J . Euh<zn,il M~c.,oh,o/.. 4X( I). 2001 pp.

62-6')
b! ?001 bq thc S r ~ i r t y of Piotomolc,

Morphological, Biochemical and Molecular Characterization of Herpetomonas samuelpessoai camargoi n. subsp., a Trypanosomatid Isolated from the Flower of the Squash Cucurbita moschata
JOAO E. FIORINI;' CARMEN S. A. TAKATA,h VIRGINIA M. TEOFILO," LUIZ C. NASCIMENTO;' PAULO M. FARIA-E-SILVA;." MAURILIO J. SOARES,' MARTA M. G. TEIXEIRAh and WANDERLEY DE SOUZA"
"Depurtamento de Cie^nciu.s Bioldgicus, UNIFENAS, 37130-000 Alfrnus, MG, tind hDepartaniento de Paru.sitologia, Instituto dr Cii.nciu.s BiomGdicus, USP, 05508-900 SGo Priulo. SP, tintl Ci&cius Biolrjgicas, EFOA, 371.30-000 Alf>nu.s, MG. und <'Luh. rlr Ultrcr-r.rtruturu Cr4 Instituto de Ric?fisicu Carlos C1rajiti.s Filho, U#-UJ, 2 I949-900Rio dc Jun(~ir~i, KJ. iintl 'Depurtumento clr Ultra-estrutura e Biologiu Celulur, lnstitutn Oswaldo Cruc/FIOCRUZ, 2 1045-900 Rio da Jtrneii-o. K J . Hru:il ABSTRACT. We report the morphological, biochemical and molecular characteristics of a trypanosoinatid isolated from the flower molecular of Cucurbitu moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of P /~~toriioiici.s-spccific markers based on spliced-leader and rihosomal genes as well as by monoclonal antibodies specific for PIi\tornontr.s argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetoiiio n a s . Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. sumuelprssoai than to any other species. However, the presence of spined flagcllates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate-specitic RAPD tingersciriiirc,l/,c,.s,sotri (wnur,qoi prints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Hei~,~,t[~i?z[iiiti.\ 11. subsp. is proposed. Key Words. Plant flagellate, RAPD analysis, ribosomal DNA, ultrastructure.

LASSIFICATION of insect and plant trypanosomatid protozoa at the species level formerly relied on morphology and host specificity. These criteria, however, are not sufficient for species descriptions due to the existence of extensive size and shape polymorphism within a single species according to both host and culture conditions. The host specificity criterion is also of little help because single species may colonize different hosts and a single host may harbor many different trypanosomatid species. For instance, since creation of the genus Phytomonas (Donovan 1909) all trypanosomatid plant species have been routinely placed in this genus. Recently, however, the other trypanosomatid genera, including Herpetomonas, Crithidia, and Leptornonas, have been found to inhabit plants (Camargo 1999; Conchon et al. 1989; Serrano et al. 1999; Teixeira et al. 1996). Besides host specificity, morphology is the traditional taxonomic criterion for trypanosomatid classification into all these genera (Wallace et al. 1983). However, morphological parameters are not of much help for ciassification because flagellates of most of these genera present indistinguishable promastigote forms. Phytomonus-specific markers, such as monoclonal antibodies (Teixeira and Camargo 1989) and ribosomal (Camargo et al. 1992; March6 et al. 1995; Teixeira, Campaner, and Camargo 1994) and spliced-leader gene sequences (Serrano et al. 1999; Teixeira et al. 1996), are now available to distinguish the genus Phytomonas from all other trypanosomatid genera. Although there is as yet no molecular marker specific for Herpetomonas, there is a set of molecular markers to enable separation of Herpetomonas from Phytomonas, Crithidia, and most Leptomonas species (Camargo et al. 1992; Teixeira et al. 1996; Teixeira et al. 1997). Adoption of biochemical and molecular criteria confirmed misclassification of trypanosomatids in genera based exclusively on traditional criteria. This fact has been especially observed for species of Herpetomonas (Camargo et al. 1992; Faria-e-Silva et al. 1994; Fiorini et al. 1989; Hollar, Lukes, and Maslov 1998; Jankevicius et al. 1993; Nunes et al. 1994; Roitman et al. 1976; Teixeira et al. 1996; Teixeira et al. 1997). Trypanosomatids have been described from the tissues and juices of several species belonging to different plant families (Camargo 1999; Camargo, Kastelein, and Roitman 1990; WalCorresponding Author: M. Soares-FAX 4434; E-mail: maurilio~ioc.tiocruz.hr number: (0055) (2 l ) 260-

