zyxwvutsrqponmlkjihgf zyxwvutsrqponmlkjih zyxwvutsrqponmlkjihg zyxwvutsrqponmlkj zyxwvutsrqponmlkjih J ProIii:oo/., 3h(3). 1989. pp 265-271 .c; 19RY by the Socieiy of Pro~ozoologir~s zyxw zyxwvu Life Cycle and Culturing of Phytomonas serpens (Gibbs), a Trypanosomatid Parasite of Tomatoes J. VITOR JANKEVICIUS,* SHIDUCA I. JANKEVICIUS,* MARTA CAMPANER,** IVETE CONCHON,** LUCIO A. MAEDA,* MARTA M. C. TEIXEIRA,** EDNA FREYMULI,ER** and E. PLESSMANN CAMARGO** *Deparramenro de Patologia Geral. Universidade Estadual de Londrina, 861 00, Londrina, PR, Brazil and **Parasiiologia. Insiituto de Ciencias BiomPdicas. C'niversidade de Siio Paulo. 05508, Siio Paulo, SP, Brazil ABSTRACT. Pure cultures of a trypanosomatid isolated from tomato fruits infected laboratory-raised tomatoes and nymphs of the hemipteran coreid Phfhiapicta.The flagellate could be transmitted back and forth from tomatoes to insects. Light and electron microscopy studies were done on culture. tomato and insect forms. Examination of enzymes of the ornithine-argininemetabolism revealed absence of arginase and presence of arginine deiminase and citrulline hydrolase. Monoclonal antibodies specific for Phytomonas spp. reacted positively with tomato and insect forms. Endonuclease digestion of the k - D N A of various Phytornonas spp. revealed a unique, distinctive pattern for the tomato flagellate. This flagellate thus seems 10constitute a separate species ofPhytornonas which we now call Phytornonas serpens (Gibbs). Key words. Arginase. arginine deiminase, citrulline, ocnithine-arginine metabolism. T RYPANOSOMATIDS of the genus Phytoinonas are etiological agents in devastating crop epiphytotics [9] but they also parasitize many plants without apparent pathogenicity [ 161. Phytomonads have also been reported in fruits [ 131 and legumes [ 17, 181. nothing being known about their pathogenicity there. The genus Phytonzonas was created by Donovan in 1909 [ 121 to accommodate trypanosomatid parasites in plants. Nevertheless, flagellates found in plants have still sometimes thereafter been assigned to Leptomonas or Herpetomontrs as was a parasite of tomatoes in South Africa, called Lepromonus serpens by Gibbs in 1957 [ 151. That flagellate, detected in the sap of tomatoes, infected the bug Nezara viridula. Cultivation, however, was not attempted, hence L. serpens never became available. Later, Podlipaev referred to the flagellate described by Gibbs as Phxlomonas serpens [2 I]. We here describe a flagellate from tomatoes in southern Brazil, its culturing, life cycle, presumed insect vector and transmission to clean tomatoes by the bite of the insect and, conversely, infection of insects upon feeding on contaminated tomatoes. Placement of the flagellate in the genus Phytornonas is our conclusion from the above evidence and also from data concerning the flagellate's biochemical constitution in enzymes of the ornithine-arginine metabolism [6] and reactivity with monoclonal antibodies specific for Phytomonas spp. [24]. MATERIALS AND METHODS Surveying of tomatoes. Fruits of Lycopersicon esrulention have been examined for flagellates. Tomatoes were picked in Rolandia, State of Parani. and Palmital, State of S5o Paulo, in southeastern Brazil. Fruits, leaves and stem were sectioned at the laboratory and their sap examined by phase microscopy. Cultures. The sap of positive fruits or organs of infected insects were inoculated in tubes containing 3 ml of a biphasic medium made of 2% agar base and rabbit blood cells plus 2 rnl of an overlay of Roitman's defined medium [ 2 3 ] containing 20 pg/nil ofampicillin. Positive cultures were cloned on blood-agar plates. Selected clones were thereafter kept on the biphasic medium. In this report, 3 cloned strains have been studied: I from tomatoes from Rolandia, strain 9T; I from Palmital, strain IOT; and 1 strain from an artificially infected insect, strain 1 5 1 . When desired, flagellates were also cultured in the liquid medium LIT [41. Cultures of flagellates from Euphorbia piwu and Jathropa macranrha were kindly given to us by Dr. M. Dollet and the isolate from E. hyssoppifolia was given by Dr. W. de Souza. These flagellates were also cultured in biphasic medium. Raising of insects and tomatoes. Tomato-plants were grown from seed in the laboratory in insect-proof cages. Adults of Phthia picla (Hemiptera, Coreidae) were collected on tomatoes in the field and bred in the laboratory. Eggs were transferred to cages containing tomatoes grown in the laboratory. Resulting nymphs and adults were kept under protected conditions and always fed on laboratory-grown tomatoes. Light and electron microscopy. Smears fixed in methanol and stained by Giemsa were used for light microscopy and drawings by camera lucida. For electron microscopy (EM) studies, salivary glands and segments of the digestive tube of P. picta and flagellates from cultures pelleted at 2,000 g for 5 min were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. at 4" C for 2 h. After several rinses in buffer, the preparations were post-fixed in 1% osmium tetroxide for 1 h at 25" C and stained overnight at 4" C in 0.5% aqueous uranyl acetate. Dehydration, embedding, sectioning and examination of the preparations were done as before [ 141. Enzyme assays. Determination of activity of arginase, citrulline hydrolase and arginine deiminase in homogenates of cultures derived from insects was done exactly as before for flagellates from plants [6]. Monoclonal antibodies assay. Reactivity towards monoclonal antibodies was determined by indirect immunofluorescence using as antigens flagellates recovered from artificially infected tomatoes or from cultures in LIT medium and fixed in 1% formaldehyde. Monoclonal antibodies were also tested against squashes of salivary glands and digestive tubes of artificially infected insects, dried on glass slides and fixed with formaldehyde. Restriction endonuclease digestion of k-DNA. Preparation of k-DNA and its digestion with restriction endonucleases were done as described [5]. Electrophoresis of the k-DNA fragments was performed on 2% agarose gels. zyxwvu RESULTS Isolation and culturing of flagellates. Ripe fruits of Lycopersicon esculentum displaying tiny yellow spots on the surface-indicative of insect biting-stood a better chance of harboring flagellates. In some instances, up to 50% of the fruits of the variety sherry were infected in backyard gardens in Rolandia County. Stems and leaves of tomato plants were never found infected. Nymphs and adults of the coreid Phthia picta were very often found feeding on tomatoes in backyard gardens. In some areas all insects harbored flagellates in the digestive tube. In commercial crops, insects were rare, likewise in infected tomatoesa fact probably related to the use of insecticides. Under light microscopy of the fruit sap, small flagellates were 265 266 zz zyxwvutsrqpo zyxwvutsrqp zyxwv g - zy J. PROTOZOOL., VOL. 36, NO. 3, MAY-JUNE 1989 Q ) 10 10 p m n 11 - 15 10 prn Fig. 1. Phytomonas serpens, strain 9T, in tomatoes and culture media: 1-10, flagellates of tomatoes; 11-15, flagellates of biphasic medium; 16-2 1, flagellates of old cultures in LIT medium. detected, mostly immobile; some presenting a rudimentary external flagellum and some with no flagellum at all as depicted in Fig. 1. Inoculation of fruit sap into the biphasic culture medium yielded rich cultures, 2 of them (9T and 10T) utilized in this study. The 2 strains seem essentially identical. They could be grown either in the biphasic or liquid LIT medium, temperature optimum 28" C. In biphasic medium, cultures grew better when the pH of the liquid overlay was acidic (down to - zyxwvutsrq z zyxwvutsrqp zyxwvuts 26 7 JANKEVICIUS ET AL.