Morphological, Biochemical and Molecular Characterization of Herpetomonas Samuelpessoai Camargoi n....

7/28/2019 Morphological, Biochemical and Molecular Characterization of Herpetomonas Samuelpessoai Camargoi n. Subsp.,

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FIORINI ET AL. -HERPE TOMONAS SAMUELPESSOAJ CAMARGOI N. SUBSP. 63Table 1. Comparison of the trypanosomatid Her/wfomonu.ssamuelpr.v.voai camargoi n. subsp. isolated from Cucwrbirti mo.cc.htittr with ref-erence-speciesof Herpetomonus and other trypanosomatid genera.

tImmuno-fluores-cenceAssay

(IF.4)

Phytomo-

RNA Small SubunitRestrictionEnzymes Mabs anti- site, Probeh SL3'

Organism Host origin Forms OCT" Arginase nus 360 Pvu 11 SSU3 SSU4 Prohe'Herpetomonus sumuelpes-soui camargoiH . saimelpessuuiH . tnariadeaneiH . samueliH . meguseliaeH . muscurumH. davidiH . roitmaniPhytomonas serpensP. franGaiP . mcgheeiCrithidia luciliae

Leptomonas sevmouri

~

Plant, flowerCucurbitu moschataHemipteran, predator2ellu.s bucogrammusFly, Muscina stubulansHemipteran, predatorZellus leucogrammusFly, Megaseliae scalarisFly, Musca domesticaPlant, latexEuphorbia cyatthophoraFly, Ornidia obesaPlant, fruitLycopersicon sculentumPlant, latexManihot esculentaPlant, seedZea maysFly, Phaeniciu serricataHemipteran, phytopha-Dvsdercus suturelusgous

PIO'P I 0PI 0PI 0P/OP/OPI 0C/OMPPPC

P

+++++++--+--

+++++++---+-

Southern blot analysis of genomic DNA digested with Pvu I1enzyme.Slot blot analysis of genomic DNA.Spliced-leader gene-derived oligonucleotide probe (Teixeira et al., 1996)."Ornithine carbamoyltransferase.' K promastigote;0,opisthomastigote; C, choanomastigote; OM, opisthomorph.

pat 28 "C in Roitman's defined medium. The percentage ofspined cells and of the different morphological stages was de-termined by counting 500 flagellates per smear. Digital imageswere printed in a Codonics NP-1600 printer (dye sublimation).Nutritional requirements and growth characteristics. Fla-gellate growth was evaluated in Roitman's defined and complexmedia (Roitman, Roitman, and Azevedo 1972), and LIT me-dium (Camargo 1964) by incubation at 28 "C or 37 "C. Nutri-tional requirements, optimal temperature, and osmolaritygrowth conditions were determined in Roitman's defined me-dium, as described before (Faria-e-Silva et al. 1994). Potassiumchloride (K Cl) was used to modify the osmolarity of the me-dium. Growth was measured by cell counts inahemocytometerafter 24, 48 and 72 h of incubation.Scanning eiectron microscopy. Two-day-old culture cellsgrown at 28 "C and 37 "C were collected by centrifugation at1,500g, fixed for 2 h with 2.5% (v/v) glutaraldehyde in 0.1Mphosphate buffer, pH 7.2, washed with the same buffer, andthen adhered to glass coverslips previously coated for 10minwith 0.1% poly-L-lysine (MW 30,000, Sigma Chemical Co,USA). Thereafter, the parasites were rinsed in buffer and post-fixed for 15 min with 1% (w/v) osmium tetroxide in 0.1Mphosphate buffer, pH 7.2. The cells were then dehydrated ingraded acetone and critical-point dried with CO,. A 20-nm-thick gold layer was deposited on the coverslips, which wereobserved with a Zeiss DSM-962 scanning electron microscope.Digital images were transferred to an IBM-PC compatible com-