lace, Roitman, and Camargo 1992). They were first described in the latex of Euphorbiaceae (Lafont 1909) and thereafter in flowers, fruit, and finally in the phloem of different plants (Camargo 1999). However, in contrast to the hundreds of later reports describing trypanosomatid species from latex, phloem, and fruits, there is only one report of an isolate found inhabiting a flower (Galli-Valerio 1921). However, this isolate was claimed to be sufficiently similar to a Herpetomonus found in the digestive tube of an hemipteran that it was considered to be the same species. No further studies based on this earlier flower trypanosomatid are available, so the finding remains puzzling. In this paper we describe the characterization of a trypanosomatid cultured after isolation from the Cucirrbita moschara flower. Upon comparing morphological, biochemical, and molecular features of this flagellate with reference-species of all other trypanosomatid genera, we classified the new isolate as Herpetomonas. After comparative analysis with several known species of this genus, we decided that it deserved the status of a new subspecies, which is herein described. MATERIALS AND METHODS Isolation of the trypanosomatid. Plants of the squash Ciccurbita moschata were collected in the vicinity of Alfenas, Minas Gerais, Brazil (21"25'45"S, 45'56'50W). Flower fragments were examined by light microscopy. Fragments positive for flagellates were inoculated into a chicken blood-agar biphasic medium overlaid with Roitman's defined medium (Roitman, Roitman, and Azevedo 1972) supplemented with antibiotics and then incubated at 25 "C. The culture obtained was cloned in agar plates as previously described (Fiorini et al. 1989). Reference-species of trypanosomatids. All reference-species utilized for comparative purposes in this study (Table 1) have been deposited at the Trypanosomatid Culture Collection of the University of SFio Paulo (TCCWSP). Organisms were cultured in LIT medium (Camargo 1964) at 28 "C. Morphological and morphometrical analysis. Smears of cultured flagellates and of sap of petals and petioles of the flower of C. rnoschata were fixed with methanol, hydrolyzed in 5 N HCl for 15 min at room temperature, stained with Giemsa (Carvalho and Deane 1974), and examined under a light microscope. Morphometrical data were obtained from camera lucida drawings of 500 flagellates from cultures grown for 48 h

62

FIORINI ET AL.-HERPETOMONAS

SAMUELPESSOAJ CAMARGOI N. SUBSP.

63

Table 1. Comparison of the trypanosomatid Her/wfomonu.ssamuelpr.v.voai camargoi n. subsp. isolated from Cucwrbirti mo.cc.htittr with reference-species of Herpetomonus and other trypanosomatid genera.
t

Immunofluorescence Assay (IF.4)

RNA Small Subunit SL3'

Prohe'

Organism
Herpetomonus sumuelpessoui camargoi H . saimielpessuui H . tnariadeanei H . samueli H . meguseliae H . muscurum H. davidi H . roitmani Phytomonas serpens P. franGai

Host origin
~

Forms PIO' PI0 PI0 PI0 P/O P/O PI0 C/OM

Restriction Enzymes Mabs antisite, Probeh PhytomoOCT" Arginase nus 360 Pvu 11 SSU3 SSU4

P. mcgheei Crithidia luciliae

Leptomonas sevmouri

Plant, flower Cucurbitu moschata Hemipteran, predator 2ellu.s bucogrammus Fly, Muscina stubulans Hemipteran, predator Zellus leucogrammus Fly, Megaseliae scalaris Fly, Musca domestica Plant, latex Euphorbia cyatthophora Fly, Ornidia obesa Plant, fruit Lycopersicon sculentum Plant, latex Manihot esculenta Plant, seed Zea mays Fly, Phaeniciu serricata Hemipteran, phytopha-

