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http://www.elsevier.de/protisPublished online date 20 January 2006

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& 2005 Elsevdoi:10.1016/j

157, 13—19, January 2006

Protist, Vol.

ORIGINAL PAPER

Ultrastructural Study of the Gametocytes andMerogonic Stages of Fallisia audaciosa(Haemosporina: Garniidae) that Infect Neutrophilsof the Lizard Plica umbra (Reptilia: Iguanidae)

Edilene O. Silvaa,1, Jose Antonio P. Dinizb, Ralph Lainsonc, Renato A. DaMattad, andWanderley de Souzae

aLaboratorio de Parasitologia, Departamento de Patologia, Centro de Ciencias Biologicas, UniversidadeFederal do Para, Av. Augusto Correa s/n, Bairro Guama, 66075-110 Belem, Para, Brazil

bUnidade de Microscopia Eletronica, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco,66090-000 Belem, Para, Brazil

cLaboratorio de Coccıdios, Departamento de Parasitologia, Instituto Evandro Chagas,Av. Almirante Barroso 492, Bairro Marco, 66090-000 Belem, Para, Brazil

dLaboratorio de Biologia Celular e Tecidual, Centro de Biociencias e Biotecnologia, Universidade Estadual doNorte Fluminense, Avenida Alberto Lamego 2000, 28013-600 Campos dos Goytacazes, RJ, Brazil

eLaboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofısica Carlos Chagas Filho, UniversidadeFederal do Rio de Janeiro, Ilha do Fundao, 21941-590 Rio de Janeiro, RJ, Brazil

Submitted June 10, 2005; Accepted October 30, 2005Monitoring Editor: C. Graham Clark

Little is known regarding the ultrastructure of the genus Fallisia (Apicomplexa: Haemosporina:Garniidae). This report describes the fine structure of some developmental stages of Fallisiaaudaciosa that infect neutrophils in the peripheral blood of the Amazonian lizard Plica umbra (Reptilia:Iguanidae). The parasites lie within a parasitophorous vacuole and exhibit the basic structures ofmembers of the Apicomplexa, such as the pellicle and the cytostome. Invaginations of the innermembrane complex were seen in the gametocytes and may be concerned with nutrition. The merontswere irregularly shaped before division, a feature unusual among members of the Apicomplexa. Theunusual presence of a parasitic protozoan within neutrophils, in some way interfering with ormodulating the microbicidal activity of such cells, is discussed.& 2005 Elsevier GmbH. All rights reserved.

Key words: Fallisia audaciosa; Garniidae; lizard; neutrophil; Plica umbra; ultrastructure.

Introduction

Species of the genus Fallisia are apicomplexanparasites of lizards (Lainson et al. 1971, 1975) and,

g author;01 7601.@ufpa.br (E.O. Silva)

ier GmbH. All rights reserved..protis.2005.10.003

more rarely, birds (Gabaldon et al. 1985). All theirdevelopmental stages occur in leucocytes orthrombocytes of their vertebrate hosts (Lainsonet al. 1971). Fallisia audaciosa infects the neutro-phils of the iguanidae lizard Plica umbra (Lainsonet al. 1975). Neutrophils, immune cells involved inthe first line of defense, are capable of migratingto inflamed tissue to destroy bacteria, fungi, and

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14 E.O. Silva et al.

protozoa by an impressive array of microbicidalmechanisms, such as production of reactiveoxygen species and the release of cytotoxicmolecules stored in their cytoplasmic granules(Faurschou and Borregaard 2003; Kobayashi et al.2003). Although some reports describe thatneutrophils are infected by a few microbialpathogens (Chen et al. 1994; Laskay et al. 2003;Laufs et al. 2002; Yoshiie et al. 2000), the onlydescription of a protozoan multiplying in neutro-phils appears to be that of F. audaciosa (Lainsonet al. 1975).

Although the morphology of F. audaciosa in thelizard host has been well documented by lightmicroscopy (Lainson et al. 1975), there have beenno studies of its fine structure. In this study, wehave confirmed that the host cell of F. audaciosa isthe neutrophil and describe the ultrastructure ofthe macrogametocyte and merogonic stages ofthe protozoan.

Figure 1. Giemsa-stained blood film showing aneutrophil of Plica umbra infected by Fallisia auda-ciosa. A. Infected neutrophils showing gametocytes(arrow). Note the lobulated nuclei of the host cells.B. Early meront (arrow). C. A mature meront (arrow) andan uninfected neutrophil (arrowhead). Bar ¼ 10mm.