puter coupled to the microscope and then printed in a CodonicsNP-1600 printer (dye sublimation).Transmission electron microscopy. Cells grown for 48 hat 28 "C and 37 "C were collected by centrifugation at 1,500gand fixed for 2 h with 1.5% glutaraldehyde and 4% parafor-maldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 1 .OmM CaCI,. Cells were then rinsed in cacodylate buffer andpost-fixed for 1h with 1% osmium tetroxide and 0.8% potas-sium ferricyanide in 0.1M cacodylate buffer, pH 7.2 containing1.0mM CaCI,. After rinsing in buffer, the parasites were de-hydrated in graded acetone and embedded in Epon. Ultrathinsections were stained with uranyl acetate and lead citrate andobserved with a Zeiss EM-900 transmission electron micro-scope operating at 80 kV .Enzyme assays and indirect immunofluorescence assay(IFA) with monoclonal antibodies (MAbs). Enzyme activitiesin crude extracts of cell homogenates were assayed as previ-ously described (Camargo et al. 1987). Extracts of Lepromonusseymouri and H. samueLpe.woni were run as positive controlsfor arginase and ornithine carbamoyltransferase (OCT) enzymeassays, respectively. Formaldehyde-fixed flagellates were testedby IFA with a pool of genus-specific anti-Phvtomonus MAbsaccording to Teixeira and Camargo (1989).Ribosomal DNA (rDNA) restriction analysis. Total geno-mic DNA was extracted with phenol-chloroform according toclassical methods, digested with Pvu I 1 restriction enzyme, elec-trophoresed in 0.8% agarose, and transferred to nylon mem-


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64 J . EUKARY OT. MICROBIOL., VOL. 48, NO. 1. JANUARY-FEBRUARY 2001

Fig. 1-3. Light microscopy of Giemsa-stained Herperomonas sunzueZpe.ssouicarnurgoi n. subsp. isolated from squash (Circurhirtr m o . s c h r u )flowers. 1.Typical promastigote (short arrow) and opisthomastigote (long arrow) forms. The arrows point to the kinetoplast. 2. Spined promastigoteform. The rod-shaped expansion (arrows) could usually be observed when the cells were kept for several days at 4 " C , as wcII as when theflagellates were grown at 37 "C . 3. Spined opisthomastigote form. Bar = 1 pm.Scanning electron microscopy of Herpetornonus sumuel pessoui cumurgoi n. subsp. 4. Cell with the rod-chapetl extension emergingclose to the flagellar pocket opening. 5. Cell with the lateral extension emerging at the medial portion of the cell body. Bar = I prn.Fig. 4-5.

branes for Southern blotting. Hybridization was done with thepTcl8S probe derived from the small subunit (SSU) rRNA geneof Trypanosoma cruzi as described (Teixeira et al. 1997).Hybridization with ribosomal and spliced leader (SL )gene-derived probes. Culture flagellates (lohcells) were dot-ted onto nylon membranes as described (Camargo et al. 1992).Membranes were hybridized with 20-nt oligonucleotides com-plementary to the sequences flanking a Pvu I 1 site in the smallsubunit (SSU) of the rRNA of H . samuelpessoai (SSU3) or H .megaseliae (SSU4), as previously reported (Teixeira et al.1997). SL3' is an 18-nt oligonucleotide complementary to aPhytomonas-specific spliced leader sequence (Teixeira et al.1996). Slot blots were hybridized with oligonucleotide probes5'-end-]abeled with [?*P]-dATPby T4 polynucleotide kinaseaspreviously described (Teixeira et al. 1996; Teixeira et al. 1997).PCR amplification and RFLP analysis of internal tran-scribed spacer (ITS) of the ribosomal DNA. Primers em-ployed to amplify ITSs were based on highly conserved se-quences of 3'-SSU and 5' large subunit (LSU) of the trypano-somatid rDNA (Cupolillo et al. 1995). Reactions mixtures of50 ~1 were prepared using 100 ng of template DNA, 2.5 unitsof Taq DNA Polymerase, 0.2 mM each dNTP, and 200 pMeach primers. The mixtures were cycled 30 times through thefollowing scheme: 1 min at 94 "C, 1 min at 55 "C,and 2 minat 72 "C.The amplified products were digested with restriction

enzymes, separated on 2.0% agarose gel and stained with eth-idium bromide.RAPD analysis. Sixteen decameric oligonucleotide primerswere employed for the characterization of the squash isolatebyRAPD patterns. For the RAPD reactions, mixtures of 50 pcontaining 100ng of DNA, 2.5 units of Taq DNA Polymerase,enzyme buffer, 15 mM MgCl,, 0.2 mM each dNTP, and 200pM of primer were cycled 34 times for 1 min at 95 "C , I minat 37 "C, and 2 min at 72 "C. The amplified products wereseparated on 2.0% agarose gel and stained with ethidium bro-mide.RESULTS