-PHYTOMONAS SERPENS IN TOMATOES zyxwvut zyxwvuts zyxwvuts Table 1. Measurements (pm) of tomato, insect and culture forms of Phytornonas serpens. Parasites present in Body length Body width ~~ Tomato Culture (9 T) Salivary glands Digestive tract (A) (B) (A) (B) 6 . 9 " f 1.4b (4.3-11.4y 17.7 f 7.0 (8.4-59.3) 5.7 k 0.9 (4.0-7.7) 24.0 17.3(7.9-79.1) 4.1 f 0.8 (2.9-4.9) 16.6 f 4.3 (9.8-26.9) 5.6 -C 1.0 (4.1-7.7) * 1.6 2.2 1.8 2.1 2.0 2.2 1.8 f 0.2(1.1-2.3) f 0.5 (1.2-3.5) i 0.3 (1.4-2.6) k 0.4(1.4-2.8) f 0.5 (1.3-2.4) f 0.4 (1.3-2.9) Flagellum length Distance from nucleus to anterior end Distance from kinetoplast to anterior end 0.6 f 2.9(0-4.3) 15.8 f 7.0 (0-26.3) 0.2 f 0.5 (0-2.1) 5.9 7.8(0-33.5) 1.8 k 3.7 (0-7.3) 13.3 f 6.6 (0-31.3) 0 1.9 f O.S(l.1-3.9) 5.5 2.3 (2.6-22.3) 2.2 f 0.4 (1.2-3.0) 4.5 f 4.5(0.9-20.7) 1.6 k 0.3 (1.3-2.0) 5.6 1.8 (2.7-10.9) 2.9 f 0.9 (1.44.8) 0.8 f 0.4(0-2.3) 2.1 f 1 . 1 (1.2-8.6) 1.3 k 0.5 (0.7-3.5) 2.3 k l.S(l.2-7.0) 0.9 f 0.3 (0.7-1.3) 2.7 k 1.3 (0-6.9) 1.9 f 1.0 (0.2-3.2) * * * f 0.3 (1.4-2.5) Urine In each case measurements correspond to the mean value p), standard deviation (b) and range c) of 50 randomly selected flagellates. In salivary glands and digestive tract of insects, the flagellates were grouped according to their size in 2 distinct populations (A and B). Measurements of body length do not include the flagellum. pH 3.5). The average generation time of a culture in biphasic medium at 28" C and pH 6 was 14 h, and maximum cell concentration was 1.2 x lo8 flagellatedml. Flagellates from culture 9T are depicted in Fig. 1. Due to the great variation in size of culture forms (Table l), we found it more informative to consider the measurements of 2 arbitrarily chosen sub-populations, 1 of small flagellates and the other of large ones. Regardless of their size, flagellates were always of the promastigote type but some of them, besides being very large, presented 2 to 4 body torsions. The percentage of twisted-body promastigotes in a culture seemed to depend on medium composition and culture age, being noticeably more abundant in old cultures in LIT medium where they could account for up to 20-30% of the total forms in the culture. We have been unable to trace any developmental relationship between the forms appearing in culture media, hence the precise sequence of events leading to the marked pleomorphism of the flagellate's life cycle remains obscure. That polymorphism did actually result from mixed cultures of different organisms is rendered unlikely by the cloning and recloning of strain 9T. Opisto- or trypomastigotes have never been found in cultures or in the flagellate hosts. Experimental infection of insects. Nymphs and adults of P. picta raised from eggs and fed on clean tomatoes were always free of flagellates. When the insects were fed on tomatoes which were either naturally or artificially infected, they became infected after 10-1 5 days, displaying flagellates in feces, urine, digestive tube and salivary glands. Insect forms, which exhibit great polymorphism, are sketched in Fig. 2; measurements are given in Table 1. Experimental infection of tomatoes. Tomato plants grown from seed under protected laboratory conditions always produced flagellate-free fruits. When desired, fruits were infected either through needle inoculation of culture forms or through bite of infected insects. Tomatoes could be easily infected either way. Once infected, they so remained until rotting. The flagellates multiplied actively in the fruits' sap: inoculation of 5 x 1O5 flagellates yielded in 10 days a n average of 1 x 1O9 organisms per fruit. Figure 1 depicts the forms found in tomatoes artificially infected. Life cycle. As noted, flagellates could be readily transmitted from tomatoes to insects and back to tomatoes. They were also culturable from insects or tomatoes. Culture forms, in turn, could also infect tomatoes or insects. In evey case the flagellates recovered were of the