A Parasite of the Lizard Neusticurus Bicarinatus(Lacertilia: Teiidae)

Reports

algaeas biological indicatorsofheavy metal Amado Filho 1994 Trace metal accumula- lI. Mariani P,C Tolomio, B Baldan,P Braghetta pollution in Sorfjorden, Norway. Environ tion by algae in Sepetiba Bay, Brazil. 1990 Cell wall ultrastructureand cation 10- Pol/ut 15: 101-107 Environ Pollut 83: 351-356 calization in some benthic marine algae. 2.Seeliger U, C Cordazzo 1982 Field and 7. Karez CS, GM AmadoFilho, DM Moll, WC Phycologia 29: 253-262 experimental evaluation of Enteromorpha Pfeiffer 1994 Concentraçõesde metaisem 12.B9rowitzka MA, AWD Larkum, CE sp. asa quali-quantitativemonitoring organ- algasmarinhas bentônicas de três regiõesdo Nickolds 1974 A scanningelectron micro- ism for copper and mercury in estuaries. Estadodo Rio de Janeiro.An Acad Bras Ci scope study of the structure and organiza- Environ Pol/ut 29: 197-206 66: 205-211 tion of the calcium carbonate deposits of 3.Bryan GW 1983 Brown seaweed,Fucus 8. Karez CS, GM Amado Filho, RP Louro, M algae. Phycologia 13: 195-203 vesiculosus, and the gastropod, Littorina Farina 1995 Accumulation of zinc by the 13.Ragan MA, O Smidsrod, B Larsen 1979 littoralis, as indicators of trace metal avail- brown algae Padina gymnospora.Buli Inst Chelation of divalent metal ions by brown ability in estuaries. Sci Total Environ 28: OceanogMonaco -In press algal polyphenols. Mar Chem 7: 265-271 91-104 9.Pellegrini L, M Pellegrini, S Delivopoulos, 14.Ragan MA, KW Glombitza 1986 4. SoederlungS, A Forsberg,M Pedersen1988 G Berail 1991 The effects of cadmium on Phlorotannins,brown algal polyphenols, p Concentration of cadmium and other met- the fine structure of the brown alga 129-241. ln Progress in phycological re- aIs in Fucus vesiculosusL. and Fontinalis Cytoseira barbata forma repensZinova et search.Round and Chapmaneds, Biopress, dalecarlica Br. Eur. from the northern Bal- Kalugina. Br PhycolJ 26: 1-8 Bristol tic Sea and the southern Bothnian Sea. 10. Yoneshigue-BragaY, MHB Neves 1981 15. Brinkhuis B, IK Chung 1986 The effects Environ Pol/ut 51: 197-212 Preliminary studies on mass culture of of copper on the fine structure of the kelp 5. Lacerda LD, WC Pfeiffer, M Fizman 1987 Gracilaria sp. using different nutrient me- Laminaria saccharina (L.) Lamour. Mar Heavy metal distribution, availability and dia, p 83-90. In Proceedings Xth Interna- Environ Res 19: 205-223 Catein Sepetiba Bay, S.E. Brazil. Sci Total tional SeaweedSymposium, Goteborg, Swé- Environ 65: 163-173 deDoT Levring ed, Walter de Gruyter, Ber- Received03 November 1994 6. Karez CS, VF Magalhães,WC Pfeiffer, GM lin Accepted28 November 1995

Plasmodium neusticuri n.sp. (Apicomplexa: Plasmodiidae), a parasite of the lizard Neusticurus bicarinatus (Lacertilia: Teiidae) in Amazonian Brazil

RALPH LAINSON1, lLAN PAPERNA2

ISeçãode Parasi.ologia, Instituto Evandro Chagas,Fundação Nacional de Saúde,Belém, PA 66017-970,Brasil and 2Faculty ofAgricu"lture, The Hebrew University ofJerusalem, Rehovot 76-100, Israel

Plasmodium neusticuri n.sp. (Apicomplexa: Plasmodiidae) is described in the blood of the liz- ardNeusticurus bicarinatus (Lacertilia: Teiidae) from Amazonian Brazil. Ali stagesofthe para- site occupy a polar position in the erythrocyte and produce no enlargement of the host cell and little or no distortion of its nucleus. Trophozoites commonly display a variable number of long filopodia, and quickly produce a small number of dark, yellow-green pigment granules. Meronts are s.maller than the host-cell nucleus. They are broadly ellipsoidal to spherical, produce 4-8 (usually 6) merozoites disposed in an irregular manner, and have some 5-10 pigment granules forming a loose clump. Macrogametocytes and microgametocytes are broadly ellipsoidal, fre- quently "kidney-shaped", rarely spherical. They are generally smaller than the host-cell nu- cleus, averaging 5.8 x 4.1 J1mand 5.8 x 4.4 J1m,respectively. Both usually exhibit a conspicuous cytoplasmic vacuole, and their pigment granules are more abundant and scattered than those of the meronts. Rare examples of extraerythrocytic meronts were encountered in monocytes. The v~ctor of the parasite is at present unknown. "- Correspondenceto: Ralph Lainson, Seçãode Parasitologia,Instituto Evandro Chagas,Fundação Nacional de Saúde,Caixa Postal 691, Belém, PA 66017-970, Brasil

