Some Blood Parasites of the Brazilian Lizards Plica Umbra and Uranoscodon Superciliosa (Iguanidae)

Parasitology (197.5), 70, 119-141 1 With 6 plates and 54 figures

Some blood parasites of the Brazilian lizards Plica umbra and Uranoscodon superciliosa (Iguanidae)

R. LAINSON,* J. J. SHA W* and I. LANDAUt * Tke WellcomeParasitology Unit, Caixa Postal3, Instituto Evandro Chagas,Belém, Pará, Brazil t MuséumNational d'Histoire Naturelle,Laboratoire de Zoologie(Vers), 57, Rue Cuvier,Paris Ve,France

(Received 19 August 1974)

SUMMARY An examination of 235Plica umbra and 167 Uranoscodonsuperciliosa (Sauria: Iguanidae) from Belém,Pará, Brazil, has revealeda number of hitherto undescribedblood parasites. These include Fallisia audaciosa, F. simplex,Garnia multiformis,Plasmodium vacuolatum and Trypanosoma plicae spp.nov., alI in P. umbra,and G. uranoscodonisp.nov. in U. superciliosa. In addition, both speciesof lizards showed occasionalEpery- throzoon and microfilaria infections. Among the haemosporines, F. audaciosais particularly interesting in its restriction to the neutrophils; and G. multiformis for its early developmentin erythroblasts of the bone-marrow, and its variable morphology in subsequentstages of the infection in the peripheralblood. Mixed infections of the different haemo- sporinesresulted in 18 combinationsof parasitesin P. umbra,indicating the importance of examining a large number of theseanimaIs when pre- paring descriptions of the individual organisms. T. plicae is a large, broad trypanosome,averaging '" 57.0 x 16.0p,m. Isolation ofthe parasite was made from four out of six P. umbra, after the culture of heart blood in NNN medium: the fact that none of the four positive lizards showed detectabletrypanosomes in the peripheralblood suggestsa high incidence of occult infections. The incidence amongthe remaining 229P. umbra, as given by the direct examination of blood-films, was only 10.0%.

INTRODUCTION During the past 8 years we have examined the blood of 235 specimens of Plica umbra (Linnaeus) and 167 Uranoscodonsuperciliosa (Linnaeus), alI from the neigh- bourhood ofBelém, Pará State, north Brazil. OftheP. umbra 114 (48.5%) showed one or more parasites in the peripheral blood, including a trypanosome, microfilaria, Eperythrozoon, hitherto undescribed Plasmodium and Garnia species and two new species of the recently described haemosporine Fallisia. Among the U. superciliosa, 59 (35.3 %) showed blood parasites: these were limited to a single new Garnia species, Epe.rythrozoonand a microfilaria. Mixed infections of haemosporines in lizards can cause considerable difficulties in interpretation of the blood cycle of the di:fferent parasites (Lainson, Landau &

19 LAINSON, J. J. SHAW AND I. LANDAU Shaw, 1974b).ln the presentstudies, many P. umbrashowed various combinations of the Plasmodium,Garnia and Fallisia infections (Table 1),while others had such scanty parasitaemiasof theseparasites that a confidentdiagnosis was impossible. Similarly, although U. superciliosapresented but a single haemosporinein alI the positive specimensexamined, the majority of the infections was extremely light and often appeareddevoid of gametocytes.lt was for these reasonsthat it was necessaryto examine suchlarge numbers of both lizards before sufficiently heavy infections of the single parasitescould be found.

MATERIALS AND METHODS P. umbra and U. 8upercilio8a are both arboreal iguanids, although they do frequently descend to ground leveI. They are easily caught by hand, or with a poose on a stick, as they spend much of the day on the trunks of the trees. . Ability to maintain lizardsalive in the laboratory is important in order to follow the course of infections: neither species ofrers much difficulty, being best kept in large mosquito cages and fed on a mixed diet of grasshoppers and other insects which are readily obtained with a sweep-net. In the absence of sunlight, inter- mittent heat may be provided by placing a 40 W lamp a few inches from one side of the cage: this seems especially important for some time after the animaIs have been fedo The laboratory environment is usually very much drier than their natural habitat, and the lizards should be provided with a constant water supply in shallow dishes. Lizards were bled from the orbital sinus, by capillary pipette, and blood films dried as quickly as possible before staining with Giemsa: tissue smears were either treated in the garoe manner or stained by a modified Giemsa method after fi:x:.ation in aqueous Bouin's fluido The techniques have been given elsewhere (Lainson, 1959; Lainson, Landau & Shaw, 1971). When possible, blood films were prepared at 2- or 3-day intervals, for periods up to a month or more, in order to obtain a better idea of the developing infections. Measurements of the haemosporines were based on 50 randomly selected parasites of the various asexual and sexual stages, using an eyepiece micrometer or camera-Iucida drawings. Whenever possible they were made from lizards which were, as far as we could see, infected with but a single parasite species. The trypanosoriles were measured by a photographic method, already described for Trypano8oma vivax (Shaw & Lainson, 1971). Photomicrographs were taken with a Zeiss W. L. research microscope, using Adox KB 14 and 17, 35 mm film. AlI measurements are recorded in pm.

DESCRIPTION OF THE PARASITES The name Fallisia has been given to some haemosporineparasites of lizards which undergo both their schizogonyand gametogonyin leucocytesor thrombocytes of the peripheral blood (Lainson, Landau & Shaw, 1974a).There is no developmentin the red blood cell series, by which character the organismsare

120R. Blood parasites 01 Brazilian lizards distinguishedfrom Garnia,the other known genusin the family Garniidae (Lainson et alo 1971). Two distinctly different speciesof Fallisia were encountered in P. umbra during the presentstudies.

Fallisia audaciosa sp. nov. (Figs. 1-7; PI.. 1A-I) The remarkable feature of this haemosporine is that development in the peripheral blood appears to be exclusively within neutrophils -cells which should play a predominant role in destroying the parasite. Four separate infections were studied and the course of infection followed in one of the lizards for 16 days, when the animal died. On no occasion was the parasite seen to invade other blood cells, and tissue smears of the spleen, liver, lung, kidney, bone-marrow and brain revealed no other division processo When the animal was first examined, immature gametocytes were akeady abundant in the blood but schizogony was at a relatively low leveI..The number of schizonts increased steadily over the next 7 days, when the parasitaemia reached massive proportions with almost alI the neutrophils containing one or more parasites. During the subsequent week the schizogony slowly waned, although schizonts were still abundant when the lizard died on day 16. Multiple infection of the cells was extremely common (Fig. 7; PI.. 1A, B, G, H), sometimes with as many as five parasites in a single neutrophil.

Schizogony The youngestparasites seen measured -2.0 x 1.0 (Fig. 1) and were more or less oval in shape.With growth, they showedno amoeboidactivity and an oval-round forro was maintained throughout subsequentnuclear division. As the schizonts increasedin size the cytoplasm stained a bright blue, with the nuclei very well deftned: azurophilic granules were rarely seen, and there was no conspicuous cytoplasmic vacuolation (Fig. 2; PI. lA, B). Unruptured, mature schizontswere spherical or oval, often filling the entire host cell (Fig. 3): they measuredfrom 13.0 x 11.0 (- 100 merozoites)to 18.0x 18.0 (- 150merozoites), with an average of 14.3x 13.2. The number of merozoites,however, varied from 50 to 150, as determined by the numerous ruptured, mature schizonts seen at the height of schizogony(PI. 1 C, D). They varied greatly in shape,probably due to distortion in making the blood films, and the normal forro is probably tear-shaped,as seenin someunruptured cells (Fig. 4). They measured-2.0 x 1.0.

Gametogony Young gametocyteswere distinguishedas malesand femaleswhen measuringas little as 4.0 x 3.0, although at this stage they stained rather faintly (PI. 1 E, F). The cytoplasmofthe developingmicrogametocyte was a delicategrey-blue, with the nuclear material already diffuse. The young macrogametocyteswere more conspicuous,their cytoplasm being brighter blue and the nucleusmore compacto At this early stageazurophilic granuleswere very inconspicuous,or not presentat

121 122 R. LAINSON, J. J. SHA W AND I. LANDAU alI: they increasedin number and size with growth of the gametocytesand were always very much more prominent in the female parasite. Apparently mature gametocytes (Figs. 5-7; PI. lI) were oval or rounded, frequently with a smalI, sphericalvacuole (rarely 2) which was commonlyplaced towards one pole of the more oval parasites.The cytoplasm of the female now stained a bright blue, the nucleus most usualIy being in the forro of a band, with a conspicuousendosome. In contrast, the male gametocytes stained an overalI pink. Macrogametocytes ranged from 7.8x 7.8 to 10.4x 7.8, average 9.5 x 7,5: microgametocyteswere slightly smalIer, from 7.8x 6.5 to 9.1 x 7.8, average9.0x 7.0.