A trypanosomatid was isolated from the sap of petals andpetioles of a Cucurbira moschata flower collected in Alfenas,State of Minas Gerais, Brazil. This isolate was deposited atUNIFENA S and in the Trypanosomatid Culture Collection(TCC) of the University of SBo Paulo.Light microscopy of stained preparations showed that flowersmearsof the squash trypanosomatid or exponentially growingcultures predominantly exhibited promastigotes (Fig. 1). Mor-phometrical analysis of the flagellates (total length: 27.7 2 8.1km; body length: 16.7 + 2.9 Fm; body width: 2.6 t 0.3 km;free flagellum: 11.42 4.8 pn) showed morphological similar-


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FIORINI ET AL . -HEKPETOMONAS SAMUELPESSOAl CAMARGOI N. SUBSP 65

Fig. 6-8. Transmission electron microscopy of Herpetomonas samuel pessoui cutnurgoi n. subsp. Bar = 0.25 pm. 6. The x r v w points to abulgy structure at the cell surface. Theseunusual structures were formed by large lipid droplets (L ) opposed to the plasma membrane. 7. Detailof a rod-shaped extension (E) emerging at the flagellar pocket (FP) opening. The extension is filled with cytoplasm, but cytoplasmic organellesare absent. F, flagellum. 8. The lateral extension (E ) is enveloped by the plasma membrane underlain by subpellicular nlicrvtubules. Note thatsome microtubules cross from the cell to the rod-shaped expansion. FP, flagellar pocket; G, Golgi complex: N, nucleus.

ity to the generaPhytomonas, Herpetomonas, and Leptomonas.The proportion of the different morphological types varied ac-cording to time and culture media. A low percentage (0.2-0.4%) of typical opisthomastigotes was observed in exponentialcultures in all media tested. This stage was more abundantlydetected at the stationary phase of LIT cultures (after day 7) orby incubating cultures at 37 "C (Fig. 1). A thin rod-shapedexpansion originating near the anterior end of some cells wasobserved in both pro- and opisthomastigotes (Fig. 2, 3).A larg-er number of spined cells was detected in cultures maintainedat 37 "C (11%) or at 4 "C (5%).Scanning electron microscopy also revealed a rod-shaped ex-pansion emerging near the anterior end of the flagellate (Fig. 4,5). Transmission electron microscopy showed that the lateral

extension was covered by the plasma membrane and subpelli-cular microtubules (Fig. 7, 8).Other features typical of trypa-nosomatids including nuclei, kinetoplasts, and mitochondriawere never observed in these expansions. No cytoplasmic en-dosymbionts were seen. Howtver, large lipid droplets were fre-quently observed opposed to the plasma membrane and formingbulges on the surface of the cell (Fig. 6) that could also beobserved by scanning electron microscopy (Fig. 4, 5) .Optimal growth of flagellates was obtained by incubation at28 "C in Roitman's complex and LIT media, with cell densityreaching loxcells/ml after 72h of incubation. Analysis in Roit-man's defined medium showed that hemin and adenin, as wellas all vitamins and amino acids of this defined medium, wereessential for flagellate growth. In this medium the optimal


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J . EUKARYOT. MICROBIOL., VOL. 48, NO. 1, JA NUARY -FEBRUARY 2001

1

ssu4

2 1

SL3'

2Fig. 9. Slot blot hybridization of whole organisms (lo6cells) withradioactively-labeled probes derived from small subunit rDNA sequenc-esof Herpetomonus samuelpessoai (SSU3) orHerpetornonus muscarum(SSU.1) and Phytomonas spliced-leader gene-sequence (SL3'). la- Her-petornonus .samuelpessoui; 1b- Herpetomonas mariadeanei; 1c- Her-petomonus samueli; 1d- Herpetomonas samuelpessoai camargoi n.subsp.; 2a- Herpetomonas megaseliae; 2b- Herpetomonas muscarum;2c- Herpetomonas davidi; 2d- Phytomonas mcgheei.