+ + + + + + +
-

+ + + + +
-

P
P P C P

gous
Dvsdercus suturelus

Southern blot analysis of genomic DNA digested with Pvu I1 enzyme. 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 of spined cells and of the different morphological stages was determined by counting 500 flagellates per smear. Digital images were printed in a Codonics NP-1600 printer (dye sublimation). Nutritional requirements and growth characteristics. Flagellate growth was evaluated in Roitman's defined and complex media (Roitman, Roitman, and Azevedo 1972), and LIT medium (Camargo 1964) by incubation at 28 "C or 37 "C. Nutritional requirements, optimal temperature, and osmolarity growth conditions were determined in Roitman's defined medium, as described before (Faria-e-Silva et al. 1994). Potassium chloride (KCl) was used to modify the osmolarity of the medium. Growth was measured by cell counts in a hemocytometer after 24, 48 and 72 h of incubation. Scanning eiectron microscopy. Two-day-old culture cells grown at 28 "C and 37 "C were collected by centrifugation at 1,500 g, fixed for 2 h with 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, washed with the same buffer, and then adhered to glass coverslips previously coated for 10 min with 0.1% poly-L-lysine (MW 30,000, Sigma Chemical Co, USA). Thereafter, the parasites were rinsed in buffer and postfixed for 15 min with 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer, pH 7.2. The cells were then dehydrated in graded acetone and critical-point dried with CO,. A 20-nmthick gold layer was deposited on the coverslips, which were observed 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 Codonics NP-1600 printer (dye sublimation). Transmission electron microscopy. Cells grown for 48 h at 28 "C and 37 "C were collected by centrifugation at 1,500 g and fixed for 2 h with 1.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2, containing 1 .O mM CaCI,. Cells were then rinsed in cacodylate buffer and post-fixed for 1 h with 1% osmium tetroxide and 0.8% potassium ferricyanide in 0.1 M cacodylate buffer, pH 7.2 containing 1.0 mM CaCI,. After rinsing in buffer, the parasites were dehydrated in graded acetone and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss EM-900 transmission electron microscope operating at 80 kV. Enzyme assays and indirect immunofluorescence assay (IFA) with monoclonal antibodies (MAbs). Enzyme activities in crude extracts of cell homogenates were assayed as previously described (Camargo et al. 1987). Extracts of Lepromonus seymouri and H. samueLpe.woni were run as positive controls for arginase and ornithine carbamoyltransferase (OCT) enzyme assays, respectively. Formaldehyde-fixed flagellates were tested by IFA with a pool of genus-specific anti-Phvtomonus MAbs according to Teixeira and Camargo ( 1989). Ribosomal DNA (rDNA) restriction analysis. Total genomic DNA was extracted with phenol-chloroform according to classical methods, digested with Pvu I1 restriction enzyme, electrophoresed in 0.8% agarose, and transferred to nylon mem-

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

Fig. 1-3. Light microscopy of Giemsa-stained Herperomonas sunzueZpe.ssoui carnurgoi 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 promastigote form. The rod-shaped expansion (arrows) could usually be observed when the cells were kept for several days at 4 "C, as wcII as when the flagellates were grown at 37 "C. 3. Spined opisthomastigote form. Bar = 1 pm. Fig. 4-5. Scanning electron microscopy of Herpetornonus sumuelpessoui cumurgoi n. subsp. 4. Cell with the rod-chapetl extension emerging close to the flagellar pocket opening. 5. Cell with the lateral extension emerging at the medial portion of the cell body. Bar = I prn.

branes for Southern blotting. Hybridization was done with the pTcl8S probe derived from the small subunit (SSU) rRNA gene of Trypanosoma cruzi as described (Teixeira et al. 1997). Hybridization with ribosomal and spliced leader (SL) gene-derived probes. Culture flagellates ( loh cells) were dotted onto nylon membranes as described (Camargo et al. 1992). Membranes were hybridized with 20-nt oligonucleotides complementary to the sequences flanking a Pvu I1 site in the small subunit (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 a Phytomonas-specific spliced leader sequence (Teixeira et al. 1996). Slot blots were hybridized with oligonucleotide probes 5'-end-]abeled with [?*P]-dATPby T4 polynucleotide kinase as previously described (Teixeira et al. 1996; Teixeira et al. 1997). PCR amplification and RFLP analysis of internal transcribed spacer (ITS) of the ribosomal DNA. Primers employed to amplify ITSs were based on highly conserved sequences of 3'-SSU and 5' large subunit (LSU) of the trypanosomatid rDNA (Cupolillo et al. 1995). Reactions mixtures of 50 ~1 were prepared using 100 ng of template DNA, 2.5 units of Taq DNA Polymerase, 0.2 mM each dNTP, and 200 pM each primers. The mixtures were cycled 30 times through the following scheme: 1 min at 94 "C, 1 min at 55 "C, and 2 min at 72 "C. The amplified products were digested with restriction

