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Amblyomma triste (Koch, 1844) (Acari: Ixodidae)
Experimental Parasitology 116 (2007) 407–413 www.elsevier.com/locate/yexpr
Amblyomma triste (Koch, 1844) (Acari: Ixodidae) Ovaries: An ultrastructural analysis Patrı´cia Rosa de Oliveira a, Gerva´sio Henrique Bechara b, Maria Izabel Camargo Mathias b
a,*
a Departamento de Biologia, I.B., UNESP, Av. 24 A, No. 1515, Cx. Postal 199, CEP: 13506-900, Rio Claro, SP, Brazil Departamento de Patologia Veterina´ria, FCAV, UNESP, Via de Acesso Prof. Paulo Castellane, s/n, CEP: 14884-900, Jaboticabal, SP, Brazil
Received 19 April 2006; received in revised form 11 February 2007; accepted 14 February 2007 Available online 28 February 2007
Abstract This study presents an ultrastructural analysis of the ovary of the tick, Amblyomma triste. The ovary of this species is of the panoistic type that is, without nursing and follicular cells. It is composed of a layer of epithelial cells forming a wall and of germinative cells that generate the oocytes which remain attached to the external margin of this wall by a multicellular pedicel. The different developmental stages in the oocytes had been described by Oliveira et al. [Oliveira, P.R., Bechara, G.H., Camargo-Mathias, M.I., 2006. Amblyomma triste (Koch, 1844) (Acari: Ixodidae): Morphological description of the ovary and of vitellogenesis. Experimental Parasitology 113, 179–185]. The results of the investigation suggest that besides exogenous production of vitellogenic elements, endogenous production can take place simultaneously, contributing to the development and growth of the oocytes. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Amblyomma triste; Tick; Ovary; Vitellogenesis; Ultrastructure
1. Introduction The economic importance of ticks is widely acknowledged and is related to their feeding habits. During feeding, many species of ticks can transmit diseases caused by protozoa, viruses, Rickettsias and spirochetes (Rey, 1973) to man and other animals. The tick species Amblyomma triste is distributed in the Neotropics and has been reported in Equador (Keirans, 1984), Argentina (Ivancovich, 1980), Uruguay and Brazil (Sinkoc et al., 1997) infesting tapir (Kohls, 1956), dogs (Correa, 1954), capybara (Hydrochaeris hydrochaeris) (Sinkoc et al., 1997), marsh deer Blastocerus dichotomus (Szabo´ et al., 2003) and humans (Venzal et al., 2003). It has also been reported also from opossum (Didelphis marsupialis) in an endemic area for Brazilian spotted fever in Pedreiras, State of Sa˜o Paulo (Lemos et al., 1997).
*
Corresponding author. Fax: +55 19 3526 4135. E-mail address: [email protected] (M.I. Camargo Mathias).
0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.02.006
According to Till (1961), Sonenshine (1991) and Said (1992), the female reproductive system of ticks generally consists of a large U-shaped ovary located at the posterior region of the body, with a pair of oviducts, an uterus, a muscular connection tube, a vagina and a genital opening. In this report, we describe the ultrastructure of the ovary, thereby contributing to our understanding of vitellogenesis in A. triste. 2. Materials and methods Semi-feeding A. triste females from the tick colony were maintained under controlled conditions (28 °C, 80% humidity and 12 h light/dark cycle) at the Department of Animal Pathology, Veterinary College, UNESP – Jaboticabal, SP, Brazil. Twenty-five specimens maintained in the refrigerator for thermal shock anesthesia were dissected in a saline solution (NaCl 7.5 g/L, Na2HPO4 2.38 g/L and KH2PO4 2.72 g/L).
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2.1. Transmission electron microscopy (TEM) The material was fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and embedded in Epon Araldite. The material was then embedded in pure Epon resin and polymerized at 60 °C for 72 h. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Afterwards, screens containing ultrathin sections of the material were examined and photographed in a Phillips 100 Transmission Electron Microscope (TEM). 3. Results The ovary wall of the tick A. triste consists of small epithelial cells that proliferate to form the pedicel, a structure that attaches the oocytes at different stages of development to the ovary wall (Fig. 1a). The cells are small and of a pavimentous type. The large, oval nucleus occupies most of the cytoplasm which only contains a few mitochondria (Fig. 1a). A thick, mildly electron dense, basal lamina, supports this epithelium.