Results

Observations by light microscopy of blood films ofa highly infected lizard showed that 25% of theblood neutrophils were infected. Gametocytes,meronts, and merozoites were confined to neu-trophils (Fig. 1A—C). Multiple invasion of neutro-phils was not seen in the present material,although Lainson et al. (1975) noted that thiswas ‘‘y a marked feature in heavy infections’’.The nucleus of a few infected neutrophils wasaltered and the cells were enlarged, probably dueto the advanced stage of infection (Fig. 1C).

TEM examination showed that uninfected neu-trophils were irregular in shape, with delicatemembrane projections. The centrally locatedmultilobulated nucleus contained large clumps ofheterochromatin in close contact with the nuclearmembrane. The cytoplasm contained vacuolesand a few granules (data not shown).

Infected neutrophils showed some irregularprojections and had parasites within a parasito-phorous vacuole (Figs 2A,B, 3A,C,D). Only macro-gametocytes and meronts were observed byTEM. The macrogametocytes had a three-layeredpellicle composed of an outer and an innermembrane complex formed by two closely ap-posed membrane units (Fig. 2B, inset). Invagina-tions of the inner membrane complex ofgametocytes were observed (Fig. 2C—E). A largevacuole, the contents of which were similar to thatfound in the host neutrophil cytoplasm, was foundinside the parasite (Fig. 2E). In some of the

macrogametocytes, an apicoplast was seen (datanot shown).

At the beginning of segmentation, meronts wereirregular in shape (Fig. 3A,B). Mature forms hadmany nuclei (Fig. 3C). At the end of merogony, themerozoites within the parasitophorous vacuolewere of heterogeneous density (Fig. 3D,E). Insome of the developing merozoites, a structureresembling an apical complex was apparent, with

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Figure 2. Transmission electron microscopy of peripheral blood neutrophils infected with Fallisia audaciosa.A. General view of an infected neutrophil (N) showing a macrogametocyte (MA). B. Section through amacrogametocyte (MA) infecting a neutrophil (N). Note the high electron-dense pellicle (arrows), see alsoinset (2� ). C. Invagination of the inner membrane complex (arrow). D. High magnification of a typicalmembrane system showing a cytostome (CY). E. Another image showing an invagination ending in a vacuole(arrow) containing material with similar electron density to that of the neutrophil cytoplasm. Neutrophil (N),parasite (P). Bars A, B ¼ 1mm; C—E. Bar ¼ 0.5 mm.

15Ultrastructure of Fallisia audaciosa

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Figure 3. Transmission electron microscopy of the merogonic process of Fallisia audaciosa. A. Thebeginning of merogony (arrowheads). B. Higher magnification of A. C. Further stages of merogony. Note thenumerous nuclei (asterisks). D. General view of an advanced stage of merogony with individual merozoitesvisible (arrows). E. Higher magnification of the merozoites (ME) showing structures suggestive of an apicalcomplex (arrow). Note the different electron density of the merozoites. Neutrophil (N), parasite (P).Bar ¼ 1mm.

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17Ultrastructure of Fallisia audaciosa

organelles similar to rhoptries (Fig. 3E). However,polar ring and dense bodies were not observed.

Discussion

The fine structure of some other Fallisia specieshas been described in some detail (Boulard et al.1987; Silva et al. 2005). The results of this reportprovide new information regarding ultrastructuralaspects of the species F. audaciosa infecting theneutrophils of the lizard P. umbra. TEM study ofthe parasite within the parasitophorous vacuoleshowed a complex pellicle system and apicalstructure suggesting the presence of an apicalcomplex. Although these parasites can probablyingest nutrients from host cell cytoplasm througha cytostome, gametocytes also showed deepinvaginations of the membrane complex thatmay be involved in nutrient intake by the parasite.A similar structure has been reported in gameto-cytes of another species of Fallisia, F. effusa (Silvaet al. 2005), and also in Eimeria auburnensis(Hammond et al. 1967). Silva et al. (2004) alsodescribed a similar structure in a hematozoan,suspected to be a species of Lainsonia (Lainsonet al. 2003) in the monocytes of the lizard Ameivaameiva. In all cases, it was suggested that theseinvaginations might increase the cell surface fornutritional purposes.

At the beginning of segmentation, the maturemeront of F. audaciosa was seen to differ fromother apicomplexans, in which there is usuallysegmentation of the thick inner membrane and therandom appearance of rhoptries beneath andalong the plasma membrane of the mother cell(Aikawa and Sterling 1974). Fallisia audaciosa isirregularly shaped before nuclear division, sug-gesting that the merogonic process is variablewithin the phylum Apicomplexa.