promastigote type, displaying the same kind of polymorphism as depicted in Fig. 1 and 2. Electron microscopy. Electron-micrographs of flagellates from cultures in biphasic medium are presented in Fig. 3-5. The culture forms exhibited the standard characteristics of a trypanosomatid without obvious peculiarities, except that mito- chondrial cristae were very scarce while the mitochondria1 matrix was very dense-a feature displayed also by other phytomonads [2]. Also in common with other phytomonads was the abundant presence of lipid droplets in the cytoplasm of culture forms. Some of the small forms found in cultures seemed to totally lack a flagellum, including its intracellular portion (not shown) whereas others exhibited a rudimentary one, barely noticeable by light microscopy (Fig. 4). This rudimentary flagellum seen by EM revealed the regular organization of a normal flagellum, except for absence of the intraflagellar structure (IFS) present in the larger forms as well as in most trypanosomatid species [ 141. The external tip of the flagellum of the small forms had a dense, non-fibrilar material (Fig. 5) not yet reported in trypanosomatids. The lumen of the salivary glands were found to be intensely populated by flagellates which were also detected in the cytoplasm of the acinar cells. A morphological study of the salivary forms will be published elsewhere. Reactivity with monoclonal antibodies. Monoclonal antibodies specific for Phytomonas spp. [24] always yielded positive results when tested against flagellates from insects, from cultures (9T, 10T, 15T) or from tomatoes artificially infected with strains 9T and 10T. Endonuclease digestion of k-DNA. Digestion of the k-DNA of different phytomonad isolates revealed marked differences among them. When digested with the 6-cutter enzymes BamHI and EcoRI, the k-DNA of the flagellates from tomato and E . pinea failed to produce fragments. However, EcoRI readily digested the k-DNA of the isolates from J. macanthra and E . hyssopifolia, yielding fragments smaller than 2 kb. Upon digestion with the 4-cutter enzyme HpaII, k-DNA of the flagellate from E. pinea produced a single fragment the length of a minicircle, as similarly reported by Riou [22]. The k-DNA of the tomato flagellate also produced a single fragment but apparently with half the size of a minicircle, suggesting the existence of 2 symmetrical restriction sites for this enzyme at minicircle length. Digestion of the k-DNA of the other flagellates produced various fragments of different sizes. Digestion of the k-DNA of the various flagellates with Sau3A produced fragments with organism-specific electrophoretic patterns (Fig. 6 ) . For the tomato flagellate, digestion with Sau3A produced a single fragment with apparently half the size of a minicircle- again suggestive of the existence of 2 symmetrical sites for this enzyme also. zyxwvutsrqpo DISCUSSION Cultures of trypanosomatids from plants are recent [3, 10, 19, 251; and until now cultures of phytomonads from vector insects were not available. Lack of isolates and cultures of Phytomonas 268 zyx zyxwvutsrqpo zyxwvutsrqponmlk zyxwvu zyxwvu J . PROTOZOOL., VOL. 36, NO. 3, MAY-JUNE 1989 . 10 p m 8 8 8 0 8 $ ....... ...: .. .. .. ... c 10 Nrn zy zyxwvutsrqponml 30 Fig. 2. Phytomonas serpens. strain 9T, in the insect Phthia picta: 1-15, flagellates of urine; 16-30, flagellates from the digestive tract. + Fig. 3-5. Thin sections of culture forms of Phytomonas serpens. 3. Longitudinal section of the predominant culture form in biphasic medium, Longitudinal section of the smali culture form, x 20,000. 5. Longitudinal section of the flagellum of the small culture form, x 80,000, The arrow points to the tip of the flagellum. K: kinetoplast; N: nucleus; F: flagellum; M: mitochondrion. x 20,000. 