Effects on the Mst cells This was most marked in thoseneutrophils containingmature or n~arly mature schizonts, when the cell becameenlarged and its nucleusforced to the periphery (Fig. 3). In many instancesthe nucleuswas seento becomefragmented and clearly degenerate: on the other hand, neutrophils containing one or even two mature gametocytesseemed to undergo little alteration other than a slight enlargement of the cell and displacementof the host nucleus.Multiple infections enlargedcells considerably. Fallisia simplex sp.nov. (Figs. 8-15; PIso lJ-L; 2A-E) This delicate little parasite develops in the thrombocytes of P. umbra and was found in 20 of the 235 specimens examined (8.5%). Of these infections, 11 were variously mixed with the other haemosporines described here (Table 1), but 9 apparently pUle infections were encountered. Seven of these were light and com- posed almost entirely of mature gametocytes, and diagnosis was based on single blood films. The remaining 2, however, were followed for periods of 2 and 4 weeks respectively, enabling us to gain a reasonably accurate impression ofthe schizogony and gametogony, and to reei confident that we were dealing with the life-history of but a single parasite. Schizogony The early stages (Fig. 8; PI. 1 J, K) a,ssumeda variety of shapes, elongate, oval, or even stellate. As growth and nuclear division ensued, the young schizont usually assumed a crescentic shape as its e:x:pansionwas impeded by the thrombocyte nucleus (Fig. 9; PI. lL) but, as the latter was pushed to one end of the cell, the older schizonts achieved a more oval outline (Fig. 10; PI. 2A). The cytoplasm of the parasite stained a clear blue, but became lesa and lesa conspicuous as nuclear division continued: azurophilic granules were inapparent throughout development. Apparently mature schizonts were from 6.5 x 6.5 to 10.0 x 6.5, average 7.8 x 6.5: ruptured schizonts were not found and there was some ditliculty in determining the number and shape of the merozoites, which were usually pressed closely together in the cell (Fig. 11). The estimated number ranged from 8 to 28, with an average of 18. The merozoites were ,..,2.0 x 1.0. Blood parasites01 Brazilian lizards

Gametogony Development of the gametocytesfollowed the usual pattern, the sexual stages appearing as small, oval or rounded bodies, with the male and female parasites soon showing the customary colour difference. Normally rounded to oval,some gametocyteswere distorted into crescenticor 'sausage'shapes against the thrombocyte nucleus,while others maintained their smooth outline by indenting or displacing the host-cell nucleus (Figs. 12-15; PI. 2B-E). The cytoplasm of the apparently mature macrogametocytewas compact, stained a bright blue, and very frequently showed a single small 'punched-out' vacuole. The azurophilic granuleswere usually relatively bulky and stained very intensely (Fig. 13): the nucleus was coloured a delicate pink and often showeda prominent endosometowards one extremity. Maleswere of a uniform pinkish hue, with smaller and more delicately staining granules (Fig. 12): they also showeda conspicuousendosome, lying in the diffuse nuclear material. Strangely enough, they very rarely seemedto possessthe vacuole, so characteristic of the female.Macrogametocytes ranged from 6.5 x 5.2 to 10.4x 7.8, average 8.2 x 6.5, andmicrogametocytes were from 6.5 x 5.2 to 9.1 x 7.2, average7.9 x 5.6.

Effects on lhe host cell Both schizonts and gametocytes commonly distorted the thrombocytes from their normal elongate form into oval or almost spherical shapes, often with considerable enlargement of the cell. The host-cell nucleus was variously indented, displaced or squashed to the periphery. We have previously described (Lainson et alo 1974a) another intra-thrombocytic member of the Garniidae, F. effusa, in the teüd lizard N eusticurus bicarinatus. This parasite transforms the thrombocyte into a cyst-like structure, following a remarkable thickening ofthe host-cell wall. No similar hypertrophy was noted in the case of F. simplex. Garnia multijormis sp.nov. (Figs. 16-19,21-34; PIso 2F-H; 3A-I; 4A-J) This haemosporine was by far the most common parasite found in the blood of P. umbra but, at the garoe time, one of the most difficult to study by virtue of its variable morphology. Of the 235 P. umbra examined, 41 (17'5%) showed infections which could be confidently diagnosed by the characteristic gametocytes, and a further 23 showed such scanty parasitaemias in the absence of detectable gametocytes that we were forced to refer to them simply as infections with 'a Garnia sp.'. Reference to Table 1, however, indicates that these scanty parasites were almost certainly always asexual stages of G. multiformis: this would put the actual incidence, then, at 64 infected lizardsout ofthe 235 examined, or 27.2 %. Many of the animaIs infected with G. multiformis were algo showing one or more other haemosporines, including a Plasmodium species which will be described later in this paper. Fortunatelywe were able to find24 specimens with apparently pure

123 124 R. LAINSON, J. J. SHAW AND I. LANDAU infections and, although the characteristic morphological features of the various parasites precluded much risk of confusionother than in the very early asexualstages, we used these single infections for the folIowing description of G. multiformis. A comparisonof the blood ffims at first presenteda confusingpicture. Some lizards showedlight parasitaemias,with scanty schizontsand gametocytesin the mature red blood celIs: in these animaIs the blood-celI picture was essentialIy similar to that of the uninfected lizard, with a predominanceof mature erythrocytes and very smalI numbers of proerythrocytes. In other instanceslizards had heavier infections, almost entirely limited to the proerythrocytes which were present in abnormalIy large numbers (up to 4°%). FinalIy, two infections were noted in which the blood picture could only be describedas that resulting from erythroblastic hyperplasia (PI. 3A). This condition was associatedwith massive schizogony,mostly confinedto the different celIsof the erythroblast series,both in the bone-marrow (PI. 2 F-H) and the blood (Figs. 16, 18, 19; PI. 3A-C). From these observationswe think that the courseof infection is probably as folIows. Mter entry of the parasite, presumablyas sporozoitesfrom an arthropod vector, there is a period of schizogonyin the erythroblasts of the bone-marrow.This may becomesufficiently heavy to stimulate a marked marrow hyperplasia,with resulting invasion ofthe peripheralblood by alI mannerofinfected, immature red blood- celIs.As this initial wave of schizogonywanes the hyperplasiadiminishes and the erythroblasts slowly disappearfrom the circulation: the parasitesare now found predominantly in the proerythrocytes, which are presentin large numbers.As the asexualcycle decreasesstill further the blood picture reverts more to the normal, with few proerythrocytes and scantyparasites which are now mostly in the mature erythrocytes. This is the chronicphase of the infection, and the one most commonly found in the lizards we examined. Whether or not the bone-marrowschizogony still 'ticks over' at a low leveI at this stageremains to be ascertained.

Schizogony In the erythroblasts Schizonts in early and late erythroblasts were very variable in size, depending ofthe number ofmerozoites produced (Figs. 16, 18, 19; PIso 2F-H; 3A-C). Thus, those with as few as 24 averaged '" 8 x 6.5, whilst others with up to '" 150 merozoites were '" 17 x 13. The shape, toa, was variable, but usually oval to spherical: some of the schizonts appeared to lie within a vacuole in the host-cell cytoplasm. The cytoplasm of the parasite stained well, but young forms with few nuclei were difficult to make out against the bright blue of the erythroblast: we could see no azurophilic granules at any stage. The nuclei stained weakly at first, becoming more dense and compact as division proceeded. Many host cells became ruptured in the preparation of the blood or bone-marrow smears, releasing contained parasites and leaving their smudged-out nucleus in the near vicinity (Figs. 16, 18; PI. 3C). This often revealed the mature schizont to be a much more complex affair than would appear from its compact appearance in the Blood parasites of Brazilian lizards 125 unruptured host cell. The cytoplasmbecomes thrown into a network of finger-like protrusions from which the merozoitesare budded off, leaving a bulky residual body (Fig. 18; PI. 30). Very similar schizontshave beendescribed by Ayala (1970) in the developmentof exo-erythrocytic stagesof Plasmodiummexicanum in lizards from Oalifornia. We had the impressionthat the merozoitesproduced by the intra- erythroblastic schizonts of G. multiformis were smaller than those developingin proerythrocytes and erythrocytes, but size may possiblydepend on the number of merozoitesformed. They averaged1.5 x slightly less than 1.0.