enzymes, separated on 2.0% agarose gel and stained with ethidium bromide. RAPD analysis. Sixteen decameric oligonucleotide primers were employed for the characterization of the squash isolate by RAPD patterns. For the RAPD reactions, mixtures of 50 p l containing 100 ng of DNA, 2.5 units of Taq DNA Polymerase, enzyme buffer, 15 mM MgCl,, 0.2 mM each dNTP, and 200 pM of primer were cycled 34 times for 1 min at 95 " C , I min at 37 "C, and 2 min at 72 "C. The amplified products were separated on 2.0% agarose gel and stained with ethidium bromide. RESULTS A trypanosomatid was isolated from the sap of petals and petioles of a Cucurbira moschata flower collected in Alfenas, State of Minas Gerais, Brazil. This isolate was deposited at UNIFENAS and in the Trypanosomatid Culture Collection (TCC) of the University of SBo Paulo. Light microscopy of stained preparations showed that flower smears of the squash trypanosomatid or exponentially growing cultures predominantly exhibited promastigotes (Fig. 1 ). Morphometrical analysis of the flagellates (total length: 27.7 2 8.1 km; body length: 16.7 +- 2.9 Fm; body width: 2.6 t 0.3 km; free flagellum: 11.4 2 4.8 p n ) showed morphological similar-

FIORINI ET AL.-HEKPETOMONAS

SAMUELPESSOAl CAMARGOI N.SUBSP

65

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

ity to the genera Phytomonas, Herpetomonas, and Leptomonas. The proportion of the different morphological types varied according to time and culture media. A low percentage (0.20.4%) of typical opisthomastigotes was observed in exponential cultures in all media tested. This stage was more abundantly detected at the stationary phase of LIT cultures (after day 7 ) or by incubating cultures at 37 "C (Fig. 1). A thin rod-shaped expansion originating near the anterior end of some cells was observed in both pro- and opisthomastigotes (Fig. 2, 3). A larger number of spined cells was detected in cultures maintained at 37 "C (11%) or at 4 "C (5%). Scanning electron microscopy also revealed a rod-shaped expansion 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 subpellicular microtubules (Fig. 7 , 8). Other features typical of trypanosomatids including nuclei, kinetoplasts, and mitochondria were never observed in these expansions. No cytoplasmic endosymbionts were seen. Howtver, large lipid droplets were frequently observed opposed to the plasma membrane and forming bulges on the surface of the cell (Fig. 6) that could also be observed by scanning electron microscopy (Fig. 4, 5 ) . Optimal growth of flagellates was obtained by incubation at 28 "C in Roitman's complex and LIT media, with cell density reaching loxcells/ml after 7 2 h of incubation. Analysis in Roitman's defined medium showed that hemin and adenin, as well as all vitamins and amino acids of this defined medium, were essential for flagellate growth. In this medium the optimal

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

ssu3

ssu4

SL3'

a
b
C

d
1 2
1

Fig. 9. Slot blot hybridization of whole organisms (lo6 cells) with radioactively-labeled probes derived from small subunit rDNA sequences of Herpetomonus samuelpessoai (SSU3) or Herpetornonus muscarum (SSU.1) and Phytomonas spliced-leader gene-sequence (SL3'). la- Herpetornonus .samuelpessoui; 1b- Herpetomonas mariadeanei; 1c- Herpetomonus 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 optimal osmolarity was 650 mOsm (range: 50-1,500 mOsm). Enzymatic assays revealed that the isolate from the squash Rower was negative for arginase, but showed ornithine carba-

moyltransferase (OCT) activity. Furthermore, IFA using a pool of seven MAbs specific for Phytomonas species failed to recognize the isolate (Table 1). The SL3' probe, specific for the genus Phytomonas and based on the spliced leader gene, failed to hybridize to total DNA from the squash flagellate (Fig. 9, Table 1). Taken together, these data suggest that the isolate does not belong to the genus Phytomonas. Southern blot analysis of total genomic DNA of the squash flagellate, digested with Pvu I1 and hybridized with the pTcl8S probe, revealed a DNA band smaller than 2.3 kb (data not shown), which is indicative of the presence of the 36OPvu IU SSU site (Camargo et al. 1992; Teixeira et al. 1997) (Table 1). These results strongly indicate that the isolate belongs to the genus Herpetomonas. Screening for the sequence type of the 36OPvu IUSSU Ranking region, employing the SSU3 and SSU4 probes, can be useful to divide Herpetomonas spp. into subgroups A and B, respectively (Teixeira et al. 1997). Cross-hybridization analysis of total genomic DNA of the squash Ragellate showed positive hybridization only with the SSU3 probe, thus permitting us to assign this isolate to group A of Herpetomonas species (Fig. 9, Table 1). Amplification of whole ITS from rDNA showed significant length variability among Herpetomonas reference-species belonging to subgroup A, whereas there is little variability in amplicon size of subgroup B. The squash isolate and H. samuel-