The cells are connected to each other by means of interdigitations of their lateral membranes (Fig. 1a). Stage I oocytes possess a homogeneous cortex with few ribosomes, either free or attached to the membrane of the lamellar rough endoplasmic reticulum, which is little developed (Fig. 1b and c). However, large numbers of elliptical, round or elongated mitochondria were detected. They are relatively small and occupy the region around the germ vesicle. This is located in the center of the stage I oocyte, having a rounded appearance, dispersed chromatin and a large nucleolus with distinct granular and fibrous regions (Fig. 1b). Surrounding the oocyte, there is an electron dense plasmic membrane containing some microvilli, mainly in the direction of the pedicel (Fig. 1d) and the basal lamina which is in two layers: an inner, thicker layer, which is in direct contact with the microvilli, and an outer, thinner layer which has a fibrillar appearance. In the region between the inner layer of the basal lamina and the membrane of the oocyte, vesicles containing material probably
Fig. 1. (a) Ultrastructure of the epithelium from the ovary of Amblyomma triste. (b) General view of oocyte I. (c) Detail of the cytoplasm near the germ vesicle of oocyte I. (d) Peripheral region of oocytes I. bl, basal lamina; pm, plasmic membrane; ep, ovary epithelium; n, nuclei; ne, nuclear envelope; m, mitochondria; gv, germ vesicle; nu, nucleollus; lrer, lamellar rough endoplasmic reticulum; mv, microvilli. Bars: A, 2 lm; B, 2 lm; C, 2 lm; D, 2 lm.
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being transported from the pedicel cells to the oocyte, can be observed (Fig. 1d). Stage II oocytes are larger than those of stage I. The cytoplasm begins to show small yolk granules with variable morphology and content, located throughout the cytoplasm. Granules with a lipid content are electrolucent; while the protein containing granules possess high electron density (Fig. 2a and b). Mitochondria with variable shapes, distributed all over the cytoplasm, are found more frequently than other organelles. Free ribosomes are also seen, along with some myelin bodies and under-developed lamellar and vesicular rough endoplasmic reticulum (Fig. 2a and b). Stage II oocytes have more microvilli in their plasmic membrane, supported by a thick basal lamina subdivided
409
in two portions. Vesicles and electron dense material between the membrane and the basal lamina are observed (Fig. 2c). In Stage III oocytes, the germ vesicles are still seen (Fig. 2d). There are numerous rounded mitochondria throughout the cytoplasm, some with disorganization of their crests and membranes. Myelin bodies, Golgi complex and lamellar and vesicular rough endoplasmic reticulum are also present (Fig. 2e and f). In the cytoplasm, yolk granules (termed p1) are also found. They are homogeneous, with a foamy appearance, being moderately electron dense with protein content, as well as having electrolucent lipid droplets (Fig. 2e and f). Microvilli with similar characteristics to previous stages are still present in the plasmic membrane in these oocytes (Fig. 2d and f).
Fig. 2. (a) General view of oocyte II (II). (b) Detail of the central cytoplasm of oocytes II. (c) Peripheral region of oocytes II. (d) General view of oocyte III (III). (e) Detail of the central cytoplasm of oocytes III. (f) Peripheral region of oocytes III. bl, basal lamina; mv, microvilli; m, mitochondria; n, nuclei; p, pedicel; lrer, lamellar rough endoplasmic reticulum; pm, plasmic membrane; l, lipid granule; vrer, vesicular rough endoplasmic reticulum; gv, germ vesicle; g, Golgi complex; arrow, proteic yolk granules. Bars: A, 10 lm; B, 2 lm; C, 1 lm; D, 10 lm; E, 2 lm; F, 2 lm.
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In Stage IV oocytes, the germ vesicle, now with obvious irregular morphology, can still be observed, with many reentrances in its envelope, some heterochromatin as well as a highly electron dense accumulation of small granules and rRNA (nuage) around it (Fig. 3a and b). Many yolk granules with varied contents and different electron densities are found. Free or grouped electrolucent lipid droplets mainly occupy regions near the periphery (Per) (Figs. 3e and 4a, b, d). The granules with protein con-
tent occupy all the cytoplasm and are present in two forms: as moderately electro dense and homogeneous granules (p1) and, less frequently, with high density granulation, that probably contain glyco or lipoprotein complexes, called here p2 (Figs. 3c, e and 4a, b, d). Mitochondria, underdeveloped lamellar rough endoplasmic reticulum and Golgi complex are present (Figs. 3c–e and 4a–d). In the periphery of the stage IV oocyte, microvilli are found that are longer than those of previous stages
Fig. 3. (a) Cytoplasm near the germ vesicle of oocyte IV. (b) Detail of the cytoplasm near the germ vesicle of oocyte IV. (c) Peripheral region of oocytes IV. (d) Detail of the peripheral cytoplasm of oocytes IV. (e) Central cytoplasm of oocytes IV. g, Golgi complex; l, lipid granule; m, mitochondria; gv, germ vesicle; mv, microvilli; ne, nuclear envelope; ch, chorium; pm, plasmic membrane; lrer, lamellar rough endoplasmic reticulum; vrer, vesicular rough endoplasmic reticulum; head arrow, granules and nuage around the germ vesicle; arrow, proteic yolk granules. Bars: A, 2 lm; B, 1 lm; C, 1 lm; D, 1 lm; E, 2 lm.
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Fig. 4. (a) Cytoplasm of oocytes IV. (b) Detail of the central cytoplasm of oocytes IV. (c) Detail of the intermediary cytoplasm of oocytes IV, showing proteic yolk granules. (d) Detail of the peripheral cytoplasm of oocytes IV, showing lipid granules. m, mitochondria; l, lipid granule; lrer, lamellar rough endoplasmic reticulum; vrer, vesicular rough endoplasmic reticulum; arrow, proteic yolk granules. Bars: a, 1 lm; b, 1 lm; c, 1 lm; d, 1 lm.
