential alternative because interesting results were obtained when they were used as a biocide in experiments with Leuconostoc mesenteroides (Messetti et al. 2010) and in pilot tests with Rhipicephalus (Boophilus) microplus (Chierice, personal information). Arnosti et al. (2011) and Sampieri et al. (2012a, b) also reported positive results when administering the esters to the hosts through diet, which acted on the morphophysiology and ultrastructure of salivary glands and ovaries of engorged R. sanguineus. Therefore, this study brings to light new data on the action of ricinoleic acid esters from castor oil on the ultrastructure of somatic cells (ovary wall and pedicel cells) and germ cells (oocytes) of semi-engorged (4-day feeding) R. sanguineus, by testing two esters concentrations: 2 and 5 g/kg of food.
Materials and methods For this work, 120 couples of R. sanguineus (120 males and 120 females) were divided into eight groups. In each group, 30 couples were placed on a New Zealand White rabbit. Four groups were used as controls and four as treatment, where the effects of esters of castor oil in concentrations of 2 and 5 g/kg of food were tested, according to protocol developed by Arnosti et al. (2011). The ricinoleic acid esters were incorporated in NaCl and added to the rabbit food, which justified the addition of only NaCl to the rabbit food of the control groups (CGs). Esters of ricinoleic acid from castor oil were kindly provided by Prof. Dr. Gilberto Orivaldo Chierice of the Department of Chemistry and Molecular Physics at the USP Institute of Chemistry of São Carlos, SP, Brazil. Procedures performed in this study were approved by the ethics committee Comitê de Ética no Uso de Animal Protocol 006/2009. Study groups Control groups Four host rabbits were classified as control 1 (CG1), control 2 (CG2), control 3 (CG3), and control 4 (CG4). They were fed with commercial rabbit food with added NaCl. The CG1 host was given rabbit food + NaCl (2 g of NaCl/kg of food) for 7 days before being infested with 30 pairs of R. sanguineus. The CG2 host was fed the same diet given to CG1, but feeding was started concomitantly with the infestation of 30 pairs of R. sanguineus. The CG3 host was fed rabbit food + NaCl (5 g of NaCl/kg of food) for 7 days before infestation
with 30 pairs of R. sanguineus. The CG4 host was fed the same diet given to CG3, but feeding began concurrently with the infestation of 30 pairs of R. sanguineus. Treatment groups Four host animals were used in the treatment groups, identified as treatment 1 (TG1), treatment 2 (TG2), treatment 3 (TG3), and treatment 4 (TG4). They were fed with commercial rabbit food with added ricinoleic acid esters synthesized from castor oil (R. communis) at concentrations of 2 and 5 g of esters/kg of food, incorporated in NaCl. The TG1 host was fed rabbit food + NaCl + 2 g of esters/ kg of food for 7 days before infestation with 30 pairs of R. sanguineus. The TG2 individual received the same diet given to TG1, but feeding began concurrently with the infestation of 30 pairs of R. sanguineus. The TG3 host was given rabbit food + NaCl + 5 g of esters/kg food 7 days before infestation with 30 pairs of R. sanguineus. The TG4 individual received the same diet given to TG3, but feeding began concurrently with the infestation of 30 pairs of R. sanguineus. In all CGs and TGs, the diet was maintained until the female ticks were collected (4 days). Later, the female ticks were collected and anesthetized by thermal shock (in the refrigerator) and dissected. The ovaries were removed in saline solution (0.13 M NaCl, 0.017 M Na2HPO4, 0.02 M KH2PO4, pH7.2) and fixed according to the transmission electron microscopy technique. Transmission electron microscopy Ovary fragments of R. sanguineus were fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer (pH7.2) for 2 h. Two 15-min washes were carried out in the same buffer, and after fixation, they were placed in a 1 % osmium tetroxide solution at 0.1 M sodium cacodylate buffer for 2 h, in the dark and at room temperature. Subsequently, the material had two more washes (15 min each) in 0.1 M sodium cacodylate buffer (pH7.2), and it was then contrasted in 2 % uranyl acetate with 10 % acetone, for 4 h in the dark. Serial dehydration in increasing acetone concentrations of 50, 70, 90, 95, and 100 % was carried out twice, for 5 min each. Soon after, the material remained in the mixture of Fig. 1 a–f Overview and details of periphery region of oocytes II and pedicel cells of R. sanguineus semi-engorged females from CG1, where a cytoplasm with few yolk granules and organelles can be observed (h–k). Overview and details of periphery region of oocytes II and pedicel cells of R. sanguineus semi-engorged females from CG2, where the cytoplasm, as well as the CG2, shows few yolk granules and many mitochondria. BL basal lamina, GV germ vesicle, l lipid yolk, mi mitochondria, nu nucleus, oo oocyte, pc pedicel cell, Va autophagic vacuoles
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acetone and resin (Epon-Araldite) in a 1:1 ratio for 12 h. The material was embedded in pure resin with a catalyzer and placed in an oven at 60 °C for 24 h for polymerization. The blocks were then cut on an ultramicrotome Sorvall PorterBlum MT2-B. Semi-thin sections were stained with azure II (1 %) and methylene blue (1 %). Ultrathin sections were collected on copper grids and contrasted with uranyl acetate and lead citrate for 45 and 10 min, respectively. The material was observed and photographed under a PHILIPS CM 100 transmission electron microscope.