200. Ciência e Cultura Journal of the Brazilian Association for the Advancement of Science Volume 48(3) .May/June 1996 Reports

Plasmodium neusticuri n.sp. (Apicomplexa: ro pequeno de grânulos de pigmento malárico uma média de 5.8 x 4.1 Jlm e 5.8 x 4.4 Jlm, Plasmodiidae)é descrito no sanguedo lagar- de cor amarelo-verde escuro. Os esquizontes respectivamente. São, geralmente, menores to Neusticurus bicarinatus (Lacertilia: são menores que o tamanho do núcleo do que o núcleo da célula hospedeira, e possu- Teiidae), na região amazônica do Brasil. To- eritrócito, e produzem 4 a 8 (usualmente 6) em um vacúolo consp{cuo no citoplasma. Os das asfases de desenvolvimentoocupam uma merozoitos que são irregularmente distribuí- grânulos de pigmento dos gamet6citos são posição polar no glóbulo vermelho, semqual- dos na célula, mais ou menos5 a 10 grânulos mais numerosose mais espalhadosque aque- quer aumento no tamanho da célula hospe- de pigmento ficam aglomerados no les encontrados nos esquizontes. Foram en- deira, e com pouca ou nenhuma deformação citoplasma. Os macrogametócitos e os contrados raros esquizontes exoeritroc{ticos do núcleo da mesma. Os trofozoitos produ- microgametócitos são de forma elipsóide ou nos mon6citos. Até agora, o vetor do parasi- zem vários filopódios compridos e um núme- em "forma de rim", raramente redondos,com ta permanece-dest'onhecido.