(Fig. 3c and d) and having a larger space between the plasmic membrane and basal lamina. Here the chorium is being deposited through exocytic vesicles. 4. Discussion Transmission electron microscopy has confirmed the classification of the ovaries of the tick A. triste as panoistic, where all germinative cells correspond to oogonia or future oocytes. This classification was based on that adopted by Denardi et al. (2004) for Amblyomma cajennense, and for Rhipicephalus sanguineus (Oliveira et al., 2005), Boophilus microplus (Saito et al., 2005) and A. triste (Oliveira et al., 2006) . The development stage of the oocytes was described according to the proposal by Oliveira et al. (2006), where the main characteristics considered were the aspect of the cytoplasm, the localization of the germ vesicle, the presence, quantity and constitution of yolk granules and the presence of chorium. Stage I oocytes had a homogeneous cortex with many mitochondria and the absence of yolk granules. Stage II oocytes showed small yolk granules, larger amounts of mitochondria, but little rough endoplasmic reticulum, which appear under the form of cisternae of low electron density. In the beginning of the development of the oocytes (stage I) of A. triste, the mitochondria are preferentially
located around the germinal vesicle. According to Balashov (1983), these are associated with granules and fibrils of riboproteins. The function of this association of the mitochondria is still unknown. Later, the mitochondria are found throughout the cytoplasm of oocytes, pointing to a preparation of these cells for the period of exogenous yolk incorporation, in which these organelles become a prerequisite for the active transportation of material (Balashov, 1972). In the periphery next to the pedicel in stage I and II oocytes, small vesicles and microvilli are found, suggesting that at this stage there is a high rate of incorporation of extra-ovarian material into the yolk, which would be synthesized by the pedicel cells and incorporated into the oocytes through pinocytic vesicles. Balashov (1983) described the presence of similar microvilli in the surface of developing oocytes of Hyalomma asiaticum, which corroborates to the data obtained by the present study. These structures would be related to the increase of the surface of the oocytes necessary for better efficiency of the active transport of elements provided by the exogenous sources to the oocyte (Balashov, 1983). According to Balashov (1983), the exogenous sources of yolk element production in ticks, would be located in their intestine cells. However, Sonenshine (1991) suggested the participation of the fat body in tick vitellogenesis, in which the cells would secrete vitellogenic protein into the hemolymph, which would, in turn, be taken up by developing
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oocytes through endocytosis. Our results additionally suggest the participation of pedicel cells in the vitellogenesis, synthesizing and providing substances to the interior of the oocytes. In stage III oocytes many microvilli in the plasmic membrane were found. Large numbers of yolk granules of varied sizes were seen, while only a small amount of rough and smooth endoplasmic reticulum, responsible for the endogenous synthesis of lipids and proteins, respectively, were found. This suggests that, in stage III oocytes, there is more evidence that yolk elements come from extra-ovarian sources. According to Camargo-Mathias (1993) and Denardi et al. (2006), mitochondria are one of the sources for lipids in the oocytes of the ant Neoponera villosa, confirming earlier observations (Bonhag, 1958; Wigglesworth, 1964). Ranade (1933) had suggested that mitochondria would be transformed into lipid yolk bodies and that the lipid material would come from the destruction of the mitochondrial crests. Stage III oocytes of A. triste show such disorganization of the crests and membranes in the mitochondria, the presence of lipids and little smooth endoplasmic reticulum as mentioned above. The present study therefore suggests that lipid components could be derived from mitochondria as well as from an exogenous source. In stage IV oocytes, numerous yolk granules of varied appearance and electron density, with many microvilli in their plasmic membrane, and the deposition of chorium between the basal lamina and the plasmic membrane can be seen. The presence of microvilli to this last stage of development suggests that the incorporation of extra-ovarian yolk elements continues throughout vitellogenesis corroborating to the data obtained by Oliveira et al. (2005) for R. sanguineus. The germ vesicle in stage IV oocytes has many reentrances as well as nuclear pores that allow the passage of material (possibly rRNA) from the nucleus to the cytoplasm. Consequently, around the germ vesicle, there is an increase in the quantity of ribosomes, as well as formation of nuage and deposition of small electron dense granules. These could indicate endogenous synthesis of yolk in