I n lhe proerythrocytes As with the forros in the erythroblasts, the young stages (Fig. 21; PI. 3F) showedno amoeboid activity and quickly assumedan oval or rounded shape:the parasites almost always occupied a polar position in the host cell. Early nuclear division (Figs. 22-24)gave bulky but rather poorly staining masses:the cytopl~sm appeareda very pale blue and sometimescontainedone or two pinkish, amorphous bodies the nature of which remains obscure. Very scanty azurophilic granules were seenin occasionalparasites. In the older schizonts disposition of the nuclei was peripheral (Fig. 24), at the curved edge of a fan-shapedschizont (Fig. 25) or scattered throughout the cytoplasm (Fig. 26). The mature schizontsranged from 5.2 x 3.3 to 9.1 x 7.S,average 7.0 x 5.2. Again, the number of merozoiteswas very variable, but much lessthan that seenin the erythroblasts: it ranged from 6 to 40, average16. The merozoitesmeasured -2.0 x 1.5.

In the erythrocytes Again, the parasites almost always occupied a polar position within the cell(93%). Early division stages often assumedan elongate, 'cigar-shape' (PI. 4G), while mature or nearly mature schizonts tended to be irregular in form (PI. 4H-J). There was a marked drop in the number of merozoitesnow produced, which ranged from 4 to 14, average8: the mature schizontswere from 4.0 x 3.5 to 7.8 x 5.2, average5.5 x 3.8, and individual merozoiteswere '" 2.0 x 1.5. One or two azurophilic granules could be seenin '" 30% of the developingschizonts.

Gametogony Mature gametocyteswere almost always in mature erythrocytes (Figs. 17, 33, 34; PI. 4D, E) but alI stagesin their developmentwere found in both erythroblasts (PI. 3D, E) and proerythrocytes (Figs. 27-31; PIso 3G-I; 4A-C). The smallest recognizableforms were oval or spherical,with the usual colourdifference between the microgametocyteand macrogametocyte,as discussedabove. The cytoplasm of the female parasitesshowed small vacuolesand, early ou, both sexesdeveloped very conspicuousazurophilic granules.The parasitesslowly enlarged,maintaining their polar position in the proerythrocytes and their smooth, oval or round shape. Later in the infection, however,the gametocytesbecame more and more elongate and mostly moved to a lateral position: during this processthey lost their smooth contour, assumingan uneven, crenatedappearance (Figs. 33, 34; PI. 4D, E). The change in morphology is shown diagrammatically in Fig. 54: this is a series of 126 LAINSON, J. J. SHAW AND I. LANDAU camera-lucida drawings of male gametocytes, selected at random, in smeB.rsof peripheral blood from a single lizard, on the day of capture (A), 1 week later (B), 2 weeks later (O) and 3 weeks later (D). The mature gametocytes were difficult to measure, due to their variable and irregular shape. They were measured from end to end along the mid-line using camera-lucida drawings and calibrated dividers: the microgametocytes ranged from 10.0 x 4.0 to 21.0 x 3.0, average 12.3 x 3.5; the macrogametocytes were from 10.5 x 2.5 to 20.0 x 3.0, average 12.6 x 3.0. The cytoplasm of the female gametocytes stained a bright blue and was finely vacuolated: the nucleus was most often in the forro of a band, very frequently a ppearing as a pink tine running along the edge of the parasite (Fig. 33). Azurophi1ic granules were abundant, heavily stained, and very often concentrated at the extremities. The male gametocytes were an overall pink, sometimes showing more densely stained streaks. Again, the cytoplasm usually contained numerous vacuoles, and heavily stained azurophilic granules which were, however, less abundant than in the female.

Schizogony in cells other tkan the red blood series Early in the infection, the presenceof large numbers of infected early and late erythroblasts made detection of asexualforms in the leucocytesrather difficult. Scanty schizontsin monocyteswere seenin one lizard, which was otherwiseshow- ing only mature gametocytesand schizontsin mature erythrocytes. The infection appearedto be apure one and the monocytic schizontswere probably those of G. multiformis.

Effects on the host cell The erythroblast Cellscontaining singleschizonts or gametocytesdid not becomeunduly enlarged but the frequency of multiple infections during the erythroblastic schizogony resulted in grossenlargement of thesecells (PIs. 2F, H; 3A), which clearly became fragile and commonly ruptured in preparation of the smears.The nucleus of the infected erythroblast was usually pushedto one side and variably indented, sometimes being reduced to a mere band around the cell margin.

The proerythrocyte Multiple infections tended to enlarge the proerythrocyte, but the major de- formation was in the displacement and indentation of the host-cell nucleus by both schizonts and gametocytes (Figs. 22-31; PIso 3F, H; 4A).

The erythrocytes The reduced size oí the erythrocytic schizonts, compared with that oí the schizontsin the proerythrocytes, resuIted in littIe deíormation oí the host cell or nucIeus. Mature gametocytesrareIy enlargedthe cell and distortion was usually restricted to dispIacementoí the host-cellnucIeus (Figs. 33, 34; PI. 4D).

R. Blood parasites o/ Brazilian lizards 127

Plasmodium vacuolatum sp.nov. (Figs. 35-44; PI. 6A-L) Infections with this Plasmodiumspecies were noted in 31 of the P. umbrastudied (13.2%). A further 11lizards showedso few parasites that we could only refer to them as 'unidentmedPlasmodiumsp.' (Table 1). There is little doubt in our minds, however,that theseinfections were alsodue to the sameparasite, because we could find no other plasmodialinfections to accountfor them in any of the 235P. umbra examined.The morphologyofthe parasiteis distinctly different from that of other saurian Plasmodium speciesalready described.Its most characteristic feature is the prominent vacuole, containing the pigment, which is present in both the asexual and the sexual stages: we therefore propose the name of Plasmodium vacuolatumsp.nov. As usual, mixed infections with the other haemosporinesdescribed here were common (Table 1), but 14 pure infections were obtained. The morphology of P. vacuolatum,however, is very distinctive and there is little chanceof confusion with Garnia and Fallisia species:it developsin a polar position within the mature erythrocytes. Schizogony The youngest forms appeared as tiny oval bodies with a vacuole, a clearly defined ring of cytoplasm, and a small densely stained nucleus (Fig. 35; PI. 6A, B). They measured ,..,2.0 x 1.0. The trophozoites and early schizonts were rarely slightly amoeboid and, with growth and nuclear division, they soon took on a bean- or pear-shape, less often oval. Even at the 2-4 nucleated stage the vacuole was very conspicuous, occupying the broader end ofthe schizont and containing alI ofthe numerous, fine, golden-yellow pigment granules (Figs. 36-39; PI. 6 C-E). Mature or nearly mature schizonts were variously bean-shaped, oval, rounded or fan-shaped (Fig. 40; PI. 6H), with the vacuole becoming somewhat less distinct with approaching maturity: they ranged from 4.0 x 3.0 to 6.0 x 4.0, average 4.8 x 3.3. The nuclei were small and densely stained, often being disposed in a peripheral position in the fan-shaped or rounded parasites. The number ofmerozoites varied from 8 to 20, with an average 16: their size was approximately 2.0 x 1.0.

Gametogony Young female and male gametocyteswere small, compact,round or oval bodies, the two sexesbeing readily differentiated by the bright blue cytoplasm of the íemale and the pink colour of the male. Both showedthe conspicuousvacuole which contained alI the pigment granules (Figs. 41, 42; PI. 61, J). The nucleus of the macrogametocytewas compactand well stained. Older gametocytesretained the oval or rounded shape,the vacuole becoming less well defined and appearingto be squashedto the periphery oí the parasite (Figs. 43, 44; PI. 6K, L). Apparently mature macrogametocytesranged from 4,0x 3,5 to 4.5 x 4.0, average4,3 x 3.5; and the microgametocytesfrom 3.5 x 3.0 to 4,5 x 4.5, average4.0 x 3.5. 9 PAR 70 R. LAINSON, J. J. SHAW AND I, LANDAU

Exo-erythrocytic 8chizogony We found no exo-erythrocytic schizonts which could with confidencebe considered as those of P. vacuolatum.