4. zyxwvutsrqp zyxwvutsr zyxwvutsrq zyxw JANKEVICIUS ET AL. -PHYTOMONAS SERPENS IN TOMATOES 269 270 zyxwvutsr zyxwvutsrqponmlk zyxwvutsr zyxwvu z J. PROTOZOOL., VOL. 36, NO. 3, MAY-JUNE Fig. 6. Electrophoretic patterns of k-DNA fragments of different Phytomonas spp. digested with the Sau3A restriction endonuclease. 1. MW markers (A DNA digested with HindIII); 2. Phytomonas serpens; 3. Phytomonas sp. from Euphorbia pinea; 4. Phytomonas sp. from E. characias;5. Phytomonas sp. from Jathropa macrantha ;6. Phytomonas sp. from E. hyssopifolia. spp. had precluded adequate studies of the biochemical and morphological characteristics of this group of flagellates. Only recently have biochemical and EM comparative studies been done between species of Phytomonas and other Trypanosomatids [2, 3, 6, 11, 20, 261 and monoclonal antibodies raised to identify phytomonads [24]. As noted, a trypanosomatid from tomatoes had been reported much earlier [ 151. This flagellate could have been placed from the outset in Phytomonas, for, by the prevailing criterion at the time, plant parasitism sufficed to define this genus. However, Conchon et al. [7] have shown that flagellates of other genera of Trypanosomatidae can also thrive on tomatoes and then infect insects. No longer then is it to be taken for granted that trypanosomatids encountered in plants necessarily belong to Phytomonas. Utilization of additional criteria is now essential. Accordingly, morphological, biochemical and immunological studies were conducted on the forms derived from tomatoes, insects and cultures throughout the flagellate’s life cycle. Although there is no description of the complete life cycle and biochemical characteristics of another Phytomonas sp. to compare with the tomato flagellate, the following facts may justify calling this flagellate a Phytomonas: a) its life cycle comprises a plant and an insect; b) only promastigotes are found throughout the flagellate’s life cycle; c) its enzymic constitution in respect to ornithine-arginine metabolism conforms to that of other Phytomonas spp.; and d) all developmental forms of the 1989 flagellate are recognized by monoclonal antibodies specific for Phytomonas. Since the k-DNA endonuclease digestion patterns of the tomato flagellate are clearly distinct from the patterns of other Phytomonas spp., we may denote it as a separate species. Although there is no way to tell whether the present isolate indeed corresponds to Gibb’s Leptomonas serpens, we retain the species name as Phytomonas serpens as recognition of the historical link between the 2 flagellates, thereby honoring the pioneering work of Gibbs. That P. serpens was originally isolated from tomato fruits does not rule out the possibility of other plants harboring it. As for its transmission to tomatoes, the hemipteran coreid Phthia picta seems a good candidate for being its natural vector: it usually feeds on tomatoes, is frequently found in nature infected with promastigotes positive for Phytomonas-specific monoclonals [Camargo et al., unpubl.] and under laboratorial conditions was easily infected with P. serpens and thereafter able to transmit the infection to clean tomatoes. But it may not be the sole vector of P. serpens: other insects also feed on tomatoes and may serve to disseminate the parasite. Actually, preliminary experiments suggest that the bug Nezara viridula is also easily infected with culture forms of the flagellate. Thus, considering the large variety of insects that feed on tomatoes in Brazil [I, 81, and that these insects may feed alternatively on different fruits, P. serpens may well be discovered in other plants and insects. Availability of cultures and of biochemical markers for the identification of P. serpens now permits the exploration of this possibility. As for a possible pathogenicity of P. serpens to tomatoes, we have not detected any damaging effect to fruits or plants; but this may merely reflect lack of expertise there. ACKNOWLEDGMENTS This work was supported by the Brazilian agencies FAPESP, FINEP, CPG/UEL and Secretaria de CiCncia e Tecnologia, SP, and by The Commission of European Communities. LITERATURE CITED 1. Araujo e Silva, A. G., Gonqalves, C. R., Galvlo, D. M., Gonqalves, A. J. L., Gomes, J., Silva, M. N. & Simoni, L. 1968. Quarto cat&logo dos insetos que vivem nas plantas do Brasil. Laboratbrio Central de Patologia Vegetal, Ministtrio da Agricultura, Rio de Janeiro. 