stage IV oocytes. The passage of material through the nuclear pores also was observed in the oocytes of camacuto scrimp Atya scabra (Cruz-Landim, 1997). Diehl (1970) showed that vitellogenin proteins of ticks are immunologically identical to proteins from the hemolymph. This could be evidence for some extra-ovarian sources of elements for the formation of yolk. Other tissues could also synthesize these substances that would be transported by hemolymph, taken up by microvilli and absorbed through pinocytosis. Balashov (1983) suggested that in the tick H. asiaticum, the yolk would be both endogenous and exogenous but that endogenous production could occur before exogenous production. Our results contradict this, since oocytes in early development (I and II) already possess evidence such as: microvilli in the plasmic membrane, and a large quantity of
mitochondria in the contact region oocyte/pedicel cells that would suggest exogenous transport of yolk elements. Denardi et al. (2004), in oocytes of the A. cajennense and Oliveira et al. (2005), in R. sanguineus described the chorium as being subdivided into two layers: the endochorium (more internal and electro dense) and the exochorium (more external and less electron dense). These characteristics were not seen in the oocytes of A. triste. Acknowledgments We thank Miss Cristina Oishi Gridi Papp, Miss Moˆnika Iamonte, Mr. Paulo Aruana˜ Cezar, Mr. Antoˆnio Teruyoshi Yabuki and Mr. Ronaldo Del Vecchio for their technical support and CAPES for financial support. Part of this work has been facilitated through the International Consortium of Ticks and Tick-borne Diseases (ICTTD-3) Coordination Action financed by the INCO program of the European Comission (Project No. 510561). References Balashov, Y.S., 1972. A translation of bloodsucking ticks (Ixodidae) vectors of diseases of man and animals. Miscellaneous Publications of the Entomological Society of America 8, 159–376. Balashov, Y.S., 1983. The female reproductive system. In: Balashov, Y.S. (Ed.), An Atlas of Ixodid Tick Ultrastructure. Entomological Society of America, Russian, pp. 98–128. Bonhag, P.F., 1958. Ovarian structure and vitellogenesis in insects. Annual Review of Entomology 3, 137–160. Correa, O., 1954. Carrapatos determinados no Rio Grande do Sul. Biologia, patologia e controle. Boletim da Diretoria de Produc¸a˜o Animal 10 (18), 38–54. Cruz-Landim, C., 1997. Cell reorganization and cell death during the secretory cycle of the hypopharyngeal gland in Meliponinae bee workers (Hymenoptera: Apidae). Acta Microscopica 6, 75–78. Camargo-Mathias, M.I., 1993. Histoquı´mica e ultra-estrutura dos ova´rios de opera´rias e rainhas de formigas Neoponera villosa (Hymenoptera: Ponerinae). Universidade Estadual Paulista, Rio Claro, 156 pp. Denardi, S.E., Bechara, G.H., Oliveira, P.R., Nunes, E.T., Saito, K.C., Camargo-Mathias, M.I., 2004. Morphological characterization of the ovary and vitellogenesis dynamics in the Amblyomma cajennense (Acari: Ixodidae). Veterinary Parasitology 125, 379–395. Denardi, S.E., Camargo-Mathias, M.I., Bechara, G.H., 2006. Amblyomma cajennense (Acari: Ixodidae): Salivary gland cells of partially engorged females ticks and the production of lipid by their mitochondria. Experimental Parasitology 113, 30–35. Diehl, P.A., 1970. Zur Oogenese bei Ornithodoros moubata (Murray) (Ixodoidea: Argasidae) unter besonderer Beru¨cksichtigung der Vitellogenese. Acta Tropical 27, 301–355. Ivancovich, J.C., 1980. Reclasificacio´n de algunas especies de garrapatas del ge´nero Amblyomma (Ixodoidea) en la Argentina. Investigaciones Agropecuarias 15, 673–682. Keirans, J.E., 1984. George Henry Faulkner Nuttall and the Nuttall tick catalogue. United States Department of Agriculture Research Series Miscellaneous Publications 1438, 1610–1700. Kohls, G.M., 1956. Concerning the identity of Amblyomma maculatum, Amblyomma tigrinum, Amblyomma triste and Amblyomma ovatum of Koch, 1844. Proceedings of the Entomological Society of Washington 58, 143–147. Lemos, E.R.S., Machado, R.D., Coura, J.R., Guimara˜es, M.A., Freire, N.M., Amorim, M., Gazeta, G.S., 1997. Epidemiological aspects of the Brazilian spotted fever: seasonal activity of ticks collected in an
P.R. de Oliveira et al. / Experimental Parasitology 116 (2007) 407–413 endemic area in Sa˜o Paulo. Revista da Sociedade Brasileira de Medicina Tropical 30, 181–185. Oliveira, P.R., Bechara, G.H., Denardi, S.E., Saito, K.C., Nunes, E.T., Camargo-Mathias, M.I., 2005. Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Experimental Parasitology 110, 146–156. Oliveira, P.R., Bechara, G.H., Camargo-Mathias, M.I., 2006. Amblyomma triste (Koch, 1844) (Acari: Ixodidae): morphological description of the ovary and of vitellogenesis. Experimental Parasitology 113, 179–185. Ranade, V., 1933. On the cytoplasmic inclusions in the oogenesis of Periplaneta americana Linn. Allahad. University Science Bulletin 9, 85–121. Rey, L., 1973. Parasitologia. Guanabara Koogan, Sa˜o Paulo, 212 pp. Said, E.A., 1992. A contribution to the anatomy and histology of the female reproductive system of Amblyomma cajennense (Acarina: Ixodidae). Journal of the Egyptian Society of Parasitology 22, 385– 393. Saito, K.C., Bechara, G.H., Oliveira, P.R., Nunes, E.T., Denardi, S.E., Camargo-Mathias, M.I., 2005. Morphological, histological, and ultrastructural studies of the ovary of the tick Boophilus microplus