Effects on lhe Mst-cell Most of the parasites simply curved around the erythrocyte nucleus (Figs. 37, 40; PI. 6D, F G), but the larger schizontsand gametocytesdia sometimesdisplace or indent the host nucleusto a mild degree(Figs. 38, 39, 41). There was no notice- able increasein the size of the infected erythrocyte.

Garnia uranoscodoni sp.nov. (Figs. 45-53; PIso 4K, L; 5A-H) A total of 46 (27'6%) ofthe 167 U. superciliosa examined were infected with a Garnia species.Its morphology differed significantly from G. multiformis, described above, and from previously recorded species (Lainson et alo 1971), and we have accordingly named the parasite Garnia uranoscodoni sp.nov. We have encountered no other haemosporine infections in U. superciliosa and the problems of mixed infections did thus not arise. On the other hand, only 5 out ofthe 46 infected lizards showed reasonably heavy parasitaemias, the reBt showing very scanty parasites. They were principally in the proerythrocytes in the more heavily infected animaIs, and predominantly in the mature erythrocytes in the older, chronic infections. Young trophozoites and schizonts were variable in their position within the host cell, but older parasites were predominantly polar.

Schizogony The young trophozoites were very amoeboid, often throwing out long filiform pseudopodia(PI. 4K). This activity was continued by the early schizonts(Fig. 46; PIso4L; 5A, B) but the parasites soonbecame more compact, growing into oval, rectangular and other elongatedshapes (Figs. 46, 47). The cytoplasmwas finely vacuolated and stained a rather dulllilac-blue in most preparations,with rare and tiny azurophilic granules in some schizonts. The nuclei were buIky, but rather delicately stained. Mature schizonts (Fig. 50; PI. 50) were round, oval or fan-shaped,sometimes with the nuclei disposedin rosette or half-circle fashion: they ranged from 4.6 x 4.0 to 11.1 x 6.5, average 6.3 x 4.9. The number of merozoitesvaried from 6 to 16, average8, and they measured ,..,2.5 x 1.0.

Gametogony The youngest gametocytesseen occupied a polar or latero-polar position and were oval or fusiform in shape:there was alreadythe customary colourdifference betweenthe young male and female, even when they measuredas little as 4x 3, and they occasionallyshowed one or two azurophilic granulesin the cytoplasm.

128 Blood parasites of Brazilian lizards 129 These granules increased in number as the parasites grew, but always remained small and inconspicuous. The initial smooth contour of the young gametocytes was quickly lost as they entered a phase of considerable amoeboid activity, with the cytoplasm producing numerous finely pointed projections (Figs. 50, 51; PI. 5D, E). With approaching maturity, however, the parasites reassumed a smooth subspherical or oval shape. The apparently mature microgametocyte stained a delicate rose-pink colour, often with a peripheral grey-blue rim giving a rather cyst-like appearance (Fig. 52; PI. 5F): a small endosome was sometimes evident. The macrogametocytes also stained delicately (Fig. 53; PI. 5G, H), the cytoplasm maintaining the pale lilac- blue hue and sometimes showing a more dense peripheral rim, although less commonly than in the microgametocyte: the nucleus was small, usually in the form of a faintly stained band and with a small endosome. A conspicuous feature of most of the gametocytes was a large, irregular vacuole measuring -4.0 x ,3'0. This was almost always visible in the female, but less frequently seen in the male. Mature microgametocytes ranged from 7.8 x 5.8 to 10.4 x 6'5, average 8,7 x 7,0: the macrogametocytes were larger, from 9.1 x 5.2 to 11.7 x 7.8, average 10.4,x 6.5.

Exo-erythrocytic schizogony We found numerous schizonts in the thrombocytes (Fig. 49) and less frequently in lymphocytes. The absence of any other haemosporine infection in alI the U. superciliosa examined convinces us that these are part of the life-cycle of G. uranoscodoni. Those in the thrombocytes were similar to the schizonts in the red blood cells, although very young forms tended to be stellate and older ones were predominantly morular in shape. They averaged 7.8 x 5.8 and produced from 4 to 14 merozoites, average 8. Schizonts in the lymphocytes appeared somewhat larger but the absence of sufficient examples in the blood-films enables us only to say that they at least reach 10.0 x 7.0 in size, and produce up to 12 merozoites (Fig. 48).

E.ffectson the host cells The red blood cells of U. superciliosa are unusually large and the majority of asexual stages and young gametocytes did not noticeably affect their appearance. Larger schizonts and mature gametocytes, however, variably displaced or de- formed the host cell nucleus (Figs. 47, 50, 52, 53; PI. 5 H) or, more rarely, slightly enlarged the cell. The schizonts in the thrombocytes sometimes enlarged the cell slightly, but the most marked deformity was the indenting of the nucleus or its displacement to one pole (Fig. 49). Schizonts in lymphocytes invariably pressed the host-cell nucleus to one side, usually reducing it to a mere peripheral band (Fig. 48). R. LAINSON, J. J. SHAW AND I. LANDAU

Table 1. Oombinations of bloodparasites* found in 102 specimens of Plica umbra (Iguanidae) in Brazil Unidentified F. G. P. (A , Numbers auda. F. multi- vacuo- Epery- Plaa- of ciosa simplex formiB latum throzoon modium Garnia lizards FaUiBia audaciosa 1 -- - - - 1 F.simplex O 9 - - 9 Garnia multiformiB 2 2 24 28 Plasmodium O 3 6 14 23 vacuolatum Eperythrozoon o o o 1 4 - 5 Unidentified o 2 3 O 1 3 9 Plasmodium sp. Unidentified 1 1 o 3 1 2 15 23 Garnia sp. F. simplex and o _ 3 _ o o o 3 P. vacuolatum P. vacuolatum and o o _ _ 1 o o 1 G. multiformiB Total 4 17 36 18 7 5 15 102 Total number of 4 20 41 31 8 11 23 infections * To avoid undue: complication, trypanosome and microfilaria infections are not included in this table.

Otherblood parasites of P. umbra and U. superciliosa Trypanosoma plicae sp.nov. (Fig. 20; PI. 51) Trypanosomeswere foundin 23 (10'0%) oftheP. umbraby direct examination of blood films. A further four infections were detectedin haemoculturesfrom six otherlizards, inNNN mediummadewithrabbitblood (Lainson& Shaw,1968). No parasiteswere found in the blood films of these falir animaIs,and it would seem that the actual infection rate is very much higher than the recorded10 %. Measurementsof the trypanosomesare summarized in Table 2. The parasite averages56'91ong (including the free flageIlumof 16-4)x 15.9wide: the undulating membrane is weIl developed,and from 2.0 to 3.0 wide. The body may often be folded on itself, making it impossibleto determine the position and size oí the nucleus in some specimens.The posterior end is broadly truncated, while the anterior end tapers delicately along the flageIlum. The kinetoplast is marginal, or submarginal,and located at the posteriorend in a terminal or subterminalposition. The long, thin and diffusenucleus stains a faint pink and is situated in the posterior end of the body (NI = 0'45): its posterior end tapers and sometimesmay touch the conspicuousflageIlar vacuole. Presenceof cytoplasmic granules, that stain with Giemsaafter methanol fixation, was variable, but in rare instancestrypano- someswere seen in which the cytoplasm was packed with hundreds of deeply staining granules. Although the parasite was isolated in NNN medium we have

130,~! Blood parasites of Brazilian lizards 131

Table 2. Mensural data of Trypanosoma plicae sp.nov. in lhe lizard Plica umbra (Iguanidae) from Brazil (all measurementsin pm)