D Uringparasites studiesof Amazonianon the bloodliz- and mature erythrocytes ofNeusticurus straight,elongated filopodium whichis difficult to distinguish, unlike those of angled in such a manneras to givelhe ards, some twenty years ago many other lizard species. Thus, lhe parasitea strangeretort-like appear- (1), the senior authorencoun-tered nucleus of lhe great majority of red ance(Figs. 1,10-13).The maio body of infections with a species of blood cells in a given blood film were lhe trophozoite, at this stage is about Plasmodium in the semi-aquatic lizard of similar size and form, and lhe con- 2.0-3.0 ~ in diameter. Neusticurus bicarinatus (Linn.). The in- tained chromatin fiasses of very vari- fections were of such a light nature, able size and distribution. A study of Meronts. Like alI other stages,lhe however, that a full description o.f the the parasite in twelve of the infected liz- meronts are polar in position. Binucleate parasite was not possible, and it re- ards, however, led to the conclusion that forms mar retain filopodia (Figs. 1,14), mained unnamed. Since that time fur- it develops predominantly in mature but after further nuclear divisions most ther infected N. bicarinatus have been erythrocytes. parasites assume a more compact ellip- acquired, permitting the following de- Neither lhe asexual stages Dor the soidal or subspherical shape (Figs. scription of the organism as a new spe- gametocytes cause any noticable en- 3,4,15-18), with lhe pigment increasing cies of the genus Plasmodium. largement or deformation of the host to some 5-10 granules which are usu- Specimens of the lizard were cap- cell. Its nucleus, toa, usually remains a1lyclustered in a poorly defined vacuole tured by hand, using a nylon noose at- unchanged, apart from' an occasional, (Fig. 4). Mature meronts (Figs. 5,19) are tached to a fine stick, in or near small slight indentation due to mature meronts considerably smaller than lhe normal streams in the vicinity of Capanema, or gametocytes (Figs. 4,5,9). The im- erythrocyte nucleus, averaging 5.0 x 3.0 Pará State, northem Brazil. They were pression was gained, however, that ~ (15 measured). They mar produce maintained in glass aquaria containing erythrocytes containing lhe larger para- from 3 to 8 tear-shaped merozoites, but a small quantity of water and sloped to sites become more fragile and, when lhe most frequent number is 6 (Fig. 5); give the lizards an option of dry or wet compacted with neighboring uninfected these measure approximately 2.0 x 1.0 conditions. Broken tiles offered them cells, are more readily deformed (Fig. ~ and are irregularly disposed in lhe suitable hiding places, and they were fed 19) host-cell cytoplasm. No fan-shaped or with Tenebrio larvae from a laboratory- None of the infected lizards ap- cruciform arrangements of lhe meront bred colony. peared to sofrer any ill effects from the nuclei were seen. Merozoites from ma- The lizards were bled from the or- infection, which in most caseswas light lUTemeronts mar remain free in lhe cy- bital sinos, using glass Paste~r pipettes (heaviest parasitaemia, 2.7% erythro- toplasm of lhe erythrocyte without rup- drawn out to a very fine point. Blood cytes infected), gametocytes were usu- lUTe of lhe cell (Fig. 20,21). Only two, films were rapidly air-dried, fixed in ally more abundantthan meronts. Blood immature exoerythrocytic meronts were absolute methyl alcohol for three min- films made at intervals of 5 days, either seen, both in monocytes, one with six utes and stained by Giemsa's method at 8.00 h or 20 h, showed no appreci- nuclei and lhe other with three. (30 drops of stain to 15.0 rnl of distilled able differences in the intensity or com- water, buffered to pH 7.4) for 90 mino position of the parasiternia, suggesting Gametocytes.These, again, are in- Parasites wére measured using an ocu- little or no synchronicity in the asexual variably polar in their position. Young lar micrometer, x8 eyepieces and a xlOO cycle. forrns are predorninantlycrescentic or neofluar objective. The water-color il- of a low triangular shape(Figs. 7,22), lustrations were based on direct obser- Trophozoites. The youngtrophozoitesand measure about 6.0 x 3.0 ~. With vations while measurements were being are at first ovoid, compact growth theybecome broadly ellipsoidal made, and by reference to photomi- and of an evencontour, and evenat this (Figs. 6,23,24,27)or bean-shaped(Figs. crographs (Figs. 10-27). The latter were earlystage a single,dark greenish-black 8,9), due to pressurefrom the adjacent made using a Zeiss "Photomicroscope pigment granule may be present(Fig. host-cell nucleus. Spherical or 111" and Kodak TMX-402 filmo AlI 9). While still uninucleate,the parasite subspherical gametocytes were less measurements are given in micrometers now becomesamoeboid and its cyto- commonlyseen (Figs. 25,26).The male

(J.lm). plasm producesa variable number of andremate parasites are usually smaller filopodia (Figs. 2,8). Presumablyolder thanthe host-cellnucleus, occasionally Wehave found lhe proerythrocytes trophozoitesvery frequentlyhave a sin- reachingthe samesize.

Volume 48(3). May/June 1996 Ciência e Cultura Journal of the Brazi/ian Association for the Advancement of Science .201 gle Reports

The mature macrog&metocyte gives lhe parasite an overall pink color, derived from lhe generic name of lhe (Figs. 6,24,25) averages 5.8 x 4.1 ~m typical of lhe hemosporine microga- type host,Neusticurus. (range 4.4 x 3.7 -6.7 x4.4~; 25 meas- metocyte. The pigment granules are ured), with a shape index (ratio of1ength/ generally more abundant (12-25) and To our knowledge, lhe .only width) of 1.4 (range 1.0-1.8). The cyto- more widely scattered than those of lhe hemosporine previously recorded in plasm stains a clear, bright blue and is ma~rogametocyte. As in lhe female, Neusticurus .l]Jcarinatus is Fallisia highly vácuolated. Almost always there there is usually a conspicuous,round cy- e.ffusaLainson, Landau and Shaw,1974. is one vacuole which is much larger than toplasmic vacuole. - This member of lhe family Garniidae is the restoThe nucleus is compact and con- readily differentiated fromP. neusticuri spicuously stained a reddish-purple. Typehost. The lizard Neusticurus n.sp. by its development exclusively in Approximately 10-16 dark, yellow- bicarinatus(Linn.) (Lacertilia:Teiidae). thrombocytes and lymphocytes of lhe green pigment granules usually form an Type locality.Low riverine forest peripheral blood (2). eUipsoidal cluster,unassociated with any in lhe vicinity of Capanema,Pará, Bra- In distinguishingP. neusticuri n.sp. obvious vacuole. Macrogametocytes al- zil. from previously described neotropical ways outnumbered the microga- .Prevalence.Of 465 lizards exam- Plasmodium species of lizards, we can metocytes. ined, 134 (28.8%)were infected. dispense with lhe nec~ssity of compari- Mature microgametocytes (Figs. Type material. Holotype and son with those possessinglarge meronts 9,26,27) are of a similar size, averag- paratypeblood films in the seniorau- producing many merozoites and round ing 5.8 x 4.4 ~ (range 4.8 x 4.0 -7.4 x thor's collection in the Departmentof or elongated gametocytes which are 5.1 ~m; 25 measured). They have a Parasitology,Instituto EvandroChagas, larger than lhe host-cell nucleus (placed shape in4ex 9f 1.3 (range 1.0 -1.8). The Belém, Pará,Brazil. by Garnham in lhe subgenus highly diffuse chromatin of the nucleus Etymology.The specific nam~is Sauramoeba (3).