growth temperature was 28 "C (range: 10-37 "C) and optimalosmolarity was 650mOsm (range: 50-1,500 mOsm).Enzymatic assays revealed that the isolate from the squashRower was negative for arginase, but showed ornithine carba-

A

moyltransferase (OCT) activity. Furthermore, IFA using a poolof seven MAbs specific for Phytomonas species failed to rec-ognize the isolate (Table 1). The SL3' probe, specific for thegenus Phytomonas and based on the spliced leader gene, failedto hybridize to total DNA from the squash flagellate (Fig. 9,Table 1). Taken together, these data suggest that the isolatedoes not belong to the genusPhytomonas.Southern blot analysis of total genomic DNA of the squashflagellate, digested with Pvu I1 and hybridized with the pTcl8Sprobe, revealed a DNA band smaller than 2.3 kb (data notshown), which is indicative of the presence of the 36OPvu IUSSU site (Camargo et al. 1992; Teixeira et al. 1997) (Table 1).These results strongly indicate that the isolate belongs to thegenus Herpetomonas. Screening for the sequence type of the36OPvu IUSSU Ranking region, employing theSSU3and SSU4probes, can be useful to divide Herpetomonas spp. into sub-groups A and B, respectively (Teixeira et al. 1997). Cross-hy-bridization analysis of total genomic DNA of the squash Ra-gellate showed positive hybridization only with the SSU3probe, thus permitting us to assign this isolate to group A ofHerpetomonas species (Fig. 9, Table 1).Amplification of whole ITS from rDNA showed significantlength variability among Herpetomonas reference-species be-longing to subgroupA, whereas there is little variability in am-plicon size of subgroup B. The squash isolate and H . samuel-

1 2 1 2 1 2 1 2bP

2.036-.636-1.018-1

-298 1-96-344 \---pnII HhaI RsaI SphIBFig. 10. Analysis of the polymorphism of the Internal Transcribed Spacer (ITS) sequences located between the 3' SSU and 5' L SU of therRNA of Herperomonas spp. by PCR amplification and restriction patterns of the amplified DNA fragments. Agarose gels (2%)stained withethidium bromide showing: A. Length polymorphism of the PCR-amplified sequences from DNA of reference-species of the genus Hrri)cforfit,rici.s.B. Similarity of restriction patterns obtained by digestion of the amplified DNA of H. .samuelpes.soai and H. scimurl~~e.s.rorriumtrrgoi n. subsp.with the fol lowing restriction enzymes: Dpn TI , Hha I, Rsa I , and Sph I.


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FIORINI ET AL . - H E R PE T OM ON AS S AM U E L PE S SU A I C AM AR G U I N. SUBSP. 67

615A

\/I ,000\/ 00

-400 \- 00\\200-

1 2 1 2 1 2 1 2 1 2 1 2

/I 00 - - - - - -38 672 685 675 684 655BFig. 11. Agarose gels (2%) stained with ethidium bromide showing RAPD patterns generated from DNA of the H . .suI?I[~r/ /)r.s.so~rr. ur? i t r r ,qo iti. subsp. and H erpetomonas spp. selected to illustrate the genetic polymorphism among members of this genus. A . RAPD patterns generated bythe primer 615 for H erpetomonus samuelpessoai camavgoi n. sp. and seven reference-strains of the genus H rr /~~,t~~~? z[~~i ~i .s.. RAPD fingerprintsobtained using six primers (638, 672, 685, 675, 684, 655) from DNA of H . . r umuel prs soni and H . .surnurlpessoui cumtrrgoi n. subsp.

pessoai showed ITS of the same size and differed markedlyfrom the ITS of other Herpetomonas species (Fig. IOA). Fur-thermore, the flower isolate could not be distinguished fromH.samuelpessoai by restriction mapping of ITS using four en-zymes (Dpn 11, Hha I , Rsa I and Sph I, Fig. 10B).Analysis ofRAPD patterns using 10primers indicated that Herpetomonasspecies belonging to group A could be easily distinguished fromeach other (Takata et al. 1996). However, results using thisapproach showed that only three of the 10primers previouslytested plus two of six new primers generated fingerprints thatdistinguished the flower isolate from H. samuelpessoai (a typ-ica result using primer 615 is shown in Figure 11A to exem-plify the species-specificity of patterns). Thus, only five out of16 primers used in RAPD analysis clearly generated uniquefingerprints for this isolate (Fig. 11B). This result suggests thatalthough it is a very close relative, the flower trypanosomatidis not identical to H . samuelpessoai.