Number Mean (S.D.) Range measured Totallength 56.9 (1.13) 49-64 15 P-K (posterior end to kinetoplast) 2.37 (1.2) 0'5-4,5 15 K-MN (kinetoplast to middle of 9.3 (1.95) 6.0-11'5 10 nucleus) P-MN (posterior end to middle of 11.9 (1'92) 8'{}-lõ'O 10 nucleus) MN-A (middle of nucleus to 27.1 (4'8) 19.0-35.0 10 anterior tip) Free flagellum 1516-4 (3-73) 12'0-28.0 Body width 15-9 (2-8) 10.0-21.00.29-0.71 Nuclear index (P-MN/MN-A) 0-45 (0-12) Nuclear length 14-4 12-17 10 Nuclear width 2-4 2-3 10 Kinetoplast length 1.1 1.0-1'5 1" Kinetoplsst width 0-9 0'5-1'0 1515 been unable to maintain these strains in culture: no developmenttook place in Culexpipiens fatigens. Based on the shape of the nucleus, there appear to be two major groups of trypanosomesin lizards -those with a rounded, compactnucleus and others with an elongate,diffuse nucleus.The trypanosome of P. umbra clearly belongsto the latter group, which has four different species,alI easily distinguished. Thus, T. bouteiand T. perroteti, of Mrican skinks, are differentiated by the lack of a free flagellum and their smaller size (see Laveran & Mesnil, 1912). T. superciliosae Walliker 1965,of the Brazilian lizard U. superciliosa,is very much larger than the trypanosomeof P. umbra,and its posteriorend is long and pointed. It is interesting to note, at this point, that we wereunable to rediscoverT. superciliosaein spite of the examination of 167 U. superciliosa.Finally, T. servetiPalaéz & Steber 1955, from Sceloporusteapenis in Mexico, doeshave a blunt posteriorend, but measures only 25-40 x 12-20. From theseobservations we considerthe trypanosomeof P. umbrato have been previously undescribed,and name it Trypanosomaplicae sp.nov.

Eperythrozoon This organism was seen in 8 (3.4%) of the P. umbra and 5 (3.°%) of the U.superciliosa studied. Most of the infections were e:x:tremelyheavy, with as many as 98 % of the erythrocytes infected. Under the Iight microscope the parasite merely appeared as an intensely stained doubIe dot (PI. 5J): muItiple infection of the cells was uncommon, and there seemed to be no obvious damage to the erythrocyte. It has been indicated that the presence of Eperythrozoon may depress the deveIopment of concomitant haemosporine infections in rodents (Ott & Stauber, 1967) and lizards (Ayala, 1973). The relative scarcity of the organism in our lizards, and the fact that so many of these reptiles showed lowgrade haemosporine 9-2 132 LAINSON, J. J. SHAW AND I. LANDAU infections anyway, precludesus from making any commenton the possibleeffect oí Eperytkrozoonon the developmentoí the blood parasiteswe have describedhere. AlI we can sayis that we have seennormal trophozoites,schizonts and gametocytes oí G. multiformis in erythrocytes algo infected with Eperytkrozoon,in one lizard.

M icrofilariae Thesewere seenin two P. umbra (0.9%) and five U. 8uperciliosa(3'0%) and were very similar, if not identical, in the two lizard species:both were sheathed, and of short, stumpy appearance.

DEFINITIONS 1. Fallisia audaciosa sp.nov. Schizogony and gametogony in the peripheral blood confined to the neutrophils. Young trophozoites rounded or oval and showing no amoeboid activity~ Mature schizonts spherical or oval, measuring from 13 x 11 to 18 x 18, average 14.3 x 13.2, and producing 00. 50-150 merozoites. Azurophi1ic granules very scanty or absent in cytoplasm of young and nearly mature schizonts: merozoites tear-shaped and measuring ,..,2 x 1. Immature gametocytes early on differentiated as microgametocytes and macrogametocytes by the very diffuse nucleus of the male and the bright blue cytoplasm of the female: azurophilic granules appearing relatively early and becoming much more prominent in the adult forms. Apparently mature gametocytes oval or round, often with a small, spherical vacuole (rarely two) at one pole. Macrogametocytes staining bright blue (with Giemsa), with nucleus usually in the form of a band and with a conspicuous endosome: they range from 7.8 x 7.8 to 10.4 x 7.8, average 9.5 x 7.5. Microgametocytes an overall pink due to highly diffuse nucleus: theyare slightly smaller than the females, measuring from 7.8 x 6.5 to 9.1 x 7.8, average 9.0x 7.0. Multiple infection of host cell a marked feature in heavy infections. Neutrophils becoming slightly enlarged and nucleus displaced in some instances, but host-cell nucleus may become grossly distorted and fragmented in cells with multiple infections or single, large schizonts. ROST. The lizard (Iguanidae) Plioo umbra (Linnaeus). LoCALITY. Secondary, tropical rain forest: Belém, Pará State, north Brazil. TYPE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil; Professor P. C. C. Garnham, Imperial College Field Station, Silwood Park, Ascot, England.

2. Fallisia simplexsp. nov. Schizogonyand gametogonyin the peripheralblood, within the thrombocytes. Young trophozoites variable in shape, but young schizont usually becoming crescenticdue to pressure against host-cell nucleus. Older schizontsmore often round or oval and strongly indenting host-cell nucleus: apparently mature schizonts from 6.5 x 6.5 to 10.0x 6.5, average 7.8 x 6,5. Number of merozoites

R. Blood parasites01 Brazilian lizards 133 produced ranging from 8 to 28, average 18: measuring ~ 2 x 1. Azurophilic granules not apparent in alI stagesof asexualdivision. Apparently mature macrogametocytesranging from 6.5 x 5.2 to 10.4x 7.8, average 8.2 x 6,5. The compact cytoplasm stains bright blue and almost always contains a small, spherical, 'punched-out' vacuole: azurophilic granules few in number but bulky and intenselystained. The nucleus stains a delicate pink and usually shows a prominent endosome at one extremity. Microgam~tocytesa uniform pink, and witha more delicate stippling of azurophilic granules: they also showa conspicuousendosome, but the cytoplasmic vacuole oftennot appare~t. Apparently mature forros measurefrom 6.5 x 5.2 to 9.1 x 7.2, averag~7.9 x 5.6. Multiple infection of host cells not a prominent feature, but may depend on the degree of parasitaemia. Larger schizontsand mature gametocytesenlarging the thrombocyte and strongly indenting the host-cellnucleus. No noticeablethicken~ ing of the ~ll membrane. HOST.The lizard (Iguanidae)Plica umbra(Linnaeus). LOCALITY.Secondary, tropical rain forest, Belém, Pará, BraZil. TYFE MATERIAL.The Wellcome Parasitology Unit, The Instituto Evandro Chagas,Belém, Pará, Brazil; ProfessorP. C. C. Garnham, Imperial CollegeField Station, Silwood Park, Ascot, England.

3.. Garnia multiformis sp.nov. Early schizogonyand gametogonyin erythroblasts and proerythrocytes of the bone-marrow;these infected cells then flooding the peripheral circulation. As the schizogonydecreases the parasites are found more predominantly in the proerythrocytes and, later, the matureerythrocytes. No malarial pigment produced at any stage of development. Schizonts in erythroblasts very variable in size. ranging from S.Ox 6.5 (24 merozoites) to 17x 13 (,..,150 merozoites): mature schizontsusually round to oval and with no apparent azurophilic granules.Cyto- plasm developing finger-like projections from which merozoitesare budded off: merozoites,..,1.5 x 1.0. Young schizontsin proerythrocytesround to oval and polar in position: scanty azurophilic granules produced with growth, and cytoplasm often algo contains one or two pinkish, amorphousbodies. Disposition of nuclei peripheral, fan-shaped or irregular: mature schizonts ranging from 5.2 x 3.3 to 9.1 x 7.S, average 7.0 x 5.2. and producing from 6 to 40 merozoites,average 1,6. Merozoitesmeasuring ,..,2.0 x 1.5. Marked drop in number of merozoitesin older infections, when the parasite largely occupiesthe mature erythrocytes. Early erythrocytic schizonts often elongate 'cigar-shape', becominground, oval, fan- shapedor irregular. Mature schizontsfrom 4.0 x 3.5 t9 7.Sx 5.2, average5.5 x 3.S, producingfrom 4 to 14 merozoites,average S: merozoites ,..,2.0 x 1.5. One or two azurophilic granules produced in someschizonts. Mature gametocytes in erythrocytes, but developmentalstages seen in both erythroblasts and proerythrocytes. Usual haemosporinecolourdifference between male and female gametocytes. Gametocytesat first round to oval and usually polar in position: later becomingelongated, irregular in shape,and latero"polarto lateral in the erythrocyte. Apparently mature microgametocytesmeasure from

9-3 R. LAINSON, J. J. SHAW AND I. LANDAU 10.0 x 4.0 to 21.0 X 3.0, average 12.3 X 3.5, and are an overall pink in colour: azurophilic granules prominent. Macrogametocytes from 10.5 X 2.5 to 20.0 x 3.0, average 12.6 x 3.0, with bright blue cytoplasm and a band-like nucleus which is often lateral in position: azurophi1ic granules very prominent. Multiple infection of cells is common and grossly enlarges some erythroblasts, which frequently rupture. Apart from cells with multiple infections, distortion of proerythrocytes and erythrocytes largely limited to displacement or indentation of the host-cell ~ucleus. Exoerythrocytic schizogony occasionally seen in monocytes. HOST. The lizard (Iguanidae) Plica umbra (Linnaeus). LOCALITY. Secondary, tropical rain forest, Belém, Pará, Brazil. TYPE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil; Professor P. C. C. Garnham, Imperial College Field Station, Silwood Park, Ascot, England.

