Parasitol Res ,2000) 86: 971±977 Ó Springer-Verlag 2000 ORIGINAL PAPER Jose Antonio PicancË o Diniz á Edilene Oliveira Silva Ralph Lainson á Wanderley de Souza The ®ne structure of Garnia gonadati and its association with the host cell Received: 4 January 2000 / Accepted: 30 May 2000 Abstract Most ofthe studies on the ®ne structure of nized as agents ofimportant diseases aecting humans protozoa ofthe Apicomplexa group have been carried ,e.g., Plasmodium and Toxoplasma) or animals ofeco- out with members ofthe Toxoplasma, Eimeria, and nomic importance ,e.g., Eimeria). Within the phylum, Plasmodium genera. In the present study we analyzed the the suborder Haemosporina presently comprises four ®ne structure of Garnia gonadoti parasitizing the red families ,Lainson 1992): ,1) Plasmodiidae, containing blood cells ofthe Amazonian reptile Gonatodes species ofthe genus Plasmodium, the true pigmented humeralis ,Reptilia: Lacertilia). Transmission electron parasites ofreptiles, birds, and mammals; ,2) Haemo- microscopy ofthin sections showed that G. gonadoti proteidae, found in similar hosts; ,3) Leucocytozoidae, presented all structures characteristic ofthe group, encountered principally in birds and more rarely in including the apicoplast. However, four special features reptiles; and ,4) Garniidae, occurring principally in were observed: ,1) absence ofthe hemozoin ,malarial) reptiles and occasionally in birds. pigment; ,2) a group ofmicrotubules associated with the The ®ne structure ofapicomplexan parasites has been mitochondrion; ,3) a vacuole containing electron-dense extensively investigated during the past 20 years, but material, which resembled the acidocalcisome described most ofthese studies have concentrated on a fewspecies, in trypanosomatids; and ,4) a special array ofthe host- especially Plasmodium and Eimeria spp. and Toxoplasma cell endoplasmic reticulum around the parasitophorous gondii. There is little information on the ultrastructure of vacuole. members ofthe family Garniidae, which presently con- tains the genera Garnia, Fallisia ,Lainson et al. 1971, 1974), and Progarnia ,Lainson 1995), probably because these parasites are ofno medical or veterinary impor- Introduction tance and are rarely available in the laboratory. An analysis ofthe ®ne structure ofsuch organisms The phylum Apicomplexa contains a large number of remains important, however, for at least three major species ofparasitic protozoa, some ofwhich are recog- reasons. First, it is important that the phenomenon of biological diversity be studied by the examination ofa large spectrum ofspecies, especially those parasitizing poorly studied hosts. Second, it must be ascertained W. de Souza ,&) Laborato rio de Ultraestrutura Celular Hertha Meyer, whether specialized structures such as the cytostome and Instituto de Biofõ sica Carlos Chagas Filho, the plastid found in Toxoplasma, Eimeria, and Plasmo- Universidade Federal do Rio de Janeiro, CCS, Bloco G, dium also occur in members ofother genera and families. Ilha do FundaÄ o, 21941-900 Rio de Janeiro-RJ, Brazil Third, a comparative analysis should be made ofthe e-mail: [email protected] Tel.: +55-21-2602364; Fax: +55-21-2808193 type ofparasitophorous vacuole foundin dierent species ofthe Apicomplexa. J. A. P. Diniz With these points in mind, we decided to study the Unidade de Microscopia Eletroà nica, Instituto Evandro Chagas, Belem, Para , Brazil ®ne structure ofa number protozoa parasitizing the blood cells ofreptiles in the Amazon region ofBrazil. In E. O. Silva Departamento de Patologia, Centro de Cieà ncias Biolo gicas, this paper we describe the ®ne structure of Garnia Universidade Federal do Para , Bele m, Para , Brazil gonatodi, a haemosporine initially described by Telford ,1970, 1973) as P. gonatodi in the blood ofthe lizard R. Lainson Laborato rio de Coccõ deos, Instituto Evandro Chagas, Gonatodes humeralis ,Reptilia: Lacertilia) from Panama Bele m, Para , Brazil and more recently redescribed and reassigned to a new 972 genus, Garnia, on the basis ofthe observation that in no electron-dense inclusion at the periphery or in the more stage ofdevelopment is malarial pigment produced central portion ofthe organism ,Figs. 2, 12). This ,Lainson et al. 1971, 1974; Boulard et al. 1987). Its ®ne structure morphologically resembles the acidocalcisome, structure has been described as being characteristic of which has been well characterized in members ofthe the Apicomplexa ,Boulard et al. 1987), and our present Trypanosomatidae family ,Docampo et al. 1995; Lu observations provide new information regarding the et al. 1998) and recently described in T. gondii ,Moreno parasite's ultrastructure and its relationship as estab- and Zhong 1996). A cytostome was also