ARTICLE IN PRESS Protist, Vol. 156, 35—43, June 2005 http://www.elsevier.de/protis Published online 22 April 2005
ORIGINAL PAPER
Ultrastructural Aspects of Fallisia effusa (Haemosporina: Garniidae) in Thrombocytes of the Lizard Neusticurus bicarinatus (Reptilia: Teiidae) Edilene O. Silvaa,1, Jose´ Antonio P. Dinizb, Ralph Lainsonc, Renato A. DaMattad, and Wanderley de Souzae a Laborato´rio de Parasitologia, Departamento de Patologia, Centro de Cieˆncias Biolo´gicas, Universidade Federal do Para´, Av. Augusto Correˆa s/n, Bairro Guama´, 66075-110 Bele´m, Para´, Brazil b Unidade de Microscopia Eletroˆnica, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco, 66090-000 Bele´m, Para´, Brazil c Laborato´rio de Coccı´dios, Departamento de Parasitologia, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco, 66090-000 Bele´m, Para´, Brazil d Laborato´rio de Biologia Celular e Tecidual, Centro de Biocieˆncias e Biotecnologia, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego 2000, 28013-600, Campos dos Goytacazes, RJ, Brazil e Laborato´rio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Funda˜o, 21941-590 Rio de Janeiro, RJ, Brazil
Submitted May 10, 2004; Accepted September 20, 2004 Monitoring Editor: C. Graham Clark
The fine structure of the different stages of the Fallisia effusa (Haemosporina: Garniidae), infecting the thrombocytes of the semi-aquatic Amazonian lizard Neusticurus bicarinatus (Reptilia: Teiidae) is described. Gametocytes, meronts, and merozoites of Fallisia effusa were found within a parasitophorous vacuole (PV). Multiple infections of micro- and macrogametocytes were observed. A circumferential coil of microtubules was seen in the cytoplasm of the infected host cell and this microtubule array was pronounced in cells harboring gametocytes. A deep invagination of the inner membrane complex of gametocytes may be involved in nutrition. The non-pigmented parasites underwent both merogony and gametogony in thrombocytes of the peripheral blood. No infection of the erythrocytes was observed. These observations confirm that Fallisia effusa displays characteristic features distinguishing it from other members of the Haemosporidian families, and that it has the ability to modulate microtubule assembly. & 2005 Elsevier GmbH. All rights reserved. Key words: Fallisia effusa; Garniidae; lizards; thrombocytes; ultrastructure.
1 Corresponding author; fax 0055 3183 1601 e-mail
[email protected] (E.O. Silva).
& 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2004.09.002
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Introduction Some authors have considered that all reptilian haemosporines with gametocytes and meronts in leukocytes or thrombocytes are members of the family Plasmodiidae. Lainson et al. (1971, 1974), however, proposed the use of a separate family, Garniidae, for the inclusion of the nonpigmented parasites with meronts and gametocytes in erythrocytes (the genus Garnia), and those in leukocytes and thrombocytes (the genus Fallisia). Numerous species of both genera have been described in a variety of lizard species of the New World (Lainson 1992; Lainson and Naiff 1999; Lainson et al. 1971, 1974, 1975) and species of Fallisia have been described in lizards of the Old World (Paperna and Landau 1990; Telford 1986). A new genus, Progarnia Lainson, 1995 (Lainson 1995) was erected in the family Garniidae for a nonpigmented haemosporine of the South American caiman, Caiman c. crocodylus, and a single species of Fallisia has been recorded in birds in Venezuela (Gabaldon et al. 1985). The species Fallisia effusa is a parasite of thrombocytes and, more rarely, of lymphocytes found in the semi-aquatic teiid lizard Neusticurus bicarinatus. Although its development in the lizard has been well documented by light microscopy, little is known regarding the ultrastructure of the parasite apart from a description of the fine structure of the gametocytes (Boulard et al. 1987). An account is given here of additional observations on the ultrastructure of the gametocytes, stages of merogony, and changes in the morphology of the infected thrombocyte. Non-mammalian thrombocytes are functionally analogous to platelets. They are involved in homeostasis (Stalsberg and Prydz 1963) and exhibit endocytic (Pellizzon and Lunardi 2000) and low phagocytic (DaMatta et al. 1998) capacities. Little is known about the relationship of thrombocytes and parasitic infection.
Figure 1. Giemsa-stained blood film showing Fallisia effusa infected thrombocytes of the lizard Neusticurus bicarinatus. (A) Infected thrombocytes display gametocyte (arrows) and a trophozoite (arrowhead). Note the cyst-like appearance of the host cell (small arrows). (B) Early merogony (arrow). Note less electron dense pellicle compared with that of the gametocyte (small arrows) and an uninfected thrombocyte (arrowhead). (C) Mature meronts (arrows). Note an uninfected thrombocyte (arrowhead). Bar ¼ 16 mm.