1 bP
2.036 1.636 1.018

-344 -298

396 1
\

DpnII

--HhaI
RsaI SphI

Fig. 10. Analysis of the polymorphism of the Internal Transcribed Spacer (ITS) sequences located between the 3' SSU and 5' LSU of the rRNA of Herperomonas spp. by PCR amplification and restriction patterns of the amplified DNA fragments. Agarose gels ( 2 % ) stained with ethidium 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.rorri cumtrrgoi n. subsp. with the following restriction enzymes: Dpn TI, Hha I, Rsa I, and Sph I.

FIORINI ET A L . - H E R P E T O M O N A S

SAMUELPESSUAI C A M A R G U I N. SUBSP.

67

1
/ I

,000

500

\
\

-400

-300
/ I 0 0

\ 200 - - - - - 638 672 615

\ \ \

685

675

684

655

Fig. 11. Agarose gels (2%) stained with ethidium bromide showing RAPD patterns generated from DNA of the H . .suI?I[~r//)r.s.so~rr c.ur?itrr,qoi ti. subsp. and H e r p e t o m o n a s spp. selected to illustrate the genetic polymorphism among members of this genus. A. RAPD patterns generated by the primer 6 15 for Herpetomonus samuelpessoai camavgoi n. sp. and seven reference-strains of the genus H r r / ~ ~ , t ~ ~ ~ ?B. z[~ RAPD ~ i ~ i . fingerprints s. obtained using six primers (638, 672, 685, 675, 684, 655) from DNA of H . .rumuelprssoni and H . .surnurlpessoui cumtrrgoi n. subsp.

pessoai showed ITS of the same size and differed markedly from the ITS of other Herpetomonas species (Fig. IOA). Furthermore, the flower isolate could not be distinguished from H. samuelpessoai by restriction mapping of ITS using four enzymes (Dpn 11, Hha I, Rsa I and Sph I, Fig. 10B). Analysis of RAPD patterns using 10 primers indicated that Herpetomonas species belonging to group A could be easily distinguished from each other (Takata et al. 1996). However, results using this approach showed that only three of the 10 primers previously tested plus two of six new primers generated fingerprints that distinguished the flower isolate from H. samuelpessoai (a typical result using primer 615 is shown in Figure 11A to exemplify the species-specificity of patterns). Thus, only five out of 16 primers used in RAPD analysis clearly generated unique fingerprints for this isolate (Fig. 11B). This result suggests that although it is a very close relative, the flower trypanosomatid is not identical to H . samuelpessoai.

DISCUSSION In this paper we describe a trypanosomatid isolated from the flower of the squash C. moschata. Although a trypanosomatid has been previously detected in the nectar of Colchicus autumnalis (Galli-Valerio 1921), this is the first trypanosomatid to be cultured from a flower. According to traditional taxonomic criteria, this flagellate could have been immediately assigned into the Phytomonas genus. However, plant origin cannot be used as an exclusive taxonomic parameter for classification of a trypanosomatid as Phytomonas, since species of Herpetomonas, Leptomonas, and Crithidia have also been found in plants (Camargo 1999; Conchon et al. 1989; Teixeira et al. 1996). Morphological characterization of the squash flagellate dem-

onstrated that usually only promastigote forms, which are also present in the genera Phytomonas, Herpetonionus, and Leptomonus, were detected in the sap of the squash flowers and in the exponential cultures. However, under special culture conditions, typical opisthomastigotes could be observed, which according to traditional morphological criteria would assign the new isolate as Herpetomonas. Enzymes of ornithine-arginine metabolism can be correlated with the trypanosomatid genera and are considered to be of taxonomic value (Camargo et al. 1987; Figueiredo et al. 1978; Yoshida et al. 1978). The squash isolate was negative for arginase and positive for OCT-a profile comparable to that described for Herpetomonas (Yoshida et al. 1978). Lack of arginase is a common feature of Phytornonas, Herpetomonas, and some Leptomonas species (Camargo et al. 1992; Camargo et al. 1987; Figueiredo et al. 1978). Activity of the OCT is particularly detected in Herpetomonas spp., but too few Phytomonas spp. have been examined to conclude that this feature is diagnostic for Herpetomonas (Camargo et al. 1987; Jankevicius et al. 1993). Thus, even though OCT activity is present in all Herpetomonas spp., this enzyme cannot be considered as an exclusive taxonomic feature of this genus. In order to assess the presence of taxonomic Phytomonas markers, we tested the squash isolate by IFA with a pool of MAbs (Sbravate et al. 1989; Teixeira and Camargo 1989) and with the spliced-leader gene-derived SL3' probe (Nunes et al. 1995; Serrano et al. 1999; Teixeira et al. 1996), which are specific for Phytomonas. In addition, we have also investigated the presence of a site 36OPvu IVSSU rRNA, which is present in Herpetomonas, Crithidia, and Leptomonas, but has never been detected in Phytomonas (Camargo et al. 1992; Teixeira et al.