Figures 10-27. Plasmodium neusticuri n.sp. Photomicrographs of the parasite seen in Giemsa stained blood films from the lizard Neusticurus bicarinatus. 10-13. Trophozoites, showing the char- acteristic rod-like filopodium; 14. binucleate meront, still with a pronoltnced filopodium; 15-18. dividing;.-reronts with from two to Figures 1-9. Plasmodium neusticuri n.sp., as seen in Giemsa six nuclei; 19. mature meront with eight merozoites; 20-21. groups stained blood films from the lizard Neusticurus bicarinatus. 1. of three early trophozoites and three merozoites, presumably de- Binucleate meront, with pronounced filopodium; 2 amoeboid rived from precociously divided meronts producing only three trophozoites;3. young meront; 4 and 5. almost mature and mature merozoites;22. double infection of an erythrocyte with two young merat\;' 6 mature macrogametocyte; 7 immature female gametocytes; 23. young macrogametocyte; 24-25. mature macrogametocyte;8. young amoeboid trophozoite and developing macrogametocytes,note conspicuous,compact nucleus (N), clumped microgametocyte in. the some erythrocyte; 9 mature pigment granules (P) and cytoplasmic vacuole; 26 and 27. mature microgametocyteand a merozoite which has rounded up following male gametocyteswith diffuse, poorly staining chromatin, dark pig- penetration of the some erythrocyte. ment granules. ana vacuole. Bar = 10.0 Jlm.

202 .Ciência e Cultura Journal of the Brazilian Association for t~e Advancement of Science Volume48(3). May/June 1996 Reports