Results The thrombocytes of 11 lizards examined (10%) were infected with F. effusa. The level of infection was usually low and only two lizards were heavily infected. Observations by light microscopy of blood film showed infected thrombocytes containing gametocytes (Fig. 1 A), developing meronts
(Fig. 1 B) and merozoites (Fig. 1 C) with the same morphology as previously described by Lainson et al. (1974). The nucleus of infected thrombocytes was altered (Fig. 1). The surface of the host cell was thickened, particularly in thrombocytes harboring gametocytes (Fig. 1 A), but occurred to a lesser extent in thrombocytes harboring other parasite stages (Fig. 1 B).
ARTICLE IN PRESS Characterization of Fallisia effusa
Multiple infected cells were common, but there were rarely more than two different stages of the parasites in the same host cell (Fig. 1 A). Electron microscopic examination showed that uninfected thrombocytes were roughly ovoid (Fig. 2 A). The centrally located ovalshaped nucleus contained a large amount of heterochromatin and patches of euchromatin (Fig. 2 A). Vacuoles, a canalicular system as well as bundles of microtubules were observed (Fig. 2 A, inset). The parasites lie in a parasitophorous vacuole (Figs 2 B—D, 3—5). The greatest alteration in the shape of the cells was seen in those containing mature gametocytes or meronts, and in those with multiple infections (Figs 2 B, 3, 4 C, 5 A, B). One to five gametocytes were observed in the same thrombocyte (Figs 2 B—D, 3, 4 A, C, D, 5 B), grossly distorting the shape of the cell. The nucleus of the gametocyte was usually centrally located and did not exhibit a heterochromatin region (Fig. 2 C, D). A pronounced circumferential coil of microtubules was observed in thrombocytes infected with gametocytes (Fig. 3). The diameter of the microtubules was approximated 25 nm. Large uninucleate macrogametocytes were electron dense and bounded by a four-layered pellicle composed of a plasma membrane, and a threelayered inner complex, formed by three closely apposed unit membranes (Fig. 3, inset). The microgametocyte was smaller, less electron dense, and with a two-layered pellicle consisting of the plasma membrane and underlying inner membrane complex (Fig. 2 D). An invagination of the pellicle was also observed in macrogametocytes (Fig. 4 A, B). Sometimes deep invaginations were observed (Fig. 4 C) that crossed large areas of the parasite (Fig. 4 C, D). Merogony of the parasite was also observed (Fig. 5 A, B). Parasites in the initial phase of merogony possessed membrane sacs and membrane-delimited bodies (Fig. 5 A). Higher magnification of these membrane structures revealed that the former presents four membrane units suggesting that it is probably an apicoplast, and the latter a mitochondrion (Fig. 5 A, inset). A residual body was detected in late merogonic stages (Fig. 5 B) with the formation of merozoites at the final stage (Fig. 5 C). In some of the forming merozoites, large rhoptries and an apical pole were seen (Fig. 5 C, D).
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Discussion This study confirms previous observations that F. effusa infects thrombocytes and that all stages of its life cycle in the vertebrate host occur in the blood. Infection by obligate intracellular parasites generally alters the shape of the infected host cells (Adams and Bushell 1989; Beyer 1997; Beyer and Sidorenko 1984; Paterson et al. 1988; Siddall and Desser 1993). Thrombocytes infected with F. effusa were distorted and a circumferential coil of microtubules occurred in the peripheral cytoplasm. This microtuble array is more evident in infected cells harboring the gametocytes as described by Lainson et al. (1974) and further supported by Boulard et al. (1987). Using light microscopy, Lainson et al. (1974) described the membrane of the infected cell as thickened, giving it a cystlike appearance. The peculiar organization of microtubules is responsible for the impression that the plasma membrane of the infected thrombocyte is thicker than that of the uninfected cells, as previously seen in Giemsastained preparations (Lainson et al. 1974). Studies on non-mammalian thrombocytes (Lee et al. 2004) and mammalian platelets (Can˜izares et al. 1994; Cerecedo et al. 2002; White and Rao 1998) have shown that the microtubules are arranged in a circumferential coil, similar to that observed in this study, maintaining the discoid shape characteristics of unstimulated platelets and thrombocytes. The depolymerization of this cytoskeleton array following cell activation leads to loss of their discoid shape (Can˜izares et al. 1994; Cerecedo et al. 2002; Lee et al. 2004; White and Rao 1998). It is possible that F. effusa modulates the physiology of infected thrombocytes inhibiting their activation. This inhibition could probably maintain the infected thrombocyte for more time in the blood circulation, facilitating transmission of the parasite to the invertebrate vector. Additional studies are necessary to identify the mechanism that induces microtubule reorganization and its physiological role. The process of merogony observed in these parasites appears to be similar to that described in others members of the phylum Apicomplexa. Invagination in the pelliclar system of gametocytes of F. effusa may be involved in the process of nutrition. This suggestion is supported by the absence of a cytostome and the fact that these invaginations reach deep areas of the parasite cytoplasm
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Figure 2. Ultrastructure of Fallisia effusa of the lizard Neusticurus bicarinatus. (A) Uninfected thrombocyte showing typical oval shape and elongated, centrally located nucleus (N). Note vacuoles (v), canalicular system (arrowheads) and bundles of microtubules (arrows) seen with increased magnification in the inset. (B) General view of the infected thrombocyte showing multiple invasion by macrogametocytes (asterisks) and microgametocytes (stars). (C) Macrogametocyte (MA). Note the electron dense pellicle (arrows). (D) Microgametocyte (MI). Bar ¼ 1 mm.