DISCUSSIONIn this paper we describea trypanosomatid isolated from theflower of the squash C. moschata. Although a trypanosomatidhas been previously detected in the nectar of Colchicusautum-nalis (Galli-Valerio 1921), this is the first trypanosomatid to becultured from a flower. According to traditional taxonomic cri-teria, this flagellate could have been immediately assigned intothe Phytomonas genus. However, plant origin cannot be usedas an exclusive taxonomic parameter for classification of a try-panosomatid as Phytomonas, since species of Herpetomonas,Leptomonas,and Crithidiahave alsobeen found in plants (Ca-margo 1999; Conchon et al. 1989; Teixeiraet al. 1996).Morphological characterization of the squash flagellate dem-

onstrated that usually only promastigote forms, which are alsopresent in the generaPhytomonas, Herpetonionus, and Leptom-onus, were detected in the sap of the squash flowers and in theexponential cultures. However, under special culture conditions,typical opisthomastigotes could be observed, which accordingto traditional morphological criteria would assign the new iso-lateas Herpetomonas.Enzymes of ornithine-arginine metabolism can be correlatedwith the trypanosomatid genera and are considered to be oftaxonomic value (Camargo et al. 1987; Figueiredo et al. 1978;Yoshida et al. 1978). The squash isolate was negative for ar-ginase and positive for OCT-a profile comparable to that de-scribed for Herpetomonas (Y oshida et al. 1978). Lack of argi-nase is a common feature of Phytornonas, Herpetomonas, andsomeLeptomonas species (Camargo et al. 1992; Camargo et al.1987; Figueiredo et al. 1978). Activity of the OCT is particu-larly detected in Herpetomonas spp., but too few Phytomonasspp. have been examined to conclude that this feature is diag-nostic for Herpetomonas (Camargo et al. 1987; Jankevicius etal. 1993). Thus, even though OCT activity is present in allHerpetomonas spp., this enzyme cannot be considered as anexclusive taxonomic feature of this genus.In order to assess the presence of taxonomic Phytomonasmarkers, we tested the squash isolate by IFA with a pool ofMAbs (Sbravate et al. 1989; Teixeira and Camargo 1989) andwith the spliced-leader gene-derived SL3' probe (Nunes et al.1995; Serrano et al. 1999; Teixeira et al. 1996), which are spe-cific for Phytomonas. In addition, we have also investigated thepresence of a site 36OPvu IVSSU rRNA, which is present inHerpetomonas, Crithidia,and Leptomonas, but has never beendetected in Phytomonas (Camargo et al. 1992; Teixeira et al.


