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 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 a€ecting humans protozoa ofthe group have been carried ,e.g., and Toxoplasma) or animals ofeco- out with members ofthe Toxoplasma, , and nomic importance ,e.g., Eimeria). Within the , 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) , containing blood cells ofthe Amazonian reptile Gonatodes ofthe Plasmodium, the true pigmented humeralis ,Reptilia: Lacertilia). Transmission electron parasites ofreptiles, , 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) , 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, ,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 di€erent 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 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- bu€er ,pH 6.9; Schliwa and Van Blerkorm 1981). The cells were mitochondrion association is restricted to the region then washed in the bu€er; 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). and species of Plasmodium, , Previous studies ,Melo and De Souza 1997; Sinai and Eimeria ,Wilson et al. 1993; Creasey et al. 1994; et al. 1997) have shown that there is some degree of Kohler et al. 1997; Wilson and Williamson 1997; Denny association between the host-cell ER cisternae and the et al. 1998; Zhao and Duszynski 1999). PV in cells infected with T. gondii. This association is not Images oftrophozoites showed a feworganelles as evident, however, as that described herein for cells surrounded by a typical membrane and containing an infected with G. gonatodi. At present, the role played by 973

Fig. 1 Giemsa-stained blood preparation showing a large number of Fig. 3 Young schizont with two nuclei ,N) and an apicoplast ,P) parasitized erythrocytes. Young trophozoites ,large arrowhead), enveloped by four unit membranes. ´45,000 schizonts ,small arrowhead), immature microgametocytes ,short Fig. 4 High magni®cation ofa young schizont, showing a longitu- arrow), and macrogametocytes ,thin arrow) are visible. ´1,000 dinal view ofthe association ofthe microtubule with the mitochon- Fig. 2 Thin section ofa macrogametocyte, showing a dense drion. Small bridges ,arrowheads) are seen connecting the two cytoplasm and small vacuoles that contain electron-dense material structures. The circle indicates the membrane lining the PV and the resembling the acidocalcisome ,arrows). ´21,600 complex membrane that envelopes the protozoan. ´60,000 974

Fig. 5 Trophozoite showing a typical cytostome ,Cy)anda ´18,000 mitochondrion ,M) associated with microtubules ,arrows),N Nucle- Fig. 7 Young schizont with two nuclei and a mitochondrion. us). ´50,000 Intranuclear spindle microtubules are visible in a dividing nucleus. Fig. 6 Young schizont with two nuclei ,N) and a mitochondrion ,M). ´90,000 975

Figs. 8, 9 Di€erent phases ofthe schizogonic process this ER-PV association remains unclear. One possibility, Fig. 8 The beginning ofthe individualization ofmerozoites. ´18,000 which deserves further investigation, would be that the Fig. 9 A more advanced stage ofthe schizogonic process, with individualized merozoites showing the apical complex ,arrows). ´10,500 ER participates in the process ofgrowth ofthe PV as the Fig. 10 General view ofa cell infectedwith only one parasite , P). intravacuolar parasites divide and the vacuole increases ´14,000 in size. 976

Figs. 10±13 Trophozoites within the PV, showing various aspects of ´40,000 the association ofthe host-cell ER , arrows) with the vacuole Fig. 13 Demonstration ,short arrows) ofthe clear association of Figs. 11, 12 Higher magni®cations ofthe cell shown in Fig. 10. microtubules with the mitochondrion ,M). ´50,000 977