2. Attias, M, Roitman, I., Camargo, E. P., Dollet, M. & Souza, W. 1988. Comparative analysis of the fine structure of four isolates of trypanosomatids of the genus Phytomonas. J. Protozool., 39365-370. 3. Attias, M. & de Souza, W. 1986. Axenic cultivation and ultrastructural study of a Phytomonas sp. isolated from the milkweed plant Euphorbia hyssopifolia. J. Protozool., 33:84-87. 4. Camargo, E. P. 1964. Growth and differentiation of Trypanosoma cruzi. I. Origin of metacyclic trypanosomes in liquid medium. Rev. Inst. Med. Trop. Scio Paulo, 6:93-100. 5. Camargo, E. P., Mattei, D. M., Barbieri, C. L. & Morel, C. M. 1982. Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids. J. Protozool., 29:25 1258. 6. Carnargo, E. P., Silva, S., Roitman, I., Souza, W., Jankevicius, J. V. & Dollet, M. 1987. Enzymes of ornithine-arginine metabolism in trypanosomatids of the genus Phytomonas. J. Protozool., 34:439441. 7. Conchon, I., Campaner, M., Sbravate, C. & Camargo, E. P. 1989. Trypanosomatids, other than Phytomonas spp., isolated from fruits. J. Protozool., 36. (in press) 8. Costa Lima, A. 1940. Insetos do Brasil. Escola Nacional de Agronomia, Sene Didatica 6 , Rio de Janeiro. 9. Dollet, M. 1984. Plant diseases caused by flagellate protozoa (Phytomonas).Annu. Rev. Phytopathol., 22: 115-132. zyxwv zy z zyxwvutsrqp zyxwvut zyxwvutsrq zyxwvutsrqpo JANKEVICIUS ET AL.-PHYTOMONAS SERPENS IN TOMATOES 10. Dollet, M., Cambrony, D. & Gargani, D. 1982. Culture ax& nique “in vitro” de Phytomonas sp. (Trypanosomatidae) d‘Euphorbe, transmis par Stenocephalus agilis Scop. (Coreidae). C. R. Acad. Sci., ser. 111, 295547450. 1 I. Dollet, M. & Wallace, F. G. 1987. Compte rendu du premier Phytomonas workshop-Cayenne, Mars 1987. Olkagineux, 42:46 1465. 12. Donovan, C. 1909. Kala-azar in Madras, specially with regard to its connection with the dog and the bug (Conorhinus). Lancet, i: 14951496. 13. Fiorini, J. E., Silva, F. P. M., Brasil, R. P., Roitman, I., Angluster, J., Souza, W. & Esteves, M. J. G. 1986. Detection oftrypanosomatids in the Solanum giro and Solanum lycopersicon in Alfenas, MG, Brazil. Mern. Inst. Oswaldo Cruz, 81(Suppl.):33. 14. Freymuller, E. & Camargo, E. P. 1981. Ultrastructural differences between species of trypanosomatids with and without endosymbionts. J. Protozool., 28:175-182. 15. Gibbs, A. J. 1957. Leptomonas serpens n. sp. parasitic in the digestive tract and salivary glands of Nezara viridula (Pentatomidae) and in the sap of Solanum lycopersicum (tomato) and other plants. Parasitology. 47:297-303. 16. Holmes, F. 0. 1925. Non-pathogenicity of the milkweed Aagellates in Maryland. Phytopathology, 22: 1 15-1 32. 17. Jankevicius, S. I., Jankevicius, J. V., Menezes, M. C. M. D., Lima, H. & Menezes, J. R. 1987. Presence of protozoa of the genus Phytomonas in leguminous crops. Brazil. Phytopathol., 12: 152. 18. Jankevicius, S. I., Jankevicius, J. V., Menezes, M. C. M. D., Torrezan, H. C., Menezes, J. R. & Rezende, M. I. 1987. Phytomonas sp. found in leguminous crops. Mern. Inst. Oswaldo Cruz, 82(Suppl.): 35. 27 1 19. Kastelein, P. & Parsadi, M. Observations on cultures of the protozoa Phytomonas sp. (Trypanosomatidae) associated with the lactifer Allamanda cathartica L. (Apocynaceae). De Surin. Landb., 32%89. 20. Petry, K., Chottelius, J. & Dollet, M. 1987. Differentiation of Phytomonas sp. and lower trypanosomatids (Herpetomonas, Crithidia) by agglutination tests with lectins. Parasitol. Res., 74: 1-4. 