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68 J . EUK ARY OT. MI CROBIOL., VOL. 48, NO. 1, JA NUARY -FEBRUARY 20011997). Results were negative for all tests, distinguishing thenew isolate from all Phytomonas species, despite its plant ori-gin.Therefore, based on morphological, biochemical, and molec-ular features, the squash flagellate cannot be considered as aPhytomonas species, whereas all data favor its assignment tothe genus Herpetomonas. We have previously shown that akDNA probe (Nunes et al. 1994) and the rRNA-derived SSU3and SSU4 probes (Teixeira et al. 1997) divided species of thegenusHerpetomonasinto subgroups A andB, which are headedby H . samuelpessoai and H. muscarum, respectively. The DNAfromthe squash flagellate hybridized exclusively with theSSU3probe, assigning this isolate to subgroup A of Herpetomonas.The size of the amplified ITS of the squash isolate was foundto be similar to that of H. samuelpessoai, which itself differedfrom all other species of Herpetomonas (Takata et al. 1996).Moreover, restriction analysis of the amplified ITS could notdistinguish these two organisms.Comparative analysis of RAPD patterns was performed todifferentiate the squash isolate fromH. samuelpessoai. PreviousRAPD analysis differentiated most species within the genusHerpetomonas, particularly species of group A (Takata et al.1996). which also proved to be more heterogeneous than groupB based on ribosomal analysis (Hollar, L ukes, and Maslov1998; Landweber and Gilbert 1994). Five outof sixteen primersgenerated fingerprints specific for the squash isolate, distin-guishing this flagellate from H . samuelpessoai and suggestingthat the new isolate is very closely related to but not identicaltoH. samuelpessoai. In addition to these genetic features sharedby these two flagellates, the squash isolate could be cultivatedat 37 "C, thus proving it to be a thermophilic organism, whichis also a property of H . samuelpessoai but not of H. muscarum(Roitman et al. 1976).Despite its small molecular variability when compared withH . samuelpessoai, the new isolate has a morphological pecu-liarity unique among the known species of Herpetomonas: thepresence of a thin, rod-shaped, lateral expansion of the plasmamembrane. This unusual structure was only described (by lightmicroscopy) previously in a Phytomonas sp. isolated from thelatex of Allamanda cathartica (Kastelein and Parsadi 1984).Nevertheless, in contrast to the flagellate here described, thisplant isolate presented biochemical and molecular characteris-tics of Phytomonas (Camargo et al. 1992; Teixeira et al. 1996).Considering the morphological, biochemical, and molecularevidence, it could be more appropriate to consider this isolateas a subspecies of H. sarnuelpessoai, as previously done for H.muscarurn muscarum and H. rnuscarum ingenoplastis, whichhave been considered to be subspecies based on the same hostorigin and distinct structures of the kinetoplast-mitochondrioncomplex (Rogers and Wallace 1971; Wallace, Wagner, andRogers 1973) and in growth differences in the presence of ox-ygen and in the electron transport pathway, with H. m. ingen-uplastis being particularly well-adapted for anaerobiosis (Red-man and Coombs 1997).The flower-parasitizing trypanosomatid described here, be-sides being the first trypanosomatid cultured and characterizedfrom flowers, represents the only record of ultrastructural, bio-chemical, and molecular descriptions of a new species of Her-petomonas. We propose that this isolate be considered a newsubspecies of Herpetomonas samuelpessoai, which we nameHerpetomonas sarnuelpessoai camargoi n. sp., in honor of Pro-fessor Erney Plessmann Camargo, a distinguished Brazilianprotozoologist.

ACKNOWLEDGMENTSThis work was supported by CAPES, CNPq, FAPEMIG, FA-PESP, FINEP, FIOCRUZ, PRONEX, and UNIFENAS.

LITERATURE CITEDCamargo, E. P. 1964.Growth and diff ercntiation in T / ~ ~ / ~ ~ r / 7 o . ~m:i.Origin of metacyclic trypanosonies in liquid media. H c v . I t7.5t. Mrcl.Trop. Sdo Paulo, 6:93-100.Camargo, E. P. 1999. Plryfomonc~snd other trypanosomatid parasitesof plants and fruit. A d v . Paru.sito/.,42:29-1 12.Camargo, E. P. , K astelein, P. & Roitman, I. 1990. Trypanosomatid par-asites of plants (Pl7vrornonu.r).Purusitol. Tockry. 6:22-25.Camargo, E. P., Silva, S., Roitman, I ., De Souza, W., J ankevi ciu. J .