4. PIasmodium vacuolatum sp.nov. Schizogony and gametogony restricted to a polar position within the mature erythrocytes. Production of abundant malarial pigment, of a light golden yellow colour, which is confined to a conspicuous vacuole in both asexual and sexual stages. Very young trophozoites often ring-shaped, measuring ,..,2.0 x 1.0. No amoeboid activity, and later stages assuming a bean or pear-shape, with pigment-vacuole at the broader end. Mature schizonts bean-shaped, oval, round or fan-shaped with vacuole becoming less distinct, measuring from 4.0 x 3.0 to 6.0 X 4.0, average 4.8 x 3.3. Number of merozoites from 8 to 20, average 16; measuring ,..,2.0 x 1.0. Young gametocytes showing conspicuous pigment vacuole, but this becoming more difficult to see in mature forms when it becomes pressed to one side of the parasite. Males and females showing the usual pink and blue colour differentiation with Giemsa staining. Nucleus of macrogametocyte compact and not normally band-form. Mature gametocytes rounded, and measuring from 4,0 x 3-5 to 4-5 x 4.0, average 4.3 x 3.5 (females) and from 3.5 x 3,0 to 4-5 x 4'5, average 4.0 x 3'5 (males). Larger schizonts and mature gametocytes sometimes displacing or indenting the host-cell nucleus, but erythrocytes are not enlarged- Exo-erythrocytic schizogony not known. ROST. The lizard (Iguanidae) PIica umbra (Linnaeus). LOCALITY,Secondary, tropical rain forest, Belém, Pará, Brazil. TYPE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil; Professor p, C. C. Garnham, Imperial College Field Station, Silwood Park, Ascot, England.

5. Garnia urano8codonisp.nov. Schizogonyand gametogonyprincipally in the proerythrocytesin early, heavier infections; becominglargely restricted to the mature erythrocytes in older chronic infections. Young asexualstages variable in position in the cell, but older parasites predominantly polar. No malarial pigment producedat any stageof development.

134 Blood parasites of Brazilian lizards 135 Young asexual stages very amoeboid and frequently with long filopodia, but soon becoming compact oval, rectangular or other elongated shapes. Rare and tiny azurophilic granules in some schizonts: cytoplasm staining poorly and nuclei tending to be bulky and ill-defined. Mature schizonts round, oval, or fan-shaped, with nuclei often in a rosette or half-circle: ranging from 4.6 x 4.0 to 11.1 x 6.5, average 6.3 x 4.9, they produced from 6 to 16 merozoites, average 8. Merozoites measuring ",2.5 x 1.0. Gametocytes polar or latero-polar, with the usual colour difference of pink microgametocyte and blue macrogametocyte. Cytoplasm contains a steadily increasing number of azurophilic granules which, however, remain fine and inconspicuous. Young gametocytes showing marked amoeboid projections, but latterly becoming a smooth oval or round shape. Both sexes staining delicately, the male often with a greyish-blue rim: female gametocytes with a small band-form nucleus and a small endosome. Mature gametocytes both show a large irregular vacuole, up to 4.0 x 3.0, but this is much more frequently seen in the female. Mature macrogametocytes ranging from 9.1 x 5.2 to 11.7 x 7'8, average 10.4 x 6,5: microgametocytes are smaller, from 7.8 x 5,8 to 10.4 x 6'5, average 8.7 x 7,0. Red blood cells not noticeably afrected by young stages, but larger schizonts and mature gametocytes sometÍme enlarging the cell and displacing or indenting the nucleus. , Exo-erythrocytic schizogony in thrombocytes, less frequently lymphocytes. ihrombocytes not markedly enlarged, but the host-cell nucleus is usually displaced or indented. Schizonts in lymphocytes may grossly distort the cell nucleus. Mature exo-erythrocytic schizonts average 7.8 x 5.8: they are morular in shape in th~ thrombocytes and produce from 4 to 14 merozoites, average 8. ROST. The lizard (Iguanidae) Uranoscodon superciliosa (Linnaeus). LoCALITY. Secondary, tropical rain forest, Belém, Pará, Brazil. TYFE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil; Professor P. C. C. Garnham, Imperial College Field Station, Silwood Park, Ascot, England.

6. Trypanosoma plicae sp.nov. Morphology. Totallength, including free flagellum, 49.0--64.0, with a mean of 56.9; distance of kinetoplast from posterior end of body (P-K) 0.5-4.5, mean 2.37; from middle ofnucleus to middle ofkinetoplast (K-MN) 6.0--11.5, mean 9'3; from middle ofnucleus to posterior end ofbody (P-MN) 8.0--15.0, mean 11.9; distance from centre ofnucleus to anterior end ofbody (MN-A) 19.0--35.0, mean 27.1. The kinetoplast is subcircular to elliptical, in a subterminal marginal position, and with a mean ofO.9 x 1-1. Thenucleus is elongated, with a mean of2-4 x 14.4, and lies in the posterior region of the body (Nl = 0.45) with its posterior end close to the kinetoplast. Undulating membrane well developed, measuring 2.0--3.0 wide, with a free flagellum of 12.0--28-0, mean 16.4. The mean, maximum body width of the trypanosome, including the undulating membrane, is 15.9. Biological characters. ln the vertebrate host the parasitaemia varIes from an occult infection to a relatively heavy peripheral blood infection: no pattern of 136 R. LAINSON, J. J. SHAW AND I. LANDAU parasitaemia was noted, and no division forms were seen. The parasite failed to develop in laboratory-bred Oulex pipiens fatigans fed on one infected lizard. Oultivation. Initial. growth in NNN medium was reasonably good, but T. plicae could not be maintained on subsequent passage. Bost specificity. No experimental data available, but the organism has not been encountered in any other lizard species from the same arca. ROST. The lizard (Iguanidae) Plica umbra (Linnaeus). LOCALITY. Secondary, tropical rain forest, Belém, Pará, Brazil. TYFE MATERIAL. The Wellcome Parasitology Unit, The Instituto Evandro Chagas, Belém, Pará, Brazil.