2 1. Podlipaev, S. A. 1986. Phytomonas elmassiani (Mastigophora: Trypanosomatidae) from the plant Cynanchum sibiricum (Asclepiadaceae) in Central Asia and Kazakhstan. Proc. Zool. Inst. Acad. Sci. USSR, 144~61-65. 22. Riou, J. F., Dollet, M., Ahomadegbe, J. G., Coulaud, D. & Riou, G. 1987. Characterization ofPhytomonas sp. kinetoplast DNA, a plant pathogenic trypanosomal species. FEBS Letters, 213:304-308. 23. Roitman, C., Roitman, I. & Azevedo, H. P. 1972. Growth of an insect trypanosomatid at 37°C in a defined medium. J. Protozool., 19:346-349. 24. Teixeira, M. M. G. & Camargo, E. P. 1989. Monoclonal antibodies for the identification of trypanosomatids of the genus Phytomonas. J. Prolozool., 36262-264. 25. Vainstein, M. H. & Roitman, I. 1986. Cultivation of Phytomonas franqai associated with poor development of root system of cassava. J. Protozool., 33:511-513. 26. Vainstein, M. H., Silva, J. B. T., Lima, V. M. Q. G., Roitman, I., Souza, W., Dollet, M. & Camargo, E. P. 1987. Electrophoretic analysis of isoenzymes in the identification of trypanosomatids of the genus Phytomonas. J. Protozool., 24:442-444. zyxwv zyxwvutsrqponmlkjih zyxwvutsrqponmlkj zyxwvutsrqpo J. Prolozool.. 36(3), 1989, pp. 271-274 0 1989 by the Society of Protozoologists Coccidian Parasites (Apicomplexa: Eimeriidae) of Nevodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria CHRIS T. McALLISTER* and STEVE J. UPTON** *Renal-Metabolic Lab (151-G), Veterans Administration Medical Center, 4500 S. Lancaster Road, Dallas, Texas 75216 and Department of Biological Sciences, University of North Texas, Denton, Texas 76203 and **Division of Biology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506 ABSTRACT. Eimeria conanti n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N. harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape, 17.9 x 13.0 (15-2 1 x 1215) pm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2-1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyle i s absent. Sporocysts are elongate, 12.9 x 5.2 (13-15 x 5-6) pm, apparently without a true Stieda body structure. Each sporocyst contains an ellipsoid residuum, 3.9 x 3.2 (3-6 x 2 4 ) prn. and elongate sporozoites, 11.4 x 2.5 (10-14 x 2-3) prn in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts. Key words. Coccidia, Cryptosporidium sp., Eimeria spp., Eimeria conanti n. sp., eimerians, survey, water snakes. T HE blotched water snake, Nerodia erythrogaster transversa (Hallowell, 1852), is a moderately large semiaquatic colubrid commonly found from western Arkansas and Missouri westward to Kansas and south through central Texas to northeastern MCxico [3]. This taxon inhabits various aquatic sites ranging from semipermanent ditches and cattle tanks to permanent rivers and tributaries. On the other hand, the Brazos water snake, N. harteri harteri (Trapido, 194 l), a protected subspecies of Harter’s water snake, is a relatively small colubrid which is locally abundant but restricted to swift-flowing, rocky streams in the upper Brazos River watershed of 10 counties in northcentral Texas [ 5 , 71. Interestingly, both N. e. transversa and N. h. harteri, as well as the diamondback water snake, N. rhomblfera rhombifera, are sympatric at various localities along the Brazos River. Recently, Upton & McAllister [9] described 3 new species of Eimeria (Apicomplexa: Eimeriidae) from N . r. rhombifera and Upton et al. [ 101added an unnamed species of Cryptosporidium to the list of coccidial parasites harbored by N. r. rhombifera and N. h. harteri. There is a plethora of information on helminth parasites of N. erythrogaster [ I , 2, 4, 8 and others]; however, to our knowledge, N . e. transversa has not been previously surveyed for coccidia. In addition, other than the report of Cryptosporidium sp. in a single N. h. harteri [lo], nothing is known about its eimerian parasites. Between July 1987 and October 1988, we examined 37 Ne-