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(Canestrini, 1887) (Acari: Ixodidae). Veterinary Parasitology 129, 299– 311. Sinkoc, A.L., Brum, J.G.W., Mu¨ller, G., Begrow, A., Paulsen, R.M.M., 1997. Ixodideos parasitos de capivaras (Hydrochaeris hydrochaeris L., 1766) da Estac¸a˜o Ecolo´gica do Taim, Rio Grande, RS. Cieˆncia Rural 27, 119–122. Sonenshine, D.E., 1991. Biology of Ticks. Oxford University Press, New York, pp. 280–303. Szabo´, M.P.J., Labruna, M.B., Pereira, M.C., Duarte, J.M.B., 2003. Ticks (Acari: Ixodidae) on Wild Marsh-Deer (Blastocerus dichotomus) from Southeast Brazil: Infestations before and after habitat loss. Journal of Medical Entomology 40, 268–274. Till, W.M., 1961. A Contribution to the Anatomy and Histology of the Brown Ear Tick Rhipicephalus appendiculatus. Swets & Zeitlinger Publishers, Amsterdam, 124 pp. Venzal, J.M., Guglielmone, A.A., Estrada-Pena, A., Cabrera, P.A., Castro, O., 2003. Ticks (Ixodida: Ixodidae) parasitising humans in Uruguay. Annals of Tropical Medicine & Parasitology 97, 769–772. Wigglesworth, V.B., 1964. The hormonal regulation of growth and reproduction in insects. In: Beament, J.W.L., Theherne, J.E., Wigglesworth, V.B. (Eds.), Advances in Insect Physiology. Academic Press, New York, pp. 247–336.
Amblyomma triste (Koch, 1844) (Acari: Ixodidae) Ovaries: An ultrastructural analysis Patrı´cia Rosa de Oliveira a, Gerva´sio Henrique Bechara b, Maria Izabel Camargo Mathias b
a,*
a Departamento de Biologia, I.B., UNESP, Av. 24 A, No. 1515, Cx. Postal 199, CEP: 13506-900, Rio Claro, SP, Brazil Departamento de Patologia Veterina´ria, FCAV, UNESP, Via de Acesso Prof. Paulo Castellane, s/n, CEP: 14884-900, Jaboticabal, SP, Brazil
Received 19 April 2006; received in revised form 11 February 2007; accepted 14 February 2007 Available online 28 February 2007
Abstract This study presents an ultrastructural analysis of the ovary of the tick, Amblyomma triste. The ovary of this species is of the panoistic type that is, without nursing and follicular cells. It is composed of a layer of epithelial cells forming a wall and of germinative cells that generate the oocytes which remain attached to the external margin of this wall by a multicellular pedicel. The different developmental stages in the oocytes had been described by Oliveira et al. [Oliveira, P.R., Bechara, G.H., Camargo-Mathias, M.I., 2006. Amblyomma triste (Koch, 1844) (Acari: Ixodidae): Morphological description of the ovary and of vitellogenesis. Experimental Parasitology 113, 179–185]. The results of the investigation suggest that besides exogenous production of vitellogenic elements, endogenous production can take place simultaneously, contributing to the development and growth of the oocytes. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Amblyomma triste; Tick; Ovary; Vitellogenesis; Ultrastructure
1. Introduction The economic importance of ticks is widely acknowledged and is related to their feeding habits. During feeding, many species of ticks can transmit diseases caused by protozoa, viruses, Rickettsias and spirochetes (Rey, 1973) to man and other animals. The tick species Amblyomma triste is distributed in the Neotropics and has been reported in Equador (Keirans, 1984), Argentina (Ivancovich, 1980), Uruguay and Brazil (Sinkoc et al., 1997) infesting tapir (Kohls, 1956), dogs (Correa, 1954), capybara (Hydrochaeris hydrochaeris) (Sinkoc et al., 1997), marsh deer Blastocerus dichotomus (Szabo´ et al., 2003) and humans (Venzal et al., 2003). It has also been reported also from opossum (Didelphis marsupialis) in an endemic area for Brazilian spotted fever in Pedreiras, State of Sa˜o Paulo (Lemos et al., 1997).
*
Corresponding author. Fax: +55 19 3526 4135. E-mail address: [email protected] (M.I. Camargo Mathias).
0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.02.006
According to Till (1961), Sonenshine (1991) and Said (1992), the female reproductive system of ticks generally consists of a large U-shaped ovary located at the posterior region of the body, with a pair of oviducts, an uterus, a muscular connection tube, a vagina and a genital opening. In this report, we describe the ultrastructure of the ovary, thereby contributing to our understanding of vitellogenesis in A. triste. 2. Materials and methods Semi-feeding A. triste females from the tick colony were maintained under controlled conditions (28 °C, 80% humidity and 12 h light/dark cycle) at the Department of Animal Pathology, Veterinary College, UNESP – Jaboticabal, SP, Brazil. Twenty-five specimens maintained in the refrigerator for thermal shock anesthesia were dissected in a saline solution (NaCl 7.5 g/L, Na2HPO4 2.38 g/L and KH2PO4 2.72 g/L).