DISCUSSION We have already discussed (Lainson et alo 1974b) recent doubts cast on the validity ofthe genus Garnia by Telford (1973b)who proposed an amendment ofthe definition of the family Plasmodiidae to include these non-pigmented haemosporines. It is not proposed to repeat here, therefore, the reasons we have given for continuing to use the family Garniidae and the two included genera, Garnia and Fallisia. Suffice it to say that the large amount of material examined has con- vinced us of the absence of malarial pigment in alI stages of the Garnia species described in the present work. It is necessaryto stress again, however, the difficulties in untangling the developmental stages of Plasmodium and Gamia in lizard species which may commonly show mixed infections with as many as three different haemosporines (Lainson et alo 1974b): P. umbra is one such lizard. Telford (1973 b) examined 7 P. umbra, 10 P. plica and 8 U. superciliosa, alI fram near Georgetown, Guyana. N one of the U. superciliosa showed blood parasites, but he recorded P~dium tropiduri Aragão & Neiva, 1909, in 3 P. umbra,. P. minasense Carini & Rudolph, 1912, in 2 P. umbra and 1 P. plica,. P. cnemidophori Carini, 1941, in 1 P. plica; and a hitherto undescribed parasite, P. uncinatum Telford, 1973, in a single P. plica. The presence oftrypanosomes and a haemo- gregarine in some lizards was mentioned, but it is not clear in which species and they were not described. Previously, in the neotropics, P. minasense had been reported only in M abuya agilis (Scincidae) from Brazil, while P. cnemidophori was recorded only from the teiid lizards Onemidophorus lemniscatus and Ameiva ameiva (Carini, 1941; Lainson, & Shaw, 1969; Telford, 1973a). In his description of the Guyanan parasites, Telford stated that 'The P. tropiduri infection in one P. umbra was confined entirely to thrombocytes, with gametocytes far outnumbering schizonts ...AlI parasites seen in this infection lacked pigment.' We find it hard to believe that a Plasmodium species, so consistently parasitizing erythrocytes, should change host cells so drastically and completely. It is surprising that the organism was even considered to be a Plasmodium under the accepted definition of this genus, and we reei sure, from his illustrations and measurements, that the author was dealing with a Fallisia, probably F. simplex. Telford inoculated blood from the infected lizard into 2 P. plica, 1 P. umbra and 4 Tropidurus. Of these, a single Tropidurus produced 'a light infection of apparently normal P. tropiduri asexual stages... after 18 days, but the lizard was negative for 190 days Bloodparasites o/ Brazilian lizards 137 thereafter'. In our experience (Lainson et alo 1974b), the frequency of chronic, non-apparent infections of various haemosporines in wild-caught lizards renders this evidence suspect: the very process of inoculating blood from one lizard genus to another might well exacerbate a hitherto unnoticed P. tropiduri infection. In his description of P. uncinatum from the single specimen of P. plica, Telford characterlzes the parasite by 'the presence of a very prominent mass of golden pigment in immature schizonts and gametocytes, which may occupy one-third to one-half ofthe parasite's body areas ...Neithervacuoles nor cytoplasmic processes are found in asexual stages ...Gametocytes are always elongate, 8-15 x 3-6 p;m, with a majority of them curving around the host cell nucleus.' This description would effectively seem to separate P. uncinatum from P. vacuolatum. The latter parasite has round-oval gametocytes in a polar position and a very conspicuous vacuole, which contains the pigment, in both the asexual stages and the gametocytes. We are worried on two points, however. Firstly, the schizonts figured for P. uncinatum look very similar to those we have described for P. vacuolatum and Telford's photographs do appear to show the pigment lying in a distinct vacuole, contrary to his texto Secondly, the gametocytes of the parasite he figures as P. minasense are similar to those we describe here as P. vacuolatum. At present it seems best to regard the three parasites as separate entities, but we would 1ike to see a great deal more material examined from Guyana to confirm that this is so. With the present descriptions, there are now f~ur recorded species of Fallisia. Two of these, F. effusa and F. audaciosa are large parasites, producing schizonts with as manyas 150merozoites: they are, however, readily differentiated. F. effusa, as discussed above, inhabits only the thrombocytes of its host, N eusticurus bicarinatus (Teiidae), in which it causes a remarkable, cyst-1ike thickening of the cell wall. Its gametocytes are much more elongate than those of F. audaciosa, reaching -10 x 6. The development of F. audaciosa in neutrophils is, of course, the most striking differential feature. The remaining two species, F. modesta Lainson, Landau & Shaw, 1974, and F. simplex, are much smaller parasites, with schizonts giving only up to 28 merozoites; In this respect they might be difficult to differentiate but for the fact that F. modesta develops in the lymphocytes of its host Tropidurus torquatus (Iguanidae), whilst F. simplex of P. umbra is limited to thrombocytes. Gametocytes of the former are -7.5 x 5.5 and of the latter -8.2 x 6.5. Of the known Garnia species, G. multiformis has to be distinguished only from the type species, G. gonatodi (Telford, 1970)(Lainson, Landau & Shaw, 1971.)for this is the only other one possessingelongate gametocytes and relatively large schizonts. In the first place, we have never seen erythroblastic development of G. gonatodi as we have described here for G. multiformis, butthismaybebecausewedidnotcatch the infection at an early enough stage. The major difference between the two parasites, however, is in the development and ultimate morphology of the gametocytes. Early gametocytes of G. gonatodi are elongate, fusiform bodies, never round or oval as are those of G. multiformis: the mature gametocytes generally have a smooth , sausage' or 'banana' shape, with only relatively small numbers of irregularly LAINSON, J. J. SHAW AND I. LANDAU shaped forms (described as 'pre-maturation' forms by Telford): in G. multiformis this crinkled shape is assumed by virtually alI the mature gametocytes, which average only -12 x 3 compared with -16 x 4 for G. gonatodi. G. uranoscodoni resembles G. telfordi (Lainson, Landau & Shaw, 1971) in its considerable amoeboid activity and small number of merozoites. It differs in its very much larger, vacuolated gametocytes, which average -10-4 x 6,5 (females) compared with only -7.0 x 4.5 (females) in G. telfordi, and its failure ever to produce the strange lobular schizonts of the latter parasite. G. uranoscodoni undergoes much of its development in the proerythrocytes, whereas G. telfordi seemsto be restricted to the mature erythrocytes. Differentiation from G. utingensis Lainson, Landau & Shaw, 1971, can be made on similar criteria, and from G. morula (Telford, 1970) (Lainson, Landau & Shaw, 1971), by the latter parasite's complete lack of amoeboid forms and its smaller, predominantly rounded gametocytes. The development of Garnia multiformis described here in P. umbra holds a particular interest for two major reasons. Firstly, it may indicate the normal course of infection for the Garniidae as a whole, and secondly it serves as a waming that considerable variation in morphology is to be expected depending at which stage of infection the blood films are prepared. Thus, the great variation in the schizonts and gametocytes of the early and late infection of G. multiformis at first led us to think that we were dealing with two different parasites. Only the e:x:ami- nation of a large number of infeçted lizards, and following the course of infection in some of them from an early stage, enable us to correct this impressiono

These studies were made under the auspicesof the Wellcome Trust, London, the Instituto Evandro Chagas,Belém and the World Health Organization,Geneva. Particular thanks are due to Roberto Naiff, SebastiãoOliveira and Henrique Buna for their patient technical assistance.

REFERENCES AvALA, S. C. (1970). Lizard malaria in California; description of a strain of Pla8modium mexicanum, and biogeography of lizard malaria in Western N orth America. J ourYWloJ Parasitology 56, 417-25. AvALA, S. C. (1973). The phlebotomine sandfly-protozoan parasite commUIÚty of central California grasslands. The American Midland Naturalist 89, 266-80. CARINI,A. (1941). Sobre um plasmodio endoglobular e uma eimeria do lagarto Onemidophorus lemniscatuslemniscatus. Arquivos de Biologia, S. Paulo 25, 205-8. LAINSON,R. (1959). Atoxoplasma Garnham, 1950, as a synonym for LankesterellaLabbé 1889. Its life cycle in the English sparrow (Passer domesticusdomesticus Linn.) JourYWl oJ Proto- zoology6, 360-71. LAINSON,R., LANDAU,I. & SHAW,J. J. (1971). On a new family ofnon-pigmented parasites in the blood of reptiles; Garniidae fam.nov. (Coccidiida; Haemosporidiidea). Some species ofthe new genus, Gamia. International Journal oJ Parasitology 1, 241-50. LAINSON,R., LANDAU, I. & SHAW,J. J. (1974a). Further parasites ofthe family Garniidae (Coccidiida; Haemosporidiidea) in Brazilian lizards. Fallisia effusa gen.nov., sp.nov and Fallisia modestagen.nov., sp.nov. Parasitology 68, 117-25. LAINSON, R., LANDAU, I. & SHAW,J. J. (1974b). Observations on non-pigmented haemo- sporidia of Brazilian lizards, including a new species of Saurocytozoonin Mabuya mabouya (Scincidae). Parasitology 69, 215-23.