In general, it is assumed that neutrophils are thefirst line of defense in vertebrates and are highlyefficient in killing invading microorganisms(Faurschou and Borregaard 2003; Kobayashiet al. 2003). However, recent evidence has shownthat some pathogens evade the microbicidalmechanisms or inhibit the spontaneous apoptosisof neutrophils (Aga et al. 2002; Laskay et al. 2003;Laufs et al. 2002; Yoshiie et al. 2000). Aga et al.(2002) showed that Leishmania promastigotes canmodify spontaneous apoptosis of human neutro-phils, but they do not replicate inside these cells.Gamonts of Hepatozoon canis were found toinfect circulating neutrophils of dogs; however,no division stages were observed in these cells

(Droleskey et al. 1993). Only two pathogenicmicroorganisms have been shown to replicatewithin mammalian neutrophils (Herron et al. 2000;Van Zandbergen et al. 2004). One of them isAnaplasma phagocytophilum, which causes hu-man granulocytic ehrlichiosis. This obligate intra-cellular bacterium is transmitted by ixodid tickbites, and can inhibit apoptosis, phagosome—lysosome fusion, and production of reactiveoxygen species of neutrophils (Gokce et al.1999; Mott and Rikihisa 2000; Scaife et al. 2003;Yoshiie et al. 2000). The other example isChlamydia pneumoniae, which has been shownto delay apoptosis and to multiply inside neutro-phils (Van Zandbergen et al. 2004). Apoptosis is animportant mechanism in human neutrophils for theregulation of homeostasis and inflammation (Savill1997). Fallisia audaciosa seems to be the onlyrecorded protozoan that naturally infects andmultiplies exclusively inside neutrophils, anachievement which prompted Lainson et al.(1975) to name it as F. audaciosa. The mechan-isms responsible for this survival are not yetknown; however, we can speculate that F. auda-ciosa probably delays the apoptotic process ofthe lizard neutrophil and inhibits the production ofreactive oxygen species, enabling its survival inthese cells. For a better understanding of thisintriguing association, additional studies will benecessary to determine just how this parasiteevades the microbicidal response of neutrophils.

Methods

Lizards: During 2001 and 2003, 76 lizard speci-mens of Plica umbra were hand-captured from thetrunks of trees in the tropical rain forest of ParaState, north Brazil. They were maintained in ananimal house of the Instituto Evandro Chagas andkept separately in standard mouse cages at 27 to32 1C. They were provided with water and larvaeand adults of Tenebrio molitor.

Blood harvesting and leukocyte separation:Lizards were restrained manually and blood wascollected by cardiac puncture into heparinized1 ml syringes (100 U/ml). Blood smears from alllizards were fixed with absolute methanol andstained by Giemsa’s method. Blood leukocyteswere separated using a method previously de-scribed (Silva et al. 2004).

Light microscopy: The parasites were studiedin Giemsa-stained blood smears, and photo-graphed using a Zeiss Axiophote Microscope withthe immersion 100� objective.

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Transmission electron microscopy: Bloodbuffy coat was fixed in 2.5% glutaraldehyde and4% formaldehyde, in a buffer solution containing60 mM Pipes, 20 mM Hepes, 10 mM ethylenegly-col-bis- (B-aminoethylether)-N,N,N0-tetraaceticacid, 70 mM KCl, and 5 mM MgCl2 (Schliwa andvan Blerkom 1981), pH 6.9, for 1 h at roomtemperature. Afterward, cells were washed in thesame buffer and post-fixed for 1 h in a solutioncontaining 1% osmium tetroxide, 0.8% ferrocya-nide, and 5 mM calcium chloride, washed,dehydrated in graded acetone, and embedded inepoxy resin. Thin sections were stained withuranyl acetate and lead citrate and examined ina Zeiss EM 900 transmission electron microscope.

Acknowledgements

This work was supported by grants from ConselhoNacional de Desenvolvimento Cientıfico e Tecno-logico (CNPq), Financiadora de Estudos e Proje-tos (FINEP), Programa de Nucleos de Excelencia(PRONEX), Instituto Evandro Chagas/SVS/MS,Fundac- ao de Coordenac- ao de Pessoal de NıvelSuperior (CAPES), Programa Nacional deCooperac- ao Academica (CAPES-PROCAD),Fundac- ao Carlos Chagas Filho de Amparo aPesquisa do Estado do Rio de Janeiro (FAPERJ),and the Wellcome Trust, London (RL). The authorsthank Manoel CM de Souza and Antonio J deOliveira Monteiro for their technical assistanceand animal care, and Marcia A. Dutra and EliandroLima for their assistance with the photographicwork. The experiments comply with currentBrazilian animal protection laws (IBAMA doc.02018.000301/02-13).

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