detected, with a lished in the host cell. more electron-dense region ofthe cytoplasm being seen near its opening. Electron-dense material within the cytostome was similar to that found in the host ery- Materials and methods throcyte cytoplasm ,Fig. 5). In addition to the subpellicular microtubules, another Specimens of Gonatodes humeralis were collected by hand from the group ofmicrotubules was seen to be closely associated trunks oftrees in an area ofriverine forestin Capanema, Para with the mitochondrion ,Figs. 4, 5, 11, 13). These State, northern Brazil, and were maintained in the laboratory on a diet oflarval ¯our beetles , Tenebrio molitor). Blood was collected structures may correspond to subpellicular microtubules from the orbital sinus using a ®nely drawn-out glass pipette, and that have changed their position and become associated thin blood ®lms were rapidly air-dried, ®xed in absolute methyl with the mitochondrion. A similar process has been alcohol, and stained with Giemsa. For transmission electron mi- observed in trypanosomatids harboring a bacterium-like croscopy ,TEM) the blood was ®xed for 120 min at room tem- endosymbiont ,Freymuller and Camargo 1981). In the perature ,approximately 250 °C) in a solution containing 2.5% glutaraldehyde, 2% freshly prepared formaldehyde in PHEM case ofthe trypanosomatids, however, the microtubule- buer ,pH 6.9; Schliwa and Van Blerkorm 1981). The cells were mitochondrion association is restricted to the region then washed in the buer; post®xed in a solution containing 1% located immediately below the plasma membrane, osmium tetroxide, 0.8% ferrocyanide, and 5 M CaCl2; dehydrated whereas in the case of G. gonatodi this association was in acetone; and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss 900 also observed in other parts ofthe cell ,Fig. 5). Mi- electron microscope. For localization ofcarbohydrates, thin sec- crotubules associated with the mitochondrion were ob- tions were collected on gold grids and subjected to the periodic served in both recently formed trophozoites and young acid-thiosemicarbazide-silver proteinate technique ,Thie ry 1967). meronts containing two nuclei. As it has previously been shown that the subpellicular microtubules of Plasmodi- um disappear during the transformation of merozoites Results and discussion into trophozoites, the microtubules associated with the mitochondrion in G. gonatodi may represent a new set of We examined 90 Gonatodes humeralis, and approxi- microtubules. mately 50% were infected with Garnia gonatodi as di- The process ofmerogony observed in G. gonatodi agnosed by the Giemsa-stained blood ®lms. Usually the appears to be similar to that described in other members level ofparasitemia was low, but a fewspecimens were ofthe Apicomplexa. Intranuclear spindle microtubules heavily infected ,Fig. 1), with abundant trophozoites, were always evident ,Figs. 6, 7) and, as described for meronts, and gametocytes showing the characteristic other apicomplexans, apical organelles and the subpel- morphology previously described elsewhere ,Lainson licular microtubules appeared in the ®nal stages of et al. 1971); the absence ofmalarial pigment was readily asexual division ,data not shown). The parasite divides con®rmed microscopically using polarized light. by schizogony, with the formation of merozoites oc- TEM ofthin sections showed the parasites to lie curring at the ®nal stage ,Figs. 8, 9). within a parasitophorous vacuole ,PV) localized in the One characteristic feature seen in cells infected with erythrocyte cytoplasm and to possess an ultrastructure G. gonatodi was the disposition ofthe host-cell endo- typical ofmembers ofthe Apicomplexa, involving the plasmic reticulum ,ER). At the onset ofinfection, cis- following structures: ,1) a membrane system composed ternae ofthe ER concentrate around the PV containing ofan outer pellicle and an inner complex formedby two parasites in the process of transformation from mero- closely apposed unit membranes ,Figs. 2±5); ,2) an an- zoites to trophozoites ,Figs. 10±12). With development terior conoid with associated subpellicular microtubules ofthe trophozoite this association becomes more obvi- ,Fig. 9); ,3) a small number ofrhoptries; and ,4) the ous, and it was possible to see a connection between the apicomplast, also known as the plastid or ``adnuclear ER cisternae and the PV membrane. In cells containing organelle'', surrounded by four membranes and usually several parasites the association was so pronounced that located close to the nucleus ,Fig. 3). These nonpho- the ER cisternae seemed to connect the various parasites tosynthetic plastids have previously been described in ,Fig. 13). Toxoplasma gondii and species of Plasmodium, Theileria, Previous