Results Ultrastructural changes observed in this study occurred to a lesser degree of severity when compared to those related to fully engorged females (Sampieri et al. 2012a). Control groups The results obtained in this study (Figs. 1 and 3) corroborate those reported by Oliveira et al. (2005), who described the ultrastructure of R. sanguineus normal ovaries. Data generated from the control groups (CG1, CG2, CG3, and CG4) of this work will not be described, but only used as a reference for comparison with the results related to the treatment groups (TG1, TG2, TG3, and TG4). Treatment group 1 The oocytes from TG1 exhibit specific morphology still preserved and plasma membrane without invaginations (Fig. 2a). An apparent increase of space between the plasma membrane and the basal lamina, which shows detachment of components, is observed (Fig. 2a, c). The cytoplasm has a few small yolk granules, which are most often lipid-originated (stages I and II of development) (Fig. 2a). Several lightly electron-dense mitochondria with defined and organized cristae, as well as autophagic vacuoles, were observed (Fig. 2a, b). The region of contact between the oocyte and the pedicel cells shows to be unchanged when compared to the CG1, especially regarding the oocyte microvilli, which exhibit unaltered morphology (Fig. 2c, d). Pedicel cells do not show apparent morphological changes, but the cytoplasm has autophagic vacuoles (Fig. 2c), several multivesicular bodies, and previtellinic protein granules (Fig. 2f). Treatment group 2 The oocytes from this group show a slightly modified morphology compared to TG1 and CG2, where the plasma membrane has small invaginations. Also, at some points
along the oocytes’ periphery, there is loss of contact between the plasma membrane and the basal lamina (Fig. 2g). These oocytes possess a large number of mitochondria with morphology varying from the elongated to the elliptical. Some of them are less electron-dense than those observed in TG1 and CG2, although their cristae are still preserved (Fig. 2m). Few yolk granules of lipid origin are observed (Fig. 2h, i). Autophagic vacuoles and myelin figures are distributed on the periphery of the oocyte (Fig. 2l). Great Golgi activity is observed, where a large number of vesicles that are probably in transit through the cisternae (Fig. 2j, k). Polysomes and multivesicular bodies are also seen (Fig. 2l, m). The germinal vesicle (nucleus) has a membrane with small invaginations and the nucleolus is not observed (Fig. 2i). The region of contact between oocytes and the pedicel cells is abnormal because the microvilli are disorganized and in a smaller number, compared to the TG1 and CG2 (Fig. 2g, h). The pedicel cells show no significant morphological changes and preserved plasma membranes. The cytoplasm has some preserved mitochondria and the shape of the nucleus ranges from spherical to elliptical, with dense heterochromatin and loose euchromatin, as in CG2 (Fig. 2g, h). Treatment group 3 Oocytes from this group are spherical to elliptical and exhibit similar morphology to those from TG2, with few membrane invaginations (Figs. 4a, b). The cytoplasm shows large number of mitochondria with few cristae and lower electron density than the preceding groups, apparently involved with lipid synthesis (Fig. 4h). Few lipid-originated yolk granules (Fig. 4f, g) are observed. Autophagic vacuoles and multivesicular bodies are in greater numbers than in TG1 and TG2, and the CGs (Fig. 4b, e, f). The region of contact between the oocyte and the pedicel cells has modifications, such as fewer and shorter microvilli (Fig. 4d). The basal lamina that surrounds the oocyte and the Fig. 2 a–e Overview and details of periphery region of oocytes II and pedicel cells of R. sanguineus semi-engorged females from TG1, where the presence of mitochondria and some autophagic vacuoles can be detected. f Cytoplasm details of pedicel cells housing vesicles with pre-vitelinic material (arrows) from TG1. g–i Overview and details of periphery region of oocytes II and pedicel cells of R. sanguineus semi-engorged females from R. sanguineus from TG2, where microvilli details of contact region between oocytes and pedicel cells can be observed (arrow head) and do nuclear envelope (arrow). j–m Oocytes cytoplasm details from TG2 individuals with presence of large areas with Golgi regions surrounded by vesicles (arrow head) and lightly electron-dense mitochondria (arrows) and polysomes (pointed circle). BL basal lamina, GV germ vesicle, l lipid yolk, MF myelin figure, mi mitochondria, mv microvilli, nu nucleus, oo oocyte, pc pedicel cell, Va autophagic vacuoles
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pedicel shows an irregular surface, and at some points, the rupture of its components is observed (Fig. 4d).