V . & Dol let, M . 1987. Enzymes of orni tine-arginine metabolism intrypanosomatids of the genus P17ytomoncr.s. ./. Proto;ool.. 34:439-441.Camargo, E. P., Sbravate C., Teixeira, M . M . G.. Ul iana. S. R ., Soares.M . B . M.. A ffonso, H. T. & Floeter-Wi nter, L . 1992. RibosomalD N A restriction analysis and synthetic oli gonucleotide probing in theidentification of genera of lower trypanosomatids. J . Purusitol.. 78:4048.Carvalho, A. L . & Deane, M. P. 1974. Trypanosomatidae isolated f romZellus 1rurwgrunirnu.s (Perty. 1834) (Hemiptera, Reduviidae), wrth adiscussion on flagellates of insectivorous bugs. J . Pri )to,-ooI ..21:s-8.Conchon, I., Campaner, M., Sbravate, C. & Camargo, E. P. 1989. Try-panosomatids, other than Pl7ytornoncr.s sp., isolated and cultured fromfruit. J . Protozoo/.,36:412414.Cupolillo, E., Grimaldi, G., M omen, H. & Beverley, S . M . 1995. Inter-genic region typing (IRT): A rapid molecular approach to the char-acterization and evolution of Lei.shrncrnici. M ol . Bioc~hrrn. t r rus i to l . .73: 45-1 55.Donovan, C. 1909. K ala azar in M adras especially with regard to itsconnection with the dog and bug ( C ~ ~ n o r r l t i n ~ ~ , s ) ,t r r 7 c ~ t .177: 1495-1496.Faria-e-Silva. P. M ., Fiorini, J . E., Soares, M . J .. Alviano, C. S. . DeSouza, W. & A ngluster, J . 1994. M embrane-ass,ociated poly\accha-rides composition, nutritional requirements and cell differentiation inHrrpefotnoncrs roitnruni: influence of the endosymbiont. 1. Eukrir~.ot.Microhiol.. 41:SS-59.Figueiredo, E. N., Y oshida, N., R oitman, C. & Camargo, E. P. 1978.Enzymes of the ornithine-arginine metaboli sm of trypanoomatids ofthe genus Critliidiu. J . Prorozool.,25:546-549.Fiorini, J . E., Faria e Sil va, P. M ., Soares, M . J. & Brazil. R. P. 1989.T res novas especies de tripanosomatideos de insetns isolados em A l-fenas, M inas Gerais, B rasil. M mi . inst. Osri~if tlo'ru:. 84:69-74.Galli- V alerio, 1921. L a fl agell iase des euphorbiacees en Suisse.Schweiz. Mrd. Woc:lretisc/ir., 2: 154.

Hollar, L., L ukes, J . & M aslov, D. A . 1998. M onophyly of endo5yni-biont-containing trypanosomatids: phylogeny versus taxonomy. J .E ukapot. Microhiol.. 45:'293-297.J ankevicius, S . I., A lmeida, M . L., J ankevicius. J . V .. Cavazzana. M..A ttias, M . & Souza, W . 1993. Axenic cultivation of trypanosomatidsfound in corn (Zeumuys) and in phytophagous hemipterans (Lrpto-gl ossus zonutus: Coreidae) and their experimental transmission. J .Eukaryot. Microhiol., 40576-581.K astelein, P. & Parsadi, M . 1984.Observations on cultures of the pro-tozoa Phytomonus sp. (T rypanosomatidae) associated with the latic-ifer Allnrnandu ccrthartica (A pocynaceae). Surin. Lundh.. 32:85-89.L afont, A . 1909. Sur la presence d'un L q~fi ~rnon ri ,~.arasite de laclassedes flagelles, dans le latex de trois euphorbiacees. Ann. ln.s~. n.stc,irr.24:205-2 19 .L andweber, L. F. & Gilbert, W. 1994. Phylogenetic analysis of RNAediting: aprimitive genetic phenomenon. Proc,. Nrrrl. Act r t l . Sci. U S A .91:918-921.M arche, S. , Roth, C., Philippe, H ., Dollet, M . & Baltr, T. 1995. Char-acterization and detection of plant trypanosomat ds by sequence anal-ysis of the small subunit rihosomal R NX gene. Mol. Riochrrn. Par-usirol., 71:lS-26.Nunes, N. R ., Teixeira, M . M . G.. Camargo, E. P. & A t'fonso, M . H.T . 1994. kDNA and rD NA sequences reveal a phylogenetic clusterof species originally placed in different genera of trypanosornatids.J . E ukaryot. M icrobiol,, 41:496-500.Nunes, N. R., Teixeira, M . M . G., Carnargo, E . P. & Buck. G. A. 1995.Sequence and structural characterization of the spliced leader genesand transcripts in Phyton7onc1.s. Mol. Bioc./tein. Purusitol., 74:233-237.


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Morphological, Biochemical and Molecular Characterization of Herpetomonas Samuelpessoai Camargoi n....

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