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2.1. Transmission electron microscopy (TEM) The material was fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and embedded in Epon Araldite. The material was then embedded in pure Epon resin and polymerized at 60 °C for 72 h. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Afterwards, screens containing ultrathin sections of the material were examined and photographed in a Phillips 100 Transmission Electron Microscope (TEM). 3. Results The ovary wall of the tick A. triste consists of small epithelial cells that proliferate to form the pedicel, a structure that attaches the oocytes at different stages of development to the ovary wall (Fig. 1a). The cells are small and of a pavimentous type. The large, oval nucleus occupies most of the cytoplasm which only contains a few mitochondria (Fig. 1a). A thick, mildly electron dense, basal lamina, supports this epithelium.
The cells are connected to each other by means of interdigitations of their lateral membranes (Fig. 1a). Stage I oocytes possess a homogeneous cortex with few ribosomes, either free or attached to the membrane of the lamellar rough endoplasmic reticulum, which is little developed (Fig. 1b and c). However, large numbers of elliptical, round or elongated mitochondria were detected. They are relatively small and occupy the region around the germ vesicle. This is located in the center of the stage I oocyte, having a rounded appearance, dispersed chromatin and a large nucleolus with distinct granular and fibrous regions (Fig. 1b). Surrounding the oocyte, there is an electron dense plasmic membrane containing some microvilli, mainly in the direction of the pedicel (Fig. 1d) and the basal lamina which is in two layers: an inner, thicker layer, which is in direct contact with the microvilli, and an outer, thinner layer which has a fibrillar appearance. In the region between the inner layer of the basal lamina and the membrane of the oocyte, vesicles containing material probably
Fig. 1. (a) Ultrastructure of the epithelium from the ovary of Amblyomma triste. (b) General view of oocyte I. (c) Detail of the cytoplasm near the germ vesicle of oocyte I. (d) Peripheral region of oocytes I. bl, basal lamina; pm, plasmic membrane; ep, ovary epithelium; n, nuclei; ne, nuclear envelope; m, mitochondria; gv, germ vesicle; nu, nucleollus; lrer, lamellar rough endoplasmic reticulum; mv, microvilli. Bars: A, 2 lm; B, 2 lm; C, 2 lm; D, 2 lm.
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being transported from the pedicel cells to the oocyte, can be observed (Fig. 1d). Stage II oocytes are larger than those of stage I. The cytoplasm begins to show small yolk granules with variable morphology and content, located throughout the cytoplasm. Granules with a lipid content are electrolucent; while the protein containing granules possess high electron density (Fig. 2a and b). Mitochondria with variable shapes, distributed all over the cytoplasm, are found more frequently than other organelles. Free ribosomes are also seen, along with some myelin bodies and under-developed lamellar and vesicular rough endoplasmic reticulum (Fig. 2a and b). Stage II oocytes have more microvilli in their plasmic membrane, supported by a thick basal lamina subdivided
409
in two portions. Vesicles and electron dense material between the membrane and the basal lamina are observed (Fig. 2c). In Stage III oocytes, the germ vesicles are still seen (Fig. 2d). There are numerous rounded mitochondria throughout the cytoplasm, some with disorganization of their crests and membranes. Myelin bodies, Golgi complex and lamellar and vesicular rough endoplasmic reticulum are also present (Fig. 2e and f). In the cytoplasm, yolk granules (termed p1) are also found. They are homogeneous, with a foamy appearance, being moderately electron dense with protein content, as well as having electrolucent lipid droplets (Fig. 2e and f). Microvilli with similar characteristics to previous stages are still present in the plasmic membrane in these oocytes (Fig. 2d and f).
Fig. 2. (a) General view of oocyte II (II). (b) Detail of the central cytoplasm of oocytes II. (c) Peripheral region of oocytes II. (d) General view of oocyte III (III). (e) Detail of the central cytoplasm of oocytes III. (f) Peripheral region of oocytes III. bl, basal lamina; mv, microvilli; m, mitochondria; n, nuclei; p, pedicel; lrer, lamellar rough endoplasmic reticulum; pm, plasmic membrane; l, lipid granule; vrer, vesicular rough endoplasmic reticulum; gv, germ vesicle; g, Golgi complex; arrow, proteic yolk granules. Bars: A, 10 lm; B, 2 lm; C, 1 lm; D, 10 lm; E, 2 lm; F, 2 lm.