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

1997). Results were negative for all tests, distinguishing the new isolate from all Phytomonas species, despite its plant origin. Therefore, based on morphological, biochemical, and molecular features, the squash flagellate cannot be considered as a Phytomonas species, whereas all data favor its assignment to the genus Herpetomonas. We have previously shown that a kDNA probe (Nunes et al. 1994) and the rRNA-derived SSU3 and SSU4 probes (Teixeira et al. 1997) divided species of the genus Herpetomonas into subgroups A and B, which are headed by H . samuelpessoai and H. muscarum, respectively. The DNA from the squash flagellate hybridized exclusively with the SSU3 probe, assigning this isolate to subgroup A of Herpetomonas. The size of the amplified ITS of the squash isolate was found to be similar to that of H. samuelpessoai, which itself differed from all other species of Herpetomonas (Takata et al. 1996). Moreover, restriction analysis of the amplified ITS could not distinguish these two organisms. Comparative analysis of RAPD patterns was performed to differentiate the squash isolate from H. samuelpessoai. Previous RAPD analysis differentiated most species within the genus Herpetomonas, particularly species of group A (Takata et al. 1996). which also proved to be more heterogeneous than group B based on ribosomal analysis (Hollar, Lukes, and Maslov 1998; Landweber and Gilbert 1994). Five out of sixteen primers generated fingerprints specific for the squash isolate, distinguishing this flagellate from H . samuelpessoai and suggesting that the new isolate is very closely related to but not identical to H. samuelpessoai. In addition to these genetic features shared by these two flagellates, the squash isolate could be cultivated at 37 "C, thus proving it to be a thermophilic organism, which is also a property of H . samuelpessoai but not of H. muscarum (Roitman et al. 1976). Despite its small molecular variability when compared with H. samuelpessoai, the new isolate has a morphological peculiarity unique among the known species of Herpetomonas: the presence of a thin, rod-shaped, lateral expansion of the plasma membrane. This unusual structure was only described (by light microscopy) previously in a Phytomonas sp. isolated from the latex of Allamanda cathartica (Kastelein and Parsadi 1984). Nevertheless, in contrast to the flagellate here described, this plant isolate presented biochemical and molecular characteristics of Phytomonas (Camargo et al. 1992; Teixeira et al. 1996). Considering the morphological, biochemical, and molecular evidence, it could be more appropriate to consider this isolate as a subspecies of H. sarnuelpessoai, as previously done for H. muscarurn muscarum and H. rnuscarum ingenoplastis, which have been considered to be subspecies based on the same host origin and distinct structures of the kinetoplast-mitochondrion complex (Rogers and Wallace 1971; Wallace, Wagner, and Rogers 1973) and in growth differences in the presence of oxygen and in the electron transport pathway, with H. m. ingenuplastis being particularly well-adapted for anaerobiosis (Redman and Coombs 1997). The flower-parasitizing trypanosomatid described here, besides being the first trypanosomatid cultured and characterized from flowers, represents the only record of ultrastructural, biochemical, and molecular descriptions of a new species of Herpetomonas. We propose that this isolate be considered a new subspecies of Herpetomonas samuelpessoai, which we name Herpetomonas sarnuelpessoai camargoi n. sp., in honor of Professor Erney Plessmann Camargo, a distinguished Brazilian protozoologist. ACKNOWLEDGMENTS This work was supported by CAPES, CNPq, FAPEMIG, FAPESP, FINEP, FIOCRUZ, PRONEX, and UNIFENAS.

LITERATURE CITED
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Received: 05-24-90, 05-23-00: u c n ~ p t e d OK-30-00