Acknowledgements This work was supported by Programa de Lainson R, Landau I, Shaw JJ ,1974) Further parasites ofthe Nu cleos de Exceleà ncia ,PRONEX), Financiadora de Estudos e family Garniidae ,Coccidiida: Haemosporidiidae) in Brazilian Projetos ,FINEP), Conselho Nacional de Desenvolvimento . Fallisia e€usa gen. nov., sp. nov., and Fallisia modesta Cientõ ®co e Tecnolo gico ,CNPq), and the Wellcome Trust. gen. nov., sp. nov. Parasitology 68: 117±125 Lu H-G, Zhong L, De Souza W, Benchimol M, Moreno SNJ, Docampo R ,1998) Ca2+ content and expression ofan acid- ocalcisomal calcium pump are elevated in intracellular forms of References Trypanosoma cruzi. Mol Biol Cell 18: 2309±2323 Melo ET, De Souza W ,1997) Relationship between the host cell Boulard Y, Landau I, Baccam D, Petit G, Lainson R ,1987) endoplasmic reticulum and the parasitophorous vacuole con- Observations ultrastructurales sur les formes sanguines des taining Toxoplasma gondii. Cell Struct Funct 22: 317±323 Garniides ,Garnia gonadoti, G. uranoscodoni et Fallisia e€usa) Moreno SNJ, Zhong L ,1996) Acidocalcisomes in Toxoplasma parasites de lezards Sud-Americains. Eur J Protistol 23: 66±75 gondii tachyzoites. Biochem J 813: 655±659 Creasey A, Mendis K, Carlton J, Williamson D, Wilson I, Carter R Schliwa M, Van Blerkorm J ,1981) Structural interaction ofcyto- ,1994) Maternal inheritance ofextrachromosomal DNA in skeletal components. J Cell Biol 90: 222±235 parasites. Mol Biochem Parasitol 65: 359±363 Sinai AP, Webster P, Joiner KA ,1997) Association ofhost cell Denny P, Preiser P, Williamson DH, Wilson I ,1998) Evidence for a endoplasmic reticulum and mitochondria with the Toxoplasma single origin ofthe 35 kb plastid DNA in apicomplexans. gondii parasitophorous vacuole membrane: a high anity in- Protist 149: 51±59 teraction. J Cell Sci 110: 2117±2128 Docampo R, Scott DA, Vercesi AE, Moreno SNJ ,1995) Intra- Telford SR ,1970) Saurian parasites in eastern Panama. J Proto- cellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi. zool 17: 566±574 Biochem J 310: 1005±1012 Telford SR ,1973) Saurian malarial parasites from Guyana: their Freymuller E, Camargo EP ,1981) Ultrastructural di€erences be- e€ect upon the validity ofthe familyGarniidae and the genus tween species oftrypanosomatids with and without endo- Garnia, with a description oftwo new species. Int J Parasitol 3: symbionts. J Protozool 28: 175±182 829±842 Kohler S, Delwich CF, Denny PW, Tilney LG, Webster P, Wilson Thie ry JP ,1967) Mise en e vidence des polysaccharides sur coupes RJM, Palmer JD, Roos D ,1997) A plastid ofprobable green ®nes en microscopie e lectronique. J Microsc 6: 987±1018 algal origin in apicomplexan parasites. Science 275: 1485±1489 Torii M, Aikawa M ,1998) Ultrastructure ofasexual stages. In: Lainson R ,1992) A protozoologist in Amazonia: neglected para- Sherman I ,ed) Malaria: parasite biology, pathogenesis, and sites, with particular reference to members of the protection. American Society for Microbiology, Washington, ,Protozoa: Apicomplexa). Cienc Cult ,Sao Paolo) 44: 81±93 pp 123±134 Lainson R ,1995) Progarnia archosauriae nov. gen., nov. sp. Wilson I, Gardner M, Rangachari K, Williamson DH ,1993) Ex- ,Haemosporina: Garnidae), a blood parasite of Caiman cro- trachromosomal DNA in the Apicomplexa. NATO ASI Ser H codilus crocodilus ,Archosauria: Crocodilia), and comments on 78: 51±52 the evolution ofreptilian and avian haemosporines. Parasitol- Wilson RJM, Williamson DH ,1997) Extrachromosomal DNA in ogy 110: 513±519 the Apicomplexa. Microbiol Mol Biol Rev 61: 1±16 Lainson R, Landau I, Shaw JJ ,1971) On a new family of non- Zhao X, Duszynski DW ,1999) Co-evolution ofplastid and nuclear pigmented parasites in the blood ofreptiles: Garniidae fam. DNA in ten Eimeria spp. from rodents ,abstract 17). Proceed- nov. ,Coccidiida: Haemosporidiidae). Some species ofthe new ings, annual meeting ofthe American Society forParasitology, genus Garnia. Int J Parasitol 1: 241±250 Monterrey, California, USA July 6±9, 1999