The pedicel cells show intact morphology, similar to those observed in the CGs (Fig. 3); however, it is already
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possible to observe cytoplasmic and autophagic vacuoles and numerous mitochondria (Fig. 4a, b). The nuclei have euchromatin and heterochromatin (Fig. 4a, b). Treatment group 4 Oocytes from this group show more pronounced morphological changes, with small invaginations in the plasma membrane and the loss of contact in some areas between the plasma membrane and the basal lamina (Fig. 4i). There are a large number of lightly electron-dense mitochondria with disorganized cristae and a higher number of autophagic vacuoles than in previous groups (Fig. 4j, k). Golgi vesicles and multivesicular bodies are also observed (Fig. 4j, k, n–p). The region of contact between the oocyte and pedicel cells has fewer and severely disorganized microvilli, compared to those from previous TGs and CGs (Fig. 4m). The pedicel cells show more evident interdigitations than in previous treatments, but their overall morphology was not altered (Fig. 4p). The cytoplasm has many mitochondria of various shapes and sizes, but they are lightly electron-dense and show slightly apparent disorganized cristae, as well as some autophagic vacuoles, multivesicular bodies, rough endoplasmic reticulum cisternae, and lysosomal vesicles (Fig. 4m, n–q).
Discussion The data obtained in this study corroborate and complement those reported by Arnosti et al. (2011) and Sampieri et al. (2012a, b), when studying R. sanguineus engorged females, where early alterations caused by esters of ricinoleic acid from castor oil in the vitellogenesis and ultrastructure of somatic and germ cells of the ovaries are demonstrated. In the present study, the changes observed in the ovary ultrastructure of engorged R. sanguineus indicated that, in general, the esters had a toxic effect, though to a lesser degree when compared with the results obtained from engorged females, when the oocytes were already in advanced stages of development and the chorion is in final deposition process (Sampieri et al. 2012a).
Fig. 3
a–c Overview of oocyte II and pedicel cells of R. sanguineus semi-engorged females from CG3 with microvilli details from contact region between the oocyte and pedicel cells (arrows) and basal lamina (arrow head). d Cytoplasm detail of oocyte II from CG3 showing Golgi regions and vesicles with lightly electron-dense content (arrow head). e–f Oocyte II overview of R. sanguineus semi-engorged females from CG4, showing periphery region (arrows) and cytoplasm details where some autophagic vacuoles can be observed (circle). g–h Cytoplasm detail of oocyte II from CG4 with Golgi regions surrounded by vesicles (arrow head) and mitochondria. i–k Overview of contact region between an oocyte II and a pedicel cell from CG4, with microvilli details and its interdigitations (arrows). BL basal lamina, GV germ vesicle, l lipid yolk, mi mitochondria, mv microvilli, nu nucleus, oo oocyte, pc pedicel cell, Va autophagic vacuoles
In TG1 and TG2 (2 g of esters/kg of food), a more subtle effect on the ovary cells was observed, although with signs of toxicity, such as autophagic vacuoles and changes in the plasma membrane, which occurred to a lesser degree when compared to the TGs with 5 g of esters/kg of food, corroborating Arnosti et al. (2011). In this study, different changes in the ovaries from the two treatment groups were observed. In TG2, the microvilli located in the region of contact between the oocyte and the pedicel cells were fewer and more disorganized than in TG1, confirming that the early administration of esters to the hosts, prior to infestation, makes the product more efficient against ticks, and its action over the yolk components of this specie, corroborating Sampieri et al. (2012b), when studying engorged females of the same species. Likewise, the presence of an extensive Golgi region in the oocytes from TG2, releasing enzyme vesicles which are probably hydrolytic, suggested the occurrence of intracellular lysis. In TG4, this was more evident by the presence of many autophagic vacuoles and mitochondria. In TG2 and TG4, knowing that the esters of ricinoleic acid derivatives of castor oil have the chemical property of hydrolyzing polysaccharides, it can be inferred that this organelle also act synthesizing polysaccharides, packaging and addressing proteins, could be synthesizing more actively new molecules of polysaccharides to compensate for the lytic action caused by the esters of this class of molecule, in addition to being more active in the release of lysosomes for the digestion of organelles damaged by the