On the other hand, we do need to cyte and are smalIer than the host-celI neusticuri n.sp., to warrant a compari- compare the parasite with those having nucleus and round to oval in shape. The son and, as members of the family meronts which are smaller than the host- meronts, however, may produce from 8- Teiidae are lirnited to the New World, it cell nucleus, produce 12 or less 20 merozoites (6). is extremely unlikely that this parasite merozoites, and have round-oval Plasmodium colombiense Ayala occurs in Old World lizards. gametocytes which ar~ equal to, or and Spain, 1976, of Anolis auratus (7) Finally, on morphological grounds smaller than, the size of the normal eryth- differs from P. neusticuri n.sp., in its one might speculate on a relationship rocyte nucleus (placed by Garnham in meronts, which produce from 3-14 nu- between P. rhadinurum and P. the subgenus Carinamoeba (3). clei, often with a fan-shaped disposition. neusticuri, in spite of their occurrence Of these latter parasites,subspecies Its gametocytes are round, bean-shaped in lizards of two different families, the of P. minasense Carini and Rudolph, or elongate, and larger than those of the Iguanidae and the Teiidae, respectively. 1912 sensu Telford, 1979, found in liz- latter parasite (up to 7.6 x 6.3 11m).The In this respect it is of interest thatIguana ards of the farnilies Scincidae, Iguanidae trophozoites are moderately amoeboid, iguana, Ctenosaurus similis and and Teiidae, are differentiated by their but do not produce the exaggerated Neusticurus bicarinatus are alI com- tiny meronts with a fan-shaped or filopodia seenin the presently described monly found in a riverine habitat, where cruciform arrangementof the nuclei, and parasite. they often spend a considerable time trophozoites which do not exhibit Plasmodium attenuatum Telford, submerged in the streams or rivers to filopodia (4). 1973, of the teiid lizard Ameiva a. escapepredators. 8 Subspecies of P. tropiduri (sensu ameiva, and P. uncinatum Telford, 1973, Telford, 1979), of ig~anids, have of the iguanid Plica plica, both produce meronts giving rise to 4.;~ merozoites, meronts with their nuclei arranged in a Referencesand notes trophozoites which lack prominent cy- fan-shaped fashion, and have elongated I.Lainson R 1992 A protozoologist in toplasmic processes, and pigment of a gametocyteswhich curve round the host- Amazonia:Neglected parasites, with particu- golden-yellow colar (5). celI nucleus (8). lar referenceto membersof lhe Coccidia(Pro- tozoa: Apicomplexa). Ci Cult J Braz Assoc Plasmodium marginatum Telford, Of alI the neotropical Plasmodium Adv Sci 44: 81-93 1979, ofthe iguanidAnolisfrenatus, has species of lizards, P. rhadinurum 2. LainsonR, I Landau,JJ Shaw 1974 Further meronts producing only 3-8 merozoites, Thompson and Huff, 1944, of the igua- parasitesoflhe family Garniidae(Coccidiida: but they are usually in the forms of"flat- Haemosporidiidea) in Brazilian lizards. nas 19uana iguana (9) and Ctenosaurus Fallisia effusagen.nov., sp.nov. and Fallisia tened fans". The trophozoites do often similis (3) is considereçi to,be unique, modestagen.nov., sp.nov. Parasitology68: have cytoplasmic projections, but "alI by virtue of the strange filiform cytoplas- 117-125 stages tend to lie along the host cell rnic projections of some of its asexual 3.GarnhamPCC 1966 Malariaparasitesand other haemosporidia.Blackwell Scientific margin" and are not consistently in a stages. These differ, however, from the Publications,Oxford polar position, as are those of P. filopodia commonly seen in the 4. Telford SR 1979 A taxonomic revision of neusticuri n.sp. In addition, the trophozoites of P. neusticuri n.sp., in that small neotropicalsaurian malarias aIlied to gametocytes of P. marginatum are they are longer, more thread-like and of Plasmodiumminasense. Ann Parasitol Hum Comp 54: 409-422 slightly larger (5.9 x 4.1 fJm) and often a reddish-pink colar in Giemsa-stained 5. Telford SR 1979 A taxonomicreconsidera- lateral in their position within the eryth- blood films. Differences are also seen tiDo of some Plasmodium speéies from rocyte, which is hypertrophied and of- in the meronts, those of P. rhadinurum iguanidlizards.Ann Parasitol Hum Comp54: 129-144 ten deformed (5). Another, similar para- producing only 4-6 merozoites, with 6. Lainson R, JJ Shaw,I Landau 1976 Some site was described by the same author mature parasiteshaving the nuclei in fan- bl00d parasitesof lhe Brazilian lizards Plica (5), but not named, from Anolis shaped or cruciform arrangements. umbra and Uranoscodon superciliosa limifrons; the trophozoites were de- Gametocytes of this species are rarely (Iguanidae).Parasitology 70: 119-141 7. Ayala SC, JL Spain 1976 A population of scribed as having "short cytoplasmic encountered,and they are usually at least Plasmodiumcolombiensesp.n. in lhe iguanid processes", but they occupied any part as large as the host-celI nucleus, or may lizard, Anolis auratus.J Parasitol 62: 177- of the erythrocyte. The meronts often be half again as large (4). Perhaps the 189 had their nuclei in a fan-shaped fashion, 8. Telford SR 1973 Saurianmalaria parasites greatestdifference between P. neusticuri from Guyana: Iheir effect upon lhe validity and were often lateral in their position n.sp., andP. rhadinurum, however, is the oflhe family Garniidaeand thegenusGamia, in the host cell. The gametocytes are fact that infections of the latter are al- wilh descriptionsof two new species.Int J larger (av. 10.4 x 4.8 fJIll) and moreelon- most always comprised only of asexual Parasitol 3: 829-842 9. TbompsonPE, CG Huff 1944 Saurianma- gate than those of P.neu.~!jcuri n.sp., and stages,whereas those of the former are laria parasites of lhe United States and frequently lateropolar in position. either comprised predominantly of Mexico. J Infect Dis 74: 68-79 Plasmodium vacuolatum Lainson, gametocytes (old infections) or are a 10.Acknowledgements: This work was sup- Shaw and Landau, 1975, ofthe iguanid mixture of asexual stages and ported by grant 035693 (RL) from lhe WellcomeTrust, London. Our Ihanksare due Plica umbra, is readily distinguished by gametocytes (earlier infections). to ConstânciaM Franco and Manoel C de its large pigment-containing vacuole It is not proposed to discuss Old Souzafor technicalhelp in lhe laboratoryand seen in both the meronts and the World saurian plasmodia here. We have lhe field. gametocytes. AlI stagesdo consistently been unable to find the description of Received 18 May 1995 occupy a polar position in the erythro- any parasite sufficiently similar to P. Accepted 09 June 1995

Volume 48(3). May/June 1996 Ciência e Cultura Journal of lhe Brazilian Association for lhe Advancement of Science .203