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In Stage IV oocytes, the germ vesicle, now with obvious irregular morphology, can still be observed, with many reentrances in its envelope, some heterochromatin as well as a highly electron dense accumulation of small granules and rRNA (nuage) around it (Fig. 3a and b). Many yolk granules with varied contents and different electron densities are found. Free or grouped electrolucent lipid droplets mainly occupy regions near the periphery (Per) (Figs. 3e and 4a, b, d). The granules with protein con-
tent occupy all the cytoplasm and are present in two forms: as moderately electro dense and homogeneous granules (p1) and, less frequently, with high density granulation, that probably contain glyco or lipoprotein complexes, called here p2 (Figs. 3c, e and 4a, b, d). Mitochondria, underdeveloped lamellar rough endoplasmic reticulum and Golgi complex are present (Figs. 3c–e and 4a–d). In the periphery of the stage IV oocyte, microvilli are found that are longer than those of previous stages
Fig. 3. (a) Cytoplasm near the germ vesicle of oocyte IV. (b) Detail of the cytoplasm near the germ vesicle of oocyte IV. (c) Peripheral region of oocytes IV. (d) Detail of the peripheral cytoplasm of oocytes IV. (e) Central cytoplasm of oocytes IV. g, Golgi complex; l, lipid granule; m, mitochondria; gv, germ vesicle; mv, microvilli; ne, nuclear envelope; ch, chorium; pm, plasmic membrane; lrer, lamellar rough endoplasmic reticulum; vrer, vesicular rough endoplasmic reticulum; head arrow, granules and nuage around the germ vesicle; arrow, proteic yolk granules. Bars: A, 2 lm; B, 1 lm; C, 1 lm; D, 1 lm; E, 2 lm.
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Fig. 4. (a) Cytoplasm of oocytes IV. (b) Detail of the central cytoplasm of oocytes IV. (c) Detail of the intermediary cytoplasm of oocytes IV, showing proteic yolk granules. (d) Detail of the peripheral cytoplasm of oocytes IV, showing lipid granules. m, mitochondria; l, lipid granule; lrer, lamellar rough endoplasmic reticulum; vrer, vesicular rough endoplasmic reticulum; arrow, proteic yolk granules. Bars: a, 1 lm; b, 1 lm; c, 1 lm; d, 1 lm.
(Fig. 3c and d) and having a larger space between the plasmic membrane and basal lamina. Here the chorium is being deposited through exocytic vesicles. 4. Discussion Transmission electron microscopy has confirmed the classification of the ovaries of the tick A. triste as panoistic, where all germinative cells correspond to oogonia or future oocytes. This classification was based on that adopted by Denardi et al. (2004) for Amblyomma cajennense, and for Rhipicephalus sanguineus (Oliveira et al., 2005), Boophilus microplus (Saito et al., 2005) and A. triste (Oliveira et al., 2006) . The development stage of the oocytes was described according to the proposal by Oliveira et al. (2006), where the main characteristics considered were the aspect of the cytoplasm, the localization of the germ vesicle, the presence, quantity and constitution of yolk granules and the presence of chorium. Stage I oocytes had a homogeneous cortex with many mitochondria and the absence of yolk granules. Stage II oocytes showed small yolk granules, larger amounts of mitochondria, but little rough endoplasmic reticulum, which appear under the form of cisternae of low electron density. In the beginning of the development of the oocytes (stage I) of A. triste, the mitochondria are preferentially
located around the germinal vesicle. According to Balashov (1983), these are associated with granules and fibrils of riboproteins. The function of this association of the mitochondria is still unknown. Later, the mitochondria are found throughout the cytoplasm of oocytes, pointing to a preparation of these cells for the period of exogenous yolk incorporation, in which these organelles become a prerequisite for the active transportation of material (Balashov, 1972). In the periphery next to the pedicel in stage I and II oocytes, small vesicles and microvilli are found, suggesting that at this stage there is a high rate of incorporation of extra-ovarian material into the yolk, which would be synthesized by the pedicel cells and incorporated into the oocytes through pinocytic vesicles. Balashov (1983) described the presence of similar microvilli in the surface of developing oocytes of Hyalomma asiaticum, which corroborates to the data obtained by the present study. These structures would be related to the increase of the surface of the oocytes necessary for better efficiency of the active transport of elements provided by the exogenous sources to the oocyte (Balashov, 1983). According to Balashov (1983), the exogenous sources of yolk element production in ticks, would be located in their intestine cells. However, Sonenshine (1991) suggested the participation of the fat body in tick vitellogenesis, in which the cells would secrete vitellogenic protein into the hemolymph, which would, in turn, be taken up by developing