chemical. Probably, there was a direct effect of esters on the material of the multivesicular bodies present in oocytes from the TG3 and TG4 because the content of these structures had lower electron density in these groups than in the others. Due to their glycoprotein content, esters would have acted on these molecules, hydrolyzing them. These results reinforced the view that ricinoleic acid esters from castor oil acted upon the organelles, even when administered in low concentrations. The mitochondria from TG2 and TG4 oocytes were observed within autophagic vacuoles, suggesting that their replacement in the oocytes of exposed ticks would become more urgent because the organelles would lose faster their morphological and consequently physiological characteristics. Studied R. sanguineus fully engorged females exposed to permethrin. This acaricide affected more severely the ovaries of semi-engorged females than fully engorged, because in the first, the oocytes were more susceptible to the action/ effects of external chemical agents, mainly by the absence of the chorion in the early stages of development. The present study demonstrated that, in the case of esters of ricinoleic acid from castor oil, exposure time to the product was a determining factor for an effective action on
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Fig. 4
a–e Overview of oocyte II and pedicel cells of R. sanguineus semi-engorged females from TG3 with microvilli details from contact region between the oocyte and pedicel cells and basal lamina (arrow) and cytoplasm where some autophagic vacuoles can be observed (arrow head). f–h Cytoplasm detail of oocyte II from TG3 showing great presence of autophagic vacuoles, mitochondria with lipidic drop (arrow), and multivesicular bodies (arrow). i–k Overview of oocyte II/ III and pedicel cells from TG4, showing detail of large presence of autophagic vacuoles and lysosomal vesicles (arrows). l–q Periphery region and cytoplasm details of oocytes II or III and pedicel cells from TG4 with large presence autophagic vacuoles, lightly electron-dense mitochondria, and many vesicular bodies (arrows) besides rough endoplasmic reticulum. GV germ vesicle, l lipid yolk, mi mitochondria, nu nucleus, oo oocyte, pc pedicel cells, RER rough endoplasmic reticulum, Va autophagic vacuoles
the oocytes. The fully engorged females treated with 5 g of esters/kg of food showed the most severe alterations in mature oocytes, especially in IV and V, where chorion deposition had already occurred and was compromised, thus suggesting that this process was influenced by the esters which probably acted hydrolyzing polysaccharides. Esters administered to the host animals from TG2 and TG4 (fed with enriched diets before being infested with ticks) acted with greatest severity on the oocytes. In these, major changes were observed in the oocytes, because when feeding started, esters were already present in the systemic circulation of the host, unlike in TG1 and TG3, where infestation occurred on the same day the administration of esters began, which caused a delay in esters availability in the blood. Therefore, the availability of esters in the feed cycle of R. sanguineus, which in experimental conditions lasts a maximum of 7 days, showed that if used in the control of ticks (as a food additive), they must be previously administered to the host. Thus, the present study brought more data on this natural acaricide that acted directly on the reproductive system of R. sanguineus females, affecting vitellogenesis and inhibiting the development of the new individual. At the same time, it can be inferred that esters can also act over Rickettsia-like microorganisms and its dynamic into ticks oocytes (Lewis 1979), once the substance is an effective biocide as demonstrated by Messetti et al. (2010). It also provided information that could support further studies with substance of plant origin, generating the expected development of products able to effectively control this type of urban pest and tick-borne pathogens.
Acknowledgments This work was financially supported by Fundação de Amparo à Pesquisa do Estado de São Paulo through grants no. 2009/12387-1, no. 2009/54125-3, and 2012/02384-8, and Conselho Nacional de Desenvolvimento Científico e Tecnológico through research fellowships to M.I. Camargo-Mathias. The authors thank Professor Dr. Salvador Claro Neto and Professor Dr. Gilberto Orivaldo Chierice for the technical support. Financial support Financial support was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil).
Conflict of interest There is no conflict of interest to declare.
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