ARTICLE IN PRESS Characterization of Fallisia effusa
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Figure 3. Thrombocyte containing two macrogametocytes. Note the array of cytoplasmic microtubules (arrowheads). Inset. Higher magnification showing a longitudinal view of the microtubules (MT) around the parasitophorous vacuole (PV). The circle indicates the four-layered pellicle. Bar ¼ 0:5 mm.
probably increasing the surface contact area as proposed before for a similar structure found in Eimeria auburnensis (Hammond et al. 1967). In summary, the protozoan F. effusa develops in thrombocytes, gametocytes modulate host cell microtubules and develop a deep invagination of the inner membrane complex, which is probably involved in nutrition.
Methods Lizards: During the 2001—2003 period, 112 individuals of the lizard Neusticurus bicarinatus were hand-captured along the banks of the rivers and streams in an area of tropical rain forest in Para´ State, north Brazil. They were maintained separately at the Instituto Evandro Chagas/PA animal house, in standard mouse cages at 27—32 1C and provided with water
and larvae and adults of the flour-beetle, Tenebrio molitor. Blood harvesting and leukocyte separation: Lizards were restrained manually and blood was collected by cardiac puncture into heparinized 1 ml syringes (100 U/ml). Blood smears from all lizards were fixed with methanol and stained with Giemsa for parasitemia evaluation. Blood leukocytes were separated as described before (Silva et al. 2004). Briefly, blood from uninfected or highly infected lizards was introduced into a plastic tube with a 2 mm diameter; the bottom end was sealed, and the tube centrifuged at 500 g for 4 minutes at 25 1C. The tube was cut with a scalpel blade under a stereo-microscope, and the buffy coat flushed out with Phosphate buffered saline and a Pasteur pipette for cell harvesting. Light microscopy: Giemsa-stained blood smears and fresh preparations were observed
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Figure 4. Ultrastructure of Fallisia effusa gametocytes. (A) General view of a gametocyte with a typical membrane system showing an invagination of the inner membrane complex (arrowheads). (B) Higher magnification of (A) showing the invagination (arrows). (C) Another image highlighting deep invaginations (arrows). Other developmental stages of the parasite are present (P). (D) Another example of deep invaginations (arrows). Bar ¼ 0:6 mm.
and photographed using an immersion 100 objective in a Zeiss Axiophot Microscope. Transmission electron microscopy: Cells of the blood buffy coat from highly infected lizards were fixed in 2.5% glutaraldehyde and 4%
freshly prepared formaldehyde in a buffer solution containing 60 mM Pipes, 20 mM Hepes, 10 mM ethyleneglycol-bis-(B-aminoethylether)-N,N,N0 -tetraacetic acid, 70 mM KCl, 5 mM MgCl2 (Schliwa and Van Blerkom 1981), and pH 6.9 for 1 hour at 25 1C. The cells
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Figure 5. Ultrastructure of Fallisia effusa merogony. (A) Early stage (arrowheads). Inset. Higher magnification showing a mitochondrion (star) and the apicoplast (asterisk) with four membrane units (small arrows). (B) Late stage showing merozoites (arrowheads), other stages of the parasite (P), and residual bodies (asterisk). (C) Late stage with merozoites (arrows) and other stages of the parasite (P). (D) Higher magnification of the merozoites. Note the large rhoptries (arrowheads) and the apical pole (arrows). Bar ¼ 0:6 mm; inset ¼ 0:4 mm.
were then washed in the same buffer and postfixed for 1 hour in a solution containing 1% osmium tetroxide, 0.8% ferrocyanide, and 5 mM calcium chloride, washed, dehydrated in
graded acetone, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM 900 transmission electron microscope.
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Acknowledgements We thank Manoel C.M. de Souza and Antonio Julio de O. Monteiro for their technical assistance and animal care, and Marcia A. Dutra for assistance with the photographic work. This study was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundac- a˜o de Coordenac- a˜o de Pessoal de Nı´vel Superior (CAPES), Fundac- a˜o Carlos Chagas Filho do Rio de Janeiro (FAPERJ), Instituto Evandro Chagas/SVS/MS, Programa Nacional de Cooperac- a˜o Acadeˆmica (CAPES-PROCAD), Programa de Nu´cleos de Exceleˆncia (PRONEX), and the Wellcome Trust, London (RL). The experiments performed in this work comply with current Brazilian animal protection laws (IBAMA Doc. 02018.000301/ 02-13).
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