138R. Blood parasites of Brazilian lizards

LAINSON,R. & SHAW,J. J. (1968). Leishmaniasisin Brazil. I. Observationsonenzooticrodent leishmaniasis -incrimination of Lutzomyia jlaviBcutellata (Mangabeira) as the vector in the lower Amazonian basin. Traf1.8aCtwns01 tM Royal Society01 Tropical M edicine aoo H ygiene 62, 385-95. LAINSON,R. & SHAW,J. J. (1969). New host recorda forPlaBmodium diploglossi, P. tropiduri Aragão & Neiva, 1909, and P. cnemidophori Carini, 1941. Parasitology 59,163-70. LAVERAN,A. & :M:ESNIL,F. (1912). Trypanosomeset trypanosomiasis. Paris: Masson. OTT,K. J. & STAUBER,L. A. (1967). Eperythrowon cooooides.Influence on course ofinfection of Plasmodium chabaudi in mouse. Science155, 1546--8. SHAW,J. J. & LAINSON,R. (1971). Trypanosoma vivax in Brazil. Annals 01 Tropical Medicine and Parasitology 66, 25-32. TELFORD,S. R. (1973a). Malarial parasites ofthe 'borriguerro' lizard, Ameivaameiva (Sauria: Teüdae) in Panama. Journal 01 Protowology 20,203-7. TELFORD,S. R. (1973b). Saurian malarial parasites from Guyana: their effect upon the validity of the fami1y Garniidae and the genus Gamia, with description of two new species. InteNmtÍOftal Joumal 01 Parasitology 3, 829-42.

EXPLANATION OF FIGURES AND PLATES Figs. 1-7. Fallisia audacio8a sp.nov., from the lizard Plica umbra, as seen in the neutrophils of Giemsa-stained blood films. 1, Young trophozoite; 2-4, developing and mature schizonts; 5, mature macrogametocyte showing conspicuous azurophilic granules; 6, mature microgametocyte; 7, multiple infection of neutrophil with male and female gametocytes and a young schizont. Figs. 8-15. FaUisia BÍmplex sp.nov., from the lizard Plica umbra, in thrombocytes of the peripheral blood. 8, Trophozoite; 9, 10, developing schizonts; 11, mature schizont; 12, mature microgametocyte; 13, mature macrogametocyte; 14, 15, single and double infections with gametocytes, showing deforroation due to pressure against host-cell nucleus. Figs. 16-19. Garnia multiformi8 sp.nQv., from the lizard Plica umbra, as seen in blood films during early phase of infection. 16, ruptured erythroblast with liberated parasites, including young female gametocytes and developing and mature schizonts; 17, older, but immature macrogametocyte from a ruptured erythroblast, showing abundant az~ophilic granules; 18, almost mature schizont from a ruptured erythroblast, with merozoites budding off from cytoplasmic protrusions; 19, almost mature schizont in intact erythroblast. Fig. 20. Trypaoo8oma plicae sp.nov., in the lizard Plica umbra. Figs. 21-34. Garnia multiformi8 sp.nov., in Plica umbra. Later infection, with development in proerythrocytes (21-31) and erythrocytes (32-34). 21, Young trophozoite; 22-26, developing and mature schizonts; 27,28, young remate and male gametocytes. Note very prominent azurophilic granules; 29, 30, older, but still immature, remate and male gametocytes; 31, elongated, young macrogametocyte; 32, maturing microgametocyte in erythrocyte, showing migration to a lateral position and beginning of irregular outline; 33, 34, late infection, with mature macro- and microgametocytes in erythrocytes. Note highly irregular shape and growth around host-cell nucleus. Figs. 35-44. PlaBmodium vacuolatum sp.nov., in erythrocytes of the lizard Plica umbra. 35, Young, ring-stage trophozoite; 36-39, developing schizonts, showing prominent vacuole containing the pigment; 40, almost mature, fan-shaped schizont; 41, 42, young male and femalegametocytes with prominent pigment vacuole; 43, 44, mature remate and male gametocytes. Figs. 45-53. Garnia uraoo8codoni sp.nov., in the lizard Uraoo8codon supercilw8a. 45, Young trophozoite; 46, two developing schizonts in same cell, showing amoeboid early stage and later compact forro; 47, nearly mature schizont; 48, exo-erythrocytic schizont in lymphocyte; 49, exo-erythrocytic schizont in thrombocyte; 50, mature, fan-shaped schizont and developing, amoeboid microgametocyte; 51, developing, amoeboid macrogametocyte; 52, 53, mature male and female gametocytes. Note conspicuous vacuole in the latter.

139 140 LAINSON, J. J. SHAW AND I. LANDAU Fig. 54. Gamia multiformiB sp.nov., in the lizard Plica umbra. Camera.-lucida,outline drawings of male gametocytes: A, On day of capture; B, 1 week later; C, at 2 weeks; D, at 3 weeks. Note the predominaritly polar position and rounded forro of young gametocytes, in proerythrocytes, during the first 2 weeks. During the latter phase of the infection these grow round the nucleus to assume a lateral position within the erythrocytes, and become elongate and irregular in outline.

PLATE 1 (x 1364) FalliBia audacioaa sp.nov., a parasite in the neutrophils of the lizard Plica umbra. A, Two young schizonts and a young femaJegametocyte; B, two older schizonts and a young macrogametocyte in the same ce11;C,D, mature, rupturing schizonts; E, F, very young female and male gametocytes; G, neutrophils with nearly mature gametocytes, showing prominent vacuoles; H, neutrophil containing two male and two female gametocytes; I, mature macrogametocyte with we11-developedazurophilic granules. FalliBia aimplex sp.nov., in the lizard Plica umbra. J, Trophozoite; K, L, developing schizonts.

PLATE 2 ( x 1364) FaUisia simplex sp.nov., in the lizardPlicaumbra, as seenin the thrombocytes ofthe peripheral blood. A, N early mature schizont; B, C, mature fema.1egametocytes ; D, mature male ~etocyte; E, single thrombocyte containing a female and male gametocyte. Note displacementof host-cell nucleus (top), the squashed microgametocyte (centre) and the flattened macro- ~etocyte (below). Gamia multiformiB sp.nov., in the lizard Plica umbra. F-H, Bone-marrow smears in early infections, showing large numbers of developing schizonts in erythroblasts.

PLATE 3 (A, x 1035; B-I, x 1364) Gamia m~ltiformis Bp.nov., in the peripheral blood of the lizard Plica ~mbra. A, Early infection: a low-power view of blood film Bhowing abnorma11ylarge number of erythroblaetB, Bome of which contain developing paraeiteB; B, intact erythroblaet with a nearly mature Bchizont; C, ruptured erythroblaet liberating a nearly mature Bchizont, which BhoWBmerozoites budding off the cytoplaemic network; D, young female gametocyte in an erythroblaet. Note well-developed azurophilic granules; E, two young female gametocyteB in a Bingle erythroblaet; F, proerythrocyte with nearly mature Bchizont of early infection; G, H, developing female and Inale gametocyteB in proerythrocyteB ; I, double infection of a proerythrocyte with two microgametocyteB. PLATE 4 ( x 1364) Garnia multiformiB sp.nov., in the periphera.l blood of the Iizard Plica umbra. A-E, Later infection, showing transformation of the developing gametocytes from smooth rounded shapes (PI. 3, G-I) into elongate crinkled forros. This is accompanied by growth round the host-cell nucleus and assumption of a lateral position by mature gametocytes in erythrocytes. A, C and Dare microgametocytes, B and E are macrogametocytes. Note very conspicuous azurophilic granUles. F -J, Developing schizonts in erythrocytes, during late infection: note , cigar-shaped' young forros and much reduced number of merozoites compared with earlier schizogony (Fig. 26; PI. 3A-C, F). Garnia urano8codonisp.nov., in the peripheraI blood of the Iizard Urano8codonsupercilio8a. K, L, Amoeboid trophozoite and young schizont with Iong filopodium, in proerythrocytes.

R. Blood parasites o/ Brazilian lizards

PLATE 5 ( x 1364) Garnia urano8codoni sp.nov., in the lizard Urano8codonsupercüio8a. A, B, Young amoeboid schizonts in proerythrocytes; C, compact, older schizont in proerythrocyte; D, E, young amoeboid female and Inale gametocyteB in erythrocytes; F, mature Inicrogametocyte in erythrocyte; G, H, znature znacrogametocytes in erythrocytes. Note conspicuouscytoplasInic vacuole in female parasites. I, Trypano8oma plicae sp.nov., in the lizard Plica umbra. J, Eperythrozoon sp., in the lizard Plica umbra.

PLATE 6 ( x 1364) Plasmodiut)} vacuolatum sp.nov., in the erythrocytes of the lizard Plica umbra. A, B, Young ring-stage trophozoites, showiIig conspicuousvacuole; C-G, developing schizonts, alI showing the large pigment-vacuole at one pole; H, nearly mature, rounded schizont with peripheral ring of nuclei and central vacuole with pigment; I, J, young female and male gametocytes showing pigment-vacuole at top left of each parasite; K, L, mature female and male gametocytes.

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