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oocytes through endocytosis. Our results additionally suggest the participation of pedicel cells in the vitellogenesis, synthesizing and providing substances to the interior of the oocytes. In stage III oocytes many microvilli in the plasmic membrane were found. Large numbers of yolk granules of varied sizes were seen, while only a small amount of rough and smooth endoplasmic reticulum, responsible for the endogenous synthesis of lipids and proteins, respectively, were found. This suggests that, in stage III oocytes, there is more evidence that yolk elements come from extra-ovarian sources. According to Camargo-Mathias (1993) and Denardi et al. (2006), mitochondria are one of the sources for lipids in the oocytes of the ant Neoponera villosa, confirming earlier observations (Bonhag, 1958; Wigglesworth, 1964). Ranade (1933) had suggested that mitochondria would be transformed into lipid yolk bodies and that the lipid material would come from the destruction of the mitochondrial crests. Stage III oocytes of A. triste show such disorganization of the crests and membranes in the mitochondria, the presence of lipids and little smooth endoplasmic reticulum as mentioned above. The present study therefore suggests that lipid components could be derived from mitochondria as well as from an exogenous source. In stage IV oocytes, numerous yolk granules of varied appearance and electron density, with many microvilli in their plasmic membrane, and the deposition of chorium between the basal lamina and the plasmic membrane can be seen. The presence of microvilli to this last stage of development suggests that the incorporation of extra-ovarian yolk elements continues throughout vitellogenesis corroborating to the data obtained by Oliveira et al. (2005) for R. sanguineus. The germ vesicle in stage IV oocytes has many reentrances as well as nuclear pores that allow the passage of material (possibly rRNA) from the nucleus to the cytoplasm. Consequently, around the germ vesicle, there is an increase in the quantity of ribosomes, as well as formation of nuage and deposition of small electron dense granules. These could indicate endogenous synthesis of yolk in stage IV oocytes. The passage of material through the nuclear pores also was observed in the oocytes of camacuto scrimp Atya scabra (Cruz-Landim, 1997). Diehl (1970) showed that vitellogenin proteins of ticks are immunologically identical to proteins from the hemolymph. This could be evidence for some extra-ovarian sources of elements for the formation of yolk. Other tissues could also synthesize these substances that would be transported by hemolymph, taken up by microvilli and absorbed through pinocytosis. Balashov (1983) suggested that in the tick H. asiaticum, the yolk would be both endogenous and exogenous but that endogenous production could occur before exogenous production. Our results contradict this, since oocytes in early development (I and II) already possess evidence such as: microvilli in the plasmic membrane, and a large quantity of
mitochondria in the contact region oocyte/pedicel cells that would suggest exogenous transport of yolk elements. Denardi et al. (2004), in oocytes of the A. cajennense and Oliveira et al. (2005), in R. sanguineus described the chorium as being subdivided into two layers: the endochorium (more internal and electro dense) and the exochorium (more external and less electron dense). These characteristics were not seen in the oocytes of A. triste. Acknowledgments We thank Miss Cristina Oishi Gridi Papp, Miss Moˆnika Iamonte, Mr. Paulo Aruana˜ Cezar, Mr. Antoˆnio Teruyoshi Yabuki and Mr. Ronaldo Del Vecchio for their technical support and CAPES for financial support. Part of this work has been facilitated through the International Consortium of Ticks and Tick-borne Diseases (ICTTD-3) Coordination Action financed by the INCO program of the European Comission (Project No. 510561). References Balashov, Y.S., 1972. A translation of bloodsucking ticks (Ixodidae) vectors of diseases of man and animals. Miscellaneous Publications of the Entomological Society of America 8, 159–376. Balashov, Y.S., 1983. The female reproductive system. In: Balashov, Y.S. (Ed.), An Atlas of Ixodid Tick Ultrastructure. Entomological Society of America, Russian, pp. 98–128. Bonhag, P.F., 1958. Ovarian structure and vitellogenesis in insects. Annual Review of Entomology 3, 137–160. Correa, O., 1954. Carrapatos determinados no Rio Grande do Sul. Biologia, patologia e controle. Boletim da Diretoria de Produc¸a˜o Animal 10 (18), 38–54. Cruz-Landim, C., 1997. Cell reorganization and cell death during the secretory cycle of the hypopharyngeal gland in Meliponinae bee workers (Hymenoptera: Apidae). Acta Microscopica 6, 75–78. Camargo-Mathias, M.I., 1993. Histoquı´mica e ultra-estrutura dos ova´rios de opera´rias e rainhas de formigas Neoponera villosa (Hymenoptera: Ponerinae). Universidade Estadual Paulista, Rio Claro, 156 pp. Denardi, S.E., Bechara, G.H., Oliveira, P.R., Nunes, E.T., Saito, K.C., Camargo-Mathias, M.I., 2004. Morphological characterization of the ovary and vitellogenesis dynamics in the Amblyomma cajennense (Acari: Ixodidae). Veterinary Parasitology 125, 379–395. Denardi, S.E., Camargo-Mathias, M.I., Bechara, G.H., 2006. Amblyomma cajennense (Acari: Ixodidae): Salivary gland cells of partially engorged females ticks and the production of lipid by their mitochondria. Experimental Parasitology 113, 30–35. Diehl, P.A., 1970. Zur Oogenese bei Ornithodoros moubata (Murray) (Ixodoidea: Argasidae) unter besonderer Beru¨cksichtigung der Vitellogenese. Acta Tropical 27, 301–355. Ivancovich, J.C., 1980. Reclasificacio´n de algunas especies de garrapatas del ge´nero Amblyomma (Ixodoidea) en la Argentina. Investigaciones Agropecuarias 15, 673–682. Keirans, J.E., 1984. George Henry Faulkner Nuttall and the Nuttall tick catalogue. United States Department of Agriculture Research Series Miscellaneous Publications 1438, 1610–1700. Kohls, G.M., 1956. Concerning the identity of Amblyomma maculatum, Amblyomma tigrinum, Amblyomma triste and Amblyomma ovatum of Koch, 1844. Proceedings of the Entomological Society of Washington 58, 143–147. Lemos, E.R.S., Machado, R.D., Coura, J.R., Guimara˜es, M.A., Freire, N.M., Amorim, M., Gazeta, G.S., 1997. Epidemiological aspects of the Brazilian spotted fever: seasonal activity of ticks collected